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

被引：155

作者：

Korlach, Jonas ^{[1
]}

Marks, Patrick J. ^{[1
]}

Cicero, Ronald L. ^{[1
]}

Gray, Jeremy J. ^{[1
]}

Murphy, Devon L. ^{[1
]}

Roitman, Daniel B. ^{[1
]}

Pham, Thang T. ^{[1
]}

Otto, Geoff A. ^{[1
]}

Foquet, Mathieu ^{[1
]}

Turner, Stephen W. ^{[1
]}

机构：

[1] Pacific Biosci, Menlo Pk, CA 94025 USA

来源：

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA | 2008年 / 105卷 / 04期

关键词：

fluorescence; metal passivation; microscopy; polyvinyl phosphonic acid; single molecule;

D O I：

10.1073/pnas.0710982105

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

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 approximate to 30% 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.

引用

页码：1176 / 1181

页数：6