Joseph Wang · 2020. 1. 17. · The Author Prof. Joseph Wang University of California San Diego...

Joseph Wang

Nanomachines

Pompe, W., Rödel, G., Weiss, H., Mertig, M.

Bio-NanomaterialsDesigning materials inspired by nature

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ISBN: 978-3-527-41015-6

He, F., Qian, X., Yang, P., Poon, T. (eds.)

The Liver Proteome

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Samori, P., Cacialli, F. (eds.)

Functional Supramolecular Architecturesfor Organic Electronics and Nanotechnology

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ISBN: 978-3-527-32611-2

Sauvage, J., Gaspard, P. (eds.)

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ISBN: 978-3-527-32277-0

Ceroni, P., Credi, A., Venturi, M. (eds.)

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ISBN: 978-0-470-25557-5

Vogel, V. (ed.)

NanotechnologyVolume 5: Nanomedicine

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ISBN: 978-3-527-31736-3

Related Titles

Joseph Wang

Nanomachines

Fundamentals and Applications

The Author

Prof. Joseph WangUniversity of California San DiegoDepartment NanoEngineeringGilman Drive 9500La Jolla, CA 92093USA

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Contents

Preface IX

1 Fundamentals–Small-ScalePropulsion 11.1 Introduction 11.2 Nanomachines History 31.3 Challenges to Nanoscale Propulsion 41.4 Low Reynolds Number Hydrodynamics 7 References 9

2 MotionofNaturalNanoswimmers 132.1 Introduction 132.2 Chemically Powered Motor Proteins 142.2.1 Biological Motors: Active Workhorses of Cells 152.2.2 Protein Motors: Basic Operation 162.2.3 Kinesins 172.2.3.1 Function and Structure 172.2.3.2 Kinesin Movement 182.2.4 Myosins 192.2.5 Dyneins 212.2.6 Biomotor-based Active Nanoscale Transport in Microchip

Devices 212.3 Rotary Biomotors 242.4 Swimming Microorganisms 262.4.1 Bacterial Flagella – Escherichia coli 272.4.2 Sperm Motility 282.4.3 Cilia-Driven Swimming of Paramecium 292.4.4 Bacteria Transporters and Actuators 30 References 31

3 MolecularMachines 353.1 Stimuli-Responsive Rotaxane, Pseudorotaxane, and Catenane

Nanomachines 373.2 Molecular Rotary Motors 43

V

VI Contents

3.3 Light-Driven Molecular Machines based on cis–trans Photoisomerization 44

3.3.1 Azobenzene-based Nanomachines 453.4 Nanocars 473.5 DNA Nanomachines 503.5.1 Autonomous Enzyme-Assisted DNA Nanomachines 533.5.2 DNA Spiders 543.5.3 pH and Light Switchable DNA Machines 55 References 57

4 Self-PropellingChemicallyPoweredDevices 614.1 Self-Propelling Catalytic Nanowires 634.1.1 Propulsion Mechanism of Catalytic Nanowire Motors 674.1.2 Magnetically Directed Movement of Catalytic Nanowire Motors 684.2 Catalytic Tubular Microengines 694.2.1 Bubble-Propulsion Mechanism of Tubular Microengines 714.2.2 Preparation of Tubular Microengines 734.2.2.1 Rolled-up Fabrication of Tubular Microengines 734.2.2.2 Membrane-Template Electrodeposition of Tubular Microengines 754.3 Catalytic Janus Microparticles: Spherical Motors 764.3.1 Preparation of Catalytic Janus Particle Motors 774.3.1.1 Janus Capsule Motors 794.3.2 Propulsion Mechanisms of Catalytic Janus Spherical Motors 794.4 Controlled Motion of Chemically Powered Nano/Microscale

Motors 814.4.1 Thermally Controlled Nanomotors 824.4.2 Light Control of Catalytic Motors 834.4.3 Potential Control of Catalytic Motors 844.5 Alternative Fuels for Chemically Powered Micro/Nanoscale Motors 844.6 Collective Behavior: Toward Swarming and Chemotaxis 864.6.1 Triggered Self-Organization of Microparticles 864.6.2 Chemotaxis: Movement along Concentration Gradients 894.7 Biocatalytic Propulsion 914.8 Motion Based on Asymmetric Release of Chemicals 934.9 Polymerization-Induced Motion 95 References 95

5 ExternallyPoweredNanomotors–Fuel-FreeNanoswimmers 1015.1 Magnetically Driven Nanomotors 1015.1.1 Helical Propellers 1025.1.2 Flexible Swimmers 1065.1.3 Surface Walkers 1075.1.4 Magnetically Actuated Artificial Cilia Array 1095.2 Electrically Driven Nanomotors 1105.2.1 Motion of Miniature Diodes 110

Contents VII

5.2.2 Micromotors Driven by Bipolar Electrochemistry 1105.3 Ultrasound-Actuated Micromotors 1125.4 Light-Driven Micromotors 1135.5 Hybrid Nanomotors 114 References 115

