micro and nano technologies in bioanalysis: methods and protocols

M E T H O D S I N M O L E C U L A R B I O L O G Y

For other titles published in this series, go to www.springer.com/series/7651

Series EditorJohn M. Walker

School of Life SciencesUniversity of Hertfordshire

Hatfield, Hertfordshire, AL10 9AB, UK

M E T H O D S I N M O L E C U L A R B I O L O G Y

Micro and Nano Technologies in Bioanalysis

Methods and Protocols

Edited by

James Weifu Lee and Robert S. Foote

Oak Ridge National Laboratory, Oak Ridge, TN, USA

EditorsJames Weifu Lee Robert S. FooteOak Ridge National Laboratory Oak Ridge National LaboratoryOak Ridge, Oak Ridge, TN, USA TN, USA

ISSN: 1064-3745 e-ISSN: 1940-6029ISBN: 978-1-934115-40-4 e-ISBN: 978-1-59745-483-4DOI: 10.1007/978-1-59745-483-4Springer Dordrecht Heidelberg London New York

Library of Congress Control Number: 2009929345

Humana Press, a part of Springer Science+Business Media, LLC 2009All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is for-bidden.The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with re-spect to the material contained herein.

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Preface

v

This book provides current information on the development of microfluidics, nanotech-nologies, and physical science techniques for the separation, detection, manipulation, and analysis of biomolecules, and should be useful to a wide audience, including molecular and cell biologists, biochemists, microbiologists, geneticists, and medical researchers. Chapters cover a variety of topics and techniques ranging from lab-on-chip technologies and microfluidics-coupled mass spectrometry for separation and detection of biomol-ecules, including proteins and nucleic acids, to manipulating and probing biomolecules with nanopores, nanochannels, optical, and other physical means, with the possibility of isolation and analysis of individual biomolecules from a single cell, and to structural and functional analysis of biomolecules with liquid nuclear magnetic resonance, X-ray and neutron scattering techniques. The book presents emerging nanotechnologies including quantum dots and molecular fluorescence for imaging and tracking of biomolecules and nanotechnologies for biomolecular delivery, gene therapy, and gene-expression control. Each chapter describes a specific technology with its fundamental mechanism and practical applications for a particular subject area, so that a competent scientist who is unfamiliar with the technology can understand its capabilities and basic procedures. In many cases, a reader should be able to carry out the techniques successfully at the first attempt by simply following the detailed practical procedures (protocols) and/or information (including useful notes) provided in the book. For sophisticated technologies such as neutron scattering, the book describes their physical concepts and discusses the new opportunities that these new technologies may bring for both basic and applied research in the fields of molecular biology and biotechnology. This book consists of 41 chapters that are organ-ized into four parts. The chapters were contributed by nearly 100 authors worldwide, who are among the worlds prominent scientists in their fields.

The first half of the volume covers microfluidic and physical methods of bioanalysis. It consists of Part I on applications of microfluidics and nanopores in separation, manipu-lation, detection, and analysis of biomolecules, and Part II on technologies of physical science in detection and analysis of biomolecules. It contains valuable protocols on micro-fluidics and physical science-related technologies that may benefit the field of molecular biology. Chapter topics are briefly described below.

Part I consists of Chaps. 110: Chap. 1 describes a commercially available nanoflow analytical technology conducted on a microfabricated chip that allows for highly efficient HPLC separation and superior sensitivity for MS detection of complex proteomic mix-tures; Chaps. 24 describe fabrication of nanofluidic channels for manipulation of DNA molecules, a single-molecule barcoding system using nanoslits for DNA analysis, and microfluidic devices with photodefinable pseudovalves for protein separation, respectively; Chap. 5 introduces specific antibody detection by using a microbead-based assay with quantum dot (QD) fluorescence on a microfluidic chip; Chap. 6 describes a biomolecular sample-focusing method based on a device design incorporating arrays of addressable on-chip microfabricated electrodes that can locally increase the concentration of DNA

in solution by electrophoretically sweeping it along the length of a microchannel; Chap. 7 describes a solid-state nanopore technique for detecting individual biopolymers, and Chap. 8 reports a method of inserting and manipulating DNA in a nanopore with optical tweezers; Chaps. 9 and 10 describe techniques of forming an -hemolysin nanopore for single-molecule analysis and for nanopore force spectroscopy of DNA duplexes.

Part II consists of Chaps. 1122: Chap. 11 describes an electrochemical method for quantitative chemical analysis of neurotransmitter release from single cells; Chaps. 1214 introduce techniques for trapping and detection of single molecules in water, ZnO nano-rods as an intracellular sensor for pH measurements, and analysis of biomolecules using surface plasmons; Chap. 15 reports use of residual dipolar couplings in structural analysis of proteinligand complexes by solution NMR spectroscopy; Chaps. 16 and 17 report Raman-assisted X-ray crystallography for the analysis of biomolecules and methods and software for diffuse X-ray scattering from protein crystals, and Chaps. 1820 describe deuterium labeling for neutron structurefunctiondynamics analysis, the basics and instrumentation of small-angle neutron scattering for molecular biology, and small-angle scattering and neutron contrast variation for studying biomolecular complexes, respec-tively; Chap. 21 describes the application of tandem mass spectrometry to identification of protein biomarkers of disease, and Chap. 22 describes the use of hyphenated MS tech-niques for comprehensive metabolome analysis.

The second half of the volume covers nanotechnologies for biosystems, and consists of Part III on applications of quantum dots and molecular fluorescence in detection, tracking, and imaging of biomolecules, and Part IV on nanotechnologies for biomolecular delivery, gene therapy, and expression control. It contains valuable information on nanoscience-empowered molecular biotechnologies.

Part III consists of Chaps. 2332: Chaps. 2325 describe multicolor detection of combed DNA molecules using quantum dots, quantum dot molecular beacons for DNA detection, and a gel electrophoretic blotting technique for identifying quantum dotprotein/proteinprotein interactions; Chaps. 26 and 27 present techniques for in vivo imaging of quantum dots and efficient biolabels in cancer diagnostics, respectively; Chap. 28 describes monitoring and affinity purification of proteins using dual tags with tetracysteine motifs, and Chap. 29 reports use of genomic DNA as a reference in DNA microarray analyses; Chap. 30 describes single-molecule imaging of fluorescent proteins expressed in living cells; Chap. 31 describes micropositron emission tomography (PET), single-photon emis-sion computed tomography (SPECT), and near-infrared (NIR) fluorescence imaging of biomolecules in vivo, which could lead to a number of exciting possibilities for biomedi-cal applications, including early detection, treatment monitoring, and drug development; Chap. 32 reports a revolutionary photo-based imaging technology: the ultrahigh resolu-tion imaging of biomolecules by fluorescence photoactivation localization microscopy (FPALM) that can now image molecular distributions in fixed and living cells with mea-sured resolution better than 30 nm, which likely represents a breakthrough technology that has now shattered the classic limit of light microscopy resolution associated with the wavelength-dependent light diffraction barrier, thought to be unbreakable for more than 100 years.

In Part IV, Chaps. 3341 describe nanotechnologies with potential biomedical appli-cations. Specifically, Chap. 33 describes real-time imaging of gene delivery and expression with DNA nanoparticle technologies and Chap. 34 reports nanoparticle-mediated gene delivery. Chapters 35 and 36 describe magnetic nanoparticles for local drug delivery using magnetic implants and functionalized magnetic nanoparticles as an in vivo delivery

vi Preface

Preface vii

system, and Chap. 37 reports formulation/preparation of functionalized nanoparticles for in vivo targeted drug delivery; Chap. 38 reports detection of mRNA in single living cells using atomic force microscopy nanoprobes; Chap. 39 describes a gene transfer tech-nique through reverse transfection using gold nanoparticles; Chap. 40 presents custom-designed molecular scissors for site-specific manipulation of the plant and mammalian genomes, and Chap. 41 describes a technique for determining DNA sequence specificity of natural and artificial transcription factors by cognate site identifier analysis, both of which could lead to modern applications in molecular biology and biomedicine.

Oak Ridge, TN James Weifu LeeRobert S. Foote

Acknowledgments

The editors, James Weifu Lee and Robert S. Foote, thank the nearly 100 authors through-out the world for their contributions and collaboration on this book project. The editing work of this volume was accomplished using significant amounts of the editors spare time including their family time. Therefore, the editors also wish to thank their respective families: the Lee family and the Foote family, for their wonderful support and understanding.

ix

Contents

Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vAcknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ixContributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv

Part I Applications of Microfluidics and Nanopores in Separation, Detection, Manipulation, and Analysis of Biomolecules

1 HPLC-Chip/MS Technology in Proteomic Profiling . . . . . . . . . . . . . . . . . . . . . . . 3Martin Vollmer and Tom van de Goor

2 Nanofluidic Channel Fabrication and Manipulation of DNA Molecules . . . . . . . . . 17Kai-Ge Wang and Hanben Niu

3 A Single-Molecule Barcoding System using Nanoslits for DNA Analysis: Nanocoding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29Kyubong Jo, Timothy M. Schramm, and David C. Schwartz

4 Microfluidic Devices with Photodefinable Pseudo-valves for Protein Separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43Z. Hugh Fan

5 Microfluidic Chips Designed for Measuring Biomolecules Through a Microbead-Based Quantum Dot Fluorescence Assay . . . . . . . . . . . . . . . 53Kwang-Seok Yun, Dohoon Lee, Hak-Sung Kim, and Euisik Yoon

6 DNA Focusing Using Microfabricated Electrode Arrays . . . . . . . . . . . . . . . . . . . . . 69Faisal A. Shaikh and Victor M. Ugaz

7 Solid-State Nanopore for Detecting Individual Biopolymers . . . . . . . . . . . . . . . . . 81Jiali Li and Jene A. Golovchenko

8 Inserting and Manipulating DNA in a Nanopore with Optical Tweezers . . . . . . . . . 95U. F. Keyser, J. van der Does, C. Dekker, and N. H. Dekker

