Nanoscience (eBook)

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

Contents 12
Part I Biological Nano-Objects 37
1 Structural and Functional Regulation of DNA:Geometry, Topology and Methylation 38
1.1 Geometry of the DNA Double Helix 39
1.2 The Z Conformation of DNA 42
1.3 Supercoiled DNA 47
1.4 Methylation of DNA 52
1.4.1 Methylation of Cytosine 54
1.4.2 CpG Sequences 56
1.4.3 Structure of Methylated CpG Dinucleotides 57
1.4.4 Specific Recognition of Symmetric Methylationby Proteins 58
1.5 Conclusion 60
References 60
2 Protein–Lipid Assemblyand Biomimetic Nanostructures 63
2.1 Introduction: Biological Membranes 63
2.2 Lipid Membranes: Structure and Properties 65
2.2.1 The Main Classes of Lipid Membranes 65
Glycerophospholipids 65
Glyceroglycolipids 68
Sphingolipids 68
Sterols 69
Minor Components 70
2.2.2 Self-Assembly 70
2.2.3 Lipid Polymorphism 73
2.2.4 Lipid Shapes 78
2.3 Models and Methods for Characterising Membranes 80
2.3.1 Liposomes 80
Methods for Synthesising Liposomes 81
Properties of Liposomes 83
2.3.2 Langmuir Monolayers 84
Forming an Insoluble Langmuir Monolayer 84
Isotherms of a Langmuir Monolayer 86
Uses of Langmuir Monolayers 89
2.3.3 Supported Membranes 91
Supported Lipid Bilayers 91
Surface Nanopatterning by Supported Lipid Bilayers 96
Langmuir–Blodgett (LB) Films 96
2.3.4 Suspended Membranes 106
2.3.5 Bilayer Lipid Membranes (BLM) 109
2.4 Protein–Lipid Assembly 111
2.4.1 Functionalising Langmuir–Blodgett Films 112
Inserting Proteins in the Interfacial MonolayerBefore Transfer to a Solid Substrate 113
Protein Association on Previously Formed LB Lipid Films 114
Oriented Insertion of Proteins in LB Films 116
2.4.2 Two-Dimensional Organisation of Proteins on Lipid Surfaces 119
Two-Dimensional Crystallisation of Soluble Proteinsin Lipid Monolayers 120
Two-Dimensional Organisation of Proteinsin Supported Lipid Bilayers 123
2.4.3 Reconstitution of Membrane Proteinsin Supported Lipid Bilayers 125
2.5 Applications of Biomimetic Membranesin Nanobiotechnology 126
2.5.1 Bio-Optoelectronic Micro- and Nanosensors 126
2.5.2 Composite Assemblies 129
References 129
3 Supramolecular Complexes of DNA 135
3.1 Introduction 135
3.2 Different Stages of Gene Transfer 139
3.2.1 Presentation 139
3.2.2 Condensation and Protection of DNA 139
3.2.3 Circulation in a Multicellular Organism 140
3.2.4 Cell Adhesion and Crossing of thePlasma Membrane 141
3.2.5 Intracellular Circulation and Entry into the Nucleus 143
3.2.6 State of the Art 144
3.3 Polymolecular DNA Assemblies:Synthesis, Characterisation and Properties 144
3.3.1 Polyplexes 144
Introduction and Structure 144
Synthesis of Polyplexes 145
Stability of Polyplexes 146
Using DNA/PEI Complexes for in Vitro Gene Transfer 147
3.3.2 Lipoplexes 149
Introduction and Structure 149
Synthesis of Lipoplexes 150
Structure and Characterisation of Lipoplexes 151
DNA Lipoplexes for Gene Transfer 152
Additives for Improving Lipoplex Properties 154
3.3.3 Modification of Polyplexes and Lipoplexesfor in Vivo Gene Transfer 154
3.4 Monomolecular DNA Assemblies (Nanoplexes):Synthesis, Characterisation, and Properties 156
3.4.1 Monomolecular Condensation of DNA 156
3.4.2 Chemical Synthesis 158
3.4.3 Synthesis and Characterisation of Nanoplexes 158
3.4.4 Nanoplex Modification for in Vivo Gene Transfer 160
3.5 Conclusion and Prospects 161
References 161
4 Functionalised Inorganic Nanoparticlesfor Biomedical Applications 163
4.1 Synthesis and Chemical Surface Modificationof Inorganic Nanoparticles 164
4.1.1 The Main Strategies 164
4.1.2 Iron Oxide Nanoparticles 167
Core Synthesis and Description of the Surface 167
Chemical Modification by Organic and Organometallic Molecules 170
Encapsulation by a Corona of Hydrophilic Macromolecules 173
Encapsulation by a Silica Shell 174
4.1.3 Semiconductor CdSe Colloids 175
Fabricating Semiconductor Cores 176
Improving Light Emission by Surface Passivation 176
Transferring Nanoparticles to an Aqueous Medium 177
4.1.4 Noble Metal Nanoparticles: Gold and Silver 179
4.2 Biological Tagging in Vitro and in Animals 180
4.2.1 Biological Tagging by Semiconductor Colloids 181
4.2.2 Biological Tagging by Metal Colloids 185
4.3 In Vivo Applications 188
4.3.1 Fate of Particles in the Blood Compartment 188
Mononuclear Phagocyte Systemand Hepatosplenic (Passive) Targeting 188
Designing Particles with Prolonged Intravascular Lifetime 190
Active Targeting Via Molecular Recognition Ligands 192
4.3.2 Tools for Medical Diagnosis: MRI Contrast Agents 193
Magnetic Resonance Imaging 193
Paramagnetic Contrast Agents (T1 Agents) 194
Paramagnetic Metal Chelates Trapped or Grafted onto Particles 194
Magnetic Susceptibility Contrast Agents (T2 Agents) 195
4.3.3 Therapeutic Tools 198
Magnetic Hyperthermia 199
Photothermal Treatment 201
4.4 Conclusion 201