6 ApplicationsofNano/MicroscaleMotors 1196.1 Cargo Towing: Toward Drug Delivery 1196.1.1 Cargo-Loading Schemes 1196.1.2 Cargo Release Strategies 1226.1.3 Drug Delivery: Realizing the Fantastic Voyage Vision 1246.2 Biosensing and Target Isolation 1266.2.1 Biomotor-Driven Sensing: Toward “Smart Dust” Devices 1266.2.2 Motion-based Signal Transduction 1286.2.3 Isolation of Biological Targets: “Swim-Catch-Isolation” 1306.3 Active Nanoscale Transport by Synthetic Motors in Microchip

Devices 1346.4 Nanomotor-based Surface Patterning and Self-Assembly 1356.5 Use of Micro/Nanoscale Motors for Environmental Monitoring and

Remediation 137 References 138

7 ConclusionsandFutureProspects 1417.1 Current Status and Future Opportunities 1417.1.1 Future Micro/Nanoscale Machines in Medicine 1437.2 Future Challenges 1447.3 Concluding Remarks 146 References 147

Glossary 149 Index 155

IX

Preface

The development of synthetic nanoscale motors, capable of converting energy into movement and forces, represents one of the most fascinating topics of nanotech-nology. Such motion of nanoscale objects through fluid environments is of con-siderable interest both fundamentally and practically, and has thus stimulated substantial research efforts. Research groups around the world are actively pursu-ing the dream of designing synthetic nanomachines that mimic biological motors and perform demanding tasks such as transporting therapeutic cargo and assem-bling nanostructures and devices.

Making a nanoscale motors has been a dream of many researchers in the field since the late 1950s and 1960s. Richard Feynman, Nobel Laureate in Physics, first suggested molecular-scale mechanical nanomachines in a famous lecture at the 1959 Meeting of the American Society of Physics entitled “There is plenty of room at the bottom.” The idea of tiny machines that can perform such complex opera-tions has been a major part of science fiction since the 1966 movie the Fantastic Voyage. In this movie, medical personnel boarded a submarine that was shrunk to microscopic size and entered the bloodstream of a wounded diplomat to save his life.

The Fantastic Voyage vision and challenge are currently being addressed in an interdisciplinary research activity across the globe involving the design of new functionalized nano/microscale motors that rely on different propulsion mecha-nisms and advanced schemes for navigating them toward their destination.

Movement is essential for life in the nanoscopic and macroscopic scales. For example, animals run away fast from dangers while protein nanomotors shut-tle cargo along intracellular microtubule tracks. Such tiny biomotors display remarkable motion capabilities, with an advanced directional movement and speed regulations. The sophisticated operation of biological nanomotors has inspired scientists and engineers to design artificial nano/microscale machines, with enhanced functionalities and capabilities, and address the challenge of con-verting nature-inspired swimming mechanisms into man-made nanoswimmers. Researchers have turned to nature, especially to microorganisms, for inspiration, resulting in artificial nano/microscale swimmers that emulate these natural swim-mers and molecular biomotors. Understanding the remarkable underlying prin-ciples of nature’s remarkable biomotors has thus provided researchers with new

X Preface

insights into how to impart greater sophistication onto the design and operation of new artificial nanomachines. Although the research in the area is at its infancy, major scientific and technological advances have already led to substantial progress over the past decade toward addressing the major challenges of scaling of conven-tional machine designs to nano/microscale dimensions and providing these tiny machines with power.

Synthetic nanomachines hold great promise for major advances in diverse applications, meeting a wide range of future technological and biomedical needs and providing unlimited possibilities based on one’s imagination. Artificial nano-scale and microscale machines could thus perform different functions, similar to nature nanomotors found in living cells, including transporting molecules or facilitat ing chemical reactions by pumping protons through membranes. Recent progress in the field of self-propelled man-made nano/microscale machines has led to major advances in the power, efficiency, directionality, motion control, functionality, and versatility of such synthetic nanomotors. Nano/microscale machines hold great promise for performing diverse operations and important tasks that include directed drug delivery, biosensing of nucleic acids or proteins, cell sorting, micropatterning, nanosurgery, exploring hazardous situ-ations, and micromanipulation. This exciting area of research is thus expected to make important contributions to diverse fields with the new powerful machines, leading to new capabilities that are currently beyond our reach and bringing major benefits to our quality of life.

My goal is to convey a realistic picture of the latest advances in the design and operation of nano/microscale machines, and to promote activity across the field of small-scale motors toward the development of advanced machines, capable of performing different important tasks that are beyond our current reach. The book is suitable for a graduate-level course in nanomachines or as a supplement to high-level undergraduate courses in nanoengineering, nanoscience, or nanotech-nology. It should also be extremely useful to those considering the use of nanomo-tors in their laboratories and to researchers in the areas of nanobiotechnology, nanomedicine, and nanoengineering, in general. Given the interdisciplinary nature of this exciting topic, I have tried to make the book a self-contained starting point for the interested student, scientist, or engineer.

