Edited by Sandra J. Rosenthal David W. Wright NanoBiotechnology Protocols METHODS IN MOLECULAR BIOLOGY ™ 303 Edited by Sandra J. Rosenthal David W. Wright NanoBiotechnology Protocols NanoBiotechnology 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 ™ John M. Walker, SERIES EDITOR 316. Bioinformatics and Drug Discovery, edited by Richard S. Larson, 2005 315. Mast Cells: Methods and Protocols, edited by Guha Krishnaswamy and David S. Chi, 2005 314. DNA Repair Protocols: Mammalian Systems, Second Edition, edited by Daryl S. Henderson, 2005 313. Yeast Protocols: Second Edition, edited by Wei Xiao, 2005 312. Calcium Signaling Protocols: Second Edition, edited by David G. Lambert, 2005 311. Pharmacogenomics: Methods and Applications, edited by Federico Innocenti, 2005 310. Chemical Genomics: Reviews and Protocols, edited by Edward D. Zanders, 2005 309. RNA Silencing: Methods and Protocols, edited by Gordon Carmichael, 2005 308. Therapeutic Proteins: Methods and Protocols, edited by C. Mark Smales and David C. James, 2005 307.Phosphodiesterase Methods and Protocols, edited by Claire Lugnier, 2005 306. Receptor Binding Techniques: Second Edition, edited by Anthony P. Davenport, 2005 305.Protein–Ligand Interactions: Methods and Protocols, edited by G. Ulrich Nienhaus, 2005 304. Human Retrovirus Protocols: Virology and Molecular Biology, edited by Tuofu Zhu, 2005 303. NanoBiotechnology Protocols , edited by Sandra J. Rosenthal and David W. Wright, 2005 302. Handbook of ELISPOT: Methods and Protocols , edited by Alexander E. Kalyuzhny, 2005 301. Ubiquitin–Proteasome Protocols, edited by Cam Patterson and Douglas M. Cyr, 2005 300. Protein Nanotechnology: Protocols, Instrumenta tion, and Applications, edited by Tuan Vo-Dinh, 2005 299. Amyloid Proteins: Methods and Protocols, edited by Einar M. Sigurdsson, 2005 298. Peptide Synthesis and Application, edited by John Howl, 2005 297. Forensic DNA Typing Protocols, edited by Angel Carracedo, 2005 296. Cell Cycle Protocols, edited by Tim Humphrey and Gavin Brooks, 2005 295. Immunochemical Protocols, Third Edition, edited by Robert Burns, 2005 294. Cell Migration: Developmental Methods and Protocols, edited by Jun-Lin Guan, 2005 293. Laser Capture Microdissection: Methods and Protocols, edited by Graeme I. Murray and Stephanie Curran, 2005 292. DNA Viruses: Methods and Protocols, edited by Paul M. Lieberman, 2005 291. Molecular Toxicology Protocols, edited by Phouthone Keohavong and Stephen G. Grant, 2005 290. Basic Cell Culture, Third Edition, edited by Cheryl D. Helgason and Cindy Miller, 2005 289. Epidermal Cells, Methods and Applications, edited by Kursad Turksen, 2005 288. Oligonucleotide Synthesis, Methods and Appli- cations, edited by Piet Herdewijn, 2005 287. Epigenetics Protocols, edited by Trygve O. Tollefsbol, 2004 286. Transgenic Plants: Methods and Protocols, edited by Leandro Peña, 2005 285. Cell Cycle Control and Dysregulation Protocols: Cyclins, Cyclin-Dependent Kinases, and Other Fac- tors, edited by Antonio Giordano and Gaetano Romano, 2004 284. Signal Transduction Protocols, Second Edition, edited by Robert C. Dickson and Michael D. Mendenhall, 2004 283. Bioconjugation Protocols, edited by Christof M. Niemeyer, 2004 282. Apoptosis Methods and Protocols, edited by Hugh J. M. Brady, 2004 281. Checkpoint Controls and Cancer, Volume 2: Activation and Regulation Protocols, edited by Axel H. Schönthal, 2004 280. Checkpoint Controls and Cancer, Volume 1: Reviews and Model Systems, edited by Axel H. Schönthal, 2004 279. Nitric Oxide Protocols, Second Edition, edited by Aviv Hassid, 2004 278. Protein NMR Techniques, Second Edition, edited by A. Kristina Downing, 2004 277. Trinucleotide Repeat Protocols, edited by Yoshinori Kohwi, 2004 276. Capillary Electrophoresis of Proteins and Peptides, edited by Mark A. Strege and Avinash L. Lagu, 2004 275. Chemoinformatics, edited by Jürgen Bajorath, 2004 274. Photosynthesis Research Protocols, edited by Robert Carpentier, 2004 273. Platelets and Megakaryocytes, Volume 2: Perspectives and Techniques, edited by Jonathan M. Gibbins and Martyn P. Mahaut- Smith, 2004 272. Platelets and Megakaryocytes, Volume 1: Functional Assays, edited by Jonathan M. Gibbins and Martyn P. Mahaut-Smith, 2004 271. B Cell