9 Forming an -Hemolysin Nanopore for Single-Molecule Analysis. . . . . . . . . . . . . . 113Nahid N. Jetha, Matthew Wiggin, and Andre Marziali

10 Nanopore Force Spectroscopy on DNA Duplexes. . . . . . . . . . . . . . . . . . . . . . . . . . 129Nahid N. Jetha, Matthew Wiggin, and Andre Marziali

Part II Technologies of Physical Science and Chemistry in Detection and Analysis of Biomolecules

11 Quantitative Chemical Analysis of Single Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153Michael L. Heien and Andrew G. Ewing

12 Trapping and Detection of Single Molecules in Water. . . . . . . . . . . . . . . . . . . . . . . 163M. Willander, K. Risveden, B. Danielsson, and O. Nur

13 ZnO Nanorods as an Intracellular Sensor for pH Measurements . . . . . . . . . . . . . . . 187M. Willander and Safaa Al-Hilli

xi

xii Contents

14 Analysis of Biomolecules Using Surface Plasmons . . . . . . . . . . . . . . . . . . . . . . . . . . 201M. Willander and Safaa Al-Hilli

15 Use of Residual Dipolar Couplings in Structural Analysis of ProteinLigand Complexes by Solution NMR Spectroscopy . . . . . . . . . . . . . . . . 231Nitin U. Jain

16 Raman-Assisted X-Ray Crystallography for the Analysis of Biomolecules . . . . . . . . . 253Dominique Bourgeois, Gergely Katona, Eve de Rosny, and Philippe Carpentier

17 Methods and Software for Diffuse X-Ray Scattering from Protein Crystals . . . . . . . 269Michael E. Wall

18 Deuterium Labeling for Neutron StructureFunctionDynamics Analysis . . . . . . . . 281Flora Meilleur, Kevin L. Weiss, and Dean A.A. Myles

19 Small-Angle Neutron Scattering for Molecular Biology:Basics and Instrumentation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293William T. Heller and Kenneth C. Littrell

20 Small-Angle Scattering and Neutron Contrast Variation for Studying Bio-Molecular Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307Andrew E. Whitten and Jill Trewhella

21 Protein Sequencing with Tandem Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . 325Assem G. Ziady and Michael Kinter

22 Metabolic Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343Vladimir V. Tolstikov

Part III Applications of Quantum Dots and Molecular Fluorescence in Detection, Tracking and Imaging of Biomolecules

23 Multicolor Detection of Combed DNA Molecules Using Quantum Dots . . . . . . . . 357Christophe Escud, Bndicte Gron-Landre, Aurlien Crut, and Pierre Desbiolles

24 Quantum Dot Molecular Beacons for DNA Detection . . . . . . . . . . . . . . . . . . . . . . 367Nathaniel C. Cady

25 Quantum Dot Hybrid Gel Blotting: A Technique for Identifying Quantum Dot-Protein/Protein-Protein Interactions . . . . . . . . . . . . . . . . . . . . . . . 381Tania Q. Vu and Hong Yan Liu

26 In Vivo Imaging of Quantum Dots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393Isabelle Texier and Vronique Josserand

27 Semiconductor Fluorescent Quantum Dots: Efficient Biolabels in Cancer Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407Patricia M. A. Farias, Beate S. Santos, and Adriana Fontes

28 The Monitoring and Affinity Purification of Proteins Using Dual Tags with Tetracysteine Motifs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421Richard J. Giannone, Yie Liu, and Yisong Wang

29 Use of Genomic DNA as Reference in DNA Microarrays . . . . . . . . . . . . . . . . . . . . 439Yunfeng Yang

30 Single-Molecule Imaging of Fluorescent Proteins Expressed in Living Cells . . . . . . 451Kayo Hibino, Michio Hiroshima, Masahiro Takahashi, and Yasushi Sako

Contents xiii

31 MicroPET, MicroSPECT, and NIR Fluorescence Imaging of Biomolecules In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461Zi-Bo Li and Xiaoyuan Chen

32 Ultrahigh Resolution Imaging of Biomolecules by Fluorescence Photoactivation Localization Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483Samuel T. Hess, Travis J. Gould, Mudalige Gunewardene, Joerg Bewersdorf, and Michael D. Mason

Part IV Nanotechnologies for Biomolecular Delivery, Gene Therapy and Expression Control

33 Real-Time Imaging of Gene Delivery and Expression with DNA Nanoparticle Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525Wenchao Sun and Assem G. Ziady

34 Nanoparticle-Mediated Gene Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547Sha Jin, John C. Leach, and Kaiming Ye

35 Magnetic Nanoparticles for Local Drug Delivery Using Magnetic Implants . . . . . . 559Rodrigo Fernndez-Pacheco, J. Gabriel Valdivia, and M. Ricardo Ibarra

36 Functionalized Magnetic Nanoparticles as an In Vivo Delivery System . . . . . . . . . . 571Shu Taira, Shinji Moritake, Takahiro Hatanaka, Yuko Ichiyanagi, and Mitsutoshi Setou

37 Formulation/Preparation of Functionalized Nanoparticles for In Vivo Targeted Drug Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589Frank Gu, Robert Langer, and Omid C. Farokhzad

38 Detection of mRNA in Single Living Cells Using AFM Nanoprobes. . . . . . . . . . . . 599Hironori Uehara, Atsushi Ikai, and Toshiya Osada

39 Reverse Transfection Using Gold Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . 609Shigeru Yamada, Satoshi Fujita, Eiichiro Uchimura, Masato Miyake, and Jun Miyake

40 Custom-Designed Molecular Scissors for Site-Specific Manipulation of the Plant and Mammalian Genomes . . . . . . . . . . . . . . . . . . . . . . . 617Karthikeyan Kandavelou and Srinivasan Chandrasegaran

41 Determining DNA Sequence Specificity of Natural and Artificial Transcription Factors by Cognate Site Identifier Analysis . . . . . . . . . . 637Mary S. Ozers, Christopher L. Warren, and Aseem Z. Ansari

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 655

Contributors

SAFAA AL-HILLI Department of Physics, Gothenburg University, Gothenburg, SwedenASEEM Z. ANSARI Department of Biochemistry, and the Genome Center, University

of Wisconsin-Madison, Madison, WI, USAJOERG BEWERSDORF Institute for Molecular Biophysics, The Jackson Laboratory,

Bar Harbor, ME, USADOMINIQUE BOURGEOIS Institut de Biologie Structurale Jean-Pierre Ebel, Grenoble,

FranceNATHANIEL C. CADY College of Nanoscale Science and Engineering, University at

Albany, Albany, NY, USAPHILIPPE CARPENTIER Institut de Biologie Structurale Jean-Pierre Ebel, Grenoble,

FranceSRINIVASAN CHANDRASEGARAN Department of Environmental Health Sciences, Johns

Hopkins University, Baltimore, MD, USAXIAOYUAN CHEN Department of Radiology and Bio-X Program, Stanford

University, Stanford, CA, USAAURLIEN CRUT Laboratoire Kastler Brossel, Dpartement de Physique, Ecole

Normale Suprieure, Paris, FranceB. DANIELSSON Department of Pure and Applied Biochemistry, Lund University,

Lund, SwedenCEES DEKKER Kavli Institute of Nanoscience, Delft University of Technology, Delft,

The NetherlandsNYNKE H. DEKKER Kavli Institute of Nanoscience, Delft University of Technology,

Delft, The NetherlandsEVE DE ROSNY Institut de Biologie Structurale Jean-Pierre Ebel, Grenoble, FrancePIERRE DESBIOLLES Laboratoire Kastler Brossel, Dpartement de Physique, Ecole

Normale Suprieure, Paris, FranceCHRISTOPHE ESCUD Musum National dHistoire Naturelle, Paris, FranceANDREW G. EWING Department of Chemistry, The Pennsylvania State University,

University Park, PA, USA, Department of Chemistry, Gteborg University, Gteborg, Sweden

Z. HUGH FAN Department of Mechanical and Aerospace Engineering, Department of Biomedical Engineering, University of Florida, Gainesville, FL, USA

PATRICIA M.A. FARIAS Department of Biophysics and Radiobiology, Federal University of Pernambuco, Cidade Universitria, Recife, PE, Brazil

OMID C. FAROKHZAD Harvard-MIT Center for Cancer Nanotechnology Excellence, Massachusetts Institute of Technology, Cambridge, MA, USA, Laboratory of Nanomedicine and Biomaterials, Brigham and Womens Hospital, Boston, MA, USA

xv

xvi Contributors

RODRIGO FERNNDEZ-PACHECO Instituto Universitario de Investigacin en Nanociencia de Aragn (INA), Universidad de Zaragoza, Zaragoza, Spain

ADRIANA FONTES Department of Biophysics and Radiobiology, Federal University of Pernambuco, Cidade Universitria, Recife, PE, Brazil

SATOSHI FUJITA Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan

BNDICTE GRON-LANDRE Musum National dHistoire Naturelle, Paris, FranceRICHARD J. GIANNONE Biosciences Division, Oak Ridge National Laboratory, Oak

Ridge, TN, USA, Graduate School of Genome Science and Technology, University of Tennessee-Oak Ridge National Laboratory, Knoxville, TN, USA

JENE A. GOLOVCHENKO Department of Physics, Division of Engineering and Applied Sciences, Harvard University, Cambridge, MA, USA

TRAVIS J. GOULD Department of Physics and Astronomy, and Institute for Molecular Biophysics, University of Maine, Orono, ME, USA

FRANK GU Harvard-MIT Center for Cancer Nanotechnology Excellence, Massachusetts Institute of Technology, Cambridge, MA, USA

Laboratory of Nanomedicine and Biomaterials, Brigham and Womens Hospital, Boston, MA, USA

MUDALIGE GUNEWARDENE Department of Physics and Astronomy, and Institute for Molecular Biophysics, University of Maine, Orono, ME, USA

