Boisseau / Lahmani Nanoscience

Nanobiotechnology and Nanobiology
2009
ISBN: 978-3-540-88633-4
Verlag: Springer Berlin Heidelberg
Format: PDF
Kopierschutz: 1 - PDF Watermark

E-Book, Englisch, 1220 Seiten

Nanobiotechnology is a rapidly developing field of research with new applications constantly emerging. This book presents the basics, fundamental results and latest achievements of nanobiotechnological research. It extends as far as promising applications of new nanomaterials and newly discovered nanoeffects. Additionally, it presents a large variety of nanobio-analysis methods.

Patrick Boisseau - Coordinator of the Nano2life European project - Specialist in Biotechnology-CEA Philippe Houdy - University Professor of Physics of solids, Specialist in nanometric media and nanomaterials Marcel Lahmani - Vice president of the French NanoMicrotechnology Club - Physicist and Docteur of Science

Autoren/Hrsg.

Boisseau, Patrick

Lahmani, Marcel

Inhaltsverzeichnis

1;Contents;12
2;Part I Biological Nano-Objects;37
2.1;1 Structural and Functional Regulation of DNA:Geometry, Topology and Methylation;38
2.1.1;1.1 Geometry of the DNA Double Helix;39
2.1.2;1.2 The Z Conformation of DNA;42
2.1.3;1.3 Supercoiled DNA;47
2.1.4;1.4 Methylation of DNA;52
2.1.4.1;1.4.1 Methylation of Cytosine;54
2.1.4.2;1.4.2 CpG Sequences;56
2.1.4.3;1.4.3 Structure of Methylated CpG Dinucleotides;57
2.1.4.4;1.4.4 Specific Recognition of Symmetric Methylationby Proteins;58
2.1.5;1.5 Conclusion;60
2.1.6;References;60
2.2;2 Protein–Lipid Assemblyand Biomimetic Nanostructures;63
2.2.1;2.1 Introduction: Biological Membranes;63
2.2.2;2.2 Lipid Membranes: Structure and Properties;65
2.2.2.1;2.2.1 The Main Classes of Lipid Membranes;65
2.2.2.1.1;Glycerophospholipids;65
2.2.2.1.2;Glyceroglycolipids;68
2.2.2.1.3;Sphingolipids;68
2.2.2.1.4;Sterols;69
2.2.2.1.5;Minor Components;70
2.2.2.2;2.2.2 Self-Assembly;70
2.2.2.3;2.2.3 Lipid Polymorphism;73
2.2.2.4;2.2.4 Lipid Shapes;78
2.2.3;2.3 Models and Methods for Characterising Membranes;80
2.2.3.1;2.3.1 Liposomes;80
2.2.3.1.1;Methods for Synthesising Liposomes;81
2.2.3.1.2;Properties of Liposomes;83
2.2.3.2;2.3.2 Langmuir Monolayers;84
2.2.3.2.1;Forming an Insoluble Langmuir Monolayer;84
2.2.3.2.2;Isotherms of a Langmuir Monolayer;86
2.2.3.2.3;Uses of Langmuir Monolayers;89
2.2.3.3;2.3.3 Supported Membranes;91
2.2.3.3.1;Supported Lipid Bilayers;91
2.2.3.3.2;Surface Nanopatterning by Supported Lipid Bilayers;96
2.2.3.3.3;Langmuir–Blodgett (LB) Films;96
2.2.3.4;2.3.4 Suspended Membranes;106
2.2.3.5;2.3.5 Bilayer Lipid Membranes (BLM);109
2.2.4;2.4 Protein–Lipid Assembly;111
2.2.4.1;2.4.1 Functionalising Langmuir–Blodgett Films;112
2.2.4.1.1;Inserting Proteins in the Interfacial MonolayerBefore Transfer to a Solid Substrate;113
2.2.4.1.2;Protein Association on Previously Formed LB Lipid Films;114
2.2.4.1.3;Oriented Insertion of Proteins in LB Films;116
2.2.4.2;2.4.2 Two-Dimensional Organisation of Proteins on Lipid Surfaces;119
2.2.4.2.1;Two-Dimensional Crystallisation of Soluble Proteinsin Lipid Monolayers;120
2.2.4.2.2;Two-Dimensional Organisation of Proteinsin Supported Lipid Bilayers;123
2.2.4.3;2.4.3 Reconstitution of Membrane Proteinsin Supported Lipid Bilayers;125
2.2.5;2.5 Applications of Biomimetic Membranesin Nanobiotechnology;126
2.2.5.1;2.5.1 Bio-Optoelectronic Micro- and Nanosensors;126
2.2.5.2;2.5.2 Composite Assemblies;129
2.2.6;References;129
2.3;3 Supramolecular Complexes of DNA;135
2.3.1;3.1 Introduction;135
2.3.2;3.2 Different Stages of Gene Transfer;139
2.3.2.1;3.2.1 Presentation;139
2.3.2.2;3.2.2 Condensation and Protection of DNA;139
2.3.2.3;3.2.3 Circulation in a Multicellular Organism;140
2.3.2.4;3.2.4 Cell Adhesion and Crossing of thePlasma Membrane;141
2.3.2.5;3.2.5 Intracellular Circulation and Entry into the Nucleus;143
2.3.2.6;3.2.6 State of the Art;144
2.3.3;3.3 Polymolecular DNA Assemblies:Synthesis, Characterisation and Properties;144
2.3.3.1;3.3.1 Polyplexes;144
2.3.3.1.1;Introduction and Structure;144
2.3.3.1.2;Synthesis of Polyplexes;145
2.3.3.1.3;Stability of Polyplexes;146
2.3.3.1.4;Using DNA/PEI Complexes for in Vitro Gene Transfer;147
2.3.3.2;3.3.2 Lipoplexes;149
2.3.3.2.1;Introduction and Structure;149
2.3.3.2.2;Synthesis of Lipoplexes;150
2.3.3.2.3;Structure and Characterisation of Lipoplexes;151
2.3.3.2.4;DNA Lipoplexes for Gene Transfer;152
2.3.3.2.5;Additives for Improving Lipoplex Properties;154
2.3.3.3;3.3.3 Modification of Polyplexes and Lipoplexesfor in Vivo Gene Transfer;154
2.3.4;3.4 Monomolecular DNA Assemblies (Nanoplexes):Synthesis, Characterisation, and Properties;156
2.3.4.1;3.4.1 Monomolecular Condensation of DNA;156
2.3.4.2;3.4.2 Chemical Synthesis;158
2.3.4.3;3.4.3 Synthesis and Characterisation of Nanoplexes;158
2.3.4.4;3.4.4 Nanoplex Modification for in Vivo Gene Transfer;160
2.3.5;3.5 Conclusion and Prospects;161
2.3.6;References;161
2.4;4 Functionalised Inorganic Nanoparticlesfor Biomedical Applications;163
2.4.1;4.1 Synthesis and Chemical Surface Modificationof Inorganic Nanoparticles;164
2.4.1.1;4.1.1 The Main Strategies;164
2.4.1.2;4.1.2 Iron Oxide Nanoparticles;167
2.4.1.2.1;Core Synthesis and Description of the Surface;167
2.4.1.2.2;Chemical Modification by Organic and Organometallic Molecules;170
2.4.1.2.3;Encapsulation by a Corona of Hydrophilic Macromolecules;173
2.4.1.2.4;Encapsulation by a Silica Shell;174
2.4.1.3;4.1.3 Semiconductor CdSe Colloids;175
2.4.1.3.1;Fabricating Semiconductor Cores;176
2.4.1.3.2;Improving Light Emission by Surface Passivation;176
2.4.1.3.3;Transferring Nanoparticles to an Aqueous Medium;177
2.4.1.4;4.1.4 Noble Metal Nanoparticles: Gold and Silver;179
2.4.2;4.2 Biological Tagging in Vitro and in Animals;180
2.4.2.1;4.2.1 Biological Tagging by Semiconductor Colloids;181
2.4.2.2;4.2.2 Biological Tagging by Metal Colloids;185
2.4.3;4.3 In Vivo Applications;188
2.4.3.1;4.3.1 Fate of Particles in the Blood Compartment;188