The material is presented in seven roughly equal chapters. Chapter 1 is devoted to fundamental aspects and challenges of nanoscale motion. Chapter 2 discusses natural (biological) nanoswimmers, while Chapter 3 gives an overview of molecu-lar and DNA machines. Chapter 4 is devoted to chemically powered catalytic nanomotors. Chapter 5 discusses fuel-free externally actuated (magnetically, elec-trically, ultrasound driven) nanomotors. Chapter 6 focuses on diverse potential applications of nano/microscale machines, ranging from drug delivery to target isolation, while the final Chapter 7 discusses future prospects, opportunities, and challenges.

I hope that you will find the content of the book highly useful, and I look forward to new exciting developments that the work described in this book is likely to inspire.

Preface XI

Finally, I wish to thank my wonderful wife, Ruth, for her great patience, love, and support; to Wei Gao, On Shun Pak, Allen Pei and other members of the UCSD nanomotor team for their help; the editorial and production staff of Wiley-VCH for their support and help; and to numerous scientists and engineers across the globe who led to the remarkable advances and to the Fantastic Voyage reported in this book. Thank you all!

Joseph WangSan Diego, USAJanuary 2013

1Fundamentals – Small-Scale Propulsion

1.1 Introduction

The motion of natural and synthetic nanoscale and microscale objects has been of considerable fundamental and practical interest and has thus stimulated sub-stantial research activity. Using nanoscale and microscale machines to perform mechanical operations represents an exciting research area. Nature has provided tremendous inspiration for designing artificial nanoscale motors and has devel-oped powerful nanoscale biomotors through millions of years of evolution. Yet, the development of synthetic nanomotors that mimic the function of nature’s amazing biomotors is only in its infancy. Scientists and engineers have been pursuing aggressively the development of advanced artificial nanomachines for only about a decade. Such development represents a major challenge when trying to mimic the essential functions of natural motors while keeping the complexity low. Synthetic nano- and microscale motors, capable of converting energy into movement and forces, represent one of the most exciting challenges facing nan-otechnology (Ebbens and Howse, 2010; Fischer and Ghosh, 2011; Mallouk and Sen, 2009; Mei et al., 2011; Mirkovic et al., 2010; Ozin et al., 2005; Paxton et al., 2006; Peyer et al., 2013; Pumera, 2010; Sengupta, Ibele, and Sen,, 2012; Wang, 2009; Wang and Gao, 2012). Recent activity in microtechnology and nanotechnol-ogy has allowed researchers to explore the microfabrication of devices capable of propulsion at the micro- and nanoscale. Powerful self-propelled and externally powered artificial nanomotors have thus been developed. Such synthetic nanoma-chines already offer an advanced performance, functionality, and capabilities along with a precise (spatial and temporal) remote motion control, and hold considerable promise for numerous transformative practical applications (Manesh and Wang, 2010; Nelson, Kaliakatsos, and Abbott, 2010; Peyer, Zhang, and Nelson, 2013; Sengupta, Ibele, and Sen, 2012; Wang and Gao, 2012).

As their name implies, nanomachines are extremely small devices. Their size is measured in nanometers (a nanometer is one-billionth of a meter), and can reach hundreds of nanometers. Larger microscale machines have size ranging from 1 to 100 μm (a micrometer is one-millionth of a meter). Such microscale machines are also covered extensively in this book, particularly in Chapters 4–6.

Nanomachines: Fundamentals and Applications, First Edition. Joseph Wang.© 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.

1

2 1 Fundamentals–Small-ScalePropulsion

Central to any molecular machine or micromachine is the motor component responsible for generating the mechanical energy. At the heart of every machine is its motor. The Oxford Dictionary of English defines a motor as “a thing that imparts motion”; work as “the operation of a force in producing movement or other physical change”; and motion as “the condition of a body, when at each successive point in time it occupies a different position or orientation in space.” A motor is the most important part of the machine as it generates the necessary power and movement by cyclically converting various energy forms (e.g., chemical, electrical, or thermal energy) into mechanical work.

This monograph will cover the generation and control of motion at the nano- and micrometer scales, including single- and multimolecule synthetic and natural motors, and particularly chemically powered and externally triggered synthetic microscale devices. The design, propulsion, and guidance of these tiny motors will be discussed along with diverse motor applications in solution and in engineered systems, and motor-driven transport systems.