Protocols, edited by Hua Gu and Klaus Rajewsky, 2004 270. Parasite Genomics Protocols, edited by Sara E. Melville, 2004 269. Vaccina Virus and Poxvirology: Methods and Protocols,edited by Stuart N. Isaacs, 2004 268. Public Health Microbiology: Methods and Protocols, edited by John F. T. Spencer and Alicia L. Ragout de Spencer, 2004 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 ™ NanoBiotechnology Protocols Edited by Sandra J. Rosenthal and David W. Wright The Department of Chemistry, Vanderbilt University, Nashville, TN © 2005 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 www.humanapress.com All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular Biology TM is a trademark of The Humana Press Inc. All papers, comments, opinions, conclusions, or recommendations are those of the author(s), and do not necessarily reflect the views of the publisher. This publication is printed on acid-free paper. ∞ ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials. Production Editor: C. Tirpak Cover design by Patricia F. Cleary Cover Illustration: From Fig. 2, Chapter 1, "Applications of Quantum Dots in Biology: An Overview," by Charles Z. 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Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1 ISSN 1064-3745 E-ISBN 1-59259-901-X Library of Congress Cataloging-in-Publication Data Nanobiotechnology protocols / edited by Sandra J. Rosenthal and David W. Wright. p. cm. (Methods in molecular biology ; 303) Includes bibliographical references and index. ISBN 1-58829-276-2 (alk. paper) 1. Nanotechnology Laboratory manuals. 2. Biotechnology Laboratory manuals. I. Rosenthal, Sandra Jean, 1966- II. Wright, David W. III. Series: Methods in molecular biology (Clifton, N.J.) ; 303. TP248.25.N35N35 2005 660.6 dc22 2005000473 v Preface Increasingly, researchers find themselves involved in discipline-spanning science that a decade ago was simply inconceivable. Nowhere is this more apparent than at the cusp of two rapidly developing fields, nanoscience and biotechnology. The resulting hybrid of nanobiotechnology holds the promise of providing revolutionary insight into aspects of biology ranging from funda- mental questions of receptor function to drug discovery and personal medi- cine. As with many fields fraught with increasing hyperbole, it is essential that the underlying approaches be based on solid, reproducible methods. It is the goal of NanoBiotechnology Protocols to provide novice and experienced re- searchers alike a cross-section of the methods employed in significant frontier areas of nanobiotechnology. In a rapidly developing field such as biotechnology, it is difficult to predict at what mature endpoint a field will arrive. Today, nanobiotechnology is mak- ing significant advances in three broad areas: novel materials synthesis, dynamic cellular imaging, and biological assays. As a testament to the true nature of interdisciplinary research involved in nanobiotechnology, each of these areas is being driven by rapid advances in the others: New materials are enabling the imaging of cellular processes for longer durations, leading to high- throughput cellular-based screens for drug discovery, drug delivery, and diag- nostic applications. NanoBiotechnology Protocols addresses methods in each of these areas. Two overview chapters are provided for perspective for those beginning inves- tigations in nanobiotechnology. Throughout this volume, there is a deliberate emphasis on the use of nanoparticles. As functionalized materials, they repre- sent one of the fundamental enabling nanoscale components for these tech- nologies. Consequently, many of the protocols highlight diverse strategies to synthesize and functionalize these probes for biological applications. Other chapters focus on the use of biological components (peptides, antibodies, and DNA) to synthesize and organize nanoparticles to be used as building blocks in larger assemblies. The methods described herein are by no means complete; vi Preface nor are they necessarily intended to be. Every day seems to produce new appli- cations of nanotechnology to biological systems. It is our hope that this volume provides a detailed, hands-on perspective of nanobiotechnology to encourage scientists