References 202
5 Living Nanomachines 205
5.1 Introduction 205
5.2 Force and Motion by Directed Assemblyof Actin Filaments 208
5.2.1 General Considerations 208
5.2.2 Assembly Dynamics of Actin in Vitro.Intrinsic Properties 211
5.2.3 Regulation of Actin Filament Assemblyin Cell Motility 213
5.2.4 Biomimetic Motility Assay 216
Motility Generated by Formationof a Branching Filament Network 216
Motility Generated by Processive Assemblyof Unbranched Filaments 217
5.2.5 Measuring the Force Producedby Directional Actin Polymerisation 217
Micromanipulation Using Optical Tweezers 217
Effect of Viscosity on the Propulsion of Functionalised Particles 218
Mechanical Measurementof the Deformation of Functionalised Membranes 218
Micromanipulation by Micropipette 219
AFM Force Measurements 221
5.2.6 Theoretical Models for Force Productionby Actin Polymerisation 221
Microscopic Models of MotionGenerated by Actin Polymerisation 222
Mesoscopic Models for Force Production 224
5.2.7 Prospects 226
5.3 Molecular Motors: Myosins and Kinesins 227
5.3.1 Introduction 227
5.3.2 Actin Filaments and Microtubules 228
Organisation of Actin Filaments and Myosin in Muscle 228
Tubulin and Microtubules 228
Similarities Between Actin and Tubulin 229
5.3.3 Motor Proteins 230
Motors Associated with Actin Filaments 230
Motors Associated with Microtubules 230
5.3.4 Motion and Forces 232
5.3.5 Motion and Structural Conformation 236
Myosin Conformations 237
Structure and Directionality of Kinesins 238
5.4 ATP Synthase:The Smallest Known Rotary Molecular Motor 240
5.4.1 Basics of ATP Synthase 240
5.4.2 How ATP Synthase Was Recognised as a Molecular Motor: A Story of Two Conceptual Leaps 242
A First Conceptual Leap: The Chemiosmotic Theory 242
A Second Conceptual Leap:From Electrochemistry to Nanomechanics 242
5.4.3 Rotation Mechanism: Current Understanding 246
5.4.4 Thermodynamics, Kinetics, and Nanomechanics 249
Mechanical Energy Produced (Consumed) by ATP Hydrolysis (Synthesis) 249
Energy Steps 250
An Old Problem Revisited: H+/ATP Stoichiometry 250
5.4.5 Conclusion 253
References 254
6 Aptamer Selection by Darwinian Evolution 257
6.1 Some Theoretical Aspects of Molecular Evolution 258
6.1.1 Darwin and the Theory of Evolution 258
6.1.2 Molecular Evolution and Properties of Nucleic Acids 259
Size and Diversity of Populations 260
Stability of Population Size and Limitation of Resources 260
Diversity and Heritability 260
6.2 Structural Features of Nucleic Acids 261
6.2.1 General Considerations: The Double Helix 261
6.2.2 Intrahelical Interaction Sites 262
6.2.3 From Secondary to Tertiary Structure: Supercoiling 263
6.2.4 Role of Cations and Water Molecules 264
6.2.5 Binding of an Aptamer to Its Target:Examples of Resolved Structures 264
6.3 SELEX 265
6.3.1 History 265
6.3.2 General Selection Principle 266
6.3.3 Chemical Modifications 269
6.4 Applications 270
6.4.1 Aptamers as Research Tools 270
Study of Nucleic Acid–Protein Interactions 270
Study of Nucleic Acids as Catalysts 271
6.4.2 Aptamers as Purification Tools 271
6.4.3 Aptamers as Detection Tools 272
Detection Method Using PCR 272
Optical Detection Method 273
Development of Aptamer Chips 273
Use of Aptamers for in Vivo Molecular Imaging 274
6.4.4 Aptamers as Regulatory Tools 274
Controlling Genetic Expression 275
Inhibition of Protein Activity 275
6.4.5 Aptamers as Therapeutic Tools 276
6.5 Conclusion 278
References 280
Part II Methods of Nanobiotechnology 284
7 Optical Tools 285
7.1 Introduction to Fluorescence Microscopy 285
7.1.1 Conventional Fluorescence Microscopy 285
Experimental Setup 285
Choice of Filter 287
7.1.2 Examples of Biological Applications 288
Localisation in Cells 288
Mobility Measurements 289
Interaction Measurements (FRET) 290
7.1.3 Confocal Microscopy 291
Principles 291
Setup 292
Example Application 292
7.1.4 Two-Photon and Multiphoton Microscopy 293
7.1.5 Conclusions and Prospects 294
7.2 Labels 294
7.2.1 Introduction 294
7.2.2 Exogenous Probes 295
Criteria for Selecting Light-Emitting Probes 295
Organic Fluorophores 297
Luminescent Lanthanide Chelates 299
Nanoparticle Probes 301
Structure. 302
Optical Properties. 302
Functionalisation. 304
Biocompatibility. 305
Applications. 307
Structure. 307
Optical Properties. 307
Functionalisation. 308
Applications. 308
Structure and Functionalisation. 309
Optical Properties. 309
Applications. 309
Structure. 311
Optical Properties. 311
Functionalisation. 311
Applications. 312
Conclusion Concerning Exogenous Probes 312
7.2.3 Endogenous Probes: Reporter Genes 313
Constructing a Reporter Gene System 314
Cell Transfection. 315
Transgenic Animal. 317
Gene Reporter Systems Using Bioluminescence 317
Application to Tumour Imaging in Small Animals. 318
Systems Based on Fluorescence: GFP and Aequorin–GFP 320
7.2.4 Conclusion 323