Designing a self-propelling micro/nanoscale object is not a simple task because as the size decreases, the influence of Brownian collisions, viscous drag, and various surface phenomena becomes dominant. Novel strategies of supplying power (or fuel) to micro/nanoscale devices are thus required to promote their propulsion. Traditional on-board power supplies, for example, batteries, cannot be scaled to these tiny dimensions. Accordingly, two main approaches have been proposed to address this challenge, including the use of external energy fields and harvesting energy from the surrounding environment. Two classes of nano/microscale motors have thus been demonstrated to date; the first – driven primarily by deformations – requires actuation by external fields; the second is fully autono-mous and powered by the asymmetric surface catalytic decomposition of solution-borne fuel molecules. These micro/nanomotors can thus be classified into two broad categories: externally powered propellers and chemically powered motors.

New nanomotors with diverse capabilities and functionalities are currently being developed by multiple research teams around the globe. This exciting research area is expected to grow rapidly as additional technological breakthroughs emerge and as nanomachines demonstrate increased capabilities and functionali-ties. These developments and capabilities will lead to a wide range of practical real-world applications, such as targeted drug delivery, microsurgery, nanoscale assembly or patterning, environmental remediation, biosensing, or cell sorting (Mallouk and Sen, 2009; Mei et al., 2011; Mirkovic et al., 2010; Nelson, Kaliakatsos, and Abbott, 2010; Peyer, Zhang, and Nelson, 2013; Sengupta, Ibele, and Sen, 2012; Wang, 2009; Wang and Gao, 2012). Similar to the dramatic evolution in electron-ics, from the pocket calculator of the 1960s to the latest iPhone 5, we expect to see a very fast evolution of present nano/microscale machines into sophisticated multifunctional nanovehicles that can perform advanced and demanding opera-tions and multiple complex tasks. Yet, in terms of miniaturization, propulsion mechanisms have not followed the remarkably fast scaling-down in size of elec-tronics over the past 50 years – described by Moore law – owing to major challenges to nanoscale locomotion (described in Section 1.3). Nevertheless, nanomachines

1.2NanomachinesHistory 3

are expected to lead to new and exciting capabilities that are currently beyond our reach, and will provide unlimited opportunities based on one’s imagination.

1.2 Nanomachines History

The implications of inertia-less to the realization of self-propulsion were recog-nized already in 1930 (Ludwig, 1930). The invention of dark field microscopy around the same time allowed observations of flagella and cilia. Back in 1951 Taylor wrote a historic paper on the swimming of microorganisms, discussing how force-free swimming is possible in a viscous medium and proposing a two-dimensional sheet as a model for flagellated cells passing traveling waves as a means of locomotion (Taylor, 1951). In 1973, Berg proved that Escherichia coli bacteria use molecular motors to rotate their helical flagella, following which helical propulsion has become an active area of research. The challenges facing microorganisms attempting to propel themselves in low Reynolds numbers regime were summarized later by Purcell in his landmark 1977 paper (Purcell, 1977).

Scientists (and science-fiction writers) have contemplated nanomachines at least since the late 1950s, when physicist Richard P. Feynman considered the scale limits for machines during his famous lecture “There is plenty of room at the bottom” at the 1959 Meeting of the American Society of Physics (Feynman, 1960). In the 1966 movie Fantastic Voyage by Harry Kleiner and a book by Issac Asimov, a team of scientists board a submarine that shrinks to a micrometer size and enters the bloodstream of a wounded diplomat to destroy a life-threatening blood clot and save his life. Despite the waves of blood that rocked the sub with every heartbeat, and antibodies that attacked it as an infection, the scientists were able to navigate their submarine through the blood stream and succeed in saving the man’s life. Feynman’s idea and the vision of Fantastic Voyage remained largely undiscussed until the mid-1980s when Eric Drexler published the book Engines of Creation, which introduced the term nanotechnology and promoted the poten-tial of molecular nanotechnology and nanomachines (Drexler, 1986). According to Drexler, the ultimate goal of nanomachine technology is the production of the “assembler,” which is a nanomachine designed to manipulate matter at the atomic level.

Pioneering contributions by Fraser Stoddart, Ben Feringa, Vincenzo Balzani, and others during the late 1990s led to a rapid progress in the development of autonomous molecular systems and shuttles that can execute complex actions (Balzani et al., 2000; Koumura et al., 1999). Active research toward understanding the operation of natural biomotors has been followed by the use of protein motors for nanoscale transport in engineering microchip systems (Hess and Vogel, 2001; Soong et al., 2000). Autonomous and stimuli-induced DNA nanomachine systems (tweezers, walkers, gears) were introduced in the early 21st century (Chen, Wang, and Mao, 2004; Yan et al., 2002; Yurke et al., 2000).


