Towards single-molecule DNA sequencing: Assays with low nonspecific adsorption

Krieg, Alexander; Ruckstuhl, Thomas; Seeger, Stefan

doi:10.1016/j.ab.2005.11.013

Cited by 12 publications

(13 citation statements)

References 27 publications

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“…The phosphonate coatings also provided low nonspecific adsorption of fluorescently labeled nucleotides to aluminum surfaces, to levels similar to fused silica or polyacrylic acid terminated polyelectrolyte multilayers (data not shown and ref. 28). …”

Section: Discussionmentioning

confidence: 99%

Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures

Korlach

Marks

Cicero

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

210

147

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Optical nanostructures have enabled the creation of subdiffraction detection volumes for single-molecule fluorescence microscopy. Their applicability is extended by the ability to place molecules in the confined observation volume without interfering with their biological function. Here, we demonstrate that processive DNA synthesis thousands of bases in length was carried out by individual DNA polymerase molecules immobilized in the observation volumes of zero-mode waveguides (ZMWs) in high-density arrays. Selective immobilization of polymerase to the fused silica floor of the ZMW was achieved by passivation of the metal cladding surface using polyphosphonate chemistry, producing enzyme density contrasts of glass over aluminum in excess of 400:1. Yields of single-molecule occupancies of Ϸ30% were obtained for a range of ZMW diameters (70 -100 nm). Results presented here support the application of immobilized single DNA polymerases in ZMW arrays for long-read-length DNA sequencing.fluorescence ͉ metal passivation ͉ microscopy ͉ polyvinyl phosphonic acid ͉ single molecule N anofabrication techniques have enabled new approaches to interrogate individual biomolecules by fluorescence techniques (reviewed in refs. 1 and 2). The extremely small size scale of the associated devices results in a drastic illumination volume reduction, allowing single-molecule investigations to take place at fluorophore concentrations increased to biologically relevant levels. In addition to higher temporal resolution and higher signal-to-noise ratios, they also provide spatial resolution beyond diffraction-limited optics.The zero-mode waveguide (ZMW) is one such nanophotonic confinement structure often consisting of a circular hole in a metal cladding film on a solid transparent substrate (3). In conjunction with laser-excited fluorescence, they provide observation volumes on the order of zeptoliters (10 Ϫ21 l), three to four orders of magnitude smaller than far-field excitation volumes. Applications of circular ZMWs have included the detection of single-molecule DNA polymerase activity by using labeled nucleotides at micromolar concentrations (3), the study of -repressor oligomerization dynamics (4), two-color crosscorrelation to rapidly screen for DNA restriction enzyme activity (5), and diffusion analysis of labeled membrane proteins in lipid bilayers of model membranes and living cells (6-11). C-shaped apertures have been described to study DNA hybridization interactions (12).ZMW technology applications have been limited by the unavailability of selective immobilization methods to position molecules exclusively in the observation volume, immediately above the transparent ZMW floor. One approach to selective immobilization exploits a feature inherent in the ZMW architecture. The transparent substrate and metal cladding are made of different materials, opening the possibility for a selective derivatization that will direct protein adsorption to the floor and not to the nearby metal walls. The nanometer size scale and three-dimensional n...

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Section: Discussionmentioning

confidence: 99%

Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures

Korlach

Marks

Cicero

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

210

147

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“…With regards to single molecule DNA sequencing, another technique is based on the use of polyelectrolyte films (Figure 5b). One report focused on the use of single polyacrilic acid layers and was shown to exhibit low tendency to attract fluorescently labeled bases [137]. Multiple films can also be used [15,138,140].…”

Section: Noise Reduction Using Microfluidic Compatible Surface Passivmentioning

confidence: 99%

Enhancing Single Molecule Imaging in Optofluidics and Microfluidics

Vasdekis

Laporte

2011

IJMS

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Microfluidics and optofluidics have revolutionized high-throughput analysis and chemical synthesis over the past decade. Single molecule imaging has witnessed similar growth, due to its capacity to reveal heterogeneities at high spatial and temporal resolutions. However, both resolution types are dependent on the signal to noise ratio (SNR) of the image. In this paper, we review how the SNR can be enhanced in optofluidics and microfluidics. Starting with optofluidics, we outline integrated photonic structures that increase the signal emitted by single chromophores and minimize the excitation volume. Turning then to microfluidics, we review the compatible functionalization strategies that reduce noise stemming from non-specific interactions and architectures that minimize bleaching and blinking.

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“…In addition, the thermal fluctuation of extended and immobilized DNA can result in nonspecific interactions between the DNA-bound proteins and the surface due to the proximity of the DNA to the surface. Although significant efforts have been dedicated to improve surface passivation for avoiding nonspecific interactions of biomolecules with the surface (8–10), the imperfection of the surface passivation commonly results in nonspecific sticking of proteins at a concentration close to the dissociation constant.…”

Section: Introductionmentioning

confidence: 99%

DNA skybridge: 3D structure producing a light sheet for high-throughput single-molecule imaging

Kim

Rashid

Cho

et al. 2019

Nucleic Acids Research

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Real-time visualization of single-proteins or -complexes on nucleic acid substrates is an essential tool for characterizing nucleic acid binding proteins. Here, we present a novel surface-condition independent and high-throughput single-molecule optical imaging platform called ‘DNA skybridge’. The DNA skybridge is constructed in a 3D structure with 4 μm-high thin quartz barriers in a quartz slide. Each DNA end is attached to the top of the adjacent barrier, resulting in the extension and immobilization of DNA. In this 3D structure, the bottom surface is out-of-focus when the target molecules on the DNA are imaged. Moreover, the DNA skybridge itself creates a thin Gaussian light sheet beam parallel to the immobilized DNA. This dual property allows for imaging a single probe-tagged molecule moving on DNA while effectively suppressing interference with the surface and background signals from the surface.

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Towards single-molecule DNA sequencing: Assays with low nonspecific adsorption

Cited by 12 publications

References 27 publications

Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures

Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures

Enhancing Single Molecule Imaging in Optofluidics and Microfluidics

DNA skybridge: 3D structure producing a light sheet for high-throughput single-molecule imaging

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