2.4.3.1.1;Mononuclear Phagocyte Systemand Hepatosplenic (Passive) Targeting;188
2.4.3.1.2;Designing Particles with Prolonged Intravascular Lifetime;190
2.4.3.1.3;Active Targeting Via Molecular Recognition Ligands;192
2.4.3.2;4.3.2 Tools for Medical Diagnosis: MRI Contrast Agents;193
2.4.3.2.1;Magnetic Resonance Imaging;193
2.4.3.2.2;Paramagnetic Contrast Agents (T1 Agents);194
2.4.3.2.3;Paramagnetic Metal Chelates Trapped or Grafted onto Particles;194
2.4.3.2.4;Magnetic Susceptibility Contrast Agents (T2 Agents);195
2.4.3.3;4.3.3 Therapeutic Tools;198
2.4.3.3.1;Magnetic Hyperthermia;199
2.4.3.3.2;Photothermal Treatment;201
2.4.4;4.4 Conclusion;201
2.4.5;References;202
2.5;5 Living Nanomachines;205
2.5.1;5.1 Introduction;205
2.5.2;5.2 Force and Motion by Directed Assemblyof Actin Filaments;208
2.5.2.1;5.2.1 General Considerations;208
2.5.2.2;5.2.2 Assembly Dynamics of Actin in Vitro.Intrinsic Properties;211
2.5.2.3;5.2.3 Regulation of Actin Filament Assemblyin Cell Motility;213
2.5.2.4;5.2.4 Biomimetic Motility Assay;216
2.5.2.4.1;Motility Generated by Formationof a Branching Filament Network;216
2.5.2.4.2;Motility Generated by Processive Assemblyof Unbranched Filaments;217
2.5.2.5;5.2.5 Measuring the Force Producedby Directional Actin Polymerisation;217
2.5.2.5.1;Micromanipulation Using Optical Tweezers;217
2.5.2.5.2;Effect of Viscosity on the Propulsion of Functionalised Particles;218
2.5.2.5.3;Mechanical Measurementof the Deformation of Functionalised Membranes;218
2.5.2.5.4;Micromanipulation by Micropipette;219
2.5.2.5.5;AFM Force Measurements;221
2.5.2.6;5.2.6 Theoretical Models for Force Productionby Actin Polymerisation;221
2.5.2.6.1;Microscopic Models of MotionGenerated by Actin Polymerisation;222
2.5.2.6.2;Mesoscopic Models for Force Production;224
2.5.2.7;5.2.7 Prospects;226
2.5.3;5.3 Molecular Motors: Myosins and Kinesins;227
2.5.3.1;5.3.1 Introduction;227
2.5.3.2;5.3.2 Actin Filaments and Microtubules;228
2.5.3.2.1;Organisation of Actin Filaments and Myosin in Muscle;228
2.5.3.2.2;Tubulin and Microtubules;228
2.5.3.2.3;Similarities Between Actin and Tubulin;229
2.5.3.3;5.3.3 Motor Proteins;230
2.5.3.3.1;Motors Associated with Actin Filaments;230
2.5.3.3.2;Motors Associated with Microtubules;230
2.5.3.4;5.3.4 Motion and Forces;232
2.5.3.5;5.3.5 Motion and Structural Conformation;236
2.5.3.5.1;Myosin Conformations;237
2.5.3.5.2;Structure and Directionality of Kinesins;238
2.5.4;5.4 ATP Synthase:The Smallest Known Rotary Molecular Motor;240
2.5.4.1;5.4.1 Basics of ATP Synthase;240
2.5.4.2;5.4.2 How ATP Synthase Was Recognised as a Molecular Motor: A Story of Two Conceptual Leaps;242
2.5.4.2.1;A First Conceptual Leap: The Chemiosmotic Theory;242
2.5.4.2.2;A Second Conceptual Leap:From Electrochemistry to Nanomechanics;242
2.5.4.3;5.4.3 Rotation Mechanism: Current Understanding;246
2.5.4.4;5.4.4 Thermodynamics, Kinetics, and Nanomechanics;249
2.5.4.4.1;Mechanical Energy Produced (Consumed) by ATP Hydrolysis (Synthesis);249
2.5.4.4.2;Energy Steps;250
2.5.4.4.3;An Old Problem Revisited: H+/ATP Stoichiometry;250
2.5.4.5;5.4.5 Conclusion;253
2.5.5;References;254
2.6;6 Aptamer Selection by Darwinian Evolution;257
2.6.1;6.1 Some Theoretical Aspects of Molecular Evolution;258
2.6.1.1;6.1.1 Darwin and the Theory of Evolution;258
2.6.1.2;6.1.2 Molecular Evolution and Properties of Nucleic Acids;259
2.6.1.2.1;Size and Diversity of Populations;260
2.6.1.2.2;Stability of Population Size and Limitation of Resources;260
2.6.1.2.3;Diversity and Heritability;260
2.6.2;6.2 Structural Features of Nucleic Acids;261
2.6.2.1;6.2.1 General Considerations: The Double Helix;261
2.6.2.2;6.2.2 Intrahelical Interaction Sites;262
2.6.2.3;6.2.3 From Secondary to Tertiary Structure: Supercoiling;263
2.6.2.4;6.2.4 Role of Cations and Water Molecules;264
2.6.2.5;6.2.5 Binding of an Aptamer to Its Target:Examples of Resolved Structures;264
2.6.3;6.3 SELEX;265
2.6.3.1;6.3.1 History;265
2.6.3.2;6.3.2 General Selection Principle;266
2.6.3.3;6.3.3 Chemical Modifications;269
2.6.4;6.4 Applications;270
2.6.4.1;6.4.1 Aptamers as Research Tools;270
2.6.4.1.1;Study of Nucleic Acid–Protein Interactions;270
2.6.4.1.2;Study of Nucleic Acids as Catalysts;271
2.6.4.2;6.4.2 Aptamers as Purification Tools;271
2.6.4.3;6.4.3 Aptamers as Detection Tools;272
2.6.4.3.1;Detection Method Using PCR;272
2.6.4.3.2;Optical Detection Method;273
2.6.4.3.3;Development of Aptamer Chips;273
2.6.4.3.4;Use of Aptamers for in Vivo Molecular Imaging;274
2.6.4.4;6.4.4 Aptamers as Regulatory Tools;274
2.6.4.4.1;Controlling Genetic Expression;275
2.6.4.4.2;Inhibition of Protein Activity;275
2.6.4.5;6.4.5 Aptamers as Therapeutic Tools;276
2.6.5;6.5 Conclusion;278
2.6.6;References;280
3;Part II Methods of Nanobiotechnology;284
3.1;7 Optical Tools;285
3.1.1;7.1 Introduction to Fluorescence Microscopy;285
3.1.1.1;7.1.1 Conventional Fluorescence Microscopy;285
3.1.1.1.1;Experimental Setup;285
3.1.1.1.2;Choice of Filter;287
3.1.1.2;7.1.2 Examples of Biological Applications;288
3.1.1.2.1;Localisation in Cells;288
3.1.1.2.2;Mobility Measurements;289
3.1.1.2.3;Interaction Measurements (FRET);290
3.1.1.3;7.1.3 Confocal Microscopy;291
3.1.1.3.1;Principles;291
3.1.1.3.2;Setup;292
3.1.1.3.3;Example Application;292
3.1.1.4;7.1.4 Two-Photon and Multiphoton Microscopy;293
3.1.1.5;7.1.5 Conclusions and Prospects;294
3.1.2;7.2 Labels;294
3.1.2.1;7.2.1 Introduction;294
3.1.2.2;7.2.2 Exogenous Probes;295
3.1.2.2.1;Criteria for Selecting Light-Emitting Probes;295
3.1.2.2.2;Organic Fluorophores;297
3.1.2.2.3;Luminescent Lanthanide Chelates;299
3.1.2.2.4;Nanoparticle Probes;301
3.1.2.2.5;Structure.;302
3.1.2.2.6;Optical Properties.;302
3.1.2.2.7;Functionalisation.;304
3.1.2.2.8;Biocompatibility.;305
3.1.2.2.9;Applications.;307
3.1.2.2.10;Structure.;307
3.1.2.2.11;Optical Properties.;307
3.1.2.2.12;Functionalisation.;308
3.1.2.2.13;Applications.;308
3.1.2.2.14;Structure and Functionalisation.;309
3.1.2.2.15;Optical Properties.;309
3.1.2.2.16;Applications.;309