TAKAHIRO HATANAKA Mitsubishi Kagaku Institute of Life Sciences, Tokyo, JapanMICHAEL L. HEIEN Department of Chemistry, The Pennsylvania State University,

University Park, PA, USAWILLIAM T. HELLER Center for Structural Molecular Biology, Oak Ridge National

Laboratory, Oak Ridge, TN, USASAMUEL T. HESS Department of Physics and Astronomy, and Institute for Molecular

Biophysics, University of Maine, Orono, ME, USAKAYO HIBINO Cellular Informatics Laboratory, RIKEN, Wako, JapanMICHIO HIROSHIMA Cellular Informatics Laboratory, RIKEN, Wako, JapanM. RICARDO IBARRA Instituto Universitario de Investigacin en Nanociencia de

Aragn (INA), Universidad de Zaragoza, Zaragoza, Spain, Instituto de Ciencia de Materiales de Aragn (ICMA), Universidad de Zaragoza-CSIC, Zaragoza, Spain

YUKO ICHIYANAGI Department of Physics, Graduate School of Engineering, Yokohama National University, Yokohama, Japan

ATSUSHI IKAI Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan

NITIN U. JAIN Biochemistry, Cellular and Molecular Biology Department, University of Tennessee, Knoxville, TN, USA

NAHID N. JETHA Department of Physics and Astronomy, University of British Columbia, Vancouver, Canada

SHA JIN DNA Resource Center, University of Arkansas, Fayetteville, AR, USA

Contributors xvii

KYUBONG JO Department of Chemistry, University of Wisconsin, Madison, WI, USA, Department of Chemistry & Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul, Korea

VRONIQUE JOSSERAND ANIMAGE, CERMEP, Lyon, FranceINSERM U823, Institut Albert Bonniot, La Tronche, FranceKARTHIKEYAN KANDAVELOU Department of Environmental Health Sciences, Johns

Hopkins University, Baltimore, MD, USAGERGELY KATONA Institut de Biologie Structurale Jean-Pierre Ebel, Grenoble,

FranceULRICH. F. KEYSER Cavendish Laboratory, University of Cambridge, UKHAK-SUNG KIM Department of Biological Sciences, KAIST, Daejeon, KoreaMICHAEL KINTER Free Radical Biology and Aging Research Program, Oklahoma

Medical Research Foundation, Oklahoma City, Oklahoma, USAROBERT LANGER Harvard-MIT Center for Cancer Nanotechnology Excellence,

Massachusetts Institute of Technology, Cambridge, MA, USAJOHN C. LEACH Biomedical Engineering Program, College of Engineering,

University of Arkansas, Fayetteville, AR, USADOHOON LEE Environment and Energy Division, Korea Institute of Industrial

Technology, Cheonan, KoreaJIALI LI Department of Physics, University of Arkansas, Fayetteville, AR, USAZI-BO LI Department of Radiology and Bio-X Program, Stanford University,

Stanford, CA, USAKENNETH C. LITTRELL Neutron Scattering Sciences Division, Oak Ridge National

Laboratory, Oak Ridge, TN, USAHONG YAN LIU Department of Biomedical Engineering, Oregon Health & Science

University, Portland, OR, USAYIE LIU Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN,

USAANDRE MARZIALI Department of Physics and Astronomy, University of British

Columbia, Vancouver, CanadaMICHAEL D. MASON Department of Chemical and Biological Engineering, and

Institute for Molecular Biophysics, University of Maine, Orono, ME, USAFLORA MEILLEUR Department of Molecular & Structural Biochemistry, North

Carolina State University, Raleigh, NC, USA, Spallation Neutron Source, Oak Ridge National Laboratory, Oak Ridge, TN, USA

JUN MIYAKE Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan

MASATO MIYAKE Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan

SHINJI MORITAKE Department of Physics, Graduate School of Engineering, Yokohama National University, Yokohama, Japan

xviii Contributors

DEAN A.A. MYLES Center for Structural Molecular Biology, Oak Ridge National Laboratory, Oak Ridge, TN, USA

HANBEN NIU Institute of Optoelectronics, Shenzhen University, Shenzhen, ChinaO. NUR Department of Science and Technology, Campus Norrkping, Linkping

University, Norrkping, SwedenTOSHIYA OSADA Department of Life Science, Graduate School of Bioscience and

Biotechnology, Tokyo Institute of Technology, Yokohama, JapanMARY S. OZERS Department of Biochemistry, University of Wisconsin-Madison,

Madison, WI, USAK. RISVEDEN Department of Pure and Applied Biochemistry, Lund University,

Lund, SwedenYASUSHI SAKO Cellular Informatics Laboratory, RIKEN, Wako, JapanBEATE S. SANTOS Department of Pharmaceutical Sciences, Federal University of

Pernambuco, Cidade Universitria, Recife, PE, BrazilTIMOTHY M. SCHRAMM Laboratory for Molecular and Computational Genomics,

Department of Chemistry, Laboratory of Genetics, and Biotechnology Center, University of Wisconsin, Madison, WI, USA

DAVID C. SCHWARTZ Laboratory for Molecular and Computational Genomics, Laboratory of Genetics, Department of Chemistry and Biotechnology Center, University of Wisconsin, Madison, WI, USA

MITSUTOSHI SETOU Mitsubishi Kagaku Institute of Life Sciences, Tokyo, Japan, National Institute for Physiological Sciences, National Institute of Natural Sciences, Aichi, Japan

FAISAL A. SHAIKH Artie McFerrin Department of Chemical Engineering, Texas A&M University, College Station, TX, USA

WENCHAO SUN Department of Pediatrics, Case Western Reserve University, Cleveland, OH, USA

SHU TAIRA Mitsubishi Kagaku Institute of Life Sciences, Tokyo, JapanMASAHIRO TAKAHASHI Cellular Informatics Laboratory, RIKEN, Wako, JapanISABELLE TEXIER Micro-Technologies for Biology and Healthcare Department, CEA

Grenoble, Grenoble, FranceVLADIMIR V. TOLSTIKOV University of California Davis Genome Center, Davis, CA,

USAJILL TREWHELLA School of Molecular and Microbial Biosciences, University of Sydney,

Sydney, NSW, AustraliaEIICHIRO UCHIMURA Research Institute for Cell Engineering, National Institute of

Advanced Industrial Science and Technology, Tokyo, JapanHIRONORI UEHARA Department of Ophthalmology & Visual Science, University of

Utah, Salt Lake City, UT, USAVICTOR M. UGAZ Artie McFerrin Department of Chemical Engineering, Texas

A&M University, College Station, TX, USA

Contributors xix

J. GABRIEL VALDIVIA Instituto Universitario de Investigacin en Nanociencia de Aragn (INA), Universidad de Zaragoza, Zaragoza, Spain, Hospital Clnico Universitario Lozano Blesa, Zaragoza, Spain

TOM VAN DE GOOR Agilent Technologies, Waldbronn, GermanyJ. VAN DER DOES Kavli Institute of Nanoscience, Delft University of Technology,

Delft, The NetherlandsMARTIN VOLLMER Agilent Technologies, Waldbronn, GermanyTANIA Q. VU Department of Biomedical Engineering, Oregon Health and Science

University, Portland, OR, USAMICHAEL E. WALL Computer, Computational, and Statistical Sciences Division,

Bioscience Division, and Center for Nonlinear Studies, Los Alamos National Laboratory, Los Alamos, NM, USA

KAI-GE WANG Institute of Photonics and Photonic Technology, Northwest University, Xian, China, Institute of Optoelectronics, Shenzhen University, Shenzhen, China

YISONG WANG Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA

CHRISTOPHER L. WARREN Department of Biochemistry, University of Wisconsin-Madison and VistaMotif LLC, Madison, WI, USA

KEVIN L. WEISS Center for Structural Molecular Biology, Oak Ridge National Laboratory, Oak Ridge, TN, USA

ANDREW E. WHITTEN Bragg Institute, Australian Nuclear Science and Technology Organisation, Lucas Heights, NSW, Australia

MATTHEW WIGGIN Department of Physics and Astronomy, and Department of Biochemistry, University of British Columbia, Vancouver, Canada

M. WILLANDER Department of Science and Technology, Linkping University, Campus Norrkping, Norrkping, Sweden, Department of Physics, Gothenburg University, Gothenburg, Sweden

SHIGERU YAMADA Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan

YUNFENG YANG Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA

KAIMING YE Biomedical Engineering Program, College of Engineering, University of Arkansas, Fayetteville, AR, USA

EUISIK YOON Department of Electrical Engineering and Computer Science, University of Michigan, Ann Arbor, MI, USA

KWANG-SEOK YUN Department of Electronic Engineering, Sogang University, Seoul, KoreaASSEM G. ZIADY Department of Pediatrics, Case Western Reserve University,

Cleveland, OH, USA

Chapter 1

HPLC-Chip/MS Technology in Proteomic Profiling

Martin Vollmer and Tom van de Goor

Summary

HPLC-chip/MS is a novel nanoflow analytical technology conducted on a microfabricated chip that allows for highly efficient HPLC separation and superior sensitive MS detection of complex proteomic mixtures. This is possible through on-chip preconcentration and separation with fluidic connection made automatically in a leak-tight fashion. Minimum precolumn and postcolumn peak dispersion and uncom-promised ease of use result in compounds eluting in bands of only a few nanoliters. The chip is fabricated out of bio-inert polyimide-containing channels and integrated chip structures, such as an electrospray emitter, columns, and frits manufactured by laser ablation technology. Meanwhile, a variety of HPLC-chips differing in design and stationary phase are commercially available, which provide a comprehen-sive solution for applications in proteomics, glycomics, biomarker, and pharmaceutical discovery. The HPLC-chip can also be easily integrated into a multidimensional separation workflow where different orthogonal separation techniques are combined to solve a highly complex separation problems. In this chapter, we describe in detail the methodological chip usage and functionality and its application in the elucidation of the protein profile of human nucleoli.