Extensive efforts over the past decade have resulted in synthetic nano/microscale motors that achieve their movement and directionality in different ways. Chemi-cally powered propulsion was demonstrated first by Whiteside’s team in 2002 (Ismagilov et al., 2002), whereas the self-propulsion of catalytic nanowire motors was demonstrated in 2004 and 2005 (Fournier-Bidoz et al., 2005; Paxton et al., 2004). Since these pioneering studies, the number of publications in this field has grown rapidly and substantially. Magnetically and electrically propelled artificial nano/microscale objects were described in 2009 (Zhang et al., 2009a, 2009b; Chang et al., 2007; Calvo-Marzal et al., 2009). Catalytically active Janus particles and bubble-propelled tubular microenegines were introduced in 2007 and 2008, respectively (Howse et al., 2007; Mei et al., 2008). The first demonstrations of micromotors transporting therapeutic payloads or sensing their surroundings were reported in 2010 (Kagan et al., 2010a; Kagan et al., 2010b). The use of ultra-sound to drive the movement of nano/microscale objects was demonstrated in 2012 (Kagan et al., 2012; Wang and Gao, 2012; Wang et al., 2012). Given the continuous flow of innovative ideas, this exciting area of research is expected to make important contributions to diverse fields, and continue to be one of the most fascinating topics in nanotechnology in the foreseeable future.

1.3 Challenges to Nanoscale Propulsion

Motion of nanoscale objects through fluid environments represents a major chal-lenge confronting nanotechnology. Specifically, achieving micro/nanoscale pro-pulsion in fluid is challenging due to the absence of inertial forces, which we all exploit for swimming at the macroscopic scale. Considering the fluid behavior on these small length scales, it is apparent that scaling of conventional machine designs to nano/microscale dimensions, and providing these tiny machines with power, face a number of major challenges. These challenges are responsible for the slow scaling down of artificial swimmers during the 20th century. In particu-lar, due to the absence of inertial effects, miniature devices cannot propel using conventional swimming mechanisms involving gliding between time-reversible movements. This was famously visualized by a single-hinged miniature “scallop” achieving no net progress by symmetrically flapping its arms (Purcell, 1977). The difficulties have been summarized by Purcell’s famous “scallop theorem” (Purcell, 1977) (discussed later in this section), which states that a reciprocal motion – based on time-reversal symmetry (i.e., a periodic back and forward dis-placement) – cannot lead to any net displacement and hence directed movement of tiny objects.

Another key factor and challenge to nanoscale motion through liquid environ-ments is the dominance of Brownian motion, named after the English botanist Robert Brown. Brownian motion involves the random (irregular) movement of microscopic particles suspended in a liquid, caused by thermally driven collisions with molecules of the surrounding solvent. These collisions can alter the trajectory

1.3ChallengestoNanoscalePropulsion 5

of moving nano/microscale motor particles and hence represent a challenge for imparting directionality on such objects. Such motion is independent of the chem-ical makeup and physical density of the particle. Brownian motion cannot be avoided and it depends on the temperature. Such motion is related to the macro-scopic measurement of diffusion characterized by the diffusion coefficient D. The diffusion of a purely Brownian particle in one dimension (x) over time (t) is given by

〈 〉 =x Dt2 2 (1.1)

Displacement of an object can thus be estimated. Unlike the movement of macroscopic motor particles, achieving directed propulsion of nanoscale objects through liquid environments requires overcoming the major difficulties posed by both the relatively strong Brownian noise and negligible inertia.

A better understanding of the role of inertia in nanoscale movement can be obtained by using the Reynolds number (Re). The Reynolds number is a dimen-sionless parameter that refers to the relative scales of the object, its inertial forces, and the viscous forces. This number is named after the British engineer Osborne Reynolds who proposed it in 1883. The Reynolds number represents the ratio of momentum to viscosity:

Re UL UL= = =ρ µ ν/ / Inertial forces/Viscous forces (1.2)

where ρ is the density of the fluid (kg/m3), μ the dynamic viscosity of the fluid, whereas U refers to the velocity of the object relative to the flow, L is the charac-teristic dimension of the object, and ν is the kinematic viscosity. The Reynolds number thus measures the significance of inertial forces relative to the viscous forces. If the Reynolds number is large, then inertia dominates. When the Rey-nolds number is very low, which could be due to small size and/or high viscosity, then hydrodynamics is governed by viscous forces.

As expected from Eq. (1.1), the extremely small dimension (L) of nanoscale objects leads to very small Reynolds numbers (Figure 1.1). Size affects the modes of motion long before reaching the nanoscale. Viscous forces dominate even at the mesoscopic dimensions of bacteria. For example, for the E. coli bacterium swimming in water (L 1–10 μm; U 10 μm/s; ρ 103 kg/m3), the Reynolds number is 10−5 to 10−4 (Figure 1.1b). In contrast to large-scale swimmers, the world of micro- and nanoscale swimmers is thus dominated by viscosity while inertial forces are negligible. The absence of inertial effects at the low Reynolds number regime rules out propulsion by a conventional swimming mechanism that cannot lead to any net displacement and hence to actual movement. Since the physics that governs mechanical dynamic processes in the two size regimes is completely different, macroscopic and nanoscale motors require fundamentally different mechanisms for controlled transport or propulsion. Movement of nano/microscale objects at this inertia-less limit (low Re number) regime thus requires the use of swimming strategies that are largely different from the flapping-like (time-reversible) symmetric strategies used by larger macro-scale swimmers (Purcell, 1977; Vandenberghe, Zhang, and Childress, 2004; Lauga and Powers, 2009).