7.3 In Vivo Detection Systems 324
7.3.1 Introduction to in Vivo Optical Imaging 324
7.3.2 Basic Principles of in Vivo Optical Imaging 325
Optical Properties of Tissues 325
Fluorescence and Bioluminescence 328
Limitations of in Vivo Optical Imaging 329
7.3.3 Experimental Setups for Fluorescence and Bioluminescence Imaging (Continuous Irradiation) 330
Typical Setup 330
Commercially Available Systems 330
Toward Optical Tomography 331
7.3.4 Applications of Fluorescence andBioluminescence Imaging 332
Detecting Lung Tumours in Mice by a (Bioluminescence) Reporter Gene System 332
Detecting Subcutaneous Tumours in Micewith an Activatable Fluorescent Probe 333
Toward Human Applications 335
7.3.5 Time-Resolved Fluorescence Imaging 335
Principles of Time-Resolved Measurement 336
Techniques for Time-Resolved Imaging Systems 336
Making Use of the Time Signal 337
Applications 338
7.4 In Vitro Detection Systems 339
7.4.1 Introduction to Biochips and Microarrays 339
Definition 339
Molecular Recognition 340
A Brief History of the Microarray 341
Special Features of Microarrays 342
Examples of Microarrays 343
Fields of Application 344
Main Methods of Fabrication 344
7.4.2 Conventional Read Instruments 345
Epifluorescence Microscopes 346
Laser Scanning Systems 348
7.4.3 Detection by Surface Plasmon Resonance (SPR) 352
Physical Basis 352
Interaction with Surface Molecules 353
Functionalisation of the Sensor 353
Measurement Configurations 354
Applications 355
Advantages and Disadvantages of SPR 355
7.4.4 Fluorescence Enhancement 356
7.4.5 Current Trends in Biological Instrumentation 358
Introduction 358
New Restrictions 358
Detection Integration 360
Optical Detection with Sensors Integrated into the Chip. 361
Simplifying the Light-Gathering Optics. 362
Optical Detection with Integrated Sources. 363
Integrating Sources and Detector. 364
Conclusion. 365
7.5 Other Detection Systems.Dynamics of Molecular Interactions 365
7.5.1 Fluorescence Recovery after Photobleaching(FRAP) and Associated Techniques 366
Fluorescent Labelling 366
Photobleaching 367
Optical Setup 368
Experimental Precautions 369
Interpreting the Data 369
Associated Techniques: iFRAP, FLIP, FLAP, PAF 371
Advantages and Disadvantages 373
7.5.2 Fluorescence Correlation Spectroscopy (FCS) 373
Principles, Theoretical Concepts, and Main Features of FCS 374
Experimental Setup 377
Spatiotemporal Resolution and Experimental Precautions 378
Experimental Applications 381
7.5.3 Tracking Single Molecules and Particles 383
Experimental Features 383
Interpreting Trajectories 383
Advantages and Disadvantages 385
Detecting Single Fluorescent Molecules 385
Metal Nanoparticles and Quantum Dots 387
7.5.4 Fluorescence Resonance Energy Transfer (FRET) 387
Introduction 387
Theory 387
Measurement Methods for FRET 388
Conclusion 392
References 393
8 Nanoforce and Imaging 406
8.1 Molecular and Cellular Imaging Using AFM 406
8.1.1 Introduction 406
8.1.2 Atomic Force Microscopy 406
8.1.3 Imaging Soluble Molecules 409
8.1.4 Membrane Imaging 411
Model Membranes 411
Subcellular Imaging and Native Membranes 413
8.1.5 AFM and Cells:Cell Imaging, Mechanical Properties, and Adhesion 415
Topography of Intact Cells 415
Mechanical Properties of Cells. Adhesion Forces 417
8.1.6 Current Limits and Future Developments 418
Reducing the Force Applied to the Sample 418
Increasing the Resolution 419
Increasing the Scan Rate 419
Identifying the Observed Structures 419
Combining AFM with Other Biophysical Techniques 419
8.1.7 Developments in Nanobiotechnology and Medecine 420
8.2 Surface Force Apparatus and Micromanipulation 421
8.2.1 Surface Force Apparatus (SFA) 421
8.2.2 Micromanipulation 428
The Biomembrane Force Probe (BFP)and the Bond-Breaking Force Between Two Molecules 428
Adhesion Force Between Living Cells 431
Micromanipulation and Adhesion Energy of Vesicles 432
8.3 Atomic Force Microscopyin Contact and Tapping Modes 433
8.3.1 Introduction 433
8.3.2 Force Measurements in Contact (Static) Mode 436
Measuring the Separation Force for Avidin–Biotin Systems 436
Force–Extension Curves for Different Polymers 438
Protein Unfolding and Images 443
8.3.3 AFM Oscillating Modes: Introduction and Definitions 444
Dynamic AFM: An Oscillating Nanotip 444
Sensitivity and Noise. An Example in FM-AFM 445
Local Rheological Properties 447
AM-AFM Mode: Influence of the Quality Factorand Investigation of Soft Materials 448
8.3.4 Oscillations in a Liquid Medium 453
8.3.5 Force Measurements and Height Images.DNA Measurements 458
To Touch or Not to Touch DNA with an Oscillating Nanotip 458
DNA Viewed from the Chromosome to the Nucleosome 464
8.4 Optical Tweezers 465
8.4.1 Basic Principles and Main Parameters 465
8.4.2 Estimating the Stiffness Constant of the Trap 467
8.4.3 Different Types of Optical Tweezers 468