3.1.2.2.17;Structure.;311
3.1.2.2.18;Optical Properties.;311
3.1.2.2.19;Functionalisation.;311
3.1.2.2.20;Applications.;312
3.1.2.2.21;Conclusion Concerning Exogenous Probes;312
3.1.2.3;7.2.3 Endogenous Probes: Reporter Genes;313
3.1.2.3.1;Constructing a Reporter Gene System;314
3.1.2.3.2;Cell Transfection.;315
3.1.2.3.3;Transgenic Animal.;317
3.1.2.3.4;Gene Reporter Systems Using Bioluminescence;317
3.1.2.3.5;Application to Tumour Imaging in Small Animals.;318
3.1.2.3.6;Systems Based on Fluorescence: GFP and Aequorin–GFP;320
3.1.2.4;7.2.4 Conclusion;323
3.1.3;7.3 In Vivo Detection Systems;324
3.1.3.1;7.3.1 Introduction to in Vivo Optical Imaging;324
3.1.3.2;7.3.2 Basic Principles of in Vivo Optical Imaging;325
3.1.3.2.1;Optical Properties of Tissues;325
3.1.3.2.2;Fluorescence and Bioluminescence;328
3.1.3.2.3;Limitations of in Vivo Optical Imaging;329
3.1.3.3;7.3.3 Experimental Setups for Fluorescence and Bioluminescence Imaging (Continuous Irradiation);330
3.1.3.3.1;Typical Setup;330
3.1.3.3.2;Commercially Available Systems;330
3.1.3.3.3;Toward Optical Tomography;331
3.1.3.4;7.3.4 Applications of Fluorescence andBioluminescence Imaging;332
3.1.3.4.1;Detecting Lung Tumours in Mice by a (Bioluminescence) Reporter Gene System;332
3.1.3.4.2;Detecting Subcutaneous Tumours in Micewith an Activatable Fluorescent Probe;333
3.1.3.4.3;Toward Human Applications;335
3.1.3.5;7.3.5 Time-Resolved Fluorescence Imaging;335
3.1.3.5.1;Principles of Time-Resolved Measurement;336
3.1.3.5.2;Techniques for Time-Resolved Imaging Systems;336
3.1.3.5.3;Making Use of the Time Signal;337
3.1.3.5.4;Applications;338
3.1.4;7.4 In Vitro Detection Systems;339
3.1.4.1;7.4.1 Introduction to Biochips and Microarrays;339
3.1.4.1.1;Definition;339
3.1.4.1.2;Molecular Recognition;340
3.1.4.1.3;A Brief History of the Microarray;341
3.1.4.1.4;Special Features of Microarrays;342
3.1.4.1.5;Examples of Microarrays;343
3.1.4.1.6;Fields of Application;344
3.1.4.1.7;Main Methods of Fabrication;344
3.1.4.2;7.4.2 Conventional Read Instruments;345
3.1.4.2.1;Epifluorescence Microscopes;346
3.1.4.2.2;Laser Scanning Systems;348
3.1.4.3;7.4.3 Detection by Surface Plasmon Resonance (SPR);352
3.1.4.3.1;Physical Basis;352
3.1.4.3.2;Interaction with Surface Molecules;353
3.1.4.3.3;Functionalisation of the Sensor;353
3.1.4.3.4;Measurement Configurations;354
3.1.4.3.5;Applications;355
3.1.4.3.6;Advantages and Disadvantages of SPR;355
3.1.4.4;7.4.4 Fluorescence Enhancement;356
3.1.4.5;7.4.5 Current Trends in Biological Instrumentation;358
3.1.4.5.1;Introduction;358
3.1.4.5.2;New Restrictions;358
3.1.4.5.3;Detection Integration;360
3.1.4.5.4;Optical Detection with Sensors Integrated into the Chip.;361
3.1.4.5.5;Simplifying the Light-Gathering Optics.;362
3.1.4.5.6;Optical Detection with Integrated Sources.;363
3.1.4.5.7;Integrating Sources and Detector.;364
3.1.4.5.8;Conclusion.;365
3.1.5;7.5 Other Detection Systems.Dynamics of Molecular Interactions;365
3.1.5.1;7.5.1 Fluorescence Recovery after Photobleaching(FRAP) and Associated Techniques;366
3.1.5.1.1;Fluorescent Labelling;366
3.1.5.1.2;Photobleaching;367
3.1.5.1.3;Optical Setup;368
3.1.5.1.4;Experimental Precautions;369
3.1.5.1.5;Interpreting the Data;369
3.1.5.1.6;Associated Techniques: iFRAP, FLIP, FLAP, PAF;371
3.1.5.1.7;Advantages and Disadvantages;373
3.1.5.2;7.5.2 Fluorescence Correlation Spectroscopy (FCS);373
3.1.5.2.1;Principles, Theoretical Concepts, and Main Features of FCS;374
3.1.5.2.2;Experimental Setup;377
3.1.5.2.3;Spatiotemporal Resolution and Experimental Precautions;378
3.1.5.2.4;Experimental Applications;381
3.1.5.3;7.5.3 Tracking Single Molecules and Particles;383
3.1.5.3.1;Experimental Features;383
3.1.5.3.2;Interpreting Trajectories;383
3.1.5.3.3;Advantages and Disadvantages;385
3.1.5.3.4;Detecting Single Fluorescent Molecules;385
3.1.5.3.5;Metal Nanoparticles and Quantum Dots;387
3.1.5.4;7.5.4 Fluorescence Resonance Energy Transfer (FRET);387
3.1.5.4.1;Introduction;387
3.1.5.4.2;Theory;387
3.1.5.4.3;Measurement Methods for FRET;388
3.1.5.4.4;Conclusion;392
3.1.6;References;393
3.2;8 Nanoforce and Imaging;406
3.2.1;8.1 Molecular and Cellular Imaging Using AFM;406
3.2.1.1;8.1.1 Introduction;406
3.2.1.2;8.1.2 Atomic Force Microscopy;406
3.2.1.3;8.1.3 Imaging Soluble Molecules;409
3.2.1.4;8.1.4 Membrane Imaging;411
3.2.1.4.1;Model Membranes;411
3.2.1.4.2;Subcellular Imaging and Native Membranes;413
3.2.1.5;8.1.5 AFM and Cells:Cell Imaging, Mechanical Properties, and Adhesion;415
3.2.1.5.1;Topography of Intact Cells;415
3.2.1.5.2;Mechanical Properties of Cells. Adhesion Forces;417
3.2.1.6;8.1.6 Current Limits and Future Developments;418
3.2.1.6.1;Reducing the Force Applied to the Sample;418
3.2.1.6.2;Increasing the Resolution;419
3.2.1.6.3;Increasing the Scan Rate;419
3.2.1.6.4;Identifying the Observed Structures;419
3.2.1.6.5;Combining AFM with Other Biophysical Techniques;419
3.2.1.7;8.1.7 Developments in Nanobiotechnology and Medecine;420
3.2.2;8.2 Surface Force Apparatus and Micromanipulation;421
3.2.2.1;8.2.1 Surface Force Apparatus (SFA);421
3.2.2.2;8.2.2 Micromanipulation;428
3.2.2.2.1;The Biomembrane Force Probe (BFP)and the Bond-Breaking Force Between Two Molecules;428
3.2.2.2.2;Adhesion Force Between Living Cells;431
3.2.2.2.3;Micromanipulation and Adhesion Energy of Vesicles;432
3.2.3;8.3 Atomic Force Microscopyin Contact and Tapping Modes;433
3.2.3.1;8.3.1 Introduction;433
3.2.3.2;8.3.2 Force Measurements in Contact (Static) Mode;436
3.2.3.2.1;Measuring the Separation Force for Avidin–Biotin Systems;436
3.2.3.2.2;Force–Extension Curves for Different Polymers;438
3.2.3.2.3;Protein Unfolding and Images;443
3.2.3.3;8.3.3 AFM Oscillating Modes: Introduction and Definitions;444
3.2.3.3.1;Dynamic AFM: An Oscillating Nanotip;444
3.2.3.3.2;Sensitivity and Noise. An Example in FM-AFM;445
3.2.3.3.3;Local Rheological Properties;447
3.2.3.3.4;AM-AFM Mode: Influence of the Quality Factorand Investigation of Soft Materials;448
