Article

Cell-surface sensors for real-time probing of cellular environments.

Center for Regenerative Therapeutics & Department of Medicine, Brigham & Women's Hospital, 65 Landsdowne Street, Cambridge, Massachusetts 02139, USA.
Nature Nanotechnology (Impact Factor: 31.17). 07/2011; 6(8):524-31. DOI: 10.1038/nnano.2011.101
Source: PubMed

ABSTRACT The ability to explore cell signalling and cell-to-cell communication is essential for understanding cell biology and developing effective therapeutics. However, it is not yet possible to monitor the interaction of cells with their environments in real time. Here, we show that a fluorescent sensor attached to a cell membrane can detect signalling molecules in the cellular environment. The sensor is an aptamer (a short length of single-stranded DNA) that binds to platelet-derived growth factor (PDGF) and contains a pair of fluorescent dyes. When bound to PDGF, the aptamer changes conformation and the dyes come closer to each other, producing a signal. The sensor, which is covalently attached to the membranes of mesenchymal stem cells, can quantitatively detect with high spatial and temporal resolution PDGF that is added in cell culture medium or secreted by neighbouring cells. The engineered stem cells retain their ability to find their way to the bone marrow and can be monitored in vivo at the single-cell level using intravital microscopy.

0 Bookmarks
 · 
306 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Many types of separation-based sensing systems have been reported for biotin assays. Although these conventional strategies provide an accurate and sensitive detection of biotin, there are still some inconveniences that exist, such as the complex sample treatment, time-consuming assay process, and technical expertise as well as the sophisticated equipment. We have addressed these limitations and report herein the proof-of-principle of a dual strategy which combines magnetic separation-enrichment with DNA-SG (SYBR Green I)-based signal amplification to develop a simple and sensitive fluorometric biotin sensing strategy. This method is based on the competition scheme where biotin and the biotinylated dsDNA compete for the binding sites of streptavidin coated on magnetic bead (SA-MB) surfaces. After separation and enrichment under the magnetic field, the fluorescence emission intensity or fluorescent images can be obtained by addition of SG, which is inversely related to biotin concentrations. Using the biotin solution as a model system, we demonstrated that our assay can detect biotin at a concentration as low as 1.19 ng mL−1 in one hour which highly excels traditional assays such as HPLC. Moreover, we also used the proposed method to measure the biotin level in actual samples, for example flour and peanuts, for which satisfactory results were obtained.
    Analytical methods 01/2014; 6(7):2091. DOI:10.1039/c3ay42128g · 1.94 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Biomolecular interactions have important cellular implications, however, a simple method for the sensing of such proximal events is lacking in the current molecular toolbox. We designed a dynamic DNA circuit capable of recognizing targets in close proximity to initiate a pre-programmed signal transduction process resulting in localized signal amplification. The entire circuit was engineered to be self-contained, i.e. it can self-assemble onto individual target molecules autonomously and form localized signal with minimal cross-talk. α-thrombin was used as a model protein to evaluate the performance of the individual modules and the overall circuit for proximity interaction under physiologically relevant buffer condition. The circuit achieved good selectivity in presence of non-specific protein and interfering serum matrix and successfully detected for physiologically relevant α-thrombin concentration (50 nM-5 μM) in a single mixing step without any further washing. The formation of localized signal at the interaction site can be enhanced kinetically through the control of temperature and probe concentration. This work provides a basic general framework from which other circuit modules can be adapted for the sensing of other biomolecular or cellular interaction of interest.
    Nucleic Acids Research 07/2014; 42(14). DOI:10.1093/nar/gku655 · 8.81 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cell membrane-anchored biochemical sensors that allow real-time monitoring of the interactions of cells with their microenvironment would be powerful tools for studying the mechanisms underlying various biological processes, such as cell metabolism and cell signaling. Despite the significance of these techniques, unfortunately, their development has lagged far behind due to the lack of a desirable membrane engineering method. In this work, we proposed a simple, efficient, biocompatible and universal strategy for one-step self-construction of cell-surface sensors using diacyllipid-DNA conjugates as the building and sensing elements. The sensors exploit the high membrane-insertion capacity of a diacyllipid tail and good sensing performance of the DNA probes. Based on this strategy, we have engineered specific DNAzymes on the cell membrane for metal ion assay in the extracellular microspace. The immobilized DNAzyme showed excellent performance for reporting and quantifying both exogenous and cell-extruded target metal ions in real time. This membrane-anchored sensor could also be used for multiple target detection by having different DNA probes inserted, providing potentially useful tools for versatile applications in cell biology, biomedical research, drug discovery and tissue engineering.
    Journal of the American Chemical Society 09/2014; 136(38). DOI:10.1021/ja5047389 · 11.44 Impact Factor

Full-text (3 Sources)

Download
102 Downloads
Available from
Jun 4, 2014