Multiple Beam Optical Traps 468
Multiforce Optical Tweezers 468
3D or Holographic Optical Tweezers 469
8.4.4 Experimental Setup 472
8.4.5 Biological Applications of Optical Tweezers 473
Cell Mechanotransduction 473
Manipulation of Whole Cells 476
Optical Measurement of Picoforces in Biology 477
8.5 Magnetic Tweezers 478
8.5.1 General Idea 478
8.5.2 A Mechanical Model for a Force Sensor:A Bead Attached to a Spring 480
8.5.3 Measuring the Bead Position withNanometric Resolution 482
Tracking a Bead in the Observation Plane 482
Tracking a Bead in the Direction Normalto the Observation Plane 483
8.5.4 Calibrating the Force Measurementby Brownian Motion 485
8.5.5 Magnets Used for Magnetic Tweezers 487
8.5.6 Advantages of Magnetic Tweezers 489
Twisting a Molecule 489
Using Magnetic Tweezers to Determine the Presence of Nicksor to Cross Two Molecules at a Single Point 491
8.5.7 Examples of Studies Using Magnetic Tweezers 492
Revealing the DNA Loop Formed by GalR 492
Observing the Separation of Two DNA Strandsby the Helicase UvrD 493
Unknotting of the DNA Molecule by Topoisomerase 494
8.5.8 Manipulating an Object with Magnetic Tweezers 497
References 498
9 Surface Methods 507
9.1 Biosensors Based on Surface Plasmon Resonance: Interpreting the Data 507
9.1.1 Introduction 507
Definition of a Biosensor 507
The Biacore Technology 507
Experimental Data or Sensorgrams 509
9.1.2 Evaluating the SPR Data 509
Interaction in Solution 510
Interaction on a Surface 511
Interaction in a Biacore Flow Cell 511
Transposing to Biacore Data 512
Complex Interaction Models 514
9.1.3 Measurements Under Mass Transport or Kinetic Conditions 514
Kinetic Experimental Conditions 514
Total Mass Transport Conditions 516
Adjusting Experimental Conditions 518
9.1.4 Other Experimental Adaptations 519
Eliminating Non-Specific Signals 519
Controlling the Molecules 523
9.1.5 Applications 525
Protein–Protein Interactions 526
SPR and Protein Structure 526
Nucleic Acid–Protein Interactions 527
Protein–Sugar Interactions 528
Interactions in a Membrane-Mimicking Environment 528
Interactions with Micro-Organisms and Eukaryotic Cells 529
RaPID Plot Isoaffinity Curves 529
Concentration Measurements 530
9.2 Ellipsometry 530
9.2.1 Introduction 530
9.2.2 Theory of Light and Polarisation 531
Description of Electromagnetic Waves 531
Properties of Electromagnetic Waves 531
Reflection of Light 534
9.2.3 Basic Principles and Possibilities of Ellipsometry 538
Underlying Principles of Ellipsometry 538
Possibilities of this Technique 539
9.2.4 Instrumentation 540
Ellipsometer Configurations 540
Detailed Description of the Phase Modulation Ellipsometer 542
9.2.5 Ellipsometric Data and Its Use 545
General Approach 545
Goodness of Fit 548
9.2.6 Applications 550
Characterising the Adsorption of Protein on a Surface 550
Kinetic Monitoring of the Adsorption of the Protein BSAon Different Surfaces 551
Characterising a DNA Layer Deposited on Gold 551
Characterising a Carbon Nanotube Sensor 552
Characterising a Photosensitive Langmuir–Blodgett (LB) Film 552
9.2.7 Conclusion 553
9.3 Optical Spectroscopy Using Waveguides 555
9.3.1 General Features of Optical Biosensors 555
9.3.2 Optical Spectroscopy of Normal ModesCoupled in a Waveguide 557
Optical Characteristics of a Film of BiomoleculesBound to an Interface 557
Principles of Waveguide Spectroscopy 558
Signal Processing 562
Consequences: Resolution and Sensitivity 563
9.3.3 Applications of Optical Waveguide Lightmode Spectroscopy 564
Antigen–Antibody Reactionsand Comparison with Other Techniques 564
Using Optical Waveguide Lightmode Spectroscopyto Monitor the Construction of Polyelectrolyte Multilayers 564
9.3.4 Conclusions 567
9.4 Vibrational Spectroscopy 570
9.4.1 General Features 570
9.4.2 Infrared Spectroscopy 571
External Reflection. Infrared Reflexion Absorption Spectroscopy (IRRAS) 571
Comment. 577
Polarisation Modulation Infrared Absorption Spectroscopy (PMIRRAS) 577
Infrared Transmission 581
9.4.3 Raman Spectroscopy 581
Basic Principles 581
Comments. 582
Methods for Enhancing the Signal 583
Comment. 584
Comment. 585
9.4.4 Prospects for Vibrational Spectroscopyin the Study of Nano-Objects 585
9.5 Brewster Angle Microscopy 586
9.6 Quartz Crystal Microbalancewith Dissipation Monitoring (QCM-D) 591
9.6.1 Introduction 591
9.6.2 Vibration of a Damped Harmonic OscillatorSubject to Forces 593
9.6.3 Crystal in Vacuum 593
9.6.4 Crystal in Contact with a Viscous Medium 594
9.6.5 Crystal Covered with a Stratified Viscoelastic Mediumin Contact with a Viscous Medium 596
9.6.6 Numerical Simulation of the QCM Response 602
9.6.7 Analysis of a Specific Experiment:Construction of a Polyelectrolyte Multilayer Film 605
9.7 Grazing IncidenceNeutron and X-Ray Reflectometry 608