Key words: HPLC-chip/MS , Nanoflow LC/MS , Multidimensional separation , Proteomics , Nucleolus

To comprehensively elucidate a complex proteome, such as that of a cell organelle, it is necessary to combine different orthogonal separation techniques. In the past, numerous techniques have been combined that exploit different chemical and physicochemical properties of the protein and peptide analytes (for recent reviews see refs. 1, 2) . Liquid-based techniques, in contrast to gel-based approaches, bear the advantage that the analytes always stay in the liquid phase. This avoids labor-intense and error-prone

1. Introduction

James Weifu Lee and Robert S. Foote (eds.), Micro and Nano Technologies in Bioanalysis, Methods in Molecular Biology, vol. 544DOI 10.1007/978-1-59745-483-4_1, Humana Press, a part of Springer Science + Business Media, LLC 2009

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4 Vollmer and Goor

extraction processes, and the final separation step can easily be directly coupled to nano electrospray, which allows for highly sensitive MS detection.

In the following study, we report the two-dimensional sepa-ration of the human nucleolus proteome, where strong cation-exchange chromatography was conducted offline in the first separation dimension, while reversed phase based HPLC-chip/MS was chosen for the last separation step. This separation scheme is similar to the Mudpit approach ( 3 ) . However, because the first and second dimensions are separated, enhanced flexibility con-cerning loading capacity and solvent compatibility is achieved.

Proteins are isolated and digested by standard procedures. Tryptic peptides are then fractionated according to their charge by using strong cation-exchange chromatography. The fractions are further separated on an HPLC-chip/MS system that contains an enrichment column for sample cleanup and concentration, and a reversed phase separation column. Because the chip separation col-umn and the electrospray emitter are both integrated on a single chip, no peak broadening occurs after the peptide analytes elute off the column and enter the chip electrospray tip. This finally results in small peak volumes and hence superior sensitivity, especially for low abundant protein species from the investigated proteome. The method described in the following section can be applied and adapted for any multidimensional proteomic workflow.

Proteomic studies usually face the dilemma that, on the one hand, sample size is limited and, on the other hand, high sensitivity and a wide dynamic range are required to identify and quantitate peptides and corresponding proteins comprehensively. High sensitivity in combination with ESI MS is best achieved by lowering the over-all HPLC flow rate to a few hundreds of nanoliters or less. This results in a decrease of the dimension of the Taylor cone and of the size of the formed droplets in the ESI spray chamber, such that, due to the higher surface tension, the overall ionization effi-ciency is increased ( 4 ) .

The drawback of nano-HPLC/MS is the occurrence of small leaks and blockages that are difficult to trace under extremely low flow conditions. Dead volumes before the separation column affect the composition of the LC gradient and the analyte elution time. Dead volumes downstream of the separation column lead to significant peak broadening and result in loss of sensitivity. Therefore, chip-based separation devices have been introduced recently that integrate the nanoflow separation and the electrospray process, such that error-prone connections susceptible to introducing dead volumes are avoided. An overview of chip-based formats used in combination with electrospray MS was recently published ( 5 ) .

Although most of the chip formats that include separation and electrospray in a single device are still academic research tools, the Agilent HPLC-chip/MS system was introduced commercially

1.1. Functionality of HPLC-Chip/MS

HPLC-Chip/MS Technology in Proteomic Profiling 5

in 2005. The system and the corresponding chips and their fabrication process have been described in detail in several reports ( 6, 7 ) .

In short, the HPLC-chip is fabricated from layers of bio-inert polyimide that are first laser-ablated to form the microfluidic chan-nels, fluidic inlet ports, column chambers, frits, and electrospray emitter. Different layers are then attached to each other by heat vacuum lamination followed by deposition of electrical contacts by metallization. The column channels of the chips are packed with standard silica-based reversed phase particles (Zorbax 300 SB-C18, 5 m m, Agilent Technologies, Waldbronn, Germany).

The chip is automatically inserted into the HPLC-chip/MS interface by a software command and clamped in a leak-tight fashion between a valve stator that bears the inlet connections of the fluidic transfer capillaries and a ceramic nano rotary valve.

HPLC-chips are available with packed separation channels of 50 m m (D) 75 m m (W) in a length of 43 mm or 150 mm. Sepa-ration of the analytes is usually performed at 200600 nL/min with the aid of a nanopump. A second column serves as enrich-ment and sample clean-up column and is available at volumes of 40 nL and 160 nL, depending on the loading capacity and com-plexity of the sample that is required for the specific separation workflow (Fig. 1 ) . The sample is loaded first onto the enrich-ment column using a capillary pump at flow rates of 4 m L/min. While salts are washed off, peptides and proteins are retained. A nano rotary valve operates directly on the surface of the chip and is turned 60 degrees to switch the enrichment column to

Fig. 1 . HPLC-chip; containing enrichment column, separation column, electrospray tip, and electrical high-voltage contacts. The chip is protected by an encapsulation and the spray tip is pushed out into the electrospray chamber in the HPLC-chip/MS interface. All fluidic connections are made automatically following a software command.

6 Vollmer and Goor

the same flow path as the separation column. Peptides elute off the enrichment column by a solvent gradient delivered from the nanopump and are transferred to the separation column (Fig. 2 ) . Analytes eluting from the separation channel travel 2 mm further into an 8 m m ID emitter channel to exit finally through the outlet hole of the electrospray tip into the ionization chamber. The chip can be used with backpressures of up to 150 bars. Typical chip lifetime exceeds 200 working hours.

The column material of the chip is retained by narrowing the chip channel on the column outlet side and a filter layer is attached at the column inlet after the packing process. HPLC-chip/MS has been successfully applied for biomarker discovery and several proteomic and glycomic research studies ( 8 13 ) . Using the described method, we were able to identify 2,024 unique peptides with high confidence that corresponded to 206 nucleolar proteins ( 11 ) .

1. Eagles minimum essential medium (Sigma Aldrich, St. Louis, MO) supplemented with 5% calf serum (Eurobio, Les Ulis, France).

2. Washing buffer, cold phosphate-buffered saline, pH 7.4.

2. Materials

2.1. Nucleolus Protein Extraction

Fig. 2 . Sample loading and analysis: The sample is loaded onto the enrichment column at 4 m L/min using the LC capillary pump. Salts and contaminants are flushed into waste while the valve is in the enrichment position ( upper panel ). The nano rotary valve, which operates directly on the surface of the chip clamped between the rotor and stator ( inter-mediate panel ), is then switched into the analytical flow path. The sample is then desorbed by opposite flow from the enrichment column using a nanopump at 300 nL /min and transferred to the analytical column where the sample is separated using a gradient of increasing organic concentration ( lower panel ).


3. Hypotonic cell buffer: 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 1 mM MgCl 2 .

4. Nonidet P-40 for cell lysis (at 0.3% final concentration) (Roche Applied Science, Mannheim, Germany).

5. Resuspension of nuclei in 0.25 M sucrose, 10 mM MgCl 2 . 6. Purification of nuclei and sonicated nucleoli fraction through

0.88 M sucrose, 0.05 mM MgCl 2 . 7. Resuspension of purified nuclei and nucleoli in 0.34 M sucrose,

0.05 mM MgCl 2 . 8. Resuspension of purified nucleoli for protein extraction in

0.34 M sucrose, 0.05 M MgCl 2 , 0.2 M magnesium acetate, addition of two volumes of glacial acetic acid for nucleic acid precipitation.

9. Dialysis in 1 M acetic acid.

1. Coomassie Plus Protein Assay Kit (Pierce, Rockford, IL) for protein concentration determination ( 14 ) .

2. 50 mM Ammonium bicarbonate for protein resuspension. 3. 100 mM DTT stock, working solution, 1 mM DTT for dena-

turation, 1 M urea ( see Note 1 ). 4. 10 mM iodoacetamide (Sigma-Aldrich) for alkylation from

100 mM stock. 5. 10 mM DTT for quenching. 6. TPCK trypsin (Pierce) (1 mg/mL stock, frozen, see Note 2 ). 7. 10% Formic acid to stop enzymatic digestion. 8. 0.1% Formic acid for resuspension of lyophilized digest.

1. Mobile phase A: 0.1% formic acid, 5% acetonitrile (HPLC grade, Merck, Darmstadt, Germany). Mobile phase B: 0.1% formic acid, 500 mM KCl, 5% acetonitrile.

2. Separation column: Agilent BioSCX series 2, 50 mm L 0.8 mm, ID (Agilent Technologies, Waldbronn, Germany, see Note 3 ).

3. HPLC configuration: Agilent 1200 Capillary LC system con-taining micro degasser, capillary pump, diode array detector (equipped with a 300-nL flow cell), thermostated m -well plate sampler and thermostated m -fraction collector ( see Note 4 ).

4. 96-conical well plates (Eppendorf, Hamburg, Germany).

1. Mobile phase A: 0.1% formic acid. Mobile phase B: 0.1% for-mic acid, 99.9% acetonitrile.

2. HPLC-chip, containing a 160-nL high-capacity enrichment column and a 150-mm separation column, packed with Zorbax SB-300 C18, 5- m m particles ( see Note 5 ).

2.2. Digestion and Alkylation of Nucleolar Proteins

2.3. Strong Cation Exchange Chromatography

2.4. Reversed Phase Separation on the HPLC-Chip with Online MS

8 Vollmer and Goor

3. HPLC configuration: Agilent 1200 LC system consisting of micro degasser, capillary pump, nanopump, thermostated m -well plate sampler, HPLC-chip/MS (cube) interface.