working in interdisciplinary fields to recognize the utility of this emerging technology. Sandra J. Rosenthal David W. Wright vii Contents Preface v Contributors ix Companion CD xii 1 Applications of Quantum Dots in Biology: An Overview Charles Z. Hotz 1 2 Fluoroimmunoassays Using Antibody-Conjugated Quantum Dots Ellen R. Goldman, Hedi Mattoussi, George P. Anderson, Igor L. Medintz, and J. Matthew Mauro 19 3 Labeling Cell-Surface Proteins Via Antibody Quantum Dot Streptavidin Conjugates John N. Mason, Ian D. Tomlinson, Sandra J. Rosenthal, and Randy D. Blakely 35 4 Peptide-Conjugated Quantum Dots: Imaging the Angiotensin Type 1 Receptor in Living Cells Ian D. Tomlinson, John N. Mason, Randy D. Blakely, and Sandra J. Rosenthal 51 5 Quantum Dot-Encoded Beads Xiaohu Gao and Shuming Nie 61 6 Use of Nanobarcodes ® Particles in Bioassays R. Griffith Freeman, Paul A. Raju, Scott M. Norton, Ian D. Walton, Patrick C. Smith, Lin He, Michael J. Natan, Michael Y. Sha, and Sharron G. Penn 73 7 Assembly and Characterization of Biomolecule–Gold Nanoparticle Conjugates and Their Use in Intracellular Imaging Alexander Tkachenko, Huan Xie, Stefan Franzen, and Daniel L. Feldheim 85 8 Whole-Blood Immunoassay Facilitated by Gold Nanoshell–Conjugate Antibodies Lee R. Hirsch, Naomi J. Halas, and Jennifer L. West 101 9 Assays for Selection of Single-Chain Fragment Variable Recombinant Antibodies to Metal Nanoclusters Jennifer Edl, Ray Mernaugh, and David W. Wright 113 10 Surface-Functionalized Nanoparticles for Controlled Drug Delivery Sung-Wook Choi, Woo-Sik Kim, and Jung-Hyun Kim 121 11 Screening of Combinatorial Peptide Libraries for Nanocluster Synthesis Joseph M. Slocik and David W. Wright 133 12 Structural DNA Nanotechnology: An Overview Nadrian C. Seeman 143 13 Nanostructured DNA Templates Jeffery L. Coffer, Russell F. Pinizzotto, and Young Gyu Rho 167 14 Probing DNA Structure With Nanoparticles Rahina Mahtab and Catherine J. Murphy 179 15 Synthetic Nanoscale Elements for Delivery of Materials Into Viable Cells Timothy E. McKnight, Anatoli V. Melechko, Michael A. Guillorn, Vladimir I. Merkulov, Douglas H. Lowndes, and Michael L. Simpson 191 16 Real-Time Cell Dynamics With a Multianalyte Physiometer Sven E. Eklund, Eugene Kozlov, Dale E. Taylor, Franz Baudenbacher, and David E. Cliffel 209 Index 224 viii Contents ix Contributors GEORGE P. ANDERSON • Center for Bio/Molecular Science and Engineering Naval Research Laboratory, Washington DC F RANZ BAUDENBACHER • Department of Physics, Vanderbilt University, Nashville, TN R ANDY D. BLAKELY • Department of Pharmacology, Vanderbilt University School of Medicine, Vanderbilt University, Nashville, TN S UNG-WOOK CHOI • Division of Chemical Engineering and Biotechnology, Yonsei University, Seoul, Korea D AVID E. CLIFFEL • Department of Chemistry, Vanderbilt University, Nashville, TN J EFFERY L. COFFER • Department of Chemistry, Texas Christian University, Fort Worth, TX J ENNIFER EDL • Department of Biochemistry, Vanderbilt University, Nashville, TN S VEN E. EKLUND • Department of Chemistry, Vanderbilt University, Nashville, TN D ANIEL L. FELDHEIM • Department of Chemistry, North Carolina State University, Raleigh, NC S TEFAN FRANZEN •Department of Chemistry, North Carolina State University, Raleigh, NC R. G RIFFITH FREEMAN • Nanoplex Technologies Inc., Menlo Park, CA X IAOHU GAO • Departments of Biomedical Engineering, Chemistry, Hematology and Oncology, Emory University, Atlanta, GA W ILHELM R. GLOMM • Department of Chemistry, North Carolina State University, Raleigh, NC E LLEN R. GOLDMAN • Center for Bio/Molecular Science and Engineering Naval Research Laboratory, Washington DC M ICHAEL A. GUILLORN • Molecular-Scale Engineering and Nanoscale Technologies Research Group, Oak Ridge National Laboratory, Oak Ridge, TN [...]