9.7.1 Reflection of X-Rays by a Plane Interface.Critical Angle and Fresnel Law 608
9.7.2 Interference Produced by a Homogeneous Filmof Nanometric Thickness 610
9.7.3 Determining the Density Profile of a Stratified Layer. Resolution 612
9.7.4 Neutron Reflectometry: Contrast Variation 614
References 616
10 Mass Spectrometry 625
10.1 Principles and Definitions 625
10.1.1 What Is Mass Spectrometry? 626
10.1.2 The Mass Spectrometer 626
10.1.3 Terminology 626
Dalton. 626
Mass Range. 626
Molecular Ion. 627
Average Mass. 627
Monoisotopic Mass. 627
Mass Accuracy. 627
Error in the Determination of m/z. 627
Loss of Accuracy due to the Ionisation Process. 627
Resolution. 627
Mass Resolution. 628
Mass Resolving Power. 628
Sensitivity. 628
10.2 Ionisation Sources for Biomolecules 628
10.2.1 Applications in Biology and Biochemistry 628
PDMS, FAB, and LSIMS 629
ESI and MALDI 630
10.2.2 Electrospray Ionisation (ESI) 631
Description of the Ionisation Process 631
Multiply Charged Species 633
Preparing the Sample 633
Limitations 634
Improving Sensitivity in ESI MS:Microspray, Nanospray, Picospray 634
10.2.3 MALDI 636
Historical Review 636
Method of Ionisation 637
10.2.4 NanoSIMS and Ion Microscopy 639
Instrumentation 640
Preparing the Sample 641
10.3 Analysers 641
10.3.1 General Considerations 641
10.3.2 Time-of-Flight Analyser 642
Linear Mode 643
Reflectron Mode 643
Comparing Linear and Reflectron Modes 645
Orthogonal Acceleration Time-of-Flight Analyser 645
10.3.3 Quadrupole Analyser 646
Theory 646
Practice 647
10.3.4 Ion Trap 649
Three-Dimensional Ion Trap 649
Linear Ion Trap 650
10.3.5 Fourier Transfer Ion Cyclotron Resonance (FT-ICR) Analyser 650
10.4 Combined Liquid Phase Separationand Mass Spectrometry 652
10.4.1 Chromatographic Techniques 652
Ion Exchange Chromatography (IEC) 652
Reversed-Phase Liquid Chromatography (RPLC) 653
10.4.2 Electrophoretic Techniques 654
Capillary (Zone) Electrophoresis (CE/CZE) 654
Capillary Electrochromatography (CEC) 654
Capillary Isoelectric Focusing (CIEF) 655
10.5 Which Mass Spectrometer Should Be Coupled with Separation Techniques: ESI or MALDI? 655
10.5.1 Combinations with HPLC 655
10.5.2 Coupling with Electrophoretic Techniques 657
10.6 Nanotechnology for the MS Interface 658
10.6.1 Microfluidic Chip Associating Chromatographyand Nanospray Tip 659
10.6.2 Nanospray Tip Array Chip 659
References 660
11 Electrical Characterisation and Dynamics of Transport 669
11.1 Ion Channels and the Patch-Clamp Technique 669
11.1.1 What Is an Ion Channel? 669
How Does an Ion Channel Work? 670
Properties of the Channels 671
11.1.2 Physiological Role of Ion Channels 674
Consequences of a Change in Channel Activity 674
Main Cell Functions 674
11.1.3 Pharmacological Dysfunction 675
Pain Channels 675
Multiple Sclerosis 675
Cystic Fibrosis 676
Myotonia 676
Cardiopathies. The Cardiac Syndrome of Prolonged QT Interval 676
Cancers 677
11.1.4 Direct Ways of Studying Ion Channels 679
Basic Concepts 679
History of the Patch Clamp 682
Experimental Implementation 683
Primary Cultures. 683
Heterologous Expression. 684
Microtransplantation. 685
Mechanical Features: Penetration of the Pipette into the Cell. 686
Electrical Features. 688
Current Recording 690
Indirect Techniques for Studying Ion Channels 696
11.1.5 Conclusion: Prospects for the Patch-ClampTechnique and the High-Throughput Revolutionin Electrophysiology 696
11.2 Amperometry 697
11.2.1 Basics of Faradaic Electrochemistry 698
Non-Faradaic Processes 699
Faradaic Processes 702
Amperometric Detection 705
11.2.2 Concentration Profiles 708
Diffusion Layer 708
Measurements Using Ultramicroelectrodes 710
11.2.3 Conclusion Regarding FaradaicElectrochemical Detection 712
11.2.4 Artificial Synapses: Biological Applicationsto Single Cells 714
Vesicular Exocytosis of Neurotransmitters 714
Detecting the Active Species of Oxidative Stress 718
Conclusion 724
11.3 Macromolecular Transport Through Naturaland Artificial Nanopores. Electrical Detection 725
11.3.1 Introduction 725
11.3.2 Electrical Detection of Particle Transport in a Pore 728
Electrical Resistance of a Pore 728
Dielectric Constant and Surface Charge Effects 729
Resistance of a Conducting Cylindrical PoreContaining an Insulating Sphere 731
11.3.3 Polymers Confined in Pores. Statics and Dynamics 733
Neutral Polymers 734
Charged Polymers 741
11.3.4 Some Natural and Artificial Systems 743
Planar Lipid Membranes and Biological Nanopores 743
Artificial Membranes and Nanopores 744
11.3.5 Conclusion and Prospects 748
11.4 Electrophoretic Techniques 749
11.4.1 Introduction 749
11.4.2 Migration of a Charged Species in Solution 750
11.4.3 Use of Polymer Matrices 751
Solutions of Entangled Polymers 751
Different DNA Migration Regimes in a Semi-Dilute Solution 752
Surface Coating 753
Examples of Polymer Matrices 755