4. LC/MSD ion trap XCT ultra (Agilent, see Note 6 ).

1. Spectrum Mill MS Proteomic workbench (Agilent) installed on a dual Xenon 2.4-GHz computer.

2. IPI human database ( http://www.ebi.ac.uk/IPI ). 3. Swiss-Prot database ( http://www.expasy.org/ch2d ).

To achieve a comprehensive profile of a cellular/subcellular proteome, it is important to optimize the workflow of a multidimensional separation such that sufficient protein digest is loaded onto the first separation column without compromising significantly the separation efficiency by overloading the separation column. To achieve the required peak capacity ( 15 ) , it is important to collect a sufficient number of fractions, which are further processed by the second orthogonal separation step. However, there is always a tradeoff in the number of collected fractions because a huge number of fractions elongates the total analysis time significantly.

For very complex proteomes, it is therefore advisable to use a prefractionation technique that either removes highly abundant proteins (such as for serum or cerebrospinal fluid [CSF] ( 16 ) ) or to use a technique that decreases the complexity without reduc-ing the overall information content of the sample, e.g., by specific enrichment for certain amino acid-containing peptides. For the described workflow, 50 m g of total protein digest is used in the first dimension. Because 24 fractions are collected, every frac-tion contains an average of 2.3 m g of digest. The collection time of the fractions is optimized by varying the collection time to ensure that the total amount of peptide in the different fractions is approximately evenly distributed ( 11 ) .

1. HeLa Cells are grown in Eagles minimum medium contain-ing 5% fetal calf serum at 37C under 5% CO 2 atmosphere to 80% confluence.

2. Cells on ice are washed with cold phosphate-buffered saline and scraped off using a Teflon cell scraper.

3. Cells are resuspended in 1215 volumes hypotonic buffer and incubated on ice for 40 min.

2.5. Data Analysis

3. Methods

3.1. Protein Isolation


4. Cell lysis ( 17 ) is initiated by addition of 0.3% Nonidet P-40; homogenization is best performed using a Dounce homog-enizer.

5. Nuclei are obtained in a pellet by centrifugation at 1,300 g using a Heraeus benchtop centrifuge, after resuspension of the homogenate in ten volumes of 0.25 M sucrose, 10 mM MgCl 2 . The supernatant containing the cytoplasm is dis-carded ( see Note 7 ).

6. Nuclei are further purified at 1,300 g for 10 min through a 0.88 M sucrose, 0.05 M MgCl 2 layer.

7. Nucleoli are obtained from nuclei by resuspension in ten volumes 0.34 M sucrose followed by five 30-s sonications on ice ( see Note 8 ).

8. Nucleoli can be separated from the remaining fraction by centrifugation at 2,000 g for 20 min through a 0.88 M sucrose, 0.05 mM MgCl 2 layer.

9. The supernatant is discarded and purified nucleoli are resus-pended in 0.34 M sucrose containing 0.05 mM MgCl 2 .

10. Protein extraction and nucleic acid removal is performed according to Madjar et al. ( 18 ) by addition of magnesium acetate to a final concentration of 0.2 M, followed by the addition of two volumes of glacial acetic acid. The solution is then incubated at 4C for 1 h. Precipitated nucleic acids are removed by centrifugation at 13,000 g . The supernatant is collected and stored at 4C and the precipitate is extracted a second time to achieve an increased yield of nucleolar pro-teins. The obtained supernatants are combined and dialyzed against 500 volumes of 1 M acetic acid.

1. The protein concentration of the dialyzed nucleoli protein solution can be determined by using the Coomassie Plus Protein Assay Kit according to the assay method described by the manufacturer. Aliquots of 50 m g protein are then evaporated to dryness using an Eppendorf SpeedVac and stored at 18C until further analysis.

2. Aliquots of 50 m g protein are resuspended in 50 mM ammo-nium bicarbonate, 1 M urea, 1 mM DTT for 1 h at 37C to denature and reduce the proteins.

3. Alkylation is performed by the addition of iodoacetamide to a final concentration of 10 mM, followed by incubation for 30 min at room temperature in the dark, followed by the addition of 10 mM DTT to quench the alkylation reaction ( see Note 9 ).

4. TPCKtrypsin is added in an enzyme/substrate ratio of 1:30. The protein solution is then incubated for 15 h at 37C in a rotary shaker at 100 rpm.

3.2. Digestion and Alkylation of Nucleolar Proteins

10 Vollmer and Goor

5. The enzymatic reaction is stopped by the addition of 10% for-mic acid until a pH of 3.0 is reached ( see Note 10 ).

6. The digest is evaporated to dryness using a SpeedVac and the resulting peptide pellet can be stored in a freezer at 18C until further processing.

7. Directly before HPLC analysis, the pellet should be resus-pended in 20 m L of mobile phase A of the strong cation exchange chromatography (5% acetonitrile, 0.1% formic acid) and stored in the thermostated m -well plate sampler of the Agilent 1200 Capillary LC system at 6C.

1. The BioSCX series 2 column (50 0.8 mm) has to be condi-tioned with 500 mM KCl, 5% acetonitrile, 0.1% formic acid, followed by flushing the column with mobile phase A until baseline stability. Detection is performed by using an Agilent 1200 diode array detector tuned at a wavelength of 222 nm ( see Note 11 ).

2. The complete sample is loaded onto the column by flushing mobile phase A at a flow rate of 20 m L/min across the cation exchanger.

3. The separation gradient can be performed under the follow-ing conditions: 0 min: 0% B; 5 min: 0% B; 8 min: 10% B; 18 min: 15% B; 29 min: 70% B; 32 min: 100% B; 38 min: 100% B ( see Note 12 ) by applying a continuous gradient.

4. Reconditioning of the column should be done for at least 15 min with 100% mobile phase A before the next run.

5. Different fraction collection times of 0.5 min, 1 min, 2 min, and 4 min are useful to distribute the total amount of peptides in the fraction more evenly (illustrated in Fig. 3 , see Note 13 ). This results in fractions between 10 and 80 m L, which are col-lected in 96-well plates with conical wells of 100 m L volume.

6. Well plates are directly transferred to the second separation dimension performed on the HPLC-chip and stored in the respective HPLC m -well plate sampler cooled to 6C.

7. Special precaution has to be taken with the HPLC equipment when using high-concentration salt solutions ( see Note 14 ).

1. 50% of the fraction is injected and loaded onto the 160-nL enrichment column of the HPLC-chip at a constant flow rate of the loading pump of 4 m L/min. The chip user interface is set automatically to enrichment during this process if the injection flush volume feature is used in the Agilent Chem-Station. An injection flush volume of 58 m L is recommended to make sure that all salts are flushed off before switching the enrichment column into the nanoflow path. The chip is oper-ated in backward flush mode to achieve optimum separation efficiency ( see Note 15 ).

3.3. Strong Cation Exchange Chromatography

3.4. Reversed Phase Separation on the HPLC-Chip with Online MS


2. After loading and desalting of the sample on the chip enrich-ment column, the latter is switched on-line by the use of the HPLC-chip interface nano rotary valve with the analytical chip column (packed with the same material as the enrichment col-umn). The sample is eluted in backward flush from the enrich-ment column and transferred to the analytical column at a flow rate of 300 nL/min by a nanopump with increasing percentage of mobile phase B.

3. A continuous linear gradient is used for separation. Gradient conditions are: 0 min: 2% mobile phase B; 10 min: 2% B; 12 min: 18% B; 42 min: 55% B; 45 min: 75% B; 48 min: 5% B; followed by a post time of 6 min for column re-equilibration ( see Note 16 ).

4. Data-dependent MS acquisition is performed on the Agilent LC/MSD Trap XCT with the following MS conditions: drying gas for solvent dissolvation at 4 L nitrogen/min and 325C; MS capillary voltage: 1,800 V ( see Note 17 ); skimmer 1: 30 V; capillary exit: 75 V; and trap drive: 85. For each precursor ion, two averages are taken. The maximum accumulation time for ions in the trap is 150 ms, with a maximum target of 125,000. MS scan range is selected in a mass-over-charge ratio range of 3002,000; Ultra scan, the fastest scanning mode of the machine, is selected for detection ( see Note 18 ).

5. Fragmentation conditions for MS/MS: A maximum of three parent ions is selected in each MS/MS cycle for fragmentation. Fragmentation amplitude for peptide fragmentation: 1.25 V; SmartFrag: on, 30200%. Spectra are actively excluded for fragmentation after four recorded spectra for 2 min to allow the detection of less-abundant coeluting compounds; doubly

Fig. 3 . UV trace of the first separation dimension on an Agilent BioSCX series 2 strong cation exchanger (0.8 mm 50 mm). The signal was recorded at a wavelength of 222 nm. Collected fractions are indicated by dashed lines . Fractions were taken with different collection times depending on the peptide concentration of the individual fractions.

12 Vollmer and Goor

charged ions are preferred for selection and further fragmen-tation; and the MS/MS scan range is between 100 and 1,800 m/z for fragment ions.

1. Database searches are performed against the IPI human data-base and on specific nucleus or nucleolus localization in the Swiss-Prot database by applying distinct auto validation cri-teria in the Agilent Spectrum Mill software (Rev. A03.02.), installed on a dual Xenon 2.4 GHz computer.

2. The following auto validation criteria are used to validate the identified proteins and peptides: Minimum score for proteins is 13. Minimum scores for spectra resulting from fragmenta-tion of 1+, 2+, 3+, and 4+ parent ions are: 8, 7, 9, and 9, respectively, with a scored peak intensity (SPI, the percentage of total peak intensity that is assigned to particular ion types) value of at least 70%. Additionally, for 2+ with a SPI greater than 90%, the minimum score is 6.

3. To minimize the number of false-positive hits, all MS/MS spectra should also be searched against the reversed entries of the IPI human database. Only spectra with a reversed score that are at least two scoring units smaller than the real score should be taken into account for the auto validation. After the auto validation, only the subset of already identified proteins is used to search the IPI database again, also allowing nonspe-cific digestion. In this case, the peptide criteria can be lowered to a score greater than 6 and an SPI greater than 50%.