... engineered bridging protein consisting of the immunoglobulin G (IgG)-binding β2 domain of streptococcal protein G modified by genetic fusion with the positively charged leucine zipper interaction domain (PG-zb), or through the use of the positively charged protein avidin A genetically engineered maltose-binding protein appended with the charged leucine zipper (MBP-zb) was used as a purification tool in. .. Center for Bio /Molecular Science and Engineering Naval Research Laboratory, Washington DC J MATTHEW MAURO • Center for Bio /Molecular Science and Engineering Naval Research Laboratory, Washington DC TIMOTHY E MCKNIGHT • Molecular- Scale Engineering and Nanoscale Technologies Research Group, Oak Ridge National Laboratory, Oak Ridge, TN IGOR L MEDINTZ • Center for Bio /Molecular Science and Engineering Naval... conjugate luminescent quantum dots with proteins extends and complements existing quantum dot-labeling methods (9,10) Conjugate preparation is simple, highly reproducible, and easily achieved We engineered proteins to interact with DHLA-capped quantum dots by appending a positively charged leucine zipper (11) interaction domain onto the C-terminus of recombinant proteins Antibodies were conjugated to quantum... relatively inert and do not impact most types of cell-based assays including immunostaining, competition binding, reporter gene, receptor internalization, and intracellular calcium release A multiplexed calcium assay for G proteincoupled receptors using quantum dots was also demonstrated The ability to spectrally encode individual cells with unique fluorescent bar codes should open new opportunities in multiplexed... dot via a short linker and characterized these probes by their interaction with serotonin transporters, electrophysiology measurements, as well as fluorescence imaging While the results for these initial conjugates show somewhat lower selectivity than high-affinity antagonists, they do show utility in the imaging of membrane proteins in living cells 3.1.5 Microplate-Based Assays Assays in microtiter plates... sulfide and zinc selenide give access to the ultraviolet Generation of far-red and near-infrared (IR) quantum dot probes will likely be extremely valuable in wholeblood assays in which absorption by hemoglobin limits the detection of shorterwavelength materials Deep tissue and in vivo imaging are other areas in which near-IR probes will find use, because scatter by tissue is minimized in this region... localized in endosomes These labeled cells were shown to be stable for as long as 12 d in culture The investigators also labeled live cells by membrane biotinylation, followed by incubation with quantum dot–avidin conjugate, although this method also resulted in quantum dot endocytosis in the cell lines studied They used the labeling procedure to study the effect of starvation on aggregation of developing... tuned to any wavelength within a range determined by the semiconductor composition Although there have been a number of reports of biological applications of quantum dots since the pioneering articles, it is clear that the use of these novel probes is still in its infancy Both protocols for using quantum dots and the methods for preparing these reagents are continually being improved Because many of... CdSe-ZnS core-shell with Fc region of the IgG (Right) In both quantum dot constructs, the MBP-zb (maltose-binding protein appended with the dimer-forming positively charged tail) serves as a purification tool for separating quantum dot–IgG conjugate away from excess IgG through affinity chromatography using crosslinked amylose resin The exact numbers of avidin, PG-zb, and MBP-zb per quantum dot are not known;... per binding event, owing to the increased absorbance and emission of the quantum dot Furthermore, there is the possibility of improved avidity compared to a dye conjugate, owing to the combined effect of many molecules of the binding ligand on the surface of the quantum dot Rosenthal et al (20) applied this concept to the study of the neurotransmitter serotonin They coupled approx 160 serotonin molecules/quantum . Series: Methods in molecular biology (Clifton, N.J.) ; 303. TP248.25.N35N35 2005 660.6 dc22 2005000473 v Preface Increasingly, researchers find themselves involved in discipline-spanning science. areas. Two overview chapters are provided for perspective for those beginning inves- tigations in nanobiotechnology. Throughout this volume, there is a deliberate emphasis on the use of nanoparticles these novel probes is still in its infancy. Both protocols for using quantum dots and the methods for preparing these reagents are continually being improved. Because many of the properties of