11.4.4 Microfluidic Systems for Separationof Long DNA Fragments 756
Microfabricated and Self-Assembled Obstacle Arrays 757
Separation by Diffusion 759
Entropic Separation 760
11.4.5 Conclusion 761
References 761
12 Microfluidics: Concepts and Applicationsto the Life Sciences 773
12.1 Introduction 773
12.2 Physics of Microfluidic Flows 775
12.2.1 Fluid Mechanics on Microscopic Scales 775
Notion of Fluid Particle 775
Fundamental Equation of Motion 775
Forces per Unit Volume 776
Dimensionless Numbers 776
Boundary Conditions 777
12.2.2 Setting the Fluid in Motion 778
Setting in Motion by Pressure Difference: Poiseuille Flow 778
Setting in Motion by an Electric Field: Electroosmosis 780
Alternative Solutions 782
12.3 Fabrication, Materials, Functions 783
12.3.1 Lithography 784
12.3.2 Different Technologies 785
Silicon 785
Glass 786
Plastic 787
12.3.3 Silicone Elastomers 788
12.3.4 Elementary Components:Pumping, Mixing, and Separating in Microvolumes 790
12.4 Applications 791
12.4.1 Crystallisation of Proteins 791
Series Approach 793
Permeability and Soft Lithography 794
Parallel Approach 794
12.4.2 Separation of DNA Molecules 794
Artificial Gels 795
Nanostructures 795
Microdielectrophoresis 797
Continuous Separations with Fixed Field 798
12.4.3 Cell Sorting 798
12.5 Conclusion 801
References 801
13 Data Processing 805
13.1 Nanobiotechnology and Data Systems 805
13.1.1 Nanobiotechnology 805
13.1.2 Data Systems 806
13.1.3 Three Examples 808
Target/Probe Hybridisation Arrays 808
Peptide Chromatography and Mass Spectrometry 810
Imaging 810
13.1.4 Technological Bottlenecks 811
13.1.5 Automated Measurements 813
13.1.6 Layout of this Chapter 813
13.2 Representing Data 814
13.2.1 Data Structures 814
13.2.2 Sampling and Quantification 815
13.2.3 Measurement Noise 815
13.2.4 Direct or Indirect Measurement 816
13.3 Correcting for Sensor Defectsand Improving the Data 816
13.3.1 Linearity and Calibration 817
13.3.2 Independence and Normalisation 818
13.3.3 Noise and Filtering 819
13.3.4 Outliers 819
13.3.5 Distortion and Geometric Corrections 820
13.4 Data Extraction 820
13.4.1 Extracting Physical Quantities 820
13.4.2 The Systems Approach 821
13.4.3 Inverse Problems 823
13.4.4 Regularised Solutions 824
13.5 Data Analysis 825
13.5.1 Selecting the Relevant Measurements 825
13.5.2 Statistical Analysis 826
13.5.3 Geometrical Analysis 826
13.5.4 Classification Methods 826
References 828
14 Molecular Dynamics. Observing Matter in Motion 832
14.1 Introduction 832
14.1.1 Relating the Microscopic to theMeso- and Macroscopic 832
14.1.2 Legitimacy of Molecular Dynamics Simulations 834
14.2 Basic Principles of Molecular Dynamics 835
14.2.1 Validity of Molecular Dynamics Simulations 835
14.2.2 Multistep Integration of the Equations of Motion 836
14.3 Potential Energy Function 837
14.3.1 Meaning of Different Terms in the Force Field 838
14.3.2 Parametrisation of Unbound Atom Terms 839
14.3.3 Beyond the Usual Force Fields 841
Classical Description of the Chemical Bond 841
Coupling Between Chemical Bond and Valence Angle 841
Beyond a Simple Set of Point Charges 842
All-Atom Versus Coarse-Grained Models 842
14.4 Integrating the Equations of Motion 843
14.4.1 Molecular Dynamics Integrators 843
Exercise. 845
14.4.2 Integration with Constraints 846
Exercise. 846
14.4.3 Molecular Dynamics at Constant Temperature 847
14.4.4 Molecular Dynamics at Constant Pressure 850
14.5 Rigorous Treatment of Electrostatic Interactions 852
14.6 Some Properties Accessible to Simulation 855
14.6.1 Structural Properties from Simulations 855
Exercise. 856
14.6.2 Dynamical Properties from Simulations 856
14.6.3 Molecular Dynamics and Free Energy 858
Exercise. 859
14.7 Molecular Dynamics and Parallelisation 860
14.8 Conclusion 863
References 864
Part III Applications of Nanobiotechnology 868
15 Real-Time PCR 869
15.1 Real-Time PCR 869
15.1.1 Polymerase Chain Reaction 869
Basics of the Chain Reaction 869
PCR Kinetics 871
Basics and Utility of Quantitative Real-Time PCR 873
15.1.2 Equipment Used for Quantitative Real-Time PCR 874
15.1.3 Fluorescence Formats 875
Fluorescent DNA Markers 875
Fluorescent Nucleic Acid Probes 877
15.2 Implementing Quantitative Real-Time PCR 881
15.2.1 Denaturation and Amplification Curves 882
15.2.2 Optimising the Annealing Temperature: Specificity 885
15.2.3 Determining the Amplification Efficiency 885
15.2.4 Relative Quantification 888
15.2.5 Multiplex PCR 889
15.3 Applications of Real-Time PCR 890
15.3.1 Real-Time PCR for the Quantificationof Viral Genomes 890
15.3.2 Real-Time PCR in Pharmacogenetics 893
Introduction to Pharmacogenetics 893
Applications in Pharmacogenetics 893
References 897
16 Biosensors. From the Glucose Electrode to the Biochip 898
16.1 Bioreceptors 899
16.1.1 Natural Bioreceptors 900
Protein Structures: Enzymes and Antibodies 900