In his famous lecture and the subsequent 1977 paper Life at Low Reynolds Number, Purcell described how a nonreciprocating motion is required for a net displacement, and proposed his “scallop theorem” (Purcell, 1977) delimiting the types of swimmer designs that are not effective on small scales. The Purcell’s scallop theorem can be stated as follows: if the sequence of shapes displayed by the swimmer is identical to the sequence of shapes displayed when seen in reverse – the so-called reciprocal motion – then the average position of the body cannot change over one period. No net translation is expected from a reciprocal motion, such as opening and closing of the “scallop” when swimming at low Reynolds numbers. This implies that swimming motions that are symmetric with respect to time reversal (i.e., a reciprocal motion) cannot lead to net displacement and cannot be used for locomotion of small-scale swimmers. As illustrated in Section 1.4, the inertia-less equations governing the surrounding fluid are linear and independent of time on very small scales (Stokes equation). Hence, any actua-tion on the fluid remaining identical under time reversal (reciprocal actuation) cannot generate any net motion.

The main message of Purcell’s paper is that tiny swimmers should deform their shapes with time in a nonreciprocal fashion in order to generate net motion (Lauga, 2011). A unique feature of propulsion of these microscale objects, com-pared with their macroscopic counterparts, is that a body striving to move must change its shape with time in a nonreciprocal fashion. The requirement of non-reciprocal body deformations adds significant complication to the design of tiny machines. Small swimmers thus require a different class of shape changes com-pared with their larger counterparts (Figure 1.1). To overcome the viscous drag forces at the low Reynolds number regime, nano/microscale swimmers must execute nonreciprocal motion, that is, require breaking of time-reversibility and

Figure 1.1 The extremely small dimension of micro/nanoscale swimmers leads to very small Reynolds numbers and requires the use of different swimming strategies that are largely different from those used in the

macroscale world. When the Reynolds number is very low due to tiny dimensions, hydrodynamics is governed by viscous forces (even in water which is not viscous fluid).

Re ~ 104

Re ~ 10–4

(a)

(b)

1.4LowReynoldsNumberHydrodynamics 7

hence escaping from the constraints of the scallop theorem. The shape changes of tiny swimmers must follow an asymmetric time sequence. Such nonreciprocat-ing motion is essential for a net displacement of micro/nanoscale objects. Accord-ing to Purcell, two approaches can be used to elude the scallop theorem: rotating a chiral arm or waving an elastic arm (Wiggins and Goldstein, 1998). To overcome the constraints of the scallop theorem, microorganisms thus swim in low Reynolds number conditions using a variety of techniques, involving an asymmetric time sequence, that are largely different from those used by macroscale swimmers. The scallop theorem thus puts a strong geometrical constraint on the type of swim-ming motion, which is effective at the low Reynolds number regime.

The Purcell’s scallop theorem can serve as a guideline for the basic requirements essential for designing nano/microscale swimmers, that is, relying on nonrecip-rocating motion for achieving net displacement. Figure 1.2 illustrates this concept using a theoretical 3-link swimmer (Becker et al., 2003; Zhang, Peyer, and Nelson, 2010). The two hinges offer two degrees of freedom, and the swimmer can go through a series of angle configuration. Such nonreciprocal series of angle con-figurations (shown as ABCDA in Figure 1.2), involving alternating movement of the front and rear links, leads to a net displacement after each single cycle.

1.4 Low Reynolds Number Hydrodynamics

To discuss the general properties of flow at low Reynolds numbers and solve for the force distribution on an organism, it is essential to solve for the flow field u and pressure p in the surrounding fluid (Lauga and Powers, 2009). The flow of fluids is commonly described by a set of nonlinear partial differential equations, known as the Navier–Stokes equations (Happel and Brenner, 1965). We treat the fluid as incompressible so that u satisfies the continuity condition ∇.u = 0. For such incompressible Newtonian flow with density ρ and viscosity μ, the flow satis-fies the Navier–Stokes equations

ρ µ∂∂

+ ⋅∇ = −∇ + ∇ ∇ ⋅ =t

pu u u u2 0, (1.3)

In low Reynolds number hydrodynamics (with Re ≪ 1) the inertial terms (left-hand side of Eq. (1.3)) can be neglected compared with the viscous terms (on

Figure 1.2 The two-hinged theoretical swimmer proposed by Purcell: the nonreciprocal series of angle configurations results in a net displacement after a whole cycle. (Reproduced with permission from Purcell, 1977.)

A B C D A


its right-hand side), resulting in a simplified expression, known as the Stokes equation

−∇ + ∇ = ∇ ⋅ =p µ 2 0 0u u, (1.4)

The motion of the medium is thus governed by the force balance given in Eq. (1.4). Low Reynolds number flow is also called Stokes flow, which occurs when inertial forces can be neglected compared with viscous forces. This equation is accurate only for Re = 0, but it is considered a good approximation for Re ≪ 1.

