Zeptomole detection of DNA nanoparticles by single-molecule fluorescence with magnetic field-directed localization

Abstract

Single-molecule fluorescence methods offer the promise of ultrasensitive detection of biomolecules, but the passive immobilization methods commonly employed require analyte concentrations in the picomolar range. Here, we demonstrate that superparamagnetic Fe3O4 nanoparticles (NPs) can be used with an external magnetic field as a simple strategy to enhance the immobilization efficiency and thereby decrease the detection limit. Inorganic nanoparticles functionalized with streptavidin were bound to biotinylated single-stranded DNA oligonucleotides, which were in turn annealed to complementary oligonucleotides labeled with a Cy3 fluorescence dye. Using an external magnetic field, the superparamagnetic nanoparticles were localized to a specific region within the flow chamber surface. From the single-molecule fluorescence time traces, single-step photobleaching indicated that the surface-immobilized NPs were primarily bound with a single Cy3-labeled oligonucleotide. This strategy gave a concentration detection limit for the Cy3-labeled oligonucleotide of 100 aM, 3,000-fold lower than that from an analogous strategy with passive immobilization. With a sample volume of 25 μL, this method achieved a mole detection limit of approximately 2.5 zeptomoles (~1500 molecules). Together, the results support that idea that single-molecule fluorescence methods could be used for biological applications, such as detection and measurements of nucleic acids from biological or clinical samples without PCR amplification.

Label‐free, zeptomole cancer biomarker detection by surface‐enhanced fluorescence on nanoporous gold disk plasmonic nanoparticles

“…By taking advantage of the distance modulation, the sensitivity of the NPGD sensor to target DNA with 28 complementary base pairs is demonstrated. Detection of short DNA target sequences (<30 base pairs) is suitably relevant to applications in micro‐RNA detection for early stage cancer diagnosis . To characterize the interplay of enhancing and quenching on NPGDs, we have compared three fluorophores on three different gold substrates.…”

New trends in plasmonic (bio)sensing

“…The unique optical, electrical, catalytic, thermal and superparamagnetic properties of magnetic nanoparticles (MNPs) have been used for a number of chemical and biochemical purposes , Moliner-Martinez et al 2011, Chen et al 2011, Tang and Lo 2013, Ambashta and Sillanpaa 2010, Oh and Park 2011, Colombo et al 2012, Huang and Juang 2011, Aguilar-Arteaga et al 2010, Beveridge et al 2011, Carregal-Romero et al 2013, Iranifam 2013, Xu and Wang 2012, Cannon et al 2012, Mani et al 2011, Tseng et al 2009, Bruls et al 2009. MNPs can work as a concentration/purification agent, as well as an amplifier for detection due to their high surface-to-volume ratios (Soelberg et al 2009, Liang et al 2012, which offers higher density of chemical bindings.…”

Hexapeptides That Inhibit Processing of Branched DNA Structures Induce a Dynamic Ensemble of Holliday Junction Conformations

“…smFRET Measurements-Junctions (40 nM) were incubated with peptide (100 M) on ice for 30 min in 10 mM Tris-Cl (pH 7.4), 25 mM NaCl and visualized using an experimental setup that has been described previously (42)(43)(44)(45). Peptide-junction complexes were formed in the absence of Mg 2ϩ and applied at a concentration of 20 -50 pM to a quartz slide (G. Finkenbeiner Inc., Waltham, MA) passivated with a 99:1 methoxy polyethylene glycol:biotinylated PEG mixture (Laysan Bio Inc., Arab, AL).…”