Whole Cells 901
16.1.2 Artificial Bioreceptors 901
Catalytic Antibodies 901
Molecularly Imprinted Polymers 902
Artificial Receptors 903
16.1.3 Using Ligand–Receptor Systems 903
16.2 Immobilisation Methods 904
16.2.1 Adsorption 904
16.2.2 Inclusion 905
16.2.3 Confinement 905
16.2.4 Crosslinking 905
16.2.5 Covalent Bonding on an Activated Substrate 906
Activation of Carboxylic Groups 906
Activation of Amine Groups 906
Activation of Hydroxyl Groups 906
Activation of Sulfhydryl Groups 906
16.3 Biosensors with Electrochemical Detection 907
16.3.1 Enzyme Electrodes 907
Electrochemical Sensors 907
Glucose Electrode 908
Urea Electrode 911
16.3.2 ENFET or Enzyme ISFET 912
16.4 Mass Transducer Biosensors 914
16.5 Enzyme Thermistors 916
16.6 Fibre Optic Biosensors 918
16.6.1 Fibre Optic Chemical Sensors 919
pH Sensors 919
NH3 Sensors 920
Oxygen Sensors 920
16.6.2 Setups for Fibre Optic Biosensors 920
16.6.3 Enzyme Fibre Optic Biosensors 921
Indirect Detection by Chemical Sensor 921
Direct Detection by Fluorescence 921
Direct Detection by Absorbance 921
16.6.4 Affinity Biosensors 922
Lectin Biosensors 922
Fibre Optic Biosensors for Detecting DNA 923
16.6.5 Biosensors Based on Chemiluminescent or Bioluminescent Detection 924
Chemiluminescent Biosensors 926
Electrochemiluminescent Biosensors 926
Bioluminescent Biosensors 926
16.7 Biochips 927
16.7.1 DNA Microarrays 930
Substrates for DNA Microarrays and Immobilisation of Probes 930
Reading the DNA Microarray 931
16.7.2 Protein and Other Microarrays 932
16.8 Conclusion 933
References 935
17 DNA Microarrays 937
17.1 Introduction 937
17.2 Analysing the Transcriptome 938
17.2.1 Basic Idea 938
17.2.2 Different Types of DNA Microarrayfor Transcriptome Analysis 939
Different Types of Support and Fabrication 939
cDNA Microarrays 941
Oligomer Microarrays 941
Third Generation Microarrays 943
17.2.3 Some Applications of DNA Microarrays 945
17.2.4 Some Remarks Concerning TranscriptomeData Analysis 945
17.2.5 Transcriptome Applications 946
17.3 Beyond the Transcriptome 949
17.3.1 CGH Microarrays 949
Basic Idea 949
Applications 951
17.3.2 ChIP on Chip 951
Basic Idea 951
Applications 952
17.3.3 When DNA Microarrays Become Cell Microarrays 954
Highly Parallel Transfection in Cell Microarrays 954
Applications 955
17.3.4 Prospects and Conclusion 956
References 956
18 Protein Microarrays 962
18.1 Overview of Proteins 962
18.2 Fabricating a Protein Array on a Flat Support 964
18.2.1 Preparation of Purified Proteins 964
Recombinant Proteins 965
In Situ Protein Production 965
Peptide Production 966
18.2.2 Substrates for Protein Microarrays 967
Flat Supports 967
Non-Flat Supports 967
18.2.3 Immobilising Proteins on the Array 968
Covalent Immobilisation 968
Non-Covalent Immobilisation 969
Capture by Affinity 969
18.2.4 Spotting Proteins 969
18.2.5 Detection Systems 972
Detection by Fluorescence 974
Detection by Surface Plasmon Resonance 974
Detection by Atomic Force Microscope 976
Detection by Mass Spectrometry 977
18.3 Other Formats for Protein Microarrays 978
18.4 Applications of Protein Microarrays 978
18.4.1 Analytical Microarrays 978
18.4.2 Functional Microarrays 981
18.5 Conclusion 983
References 984
19 Cell Biochips 989
19.1 Biochips for Analysing and Processing Living Cells 989
19.1.1 From Single Cells to Reconstituted Tissue 989
Parallel Cell Biochips 989
Series Biochips 990
Biochips for Manipulating and Analysing Single Cells 990
Tissue Models on a Chip 991
19.1.2 Cell Micromanipulation Methods 991
19.1.3 Methods for Characterising MicroculturedCells on Chip 993
19.2 Patch-Clamp Microarrays 996
19.2.1 Motivations 996
Limitations of the Patch-Clamp Techniquein the Pharmaceutical Industry 996
Locating Techniques for Studying Ion Channelsin the Drug Development Cycle 996
19.2.2 Emergence of New Patch-Clamp Platforms 998
Introduction 998
Microelectrodes and Automated Recording of Cell Currents 998
On-Chip Measurement Systems 1000
Array Chips. 1003
Rod-Shaped Chips. 1003
Transverse Chips. 1003
Choice of Material. 1003
Positioning and Capture of a Single Cell. 1006
Making the Seal: The Key Parameters. 1007
Micro- or Macrofluidics? 1008
Integration of Electrical Measurements. 1008
Compatibility of Cell Preparation and On-Chip Measurement. 1009
19.2.3 A Cultural Revolution? Prospects 1011
`Patchers Versus Screeners' 1011
Expected Technological Progress 1013
Ion Channels or Biosensors? 1015
Conclusion 1016
References 1016
20 Lab on a Chip 1022
20.1 The General Idea 1022
20.2 Implanted Functions 1024
20.2.1 Sample Preparation 1025
Filtering 1025
Separation by Transport Phenomena 1026
Solid Phase Extraction 1027
Magnetic Particles 1029
Liquid–Liquid Extraction 1029
Polymerase Chain Reaction (PCR) 1030
20.2.2 Transduction 1030
20.3 Technological Aspects 1032