3.2.3.4;8.3.4 Oscillations in a Liquid Medium;453
3.2.3.5;8.3.5 Force Measurements and Height Images.DNA Measurements;458
3.2.3.5.1;To Touch or Not to Touch DNA with an Oscillating Nanotip;458
3.2.3.5.2;DNA Viewed from the Chromosome to the Nucleosome;464
3.2.4;8.4 Optical Tweezers;465
3.2.4.1;8.4.1 Basic Principles and Main Parameters;465
3.2.4.2;8.4.2 Estimating the Stiffness Constant of the Trap;467
3.2.4.3;8.4.3 Different Types of Optical Tweezers;468
3.2.4.3.1;Multiple Beam Optical Traps;468
3.2.4.3.2;Multiforce Optical Tweezers;468
3.2.4.3.3;3D or Holographic Optical Tweezers;469
3.2.4.4;8.4.4 Experimental Setup;472
3.2.4.5;8.4.5 Biological Applications of Optical Tweezers;473
3.2.4.5.1;Cell Mechanotransduction;473
3.2.4.5.2;Manipulation of Whole Cells;476
3.2.4.5.3;Optical Measurement of Picoforces in Biology;477
3.2.5;8.5 Magnetic Tweezers;478
3.2.5.1;8.5.1 General Idea;478
3.2.5.2;8.5.2 A Mechanical Model for a Force Sensor:A Bead Attached to a Spring;480
3.2.5.3;8.5.3 Measuring the Bead Position withNanometric Resolution;482
3.2.5.3.1;Tracking a Bead in the Observation Plane;482
3.2.5.3.2;Tracking a Bead in the Direction Normalto the Observation Plane;483
3.2.5.4;8.5.4 Calibrating the Force Measurementby Brownian Motion;485
3.2.5.5;8.5.5 Magnets Used for Magnetic Tweezers;487
3.2.5.6;8.5.6 Advantages of Magnetic Tweezers;489
3.2.5.6.1;Twisting a Molecule;489
3.2.5.6.2;Using Magnetic Tweezers to Determine the Presence of Nicksor to Cross Two Molecules at a Single Point;491
3.2.5.7;8.5.7 Examples of Studies Using Magnetic Tweezers;492
3.2.5.7.1;Revealing the DNA Loop Formed by GalR;492
3.2.5.7.2;Observing the Separation of Two DNA Strandsby the Helicase UvrD;493
3.2.5.7.3;Unknotting of the DNA Molecule by Topoisomerase;494
3.2.5.8;8.5.8 Manipulating an Object with Magnetic Tweezers;497
3.2.6;References;498
3.3;9 Surface Methods;507
3.3.1;9.1 Biosensors Based on Surface Plasmon Resonance: Interpreting the Data;507
3.3.1.1;9.1.1 Introduction;507
3.3.1.1.1;Definition of a Biosensor;507
3.3.1.1.2;The Biacore Technology;507
3.3.1.1.3;Experimental Data or Sensorgrams;509
3.3.1.2;9.1.2 Evaluating the SPR Data;509
3.3.1.2.1;Interaction in Solution;510
3.3.1.2.2;Interaction on a Surface;511
3.3.1.2.3;Interaction in a Biacore Flow Cell;511
3.3.1.2.4;Transposing to Biacore Data;512
3.3.1.2.5;Complex Interaction Models;514
3.3.1.3;9.1.3 Measurements Under Mass Transport or Kinetic Conditions;514
3.3.1.3.1;Kinetic Experimental Conditions;514
3.3.1.3.2;Total Mass Transport Conditions;516
3.3.1.3.3;Adjusting Experimental Conditions;518
3.3.1.4;9.1.4 Other Experimental Adaptations;519
3.3.1.4.1;Eliminating Non-Specific Signals;519
3.3.1.4.2;Controlling the Molecules;523
3.3.1.5;9.1.5 Applications;525
3.3.1.5.1;Protein–Protein Interactions;526
3.3.1.5.2;SPR and Protein Structure;526
3.3.1.5.3;Nucleic Acid–Protein Interactions;527
3.3.1.5.4;Protein–Sugar Interactions;528
3.3.1.5.5;Interactions in a Membrane-Mimicking Environment;528
3.3.1.5.6;Interactions with Micro-Organisms and Eukaryotic Cells;529
3.3.1.5.7;RaPID Plot Isoaffinity Curves;529
3.3.1.5.8;Concentration Measurements;530
3.3.2;9.2 Ellipsometry;530
3.3.2.1;9.2.1 Introduction;530
3.3.2.2;9.2.2 Theory of Light and Polarisation;531
3.3.2.2.1;Description of Electromagnetic Waves;531
3.3.2.2.2;Properties of Electromagnetic Waves;531
3.3.2.2.3;Reflection of Light;534
3.3.2.3;9.2.3 Basic Principles and Possibilities of Ellipsometry;538
3.3.2.3.1;Underlying Principles of Ellipsometry;538
3.3.2.3.2;Possibilities of this Technique;539
3.3.2.4;9.2.4 Instrumentation;540
3.3.2.4.1;Ellipsometer Configurations;540
3.3.2.4.2;Detailed Description of the Phase Modulation Ellipsometer;542
3.3.2.5;9.2.5 Ellipsometric Data and Its Use;545
3.3.2.5.1;General Approach;545
3.3.2.5.2;Goodness of Fit;548
3.3.2.6;9.2.6 Applications;550
3.3.2.6.1;Characterising the Adsorption of Protein on a Surface;550
3.3.2.6.2;Kinetic Monitoring of the Adsorption of the Protein BSAon Different Surfaces;551
3.3.2.6.3;Characterising a DNA Layer Deposited on Gold;551
3.3.2.6.4;Characterising a Carbon Nanotube Sensor;552
3.3.2.6.5;Characterising a Photosensitive Langmuir–Blodgett (LB) Film;552
3.3.2.7;9.2.7 Conclusion;553
3.3.3;9.3 Optical Spectroscopy Using Waveguides;555
3.3.3.1;9.3.1 General Features of Optical Biosensors;555
3.3.3.2;9.3.2 Optical Spectroscopy of Normal ModesCoupled in a Waveguide;557
3.3.3.2.1;Optical Characteristics of a Film of BiomoleculesBound to an Interface;557
3.3.3.2.2;Principles of Waveguide Spectroscopy;558
3.3.3.2.3;Signal Processing;562
3.3.3.2.4;Consequences: Resolution and Sensitivity;563
3.3.3.3;9.3.3 Applications of Optical Waveguide Lightmode Spectroscopy;564
3.3.3.3.1;Antigen–Antibody Reactionsand Comparison with Other Techniques;564
3.3.3.3.2;Using Optical Waveguide Lightmode Spectroscopyto Monitor the Construction of Polyelectrolyte Multilayers;564
3.3.3.4;9.3.4 Conclusions;567
3.3.4;9.4 Vibrational Spectroscopy;570
3.3.4.1;9.4.1 General Features;570
3.3.4.2;9.4.2 Infrared Spectroscopy;571
3.3.4.2.1;External Reflection. Infrared Reflexion Absorption Spectroscopy (IRRAS);571
3.3.4.2.2;Comment.;577
3.3.4.2.3;Polarisation Modulation Infrared Absorption Spectroscopy (PMIRRAS);577
3.3.4.2.4;Infrared Transmission;581
3.3.4.3;9.4.3 Raman Spectroscopy;581
3.3.4.3.1;Basic Principles;581
3.3.4.3.2;Comments.;582
3.3.4.3.3;Methods for Enhancing the Signal;583
3.3.4.3.4;Comment.;584
3.3.4.3.5;Comment.;585
3.3.4.4;9.4.4 Prospects for Vibrational Spectroscopyin the Study of Nano-Objects;585
3.3.5;9.5 Brewster Angle Microscopy;586
3.3.6;9.6 Quartz Crystal Microbalancewith Dissipation Monitoring (QCM-D);591
3.3.6.1;9.6.1 Introduction;591
3.3.6.2;9.6.2 Vibration of a Damped Harmonic OscillatorSubject to Forces;593
3.3.6.3;9.6.3 Crystal in Vacuum;593
3.3.6.4;9.6.4 Crystal in Contact with a Viscous Medium;594
3.3.6.5;9.6.5 Crystal Covered with a Stratified Viscoelastic Mediumin Contact with a Viscous Medium;596
