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. 2009;9(7):5590-9.
doi: 10.3390/s90705590. Epub 2009 Jul 14.

Quantitative analysis of nucleic Acid hybridization on magnetic particles and quantum dot-based probes

Sun Hee Lim  1 , Felix Bestvater, Philippe Buchy, Sek Mardy, Alexey Dan Chin Yu
Affiliations

Quantitative analysis of nucleic Acid hybridization on magnetic particles and quantum dot-based probes

Sun Hee Lim et al. Sensors (Basel). 2009.

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.

Keywords: DNA; flow cytometry; fluorescence microscopy; hybridization; magnetic particles; quantum dots.

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Figures

Figure 1.
Figure 1.
Schematic procedure of the MP and QD-based hybridization assays.
Figure 2.
Figure 2.
Oligo binding on the surface of MPs and hybridization of the MP probes and target strand. A. Different quantities of oligos with varying lengths of spacers from 0 to 54 chains were conjugated to MPs. After conjugation, they were stained with OliGreen and analyzed by flow cytometry. B. Hybridization of 100-mer target DNA and MP probes. In analogy to A, PicoGreen fluorescence was measured by flow cytometry and plotted as a function of target strand amount.
Figure 3.
Figure 3.
Mobility shift assay of QD probes and DNA hybridization. M: 100 bp ladder, 1: biotinylated 25-mer oligo, 2: QD605 streptavidin, 3: QD605 streptavidin/oligo, 4: 100-mer target strand, 5: QD605 streptavidin/oligo/100-mer target, 6: Non-complementary 100-mer, 7: QD605 streptavidin/oligo/non-complementary 100-mer.
Figure 4.
Figure 4.
A. Fluorescence images of MPs after hybridization with target DNA of varying length (25-, 40-, 100-mer) and QD probes. B. The hybridization complexes were characterized and quantified by fluorescence microscopy (upper panel) and flow cytometry (bottom panel). Fluorescence intensity is plotted as a function of concentration of the target DNA.
See this image and copyright information in PMC

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