Alina Munteanu

University of Florida, Gainesville, Florida, United States

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Publications (3)14.43 Total impact

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    ABSTRACT: Study of the density, spatial distribution, and molecular interactions of receptors on the cell membrane provides the knowledge required to understand cellular behavior and biological functions, as well as to discover, design, and screen novel therapeutic agents. However, the mapping of receptor distribution and the monitoring of ligand-receptor interactions on live cells in a spatially and temporally ordered manner are challenging tasks. In this paper, we apply fluorescence correlation spectroscopy (FCS) to map receptor densities on live cell membranes by introducing fluorescently marked aptamer molecules, which specifically bind to certain cell-surface receptors. The femtoliter-sized (0.4 fL) observation volume created by FCS allows fluorescent-aptamer detection down to 2 molecules and appears to be an ideal and highly sensitive biophysical tool for studying molecular interactions on live cells. Fluorophore-labeled aptamers were chosen for receptor recognition because of their high binding affinity and specificity. Aptamer sgc8, generated for specific cell recognition by a process called cell systematic evolution of ligands by exponential enrichment, was determined by FCS to have a binding affinity in the picomolar range (dissociation constant K(d)=790+/-150 pM) with its target membrane receptor, human protein tyrosine kinase-7 (PTK7), a potential cancer biomarker. We then constructed a cellular model and applied this aptamer-receptor interaction to estimate receptor densities and distributions on the cell surface. Specifically, different expression levels of PTK7 were studied by using human leukemia CCRF-CEM cells (1300+/-190 receptors microm(-2)) and HeLa cervical cancer cells (550+/-90 receptors microm(-2)). Competition studies with excess nonlabeled aptamers and proteinase treatment studies proved the validity of the density-estimation approach. With its intrinsic advantages of direct measurement, high sensitivity, fast analysis, and single-cell measurement, this FCS density-estimation approach holds potential for future applications in molecular-interaction studies and density estimations for subcellular structures and membrane receptors.
    No preview · Article · May 2009 · Chemistry - A European Journal
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    ABSTRACT: A simple and portable flow channel optical detection system combined with bioconjugated luminescent nanoparticles allows the rapid detection of single bacterial cells without sample enrichment. The optical system is designed to have single-molecule-detection capability in a microcapillary flow channel by decreasing the laser excitation probe volume to a few picoliters, which consequently results in a low background. Specific monoclonal antibodies were immobilized on nanoparticles to form nanoparticle-antibody conjugates. The bioconjugated nanoparticles bind to the target bacteria when they recognize the antigen on the bacterium surface, thus providing a bright luminescent signal for the detection of individual bacteria cells. The high sensitivity provided by the luminescent and photostable silica nanoparticles eliminates the need for further enrichment of bacteria samples and signal amplification. This flow channel detection system is convenient and allows the detection of single bacterial cells within a few minutes.
    No preview · Article · Oct 2006 · Chemistry - An Asian Journal
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    ABSTRACT: Molecular beacons (MBs) are hairpin-shaped oligonucleotides that contain both fluorophore and quencher moieties. They act like switches and are normally in a closed state, when the fluorophore and the quencher are brought together to turn "off" the fluorescence. When prompted to undergo conformational changes that open the hairpin structure, the fluorophore and the quencher are separated, and fluorescence is turned "on." This Education will outline the principles of MBs and discuss recent bioanalytical applications of these probes for in vitro RNA and DNA monitoring, biosensors and biochips, real-time monitoring of genes and gene expression in living systems, as well as the next generation of MBs for studies on proteins, the MB aptamers. These important applications have shown that MBs hold great potential in genomics and proteomics where real-time molecular recognition with high sensitivity and excellent specificity is critical.
    Full-text · Article · Aug 2005 · The Analyst