1. DTT stock should be freshly prepared when used. An appro-priate amount of urea is weighed before use and doubly dis-tilled water is added to make up the final concentration of 1 M urea, 1 mM DTT. The solution should not be heated above 37C to avoid carbamylation ( 19 ) . Iodoacetamide should be handled with gloves because of its toxic properties.

2. Trypsin stock should be stored in small aliquots in an acidic buffer like acetate buffer and stored at 18C to prevent auto-digestion and loss of activity. Freeze-thaw cycles should be minimized.

3. A 0.3 mm 35 mm BioSCX series 2 column is also commer-cially available, however, the loading capacity of this column is between 5 and 10 m g, whereas, for the 0.8 mm 50 mm column, the loading capacity was determined to be between 50 and 100 m g. Recommended flow rates are 5 m L/min and 20 m L/min, respectively.

3.5. Data Analysis and Processing

4. Notes


4. Thermostats should be set at 6C to prevent degradation of the sample. The 96-well plate should be sealed using a plate sealer if samples need to be stored after the first separation dimension.

5. Alternatively, an HPLC-chip is available that contains a 40-nL enrichment column. This chip can be run in forward and backward flush mode. If high loading capacity is required, such as for a complex proteome sample, the 160-nL enrich-ment column chip is the preferred choice.

6. The Agilent HPLC-chip/MS system can also be equipped with a LC/MSD trap XCT and XCT plus mass spectrom-eter. However, these instruments have slower scan rates and slower electronic signal processing. Therefore, the use of the XCT ultra leads to a higher number of identified compounds in highly complex samples.

7. It is crucial to strictly follow the described centrifugation speed and sucrose concentrations to obtain a good separa-tion of cellular components.

8. Longer sonication intervals should be avoided to keep the temperature of the suspension low.

9. Quenching of the reaction is recommended to prevent alkyla-tion of the trypsin.

10. Acidification of the digest supports evaporation of the solu-tion in the SpeedVac.

11. For proper function of the BioSCX column, the following conditioning steps at 20 m L/min are recommended: 5% aceto-nitrile, 0.03% formic acid for 10 min followed by 5% acetonitrile, 0.03% formic acid, 500 mM KCl for 15 min and 5% acetonitrile, 0.03% formic acid for at least 20 min or until the baseline is flat again at a wavelength of 220 nm UV detection.

12. A flatter gradient with a slower increase of %B/min is recommended with samples that are more complex. This, however, increases the number of fractions and the total analysis time.

13. The UV signal gives a rough estimate of the overall peptide concentration in the corresponding fraction. Performing a pre-run under identical separation conditions with a low amount of sample might give a good estimate of how to adjust the collection time of the individual fractions.

14. The system needs to be flushed thoroughly for at least 2 h immediately after analysis to prevent precipitation of salts, which can cause blockage or leakage in the LC system and to avoid corrosion of stainless steel components. Organic solvents must not be used as long as salt solutions are still in the system because they cause precipitation of crystalline

14 Vollmer and Goor

salts that might damage the system. The column should be stored in a refrigerator after use. It is recommended to use an inline filter in the loading path of the system to prevent clogging of the SCX columns by sample debris and to extend the lifetime of the column. Samples should always be centri-fuged after resuspension to prevent residual sample debris from clogging the column.

15. In general, an HPLC chip can be operated in forward and backward flush mode. In forward flush mode, the loading capillary enters the chip stator side of the rotary valve at port 6, whereas the waste capillary exits at port 5 ( Fig . 2 ). For back-ward flush mode, the loading capillary is connected to port 5 whereas the waste capillary leaves on port number 6. For chips with enrichment columns larger than 40 nL, backward flush mode is recommended to preserve narrow peak width.

16. Reconditioning time can be shortened by 12 min if higher primary flows are selected for the LC pumps. However, this increases solvent consumption.

17. Capillary voltage might be variable from setup to setup and is usually in the range of 1,7501,950 V at starting condi-tions. After a few hours of operation, the voltage should be increased by 50100 V to guarantee stable spray perform-ance over a long period of time.

18. As an alternative, the operation mode standard enhanced can be used. This results in slower scanning and data processing. If higher mass resolution is required, the standard enhanced setting should be used.

References

1. Issaq , J. H. , Chan , K. C. , Janini , G. M. , Conrads , T. P. , and Veenstra , T. D. (2005) . Multidimen-sional separation of peptides for effective pro-teomic analysis . J. Chromatogr . B 817 , 35 47

2. Jandera , P. (2006). Column selectivity for two-dimensional liquid chromatography . J. Sep. Sci. 29 , 1763 1783

3. Wolters , D. A. , Washburn M. P. , and Yates , J. R. (2006). An automated multidimensional protein identification technology for shotgun proteomics . Anal. Chem. 73 , 5683 5690

4. Wilm , M. and Mann M. (1996). Analytical properties of the nanoelectrospray ion source . Anal Chem. , 68 , 1 8

5. Koster , S. and Verpoorte , E. (2007). A dec-ade of microfluidic analysis coupled with elec-trospray mass spectrometry: an overview . Lab Chip , 7 , 1394 1412

6. Yin , H. , Killeen , K. , Brennen , R. , Sobek , D. , Werlich , M. , and van de Goor T. (2005). Microfluidic chip for peptide analysis with an integrated HPLC column, sample enrichment column and nanoelectrospray tip . Anal. Chem. 77 , 527 533

7. Yin , H. and Killeen , K. (2007). The fundamen-tal aspects and applications of Agilent HPLC-Chip . J. Sep. Sci. 30 , 1427 1434

8. Fortier , M.-H. , Bonneil , E. , Goodley , P. , and Thibault , P. (2005). Integrated microfluidic device for mass spectrometry-based proteom-ics and its application to biomarker discovery programs . Anal. Chem. 77 , 1631 1640

9. Hardouin , J. , Duchateau , M. , Caron-Jou-bert , R. , and Caron , M. (2006). Usefulness of an integrated microfluidic device (HPLC-Chip-MS) to enhance confidence in protein


identification by proteomics . Rapid Commun. Mass Spectrom . 20 , 3236 3244

10. Ninonuevo , M. R. , Park , Y. , Yin , H. , Zhang , J. , Ward , R. E. , Clowers , B. H. et al. (2006). A strategy for annotating the human milk gly-come . J. Agric. Food Chem. 54 , 7471 7480

11. Vollmer , M. , Hoerth , P. , Rozing , G. , Coute , Y. , Grimm , R. , Hochstrasser , D. , and Sanchez , J.-C. (2006). Multi-dimensional HPLC/MS of the nucleolar proteome using HPLC-chip/MS . J. Sep. Sci. 29 , 499 509

12. Hoerth , P. , Miller , C.A. , Preckel , T. , and Wenz , C. (2006). Efficient fractionation and improved protein identification by peptide OFFGEL electrophoresis . Mol. Cell. Proteomics 5.10 , 1968 1974

13. Staes , A. , Timmerman , E. , Van Damme , J. , Helsens , K. , Vandekerckhove , J. , Vollmer , M. , and Gevaert , K. (2007) . Assessing a novel microfluidic interface for shotgun proteome analyses . J. Sep. Sci. 30 , 1468 1476

14. Bradford , M. M. (1976) . A rapid and sensitive method for the quantitation of microgram

quantities of protein utilizing the principle of protein-dye binding . Anal. Biochem. 72 , 248 254

15. Giddings , J. C. (1987). Concepts and com-parisons in multidimensional chromatography . J. High Res. Chromatogr. 10 , 319 323

16. Zolotarjova , N. , Martosella , J. , Nicol , B. , Bayley J. ,. and Boyes , B. (2005). Differences among techniques for high-abundant protein removal depletion . Proteomics 5 , 3004 3013

17. Scherl , A. , Coute , Y. , Deon , C. , Calle , A. , Karine , K. , Sanchez , J.-C. et al. (2002) . Func-tional proteomic analysis of human nucleolus . Mol. Biol. Cell. 13 , 4100 4109

18. Madjar , J.-J. , Arpin , M. , Buisson , M. , Reboud , J. P. (1979). Spot position of rat ribosomal proteins by four different two-dimensional electrophoreses in polyacrylamide gel . Mol. Gen. Genet. 171 , 121 134

19. Lippincott , J. and Apostol I. (1999). Car-bamylation of cysteine: a potential artifact in peptide mapping of hemoglobins in the pres-ence of urea . Anal. Biochem. 267 , 57 64

Chapter 2

Nanofluidic Channel Fabrication and Manipulation of DNA Molecules

Kai-Ge Wang and Hanben Niu

Summary

Confining DNA molecules in a nanofluidic channel, particularly in channels with cross sections compa-rable to the persistence length of the DNA molecule (about 50 nm), allows the discovery of new bio-physical phenomena. This sub-100 nm nanofluidic channel can be used as a novel platform to study and analyze the static as well as the dynamic properties of single DNA molecules, and can be integrated into a biochip to investigate the interactions between protein and DNA molecules. For instance, nanofluidic channel arrays that have widths of approximately 40 nm, depths of 60 nm, and lengths of 50 m m are cre-ated rapidly and exactly by a focused-ion beam milling instrument on a silicon nitride film; and the open channels are sealed with anodic bonding technology. Subsequently, lambda phage DNA ( l -DNA; stained with the fluorescent dye, YOYO-1) molecules are introduced into these nanoconduits by capillary force. The movements of the DNA molecules, e.g. stretching, recoiling, and transporting along channels, are studied with fluorescence microscopy.

Key words: Nanofluidic channels , Nanopore , Focused-ion beam , DNA molecules , Fluorescence microscopy

Recently, with the advancements of nanotechnologies, many sci-entists in different research areas, including both fundamental studies and applied techniques, have focused their attention on the fabrication of nanofluidic devices and the applications in the research of single biomolecules, such as DNA and protein mol-ecules ( 1 3 ) . Nanofluidic channels with critical dimensions com-parable to the size of molecules provide new possibilities for direct observation, manipulation, and analysis of single biomolecules, and

1. Introduction

James Weifu Lee and Robert S. Foote (eds.), Micro and Nano Technologies in Bioanalysis, Methods in Molecular Biology, vol. 544DOI 10.1007/978-1-59745-483-4_2, Humana Press, a part of Springer Science + Business Media, LLC 2009

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18 Wang and Niu

provide a novel technological platform as an ultrasensitive and high-resolution sensor for studying single DNA molecules.