3.3.6.6;9.6.6 Numerical Simulation of the QCM Response;602
3.3.6.7;9.6.7 Analysis of a Specific Experiment:Construction of a Polyelectrolyte Multilayer Film;605
3.3.7;9.7 Grazing IncidenceNeutron and X-Ray Reflectometry;608
3.3.7.1;9.7.1 Reflection of X-Rays by a Plane Interface.Critical Angle and Fresnel Law;608
3.3.7.2;9.7.2 Interference Produced by a Homogeneous Filmof Nanometric Thickness;610
3.3.7.3;9.7.3 Determining the Density Profile of a Stratified Layer. Resolution;612
3.3.7.4;9.7.4 Neutron Reflectometry: Contrast Variation;614
3.3.8;References;616
3.4;10 Mass Spectrometry;625
3.4.1;10.1 Principles and Definitions;625
3.4.1.1;10.1.1 What Is Mass Spectrometry?;626
3.4.1.2;10.1.2 The Mass Spectrometer;626
3.4.1.3;10.1.3 Terminology;626
3.4.1.3.1;Dalton.;626
3.4.1.3.2;Mass Range.;626
3.4.1.3.3;Molecular Ion.;627
3.4.1.3.4;Average Mass.;627
3.4.1.3.5;Monoisotopic Mass.;627
3.4.1.3.6;Mass Accuracy.;627
3.4.1.3.7;Error in the Determination of m/z.;627
3.4.1.3.8;Loss of Accuracy due to the Ionisation Process.;627
3.4.1.3.9;Resolution.;627
3.4.1.3.10;Mass Resolution.;628
3.4.1.3.11;Mass Resolving Power.;628
3.4.1.3.12;Sensitivity.;628
3.4.2;10.2 Ionisation Sources for Biomolecules;628
3.4.2.1;10.2.1 Applications in Biology and Biochemistry;628
3.4.2.1.1;PDMS, FAB, and LSIMS;629
3.4.2.1.2;ESI and MALDI;630
3.4.2.2;10.2.2 Electrospray Ionisation (ESI);631
3.4.2.2.1;Description of the Ionisation Process;631
3.4.2.2.2;Multiply Charged Species;633
3.4.2.2.3;Preparing the Sample;633
3.4.2.2.4;Limitations;634
3.4.2.2.5;Improving Sensitivity in ESI MS:Microspray, Nanospray, Picospray;634
3.4.2.3;10.2.3 MALDI;636
3.4.2.3.1;Historical Review;636
3.4.2.3.2;Method of Ionisation;637
3.4.2.4;10.2.4 NanoSIMS and Ion Microscopy;639
3.4.2.4.1;Instrumentation;640
3.4.2.4.2;Preparing the Sample;641
3.4.3;10.3 Analysers;641
3.4.3.1;10.3.1 General Considerations;641
3.4.3.2;10.3.2 Time-of-Flight Analyser;642
3.4.3.2.1;Linear Mode;643
3.4.3.2.2;Reflectron Mode;643
3.4.3.2.3;Comparing Linear and Reflectron Modes;645
3.4.3.2.4;Orthogonal Acceleration Time-of-Flight Analyser;645
3.4.3.3;10.3.3 Quadrupole Analyser;646
3.4.3.3.1;Theory;646
3.4.3.3.2;Practice;647
3.4.3.4;10.3.4 Ion Trap;649
3.4.3.4.1;Three-Dimensional Ion Trap;649
3.4.3.4.2;Linear Ion Trap;650
3.4.3.5;10.3.5 Fourier Transfer Ion Cyclotron Resonance (FT-ICR) Analyser;650
3.4.4;10.4 Combined Liquid Phase Separationand Mass Spectrometry;652
3.4.4.1;10.4.1 Chromatographic Techniques;652
3.4.4.1.1;Ion Exchange Chromatography (IEC);652
3.4.4.1.2;Reversed-Phase Liquid Chromatography (RPLC);653
3.4.4.2;10.4.2 Electrophoretic Techniques;654
3.4.4.2.1;Capillary (Zone) Electrophoresis (CE/CZE);654
3.4.4.2.2;Capillary Electrochromatography (CEC);654
3.4.4.2.3;Capillary Isoelectric Focusing (CIEF);655
3.4.5;10.5 Which Mass Spectrometer Should Be Coupled with Separation Techniques: ESI or MALDI?;655
3.4.5.1;10.5.1 Combinations with HPLC;655
3.4.5.2;10.5.2 Coupling with Electrophoretic Techniques;657
3.4.6;10.6 Nanotechnology for the MS Interface;658
3.4.6.1;10.6.1 Microfluidic Chip Associating Chromatographyand Nanospray Tip;659
3.4.6.2;10.6.2 Nanospray Tip Array Chip;659
3.4.7;References;660
3.5;11 Electrical Characterisation and Dynamics of Transport;669
3.5.1;11.1 Ion Channels and the Patch-Clamp Technique;669
3.5.1.1;11.1.1 What Is an Ion Channel?;669
3.5.1.1.1;How Does an Ion Channel Work?;670
3.5.1.1.2;Properties of the Channels;671
3.5.1.2;11.1.2 Physiological Role of Ion Channels;674
3.5.1.2.1;Consequences of a Change in Channel Activity;674
3.5.1.2.2;Main Cell Functions;674
3.5.1.3;11.1.3 Pharmacological Dysfunction;675
3.5.1.3.1;Pain Channels;675
3.5.1.3.2;Multiple Sclerosis;675
3.5.1.3.3;Cystic Fibrosis;676
3.5.1.3.4;Myotonia;676
3.5.1.3.5;Cardiopathies. The Cardiac Syndrome of Prolonged QT Interval;676
3.5.1.3.6;Cancers;677
3.5.1.4;11.1.4 Direct Ways of Studying Ion Channels;679
3.5.1.4.1;Basic Concepts;679
3.5.1.4.2;History of the Patch Cl682
3.5.1.4.3;Experimental Implementation;683
3.5.1.4.4;Primary Cultures.;683
3.5.1.4.5;Heterologous Expression.;684
3.5.1.4.6;Microtransplantation.;685
3.5.1.4.7;Mechanical Features: Penetration of the Pipette into the Cell.;686
3.5.1.4.8;Electrical Features.;688
3.5.1.4.9;Current Recording;690
3.5.1.4.10;Indirect Techniques for Studying Ion Channels;696
3.5.1.5;11.1.5 Conclusion: Prospects for the Patch-ClampTechnique and the High-Throughput Revolutionin Electrophysiology;696
3.5.2;11.2 Amperometry;697
3.5.2.1;11.2.1 Basics of Faradaic Electrochemistry;698
3.5.2.1.1;Non-Faradaic Processes;699
3.5.2.1.2;Faradaic Processes;702
3.5.2.1.3;Amperometric Detection;705
3.5.2.2;11.2.2 Concentration Profiles;708
3.5.2.2.1;Diffusion Layer;708
3.5.2.2.2;Measurements Using Ultramicroelectrodes;710
3.5.2.3;11.2.3 Conclusion Regarding FaradaicElectrochemical Detection;712
3.5.2.4;11.2.4 Artificial Synapses: Biological Applicationsto Single Cells;714
3.5.2.4.1;Vesicular Exocytosis of Neurotransmitters;714
3.5.2.4.2;Detecting the Active Species of Oxidative Stress;718
3.5.2.4.3;Conclusion;724
3.5.3;11.3 Macromolecular Transport Through Naturaland Artificial Nanopores. Electrical Detection;725
3.5.3.1;11.3.1 Introduction;725
3.5.3.2;11.3.2 Electrical Detection of Particle Transport in a Pore;728
3.5.3.2.1;Electrical Resistance of a Pore;728
3.5.3.2.2;Dielectric Constant and Surface Charge Effects;729
3.5.3.2.3;Resistance of a Conducting Cylindrical PoreContaining an Insulating Sphere;731
3.5.3.3;11.3.3 Polymers Confined in Pores. Statics and Dynamics;733
3.5.3.3.1;Neutral Polymers;734
3.5.3.3.2;Charged Polymers;741
3.5.3.4;11.3.4 Some Natural and Artificial Systems;743
3.5.3.4.1;Planar Lipid Membranes and Biological Nanopores;743
3.5.3.4.2;Artificial Membranes and Nanopores;744
3.5.3.5;11.3.5 Conclusion and Prospects;748
3.5.4;11.4 Electrophoretic Techniques;749
3.5.4.1;11.4.1 Introduction;749
3.5.4.2;11.4.2 Migration of a Charged Species in Solution;750