20.4 Conclusion 1034
References 1036
21 Polyelectrolyte Multilayers 1040
21.1 The Idea 1040
21.1.1 Construction and Properties 1040
21.1.2 Physical Origin of InteractionsBetween Polyanions and Polycations 1042
21.2 Linear Growth and Exponential Growthof Polyelectrolyte Films 1044
21.2.1 Linear Growth 1044
21.2.2 Exponential Growth 1046
21.2.3 Fabrication of Polyelectrolyte Multilayers 1049
21.3 Biological Functionalisation 1050
21.3.1 Biologically Inert Films 1050
21.3.2 Functionalisation by Protein Insertion 1052
21.3.3 Functionalisation by Peptides 1056
21.3.4 Functionalisation by Drugs 1058
21.3.5 Development of Nanoreactors 1058
21.4 Making Hollow Particles from Multilayers 1060
21.5 The Route to More Complex Architectures 1061
21.6 Prospects 1063
References 1064
22 Biointegrating Materials 1066
22.1 Cell and Tissue Engineering 1066
22.2 Modifying Material Surfaces 1068
22.2.1 Using Nanoparticles to Deliver Active Ingredients 1068
22.2.2 Macroscale Functionalisation of Biomaterial Surfaces 1071
22.2.3 The Relevance of Controlled Nanotopochemistryand Nanodomains 1075
22.3 Applications of Biointegrated Biomaterials 1077
22.3.1 Applications to Bone Tissue 1077
22.3.2 Applications to the Vascular System 1079
22.4 In Vivo Assessment of Tissue Engineering Products 1081
22.4.1 Animal Models 1081
22.4.2 Which Animal Model for Which Application? 1082
22.4.3 Standard Methods for in Vivo Evaluationof Tissue Engineering Products 1083
22.5 Investigative MethodsAssociated with Tissue Engineering 1084
References 1086
23 Viral Vectors for in Vivo Gene Transfer 1092
23.1 Introduction 1092
23.1.1 In Vivo Gene Transfer 1092
23.1.2 Viral Vectors 1094
23.2 Main Types of Viral Vector 1094
23.2.1 Retroviral and Lentiviral Vectors 1095
Retroviral Vectors 1095
Lentiviral Vectors 1097
23.2.2 Adenoviral Vectors 1100
Wild-Type Virus 1100
Recombinant Vector 1100
23.2.3 Adeno-Associated Vectors 1101
23.3 Biomedical Applications of the Viral Platform 1102
23.3.1 Gene Therapy 1103
Overexpression 1103
Inhibition of Expression by RNA Interference 1105
23.3.2 Animal Models of Human Pathologies 1108
Intracerebral Injection 1109
Transgenesis 1110
23.4 Controlling and Visualising Transgene Expression 1112
23.4.1 Controlling Transgene Expression 1112
23.4.2 Imaging Transgene Expression 1115
23.5 Prospects 1115
References 1116
24 Pharmaceutical Applications of Nanoparticle Carriers 1120
24.1 Introduction to Drug Delivery in Pharmaceutics 1120
24.2 Nanoparticle Carriers 1121
24.2.1 The Main Nanoparticle Carriers 1121
24.2.2 Carrier Characteristics 1124
Importance of Composition 1124
Importance of Size 1125
Importance of Charge. Zeta Potential 1126
The Carrier–Active Principle Association 1127
24.3 Development of Carriersfor Pharmaceutical Applications 1128
24.3.1 Thermosensitive and pH-Sensitive(Fusogenic) Liposomes 1129
24.3.2 Modifying the Carrier Surface 1129
Stealth Particles 1130
Targeting 1131
24.4 Applications of Carriers 1134
24.4.1 Medical Mycology and Parasitology 1134
24.4.2 Ophthalmology 1135
24.4.3 Infectious Diseases 1136
24.4.4 Cancerology 1136
24.5 Conclusion 1137
References 1138
25 Activatable Nanoparticles for Cancer Treatment. Nanobiotix 1143
25.1 Introduction 1143
25.2 NanoTherapeutics 1145
25.3 Different Families of Nanoparticles 1147
25.4 NanoTherapeutic Action Mechanisms 1148
25.4.1 NanoMag 1148
25.4.2 NanoPDT 1148
25.4.3 NanoXRay 1149
25.4.4 Nano(U)Sonic 1150
25.5 Synthesising NanoMag Particles 1150
25.5.1 Coating the Fe2O3 Particles with SiO2 1151
25.5.2 Adding the Spacer 1152
25.5.3 Adding the Ligand 1152
25.6 Advantages of the NanoTherapeutic Families 1152
25.6.1 NanoMag 1152
25.6.2 NanoPDT 1153
25.6.3 NanoXRay 1155
25.6.4 Nano(U)Sonic 1155
25.7 Results (NanoMag) 1155
25.7.1 In Vitro Experiments 1155
25.7.2 In Vivo Experiments 1160
25.8 Conclusion 1163
References 1163
26 The Medical, Social, and Economic Stakesof Nanobiotechnology 1164
26.1 From Current to Future Applications 1164
26.1.1 Diagnosis and Therapy 1164
In Vivo Diagnosis 1165
In Vitro Diagnosis 1165
Therapy 1166
26.1.2 Cosmetics 1170
26.1.3 Product Quality and Traceability 1171
26.1.4 Environment and Risk Prevention 1172
26.2 From Individual Players to Clusters 1173
26.2.1 Different Players Around the Worldand the Position of France 1173
26.2.2 Clusters and Other Poles of Competitivity 1173
26.3 From Funding to Industrialisation 1173
26.3.1 Patents 1173
26.3.2 Funding Nanobiotechnological Activity 1174
26.3.3 The Markets: Between Fantasy and Reality 1175
26.4 From Risks to Precautions 1176
26.4.1 New Risks and Ethical Considerations 1176
26.4.2 Science Fiction or Future Reality? 1177
26.4.3 Image and Communication 1178
26.4.4 Convergence of Nanoscience and the Life Sciences 1178
26.5 The Advent of Nanomedicine 1179
References 1182
Index 1183