The nanofluidic channel is defined as a channel with at least one cross-section dimension (depth or diameter) in the nano-meter range (one-dimension or two-dimension nanochannel) ( 4 ) . In particular, a nanopore is thought of as a channel with all three dimensions in the nanoscale range, so that the work done with a nanopore falls under the realm of nanofluidics. The poten-tial application of nanopores as detectors for ultrafast genome sequencing is its most attractive application ( 5 7 ) . Nanofluidics has great benefits for bioscience studies ( 8 ) , the practical applica-tions of nanofluidics are improvements to the state of the art of DNA separation and sequencing providing significant reductions in both time and cost. The small dimensions of the nanoscale structure reduce processing times and the amount of reagents necessary for assay, substantially reducing costs.

At present, different approaches have been undertaken to successfully fabricate nanoscale structures. In general, nanofabri-cation methods can be divided roughly into two groups ( 9 ) : top-down and bottom-up methods. Top-down methods start with patterns made on a large scale and reduce their lateral dimen-sions before forming nanostructures; these methods are mainly adopted by physics scientists. On the other hand, bottom-up methods begin with atoms or molecules to build nanostructures, in some cases, through smart use of self-organization.

Top-down methods can be classified into two categories, that is, optical masked lithography and optical maskless lithography. The focused-ion beam (FIB) milling tool is a maskless lithogra-phy technique that can image features on a lithographic surface directly ( 10 ) . This technique has the advantages of facility and celerity; the patterns created are smooth and can be easily con-trolled and faithfully reproduced for different applications.

Micro-scale and submicro-scale fluidic channel arrays have been used for studying single biomolecules for many years ( 11 ) . However, applications with sub-100 nm fluidic channel arrays in biomolecular studies are rarely reported. The limitation is partially caused by the difficulty of fabrication process and metrology ( 12 ) . In addition, the properties of DNA molecules confined in these fluidic channels and the dynamics of DNA movements under this condition are not well known.

Although there are many possible applications of nanofluidic channels for DNA study, in this chapter, we focus on the manipu-lation of single DNA molecules, e.g., stretching, recoiling, and transporting. We describe the fabrication of open nanofluidic channel arrays (40 nm width, 60 nm depth) in silicon nitride (Si 3 N 4 ) membrane surfaces using the FIB milling technique and other nanofabrication techniques. Next, we describe the sealing

Nanofluidic Channel Fabrication and Manipulation of DNA Molecules 19

of these channels with Pyrex glass by the anodic bonding tech-nique; followed by a description of nanoscale channel arrays used to study the properties of single DNA molecules with the help of fluorescence microscopy. DNA molecules (e.g., l -phage DNA molecules) stained with the fluorescent dye YOYO-1 can be driven to stretch and transport along these open nanoconduits by capillary force, and also to recoil in the enclosed nanoconduits under the force of the electrode field. Because the dimension of the channel is approximately the natural-state DNA molecule per-sistence length ( ~ 50 nm) in aqueous buffer, this nanostructured channel can provide an essential new method for detecting and analyzing single DNA molecules. Such nanoconduits can be used as one component of a lab-on-a-chip in the manipulating sin-gle biomolecules. These nanochannel systems are also expected to find significant applications in medical diagnostic systems.

1. The substrate silicon wafers are 3-inch or 4-inch diameter, n -type, 390- m m or 525- m m thick, double-sided mirror-pol-ished, single crystal oriented standard bare wafers.

2. Preclean the wafers with a H 2 SO 4 -H 2 O 2 (10:1, v/v) mixture at 120C for 20 min followed by buffered HF (BHF; NH 4 F:HF = 7:1, v/v) for 2 min at room temperature to remove surface organics and metals.

3. Rinse with doubly deionized water (Millipore S.A., Molsheim, France).

4. Dry with pure nitrogen gas.

1. Pyrex 7740 borosilicate glass (3-inch or 4-inch; 600- m m thick, Corning Inc., Corning, NY), matched with the substrate silicon wafer used. The surface roughness of glass is less than 1 nm.

2. Preclean the glass with a standard solution of H 2 SO 4 -H 2 O 2 at 120C for 20 min, and then dip into a solution of buffered HF (BHF; NH 4 F:HF = 7:1, v/v) for 2 min at room tempera-ture to remove surface organics and metals.

3. Rinse with doubly deionized water (Millipore S.A.). 4. Dry with a stream of pure nitrogen.

1. Tris-EDTA: 10 mM Tris base, 1 mM EDTA, pH 8.0. All buff-ers are made with 18.2 M water purified through the Milli-Q water Purification System (Millipore).

2. TBE: 45 mM Tris base, 1 mM EDTA, 45 mM boric acid.

2. Materials

2.1. Substrates for Nanofluidic Channels

2.1.1. Substrate Silicon Wafers

2.1.2. Encapsulating Pyrex Glasses

2.2. Biophysics Experimental Buffers

20 Wang and Niu

1. l -Phage DNA molecules (Sino-American Biotechnology Company, Beijing, China), stored in alcohol at 20C. The final DNA concentration is 1 m g/mL in buffer containing 10 mM Tris-HCl, 10 mM NaCl, and 1 mM EDTA (Sigma, St. Louis, MO, USA), pH 8.0 ( see Note 1 ).

2. DNA (1 ng/ m L) is stained with 0.25 m M fluorescent dye YOYO-1 (Molecular Probes, Carlsbad, CA, USA) at a ratio of ten base pairs per dye molecule (bp/dye = 10:1), mixing DNA complex molecules with a specific volume of freshly pre-pared 0.1 m M dye solution (10 mM Tris, 1 mM EDTA buffer, pH 8.0) ( see Note 2 ).

Nanofluidics fabrication and applications have now attracted great enthusiasm because of their brilliant prospects. Nanochan-nel fabrication techniques should be cost-effective and one should be able to control the channel dimensions precisely. With the rapid improvements of nanotechnological manufacturing, four methods are now (normally) used for fabrication of nanofluidics channels, e.g., bulk nanomachining and wafer bonding ( 13 ) , sur-face nanomachining ( 14 ) , buried channel technology ( 15 ) , and nanoimprinting lithography ( 16 ) . In general, the bulk nanoma-chining technique is the preferred approach for nanoscale fabri-cation. Among the nanomachining techniques, the FIB milling technique has many advantages ( 17 ) it is an extremely versatile technique for making arbitrary micro- and nano-structures with no essentially required preprocessing or postprocessing.

Nanofluidics channels can act as a novel basis for more pre-cisely controlling the behaviors of single DNA molecules when the diameter of the channel is comparable to or less than the persistence length of the DNA molecule ( 18 ) . At nanoscale dimensions, different biophysical phenomena start to dominate, this leads to new scientific insights and applications ( 19 ) . We can use these nanoscale structures (open nanofluidic channels and enclosed nanofluidic channels) to manipulate, detect, and analyze individual biological molecules, and can also carry out individual molecular reactions within these nanofluidic environments while electric fields are used to drive flow, move analytes, and separate ionic species.

1. 500-nm thick lower stress ( ~ 200 MPa tensile) Si 3 N 4 films are deposited on both sides of the prepared bare silicon wafer by standard low-pressure chemical vapor deposition (LPCVD, M80100, Sevenstar Electronics Co., Beijing, China). The working condition are: temperature, 800C; pressure, 200 mTorr; gas, SiCl 2 H 2 and NH 3 .

2.3. Biomolecule Sample

3. Methods

3.1. Fabrication of Nanofluidic Channels

3.1.1. Creating Free-standing Si 3 N 4 Crystal Membranes


2. Approximately 100- m m thick photoresist (ARN7500, Ger-manTech Co., Beijing, China) is spun onto the front side of the silicon wafer with the spin coater (KW-4A, XiaMen Che-mat Scientific Instrument Company, XiaMen, China) at the speed of 5,000 rpm.

3. Bake the wafer at 140C for approximately 30 min and then store at room temperature in a dust-free environment.

4. A standard photolithography process is used to pattern an appropriate square ( ~ 1,200 1,200 m m 2 ) in the photoresist layer, that is, the same square pattern is exposed on the Si 3 N 4 surface.

5. The reactive ion etching (RIE, Plasmalab 80Plus RIE, Oxford Instruments Co., Abingdon, UK) is used to open the hole in the Si 3 N 4 membrane with a SF 6 /O 2 (1:4, v/v) gas mixture for 2 min, working conditions: RF, 100 W; pressure, 110 mTorr; temperature, 100C.

6. The residual photoresist on the front surface is removed using oxygen plasma with an O 2 /CF 4 gas mixture (CF 4 is ~ 20% in total gas mixture volume); RF power, 60 W; pressure, 135 mTorr; temperature, 120C.

7. The wafer is immersed into 40% (m/v) potassium hydroxide KOH(aq) at 60C for ~ 10 min to create a 100 100- m m 2 free-standing Si 3 N 4 membrane ( see Note 3 ).

1. Vent the FIB (DB235, FEI Company, Hillsboro, OR, USA) system to mount the silicon wafer with a free-standing Si 3 N 4 membrane sample carefully and tightly and then pump down the system. When vacuum is reached, switch on the beam. Carefully move the sample in the Z-direction to get closer to the working distance.

2. On the backside of the free-standing Si 3 N 4 membrane, a stand-ard FIB milling technique is used to fabricate nanoscale fluidic channel arrays. The model of FIB drilled is single-pass with a 30 keV Ga + ion beam. The initial incident ion beam full-width half-maximum (WHFM) diameter is 20 nm.