3.5.4.3;11.4.3 Use of Polymer Matrices;751
3.5.4.3.1;Solutions of Entangled Polymers;751
3.5.4.3.2;Different DNA Migration Regimes in a Semi-Dilute Solution;752
3.5.4.3.3;Surface Coating;753
3.5.4.3.4;Examples of Polymer Matrices;755
3.5.4.4;11.4.4 Microfluidic Systems for Separationof Long DNA Fragments;756
3.5.4.4.1;Microfabricated and Self-Assembled Obstacle Arrays;757
3.5.4.4.2;Separation by Diffusion;759
3.5.4.4.3;Entropic Separation;760
3.5.4.5;11.4.5 Conclusion;761
3.5.5;References;761
3.6;12 Microfluidics: Concepts and Applicationsto the Life Sciences;773
3.6.1;12.1 Introduction;773
3.6.2;12.2 Physics of Microfluidic Flows;775
3.6.2.1;12.2.1 Fluid Mechanics on Microscopic Scales;775
3.6.2.1.1;Notion of Fluid Particle;775
3.6.2.1.2;Fundamental Equation of Motion;775
3.6.2.1.3;Forces per Unit Volume;776
3.6.2.1.4;Dimensionless Numbers;776
3.6.2.1.5;Boundary Conditions;777
3.6.2.2;12.2.2 Setting the Fluid in Motion;778
3.6.2.2.1;Setting in Motion by Pressure Difference: Poiseuille Flow;778
3.6.2.2.2;Setting in Motion by an Electric Field: Electroosmosis;780
3.6.2.2.3;Alternative Solutions;782
3.6.3;12.3 Fabrication, Materials, Functions;783
3.6.3.1;12.3.1 Lithography;784
3.6.3.2;12.3.2 Different Technologies;785
3.6.3.2.1;Silicon;785
3.6.3.2.2;Glass;786
3.6.3.2.3;Plastic;787
3.6.3.3;12.3.3 Silicone Elastomers;788
3.6.3.4;12.3.4 Elementary Components:Pumping, Mixing, and Separating in Microvolumes;790
3.6.4;12.4 Applications;791
3.6.4.1;12.4.1 Crystallisation of Proteins;791
3.6.4.1.1;Series Approach;793
3.6.4.1.2;Permeability and Soft Lithography;794
3.6.4.1.3;Parallel Approach;794
3.6.4.2;12.4.2 Separation of DNA Molecules;794
3.6.4.2.1;Artificial Gels;795
3.6.4.2.2;Nanostructures;795
3.6.4.2.3;Microdielectrophoresis;797
3.6.4.2.4;Continuous Separations with Fixed Field;798
3.6.4.3;12.4.3 Cell Sorting;798
3.6.5;12.5 Conclusion;801
3.6.6;References;801
3.7;13 Data Processing;805
3.7.1;13.1 Nanobiotechnology and Data Systems;805
3.7.1.1;13.1.1 Nanobiotechnology;805
3.7.1.2;13.1.2 Data Systems;806
3.7.1.3;13.1.3 Three Examples;808
3.7.1.3.1;Target/Probe Hybridisation Arrays;808
3.7.1.3.2;Peptide Chromatography and Mass Spectrometry;810
3.7.1.3.3;Imaging;810
3.7.1.4;13.1.4 Technological Bottlenecks;811
3.7.1.5;13.1.5 Automated Measurements;813
3.7.1.6;13.1.6 Layout of this Chapter;813
3.7.2;13.2 Representing Data;814
3.7.2.1;13.2.1 Data Structures;814
3.7.2.2;13.2.2 Sampling and Quantification;815
3.7.2.3;13.2.3 Measurement Noise;815
3.7.2.4;13.2.4 Direct or Indirect Measurement;816
3.7.3;13.3 Correcting for Sensor Defectsand Improving the Data;816
3.7.3.1;13.3.1 Linearity and Calibration;817
3.7.3.2;13.3.2 Independence and Normalisation;818
3.7.3.3;13.3.3 Noise and Filtering;819
3.7.3.4;13.3.4 Outliers;819
3.7.3.5;13.3.5 Distortion and Geometric Corrections;820
3.7.4;13.4 Data Extraction;820
3.7.4.1;13.4.1 Extracting Physical Quantities;820
3.7.4.2;13.4.2 The Systems Approach;821
3.7.4.3;13.4.3 Inverse Problems;823
3.7.4.4;13.4.4 Regularised Solutions;824
3.7.5;13.5 Data Analysis;825
3.7.5.1;13.5.1 Selecting the Relevant Measurements;825
3.7.5.2;13.5.2 Statistical Analysis;826
3.7.5.3;13.5.3 Geometrical Analysis;826
3.7.5.4;13.5.4 Classification Methods;826
3.7.6;References;828
3.8;14 Molecular Dynamics. Observing Matter in Motion;832
3.8.1;14.1 Introduction;832
3.8.1.1;14.1.1 Relating the Microscopic to theMeso- and Macroscopic;832
3.8.1.2;14.1.2 Legitimacy of Molecular Dynamics Simulations;834
3.8.2;14.2 Basic Principles of Molecular Dynamics;835
3.8.2.1;14.2.1 Validity of Molecular Dynamics Simulations;835
3.8.2.2;14.2.2 Multistep Integration of the Equations of Motion;836
3.8.3;14.3 Potential Energy Function;837
3.8.3.1;14.3.1 Meaning of Different Terms in the Force Field;838
3.8.3.2;14.3.2 Parametrisation of Unbound Atom Terms;839
3.8.3.3;14.3.3 Beyond the Usual Force Fields;841
3.8.3.3.1;Classical Description of the Chemical Bond;841
3.8.3.3.2;Coupling Between Chemical Bond and Valence Angle;841
3.8.3.3.3;Beyond a Simple Set of Point Charges;842
3.8.3.3.4;All-Atom Versus Coarse-Grained Models;842
3.8.4;14.4 Integrating the Equations of Motion;843
3.8.4.1;14.4.1 Molecular Dynamics Integrators;843
3.8.4.1.1;Exercise.;845
3.8.4.2;14.4.2 Integration with Constraints;846
3.8.4.2.1;Exercise.;846
3.8.4.3;14.4.3 Molecular Dynamics at Constant Temperature;847
3.8.4.4;14.4.4 Molecular Dynamics at Constant Pressure;850
3.8.5;14.5 Rigorous Treatment of Electrostatic Interactions;852
3.8.6;14.6 Some Properties Accessible to Simulation;855
3.8.6.1;14.6.1 Structural Properties from Simulations;855
3.8.6.1.1;Exercise.;856
3.8.6.2;14.6.2 Dynamical Properties from Simulations;856
3.8.6.3;14.6.3 Molecular Dynamics and Free Energy;858
3.8.6.3.1;Exercise.;859
3.8.7;14.7 Molecular Dynamics and Parallelisation;860
3.8.8;14.8 Conclusion;863
3.8.9;References;864
4;Part III Applications of Nanobiotechnology;868
4.1;15 Real-Time PCR;869
4.1.1;15.1 Real-Time PCR;869
4.1.1.1;15.1.1 Polymerase Chain Reaction;869
4.1.1.1.1;Basics of the Chain Reaction;869
4.1.1.1.2;PCR Kinetics;871
4.1.1.1.3;Basics and Utility of Quantitative Real-Time PCR;873
4.1.1.2;15.1.2 Equipment Used for Quantitative Real-Time PCR;874
4.1.1.3;15.1.3 Fluorescence Formats;875
4.1.1.3.1;Fluorescent DNA Markers;875
4.1.1.3.2;Fluorescent Nucleic Acid Probes;877
4.1.2;15.2 Implementing Quantitative Real-Time PCR;881
4.1.2.1;15.2.1 Denaturation and Amplification Curves;882
4.1.2.2;15.2.2 Optimising the Annealing Temperature: Specificity;885
4.1.2.3;15.2.3 Determining the Amplification Efficiency;885
4.1.2.4;15.2.4 Relative Quantification;888
4.1.2.5;15.2.5 Multiplex PCR;889
4.1.3;15.3 Applications of Real-Time PCR;890
4.1.3.1;15.3.1 Real-Time PCR for the Quantificationof Viral Genomes;890
4.1.3.2;15.3.2 Real-Time PCR in Pharmacogenetics;893
4.1.3.2.1;Introduction to Pharmacogenetics;893
4.1.3.2.2;Applications in Pharmacogenetics;893
4.1.4;References;897
4.2;16 Biosensors. From the Glucose Electrode to the Biochip;898