The Stokes equation, which governs the fluid dynamics under low Reynolds number conditions, is a linear equation resulting in flows proportional to the applied forces, ∇p = μ∇2u. The absence of time-dependent terms in this equation, combined with its linearity, implies that no net forward movement is expected for a motion that fully retracts itself (i.e., the scallop theorem). The linearity and time-independence of the Stokes equation also imply that the distance traveled by a swimmer depends only on the sequence of shape changes of the swimmer, but not on the rate at which they occur (namely rate independence) (Lauga and Powers, 2009). The Stokes equation thus emphasizes that the propulsion force in the low Reynolds number regime depends only on the relative position of the propeller. The only significant force acting on the particles is due to drag and therefore the particle shape.

The Stokes equation has several important properties:(i) Linearity: The Stokes equation is linear, meaning that the response of a

Stokes flow will be proportional to the forces applied to it. (ii) Instantaneity: A Stokes flow has no time dependence, except through its boundary conditions. (iii) Time-reversibility, that is, a time-reversed Stokes flow solves the same equations as the original Stokes flow.

Since Stokes equation is linear, there is a linear relationship between kinetics and kinematics. Specifically, if the solid body is subject to an external force F and torque τ, it will move with velocity U and rotation rate Ω, satisfying the matrix, known as the “resistance matrix” (or “propulsion matrix”) (Happel and Brenner, 1981):

F A B

B C

UTt

=

Ω

(1.5)

where A, BT, and C are second-order tensors dependent on the geometry (Kim and Karrila, 1991). Or the reverse

U M N

N O

FTΩ

=

t

(1.6)

where the matrix is known as the “mobility matrix.” The reciprocal theorem forces these matrices to be symmetric. To calculate this matrix, a solution of the Stokes equations for the specific geometry is required.

The resistance matrix of objects with simple shapes, such as a sphere, may be obtained exactly and analytically. However, for more complicated objects, the matrix is usually obtained via numerical methods such as slender-body theory

References 9

(Johnson, 1980) and the method of regularized Stokeslets (Cortez, Fauci, and Medovikov, 2005).

In the low Reynolds number regime, the thrust force of the swimmer is coun-terbalanced by its drag force (friction):

F Fthrust drag= (1.7)

For example, the driving force of a single microsphere motor swimming in water at the low Reynolds number regime (Re < 0.1), with no turbulence, depends lin-early on its velocity and can be calculated using the Stokes drag law (Probstein, 1994)

F rdrag = 6πη υ (1.8)

where η is the viscosity of the water, and r and v are the radius and the velocity of the microsphere, respectively. This expression for the drag force of spherical objects was derived in 1851 by the Irish physicist George Gabriel Stokes. As expected from the increased hydrodynamic resistance, the speed of such micro-sphere motor is inversely proportional to its size:

υ η= F rdrag/6π (1.9)

A larger propulsive force or a lower drag force is thus required for increasing the swimming speed. For swimmers of different shapes, such as rods and disks, expressions for the drag coefficient must consider the asymmetry in the viscous drag and the continuous changes of particle orientation associated with the differ-ent geometries.

The Brownian diffusion coefficient of a particle with radius r is given by the Stokes–Einstein equation

D kT r= /6πη (1.10)

where k is the Boltzmann constant and T is the absolute temperature. The Stokes–Einstein equation indicates that the diffusion of the particle moving through spe-cific media is inversely related to the friction drag and is strongly influence by the surrounding temperature.

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2Motion of Natural Nanoswimmers

2.1 Introduction

Some of the most fascinating biological systems involve motor functions, which perform mechanical transformations (Schliwa and Woehlke, 2003; van den Heuvel and Dekker, 2007; Vogel, 2005). Nature nanomotors have been created over mil-lions years of evolution to perfectly match their specific tasks and responsibilities and have provided a tremendous inspiration for the design of artificial nanoscale motors. There are many examples of nanomotors in nature. Such biomotors are the essential agents of movement in living organisms. Molecular machines are biological macromolecular systems that perform work in the cell. They are typically multiprotein complexes capable of transducing chemical energy into mechanical work in order to accomplish a complex molecular process. Within the cell, linear motors, including kinesin and myosin, DNA and RNA polymerase, dyneins, play a key role in muscle contraction, and transporting organelles and synaptic vesicles, ranscription, mitosis, and meiosis. For example, protein motors, such as kinesin and myosin, are widely used within cells for a variety of transport tasks. These biological motors perform work and are engaged in well-defined mechanical tasks such as muscle contraction or the transport of objects is apparent in all living systems. “Larger” natural swimmers, such as the microorganisms bacteria or spermatozoa, have developed a few different swimming modes to generate the nonreciprocating motion and swim at the low Reynolds number regime (Figure 2.1), none of which is found in macroscale swimmers. Understanding the remark-able motion and operation of nature’s biomotors has provided researchers with new insights into how to impart greater sophistication onto the movement of artificial nanomachines.