3. Choose appropriate working conditions to control the channels depth and width, where ion beam current, overlap, and dwell time are 10 pA, 50%, and 0.3 m s, respectively ( see Note 4 ).

4. Deposit platinum in the two reservoirs as the electrode. 5. A wafer bonder (EV501, EV Group, St. Florian, Austria)

is used to bond the Pyrex glass to the substrate wafer. The voltage applied on the glass wafer is negative with respect to that of the silicon wafer. The bonding process is approxi-mately 30 min at 350C with an applied voltage of 800 V. A sketching image of nanofluidic channel fabrication is shown in Fig. 1 . Some typical nanochannel arrays created are shown in Fig. 2 .

3.1.2. Fabricating Nanofluidic Channels

22 Wang and Niu

Fig. 1. Schematic drawing of fabricating process for nanofluidic channels .

Pyrex glass

RIE etching a Si3N4 hole

Photoresist

Si

Photolithography producing a hole on the photoresist layer

Fabricating micro/nanofluidic conduits with FIB .

Sealing the open channel array with anodic bonding technique.

KOH solution anisotropically etching Sisubstrate and producing a 100100 m2

free-standing Si3N4 membrane.

Si3N4


1. Incubate the DNA/YOYO-1 mixture solution in a dark room for ~ 30 min. In all experiments, the DNA base pair-to-dye ratio is kept at 10:1 (bp/dye = 10).

2. Dilute the DNA/YOYO-1 complex solution to 6.5 pM in a 50 mM Bis-Tris buffer (pH 7.5, Sigma).

3. Admix the buffer with 5% (v/v) b -mercaptoethanol (Sigma) as an antiphotobleaching agent and 2.5% (w/w) poly ( n -vinylpyrrolidone) (PVP, Sigma) to reduce both electroos-motic flow and nonspecific binding of DNA to channel walls.

3.2. Manipulation of DNA Molecules

3.2.1. Preparing Biomolecule Samples

Fig. 2 . SEM images of nanofluidic channel arrays at the center of the free-standing Si 3 N 4 membrane .

24 Wang and Niu

1. Carefully place the nanofluidics channel system on the lug-gage carrier.

2. With a syringe, transfer the DNA/YOYO-1 complex solution into one reservoir of the open fluidic channel system with a Digital Precision Microliter Pipette (Gilson S.A.S., Roissy en France, France). The solution migrates into and is transported along the open channels by capillary action as soon as it arrives at the channel entrance ( see Note 5 ).

3. With a syringe, transfer the DNA/YOYO-1 complex solution into one reservoir of the enclosed nanochannel system; the solution is loaded into the nanochannels via capillary action and then transported through the nanochannels by using an applied electrical field with platinum electrodes inserted into the reservoirs.

4. A 5-V bias is applied to drive a DNA molecule from the reser-voir into a nanochannel ( see Note 6 ).

5. Switch off the bias field before the DNA molecule has com-pletely entered the nanochannel. As a result, DNA molecule is driven back to the reservoir and can be observed to both recoil and unstretch simultaneously.

6. The DNA complex molecule is then driven entirely into the nanochannel, and the bias field is switched off ( see Note 7 ).

7. A DNA molecule is electrophoretically driven from the reser-voir into a nanochannel. Upon fully entering a nanochannel, the molecule begins to relax and finally reaches its equilib-rium extension length inside the channel ( see Note 8 ).

8. After the molecule has completely relaxed to its equilibrium length inside the nanochannel, it is electrophoretically driven to the exit of the channels. Once the tip of the molecule is straddling the interface, the voltage is turned off and the mol-ecule is observed to completely recoil from the nanochannel ( see Note 9 ).

1. The fluorescently stained DNA/YOYO-1 complex molecules are observed using an inverted optical microscope (1X-70, Olympus, Tokyo, Japan) by epifluorescence with a 20 objective.

2. A 100-W mercury lamp is used in combination with a U-MWB excitation cube (BP450480, dm500, BA515) for light-induced fluorescence illumination ( see Note 10 ).

3. Fluorescence light from the complex molecules is detected by a cooled charge-coupled device (CCD) camera (1,300 1,300 pixels, 12-bit digitization; Cool SNAP-HQ, Roper Sci-entific, Inc., Tucson, AZ, USA). MetaMorph software (Uni-versal Imaging Corporation, West Chester, PA, USA) is used for the system control, data acquisition, and data processing.

3.2.2. Manipulating DNA Molecules in the Nanofluidic Channels

3.2.3. Single Molecular Optical Imaging


The CCD acquisition time is 3 s ( see Note 11 ). Figure 3 shows a typical fluorescence image of the l -phage DNA mol-ecules passing along the open fluidic channels.

1. Lambda-phage DNA is a linear double-stranded helix that contains 48.502 kbp, its molecular mass is ~ 30.6 MDa, and its contour length is ~ 16.2 m m. It is widely used in life sciences.

2. When the mixing ratio (dye molecules per base pair) is below 1:8, the predominant binding mode of YOYO-1 on DNA is bis-intercalation; when the mixing ratio is above 1:8, groove association (external binding) with DNA begins to contribute significantly.

3. When the wafer is immersed into the KOH solution, the sili-con is etched at 54.7-degree angles relative to the surface nor-mal. This anisotropic etching creates a free-standing 100 100 m m 2 Si 3 N 4 membrane on the backside of the wafer.

4. These open nanochannels can be made with different shapes, e.g., uniform-linear or curvilinear. All of these nanochannels are combined by two bigger containers, which act as the solu-tion reservoirs. The linear nanofluidic channel can be cre-ated down to 40-nm width and 60-nm depth. The channel lengths are 50 m m, and the distance between two channels is 5 m m.

5. l -phage DNA molecules can be observed stretched and threaded along these open nanochannels. DNA molecules can be moved along the channels, although they move only a short distance, not through the whole conduit. In addition, it can be seen that not all channels are filled with DNA

4. Notes

Fig. 3. A typical fluorescence image of stained l -phage DNA inside fluidic channels. Scale bar, 10 m m.

26 Wang and Niu

molecules, i.e., there is only buffer liquid within some conduits.

6. This bias resulted in E = 100 V/cm in the nanochannels, which is large enough to drive DNA molecules. DNA mol-ecules carry negative charges, which prevent them from adhering to the nanofluidic channel walls, which are also negatively charged. This electrostatic repulsion effectively prevents the nonspecific binding of biomolecules to the nanofluidic channel surface.

7. Once the DNA molecule has contracted, it is slowly driven back down the nanochannel until a small portion of the mol-ecule has reached the reservoir. At this point, the field is switched off and the molecule is observed to undergo a pure recoil process.

8. Stretching is caused by the electric force pulling the mol-ecules into the nanochannel against a resistance at the entrance. The resistance at the entrance is probably caused by the entropic interface force and friction for molecules encountering the entrance edges.

9. Because molecules are allowed to reach equilibrium before beginning to recoil, this process is driven purely by the entropic recoil force and unaffected by elastic restoration.

10. YOYO-1 has an excitation maximum at 491 nm and an emis-sion maximum at 509 nm; that is, YOYO-1 molecules emit green fluorescence under the excitation of blue light.

11. YOYO-1 molecules bind strongly to doubled-strand DNA molecules and the fluorescence quantum yields of the bound dyes are very high. The amount of intercalated dye is pro-portional to the length of the molecule, therefore, meas-uring the total fluorescent intensity from a single molecule gives a direct measurement of its length.

This work is supported by grants from the National Natural Science Foundation of China (No. 60771048, No. 60025516, and No. 10334100), and the Major Project of National Science Foundation of China (No. 60138010), and partly supported by National Center for Nanoscience and Technology, China.

Acknowledgments


References

1. Tegenfeldt , J. O. , Prinz , C. , Cao , H. , Huang , R. L. , Austin , R. H. , Chou , S. Y. , Cox , E. C. , Sturm , J. C , (2004) Micro- and nanofluid-ics for DNA analy . Anal. Bioanal. Chem. 378 , 1678 1692

2. van der Heyden , F. H. J. , Stein , D. , Dekker , C. (2005) Streaming currents in a single nanoflu-idic channel . Phys. Rev. Lett. 95 , 116104

3. Baldessari , F. and Santiago , J. G. (2006) Elec-trophoresis in nanochannels: brief review and speculation . J. Nanobiotechnology . 4 , 12 16

4. Eijkel , J. C. T. and van den Berg , A. (2005) Nanofluidics: what is it and what can we expect from it? Microfluid Nanofluids. 1 , 249 267

5. Henrickson , S. E. , Misakian , M. , Robertson , B. , Kasianowicz , J. J. (2000) Driven DNA transport into an asymmetric nanometer-scale pore . Phys. Rev. Lett. 85 , 3057 3060

6. Li , J. , Stein , D. , McMullan , C. , Branton , D. , Aziz , M. J. , Golovchenko , J. A. (2001) Ion-beam sculpting at nanometre length scales . Nature . 412 , 166 169

7. Dekker , C. (2007) Solid state nanopores . Nat. Nanotechnol 2 , 209 215

8. Lin , Y. , Huang , M. , Chang , H. (2005) Nano-materials and chip-based nanostructures for capillary electrophoretic separations for DNA . Electrophoresis . 26 , 320 330

9. Mijatovic , D. , Eijkel , J. C. T. , van den Berg , A. (2005) Technologies for nanofluidic sys-tems: top-down vs. bottom-up . Lab. Chip. 5 , 492 500

10. Biance , A. L. , Gierak , J. , Bourhis , E. , Madouri , A. , Lafosse , X. , Patriarche , G. , Oukhaled , G. , Ulysse , C. , Galas , J. C. , Chen , Y. , Auvray , L. (2006) Focused ion beam sculpted mem-branes for nanoscience tooling . Microelectro. Eng. 83 , 1474 1477

11. Squires , T. M. and Quake , S. R. (2005) Microfluidics: fluid physics at th

micro and nano technologies in bioanalysis: methods and protocols

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