4.2.1;16.1 Bioreceptors;899
4.2.1.1;16.1.1 Natural Bioreceptors;900
4.2.1.1.1;Protein Structures: Enzymes and Antibodies;900
4.2.1.1.2;Whole Cells;901
4.2.1.2;16.1.2 Artificial Bioreceptors;901
4.2.1.2.1;Catalytic Antibodies;901
4.2.1.2.2;Molecularly Imprinted Polymers;902
4.2.1.2.3;Artificial Receptors;903
4.2.1.3;16.1.3 Using Ligand–Receptor Systems;903
4.2.2;16.2 Immobilisation Methods;904
4.2.2.1;16.2.1 Adsorption;904
4.2.2.2;16.2.2 Inclusion;905
4.2.2.3;16.2.3 Confinement;905
4.2.2.4;16.2.4 Crosslinking;905
4.2.2.5;16.2.5 Covalent Bonding on an Activated Substrate;906
4.2.2.5.1;Activation of Carboxylic Groups;906
4.2.2.5.2;Activation of Amine Groups;906
4.2.2.5.3;Activation of Hydroxyl Groups;906
4.2.2.5.4;Activation of Sulfhydryl Groups;906
4.2.3;16.3 Biosensors with Electrochemical Detection;907
4.2.3.1;16.3.1 Enzyme Electrodes;907
4.2.3.1.1;Electrochemical Sensors;907
4.2.3.1.2;Glucose Electrode;908
4.2.3.1.3;Urea Electrode;911
4.2.3.2;16.3.2 ENFET or Enzyme ISFET;912
4.2.4;16.4 Mass Transducer Biosensors;914
4.2.5;16.5 Enzyme Thermistors;916
4.2.6;16.6 Fibre Optic Biosensors;918
4.2.6.1;16.6.1 Fibre Optic Chemical Sensors;919
4.2.6.1.1;pH Sensors;919
4.2.6.1.2;NH3 Sensors;920
4.2.6.1.3;Oxygen Sensors;920
4.2.6.2;16.6.2 Setups for Fibre Optic Biosensors;920
4.2.6.3;16.6.3 Enzyme Fibre Optic Biosensors;921
4.2.6.3.1;Indirect Detection by Chemical Sensor;921
4.2.6.3.2;Direct Detection by Fluorescence;921
4.2.6.3.3;Direct Detection by Absorbance;921
4.2.6.4;16.6.4 Affinity Biosensors;922
4.2.6.4.1;Lectin Biosensors;922
4.2.6.4.2;Fibre Optic Biosensors for Detecting DNA;923
4.2.6.5;16.6.5 Biosensors Based on Chemiluminescent or Bioluminescent Detection;924
4.2.6.5.1;Chemiluminescent Biosensors;926
4.2.6.5.2;Electrochemiluminescent Biosensors;926
4.2.6.5.3;Bioluminescent Biosensors;926
4.2.7;16.7 Biochips;927
4.2.7.1;16.7.1 DNA Microarrays;930
4.2.7.1.1;Substrates for DNA Microarrays and Immobilisation of Probes;930
4.2.7.1.2;Reading the DNA Microarray;931
4.2.7.2;16.7.2 Protein and Other Microarrays;932
4.2.8;16.8 Conclusion;933
4.2.9;References;935
4.3;17 DNA Microarrays;937
4.3.1;17.1 Introduction;937
4.3.2;17.2 Analysing the Transcriptome;938
4.3.2.1;17.2.1 Basic Idea;938
4.3.2.2;17.2.2 Different Types of DNA Microarrayfor Transcriptome Analysis;939
4.3.2.2.1;Different Types of Support and Fabrication;939
4.3.2.2.2;cDNA Microarrays;941
4.3.2.2.3;Oligomer Microarrays;941
4.3.2.2.4;Third Generation Microarrays;943
4.3.2.3;17.2.3 Some Applications of DNA Microarrays;945
4.3.2.4;17.2.4 Some Remarks Concerning TranscriptomeData Analysis;945
4.3.2.5;17.2.5 Transcriptome Applications;946
4.3.3;17.3 Beyond the Transcriptome;949
4.3.3.1;17.3.1 CGH Microarrays;949
4.3.3.1.1;Basic Idea;949
4.3.3.1.2;Applications;951
4.3.3.2;17.3.2 ChIP on Chip;951
4.3.3.2.1;Basic Idea;951
4.3.3.2.2;Applications;952
4.3.3.3;17.3.3 When DNA Microarrays Become Cell Microarrays;954
4.3.3.3.1;Highly Parallel Transfection in Cell Microarrays;954
4.3.3.3.2;Applications;955
4.3.3.4;17.3.4 Prospects and Conclusion;956
4.3.4;References;956
4.4;18 Protein Microarrays;962
4.4.1;18.1 Overview of Proteins;962
4.4.2;18.2 Fabricating a Protein Array on a Flat Support;964
4.4.2.1;18.2.1 Preparation of Purified Proteins;964
4.4.2.1.1;Recombinant Proteins;965
4.4.2.1.2;In Situ Protein Production;965
4.4.2.1.3;Peptide Production;966
4.4.2.2;18.2.2 Substrates for Protein Microarrays;967
4.4.2.2.1;Flat Supports;967
4.4.2.2.2;Non-Flat Supports;967
4.4.2.3;18.2.3 Immobilising Proteins on the Array;968
4.4.2.3.1;Covalent Immobilisation;968
4.4.2.3.2;Non-Covalent Immobilisation;969
4.4.2.3.3;Capture by Affinity;969
4.4.2.4;18.2.4 Spotting Proteins;969
4.4.2.5;18.2.5 Detection Systems;972
4.4.2.5.1;Detection by Fluorescence;974
4.4.2.5.2;Detection by Surface Plasmon Resonance;974
4.4.2.5.3;Detection by Atomic Force Microscope;976
4.4.2.5.4;Detection by Mass Spectrometry;977
4.4.3;18.3 Other Formats for Protein Microarrays;978
4.4.4;18.4 Applications of Protein Microarrays;978
4.4.4.1;18.4.1 Analytical Microarrays;978
4.4.4.2;18.4.2 Functional Microarrays;981
4.4.5;18.5 Conclusion;983
4.4.6;References;984
4.5;19 Cell Biochips;989
4.5.1;19.1 Biochips for Analysing and Processing Living Cells;989
4.5.1.1;19.1.1 From Single Cells to Reconstituted Tissue;989
4.5.1.1.1;Parallel Cell Biochips;989
4.5.1.1.2;Series Biochips;990
4.5.1.1.3;Biochips for Manipulating and Analysing Single Cells;990
4.5.1.1.4;Tissue Models on a Chip;991
4.5.1.2;19.1.2 Cell Micromanipulation Methods;991
4.5.1.3;19.1.3 Methods for Characterising MicroculturedCells on Chip;993
4.5.2;19.2 Patch-Clamp Microarrays;996
4.5.2.1;19.2.1 Motivations;996
4.5.2.1.1;Limitations of the Patch-Clamp Techniquein the Pharmaceutical Industry;996
4.5.2.1.2;Locating Techniques for Studying Ion Channelsin the Drug Development Cycle;996
4.5.2.2;19.2.2 Emergence of New Patch-Clamp Platforms;998
4.5.2.2.1;Introduction;998
4.5.2.2.2;Microelectrodes and Automated Recording of Cell Currents;998
4.5.2.2.3;On-Chip Measurement Systems;1000
4.5.2.2.4;Array Chips.;1003
4.5.2.2.5;Rod-Shaped Chips.;1003
4.5.2.2.6;Transverse Chips.;1003
4.5.2.2.7;Choice of Material.;1003
4.5.2.2.8;Positioning and Capture of a Single Cell.;1006
4.5.2.2.9;Making the Seal: The Key Parameters.;1007
4.5.2.2.10;Micro- or Macrofluidics?;1008
4.5.2.2.11;Integration of Electrical Measurements.;1008
4.5.2.2.12;Compatibility of Cell Preparation and On-Chip Measurement.;1009
4.5.2.3;19.2.3 A Cultural Revolution? Prospects;1011
4.5.2.3.1;`Patchers Versus Screeners';1011
4.5.2.3.2;Expected Technological Progress;1013
4.5.2.3.3;Ion Channels or Biosensors?;1015
4.5.2.3.4;Conclusion;1016
4.5.3;References;1016
4.6;20 Lab on a Chip;1022
4.6.1;20.1 The General Idea;1022
4.6.2;20.2 Implanted Functions;1024
4.6.2.1;20.2.1 Sample Preparation;1025
4.6.2.1.1;Filtering;1025
4.6.2.1.2;Separation by Transport Phenomena;1026
4.6.2.1.3;Solid Phase Extraction;1027
4.6.2.1.4;Magnetic Particles;1029
