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ABSTRACT: In the present study, we report a novel separation-free method to detect and quantify avian influenza virus A (H5N1) nucleic acid without amplification, based on the alteration of photophysical parameters of quantum dot (QD) probes after hybridization with specific complementary target DNA. The target DNA was quantified in a custom-made portable device by simultaneously measuring lifetime and quenching of the QD probes. QD probes (25-mer) showed a 30% lifetime reduction and 40% fluorescence quenching when hybridized with complementary 25-mer target DNA. In comparison with a conventional QD-based assay, this assay provides a simple quantitation of nucleic acids with a single labeling step.
Analytical Chemistry 02/2010; 82(3):886-91. · 5.86 Impact Factor
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ABSTRACT: The human immunodeficiency virus (HIV) pandemic mainly affects developing countries, where the Joint United Nations Programme on HIV/AIDS (UNAIDS) estimates suggest that less than 1 in 10 people are aware of their HIV sero-status. In order to enhance epidemiological surveys, prevention programs, and therapeutic interventions, development of specific, rapid, and convenient diagnostic detection systems is still warranted. Here we report the direct detection of HIV particles using broadly HIV-1 neutralizing gp120 monoclonal antibody (gp120MAbs)-conjugated magnetic beads (MBs) and fluorescent nanosized polymeric beads (FNBs). The HIV-1 envelope glycoprotein gp120 is anchored to the viral surface through gp41 and mediates entry into target cells by interaction with the main cellular receptor (CD4) and coreceptors (e.g., CCR5 and CXCR4). FNBs conjugated to gp120MAbs (gp120MAbs-FNBs) were used to generate fluorescent signals, whereas MBs conjugated to gp120MAbs (gp120MAbs-MBs) were employed to isolate HIV-1 particles. In presence of HIV-1 particles, addition of gp120MAbs-FNBs and gp120MAbs-MBs leads to the formation of a MBs/HIV-1 particles/FNB complex, which can be easily isolated and concentrated by common magnet separation. We demonstrate the ability of detecting HIV-1 particles specifically and directly using MBs and FNBs with low sample volume (less than 100 microL) and rapidity (less than 1.5 h) without any pretreatment of test samples. The specific binding of FNBs with HIV-1 particles on the surface of MBs was confirmed by fluorescence microscopy and fluorescence-activated cell sorting (FACS). Imaging and FACS analysis revealed the specific and quantitative detection of HIV-1 particles. These results provide proof-of-principle that broadly HIV-1 neutralizing gp120 antibodies coupled to nanobeads can be employed for the direct detection of HIV-1 particles with potential implication for the development of specific, rapid, and convenient diagnostic systems.
Analytical Chemistry 03/2009; 81(6):2388-93. · 5.86 Impact Factor
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ABSTRACT: In the present study we describe sandwich design hybridization probes consisting of magnetic particles (MP) and quantum dots (QD) with target DNA, and their application in the detection of avian influenza virus (H5N1) sequences. Hybridization of 25-, 40-, and 100-mer target DNA with both probes was analyzed and quantified by flow cytometry and fluorescence microscopy on the scale of single particles. The following steps were used in the assay: (i) target selection by MP probes and (ii) target detection by QD probes. Hybridization efficiency between MP conjugated probes and target DNA hybrids was controlled by a fluorescent dye specific for nucleic acids. Fluorescence was detected by flow cytometry to distinguish differences in oligo sequences as short as 25-mer capturing in target DNA and by gel-electrophoresis in the case of QD probes. This report shows that effective manipulation and control of micro- and nanoparticles in hybridization assays is possible.
Sensors 01/2009; 9(7):5590-9. · 1.74 Impact Factor