In this chapter, we discuss the design and operation of biological nanomotors, and address several key questions: (i) How do biomotors convert chemical energy into mechanical work? (ii) How is the movement directionality of biomotors achieved? (iii) How is the movement coordinated or regulated? For recent reviews on biological molecular motors see Hess and Bachand (2005), Mickler, Schleiff, and Hugel (2008), Schliwa and Woehlke (2003), and van den Heuvel and Dekker (2007).

Nanomachines: Fundamentals and Applications, First Edition. Joseph Wang.© 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.

13

14 2 MotionofNaturalNanoswimmers

Different triggering stimuli, such as adenosine triphosphate (ATP) fuel, a pH gradient, or light signals, are used to activate biological motors. ATP is the primary fuel for most of the commonly studied biomolecular motors, since it is the major source of energy in living cells. The high chemical energy stored in the ATP molecule reflects its three negatively charged phosphate tails. The hydrolysis of ATP to adenosine diphosphate (ADP) and inorganic phosphate (Pi) results in the release of free energy (from the closely packed phosphate tails), which is available to carry out mechanical work. Biomolecular motors con-tinuously cycle through their sequence of mechanochemical reaction steps as long as the free energy obtained from the ATP hydrolysis (−12 kcal/mol) exceeds the mechanical load multiplied by the step size and the maximum motor efficiency.

2.2 Chemically Powered Motor Proteins

Most of the natural machinery is built from proteins. Proteins undergo a mechani-cal movement upon binding a particular substrate or ligand. These tiny biomotors have thus mastered the art of converting conformational changes into directed movement. These movements are usually reversible, corresponding to the binding states of the protein with its ligand as it shifts reversibly between the unbound and substrate-bound states. Such motion may range from small and localized to changes in the protein structure on the nanometer scale.

Figure 2.1 The microorganisms bacteria (a) and spermatozoa (b) swim by using flexible oar-like and screw-like nonreciprocal movements, respectively. (Reproduced with permission from Purcell, 1977.)

The flexible car

The corkscrew

(a)

(b)

2.2ChemicallyPoweredMotorProteins 15

2.2.1 Biological Motors: Active Workhorses of Cells

Efficient conversion of chemical energy into mechanical work by biological motors enables a wide range of functions in nature, ranging from intracellular transport to large-scale actuation by muscles (Mavroidis, Dubey, and Yarmush, 2004). Protein motors are key components of the cell factory as they are involved in a broad range of cellular activities. A cell, like a city, must organize its communities of macromolecules and establish the timing of transactions. Such high degree of spatial/temporal organization and transport activities are vital for the cell behavior and survival. The timing of transactions is of fundamental importance for cell behavior.

While diffusion is the primary way of transport within living cells, some cargoes need to be transported quickly, in one direction, and over a long dis-tance. Cells have thus evolved advanced molecular machinery, based on protein biomotors and microtubule filaments, to support the intracellular transport of cargo to various destinations within the cytoplasm (Schliwa and Woehlke, 2003; Vale, 2003). Such intracellular transport processes resemble the transport of goods on the roads, with the protein motors acting as transport trucks. These biomotors traffic essential chemical “packages” between the heart of the cell, the nucleus, and the cell periphery, thus keeping the cells functioning. The main mechanism for intracellular transport involves protein motors that carry cargo from one location in the cell to another along a cytoskeletal track. These tracks include microtubules filaments (in the case of kinesins or dyneins motors) and actin in the case of myosins. The cytoskeletal track networks serve as the intracellular road system (“cellular highways”) and provide the nec-essary functionality. Microtubule filaments have 200-fold larger stiffness than the actin ones.

Cytoskeletal tracks are structurally asymmetric and contain defined binding sites so that the biomotor, upon binding, can read the direction toward which the fila-ment is pointing, thus ensuring a directed transport of the motor. These motor proteins recognize and bind to the correct fiber (track) system and to the appropri-ate cargo, and carry their cellular freight and transport it along the microtubule “tracks” to well-defined destinations. Such protein motors can carry different types of cargo, including membrane vesicles, whole organelles, or messenger RNAs, while walking along their microtubules. Different cargos are typically barcoded so that they can be recognized by the corresponding protein motor (Goel and Vogel, 2008). The intracellular transport processes thus ensure the temporal and spatial demands for on-time delivery of cargos in the cell. Disruption of these transport networks and processes can cause chaos and might lead to a cell death. Motor proteins may thus be adapted to operate in crowded environments and to avoid forming traffic jams (Leduc et al., 2012).


2.2.2 Protein Motors: Basic Operation

Biomolecular nanoscale motors, also known as motor proteins, are unique stimulus-responsive polymers. Their high degree of sophistication, efficiency, and specific