4.6.2.1.5;Liquid–Liquid Extraction;1029
4.6.2.1.6;Polymerase Chain Reaction (PCR);1030
4.6.2.2;20.2.2 Transduction;1030
4.6.3;20.3 Technological Aspects;1032
4.6.4;20.4 Conclusion;1034
4.6.5;References;1036
4.7;21 Polyelectrolyte Multilayers;1040
4.7.1;21.1 The Idea;1040
4.7.1.1;21.1.1 Construction and Properties;1040
4.7.1.2;21.1.2 Physical Origin of InteractionsBetween Polyanions and Polycations;1042
4.7.2;21.2 Linear Growth and Exponential Growthof Polyelectrolyte Films;1044
4.7.2.1;21.2.1 Linear Growth;1044
4.7.2.2;21.2.2 Exponential Growth;1046
4.7.2.3;21.2.3 Fabrication of Polyelectrolyte Multilayers;1049
4.7.3;21.3 Biological Functionalisation;1050
4.7.3.1;21.3.1 Biologically Inert Films;1050
4.7.3.2;21.3.2 Functionalisation by Protein Insertion;1052
4.7.3.3;21.3.3 Functionalisation by Peptides;1056
4.7.3.4;21.3.4 Functionalisation by Drugs;1058
4.7.3.5;21.3.5 Development of Nanoreactors;1058
4.7.4;21.4 Making Hollow Particles from Multilayers;1060
4.7.5;21.5 The Route to More Complex Architectures;1061
4.7.6;21.6 Prospects;1063
4.7.7;References;1064
4.8;22 Biointegrating Materials;1066
4.8.1;22.1 Cell and Tissue Engineering;1066
4.8.2;22.2 Modifying Material Surfaces;1068
4.8.2.1;22.2.1 Using Nanoparticles to Deliver Active Ingredients;1068
4.8.2.2;22.2.2 Macroscale Functionalisation of Biomaterial Surfaces;1071
4.8.2.3;22.2.3 The Relevance of Controlled Nanotopochemistryand Nanodomains;1075
4.8.3;22.3 Applications of Biointegrated Biomaterials;1077
4.8.3.1;22.3.1 Applications to Bone Tissue;1077
4.8.3.2;22.3.2 Applications to the Vascular System;1079
4.8.4;22.4 In Vivo Assessment of Tissue Engineering Products;1081
4.8.4.1;22.4.1 Animal Models;1081
4.8.4.2;22.4.2 Which Animal Model for Which Application?;1082
4.8.4.3;22.4.3 Standard Methods for in Vivo Evaluationof Tissue Engineering Products;1083
4.8.5;22.5 Investigative MethodsAssociated with Tissue Engineering;1084
4.8.6;References;1086
4.9;23 Viral Vectors for in Vivo Gene Transfer;1092
4.9.1;23.1 Introduction;1092
4.9.1.1;23.1.1 In Vivo Gene Transfer;1092
4.9.1.2;23.1.2 Viral Vectors;1094
4.9.2;23.2 Main Types of Viral Vector;1094
4.9.2.1;23.2.1 Retroviral and Lentiviral Vectors;1095
4.9.2.1.1;Retroviral Vectors;1095
4.9.2.1.2;Lentiviral Vectors;1097
4.9.2.2;23.2.2 Adenoviral Vectors;1100
4.9.2.2.1;Wild-Type Virus;1100
4.9.2.2.2;Recombinant Vector;1100
4.9.2.3;23.2.3 Adeno-Associated Vectors;1101
4.9.3;23.3 Biomedical Applications of the Viral Platform;1102
4.9.3.1;23.3.1 Gene Therapy;1103
4.9.3.1.1;Overexpression;1103
4.9.3.1.2;Inhibition of Expression by RNA Interference;1105
4.9.3.2;23.3.2 Animal Models of Human Pathologies;1108
4.9.3.2.1;Intracerebral Injection;1109
4.9.3.2.2;Transgenesis;1110
4.9.4;23.4 Controlling and Visualising Transgene Expression;1112
4.9.4.1;23.4.1 Controlling Transgene Expression;1112
4.9.4.2;23.4.2 Imaging Transgene Expression;1115
4.9.5;23.5 Prospects;1115
4.9.6;References;1116
4.10;24 Pharmaceutical Applications of Nanoparticle Carriers;1120
4.10.1;24.1 Introduction to Drug Delivery in Pharmaceutics;1120
4.10.2;24.2 Nanoparticle Carriers;1121
4.10.2.1;24.2.1 The Main Nanoparticle Carriers;1121
4.10.2.2;24.2.2 Carrier Characteristics;1124
4.10.2.2.1;Importance of Composition;1124
4.10.2.2.2;Importance of Size;1125
4.10.2.2.3;Importance of Charge. Zeta Potential;1126
4.10.2.2.4;The Carrier–Active Principle Association;1127
4.10.3;24.3 Development of Carriersfor Pharmaceutical Applications;1128
4.10.3.1;24.3.1 Thermosensitive and pH-Sensitive(Fusogenic) Liposomes;1129
4.10.3.2;24.3.2 Modifying the Carrier Surface;1129
4.10.3.2.1;Stealth Particles;1130
4.10.3.2.2;Targeting;1131
4.10.4;24.4 Applications of Carriers;1134
4.10.4.1;24.4.1 Medical Mycology and Parasitology;1134
4.10.4.2;24.4.2 Ophthalmology;1135
4.10.4.3;24.4.3 Infectious Diseases;1136
4.10.4.4;24.4.4 Cancerology;1136
4.10.5;24.5 Conclusion;1137
4.10.6;References;1138
4.11;25 Activatable Nanoparticles for Cancer Treatment. Nanobiotix;1143
4.11.1;25.1 Introduction;1143
4.11.2;25.2 NanoTherapeutics;1145
4.11.3;25.3 Different Families of Nanoparticles;1147
4.11.4;25.4 NanoTherapeutic Action Mechanisms;1148
4.11.4.1;25.4.1 NanoMag;1148
4.11.4.2;25.4.2 NanoPDT;1148
4.11.4.3;25.4.3 NanoXRay;1149
4.11.4.4;25.4.4 Nano(U)Sonic;1150
4.11.5;25.5 Synthesising NanoMag Particles;1150
4.11.5.1;25.5.1 Coating the Fe2O3 Particles with SiO2;1151
4.11.5.2;25.5.2 Adding the Spacer;1152
4.11.5.3;25.5.3 Adding the Ligand;1152
4.11.6;25.6 Advantages of the NanoTherapeutic Families;1152
4.11.6.1;25.6.1 NanoMag;1152
4.11.6.2;25.6.2 NanoPDT;1153
4.11.6.3;25.6.3 NanoXRay;1155
4.11.6.4;25.6.4 Nano(U)Sonic;1155
4.11.7;25.7 Results (NanoMag);1155
4.11.7.1;25.7.1 In Vitro Experiments;1155
4.11.7.2;25.7.2 In Vivo Experiments;1160
4.11.8;25.8 Conclusion;1163
4.11.9;References;1163
4.12;26 The Medical, Social, and Economic Stakesof Nanobiotechnology;1164
4.12.1;26.1 From Current to Future Applications;1164
4.12.1.1;26.1.1 Diagnosis and Therapy;1164
4.12.1.1.1;In Vivo Diagnosis;1165
4.12.1.1.2;In Vitro Diagnosis;1165
4.12.1.1.3;Therapy;1166
4.12.1.2;26.1.2 Cosmetics;1170
4.12.1.3;26.1.3 Product Quality and Traceability;1171
4.12.1.4;26.1.4 Environment and Risk Prevention;1172
4.12.2;26.2 From Individual Players to Clusters;1173
4.12.2.1;26.2.1 Different Players Around the Worldand the Position of France;1173
4.12.2.2;26.2.2 Clusters and Other Poles of Competitivity;1173
4.12.3;26.3 From Funding to Industrialisation;1173
4.12.3.1;26.3.1 Patents;1173
4.12.3.2;26.3.2 Funding Nanobiotechnological Activity;1174
4.12.3.3;26.3.3 The Markets: Between Fantasy and Reality;1175
4.12.4;26.4 From Risks to Precautions;1176
4.12.4.1;26.4.1 New Risks and Ethical Considerations;1176
4.12.4.2;26.4.2 Science Fiction or Future Reality?;1177
4.12.4.3;26.4.3 Image and Communication;1178
4.12.4.4;26.4.4 Convergence of Nanoscience and the Life Sciences;1178
4.12.5;26.5 The Advent of Nanomedicine;1179
4.12.6;References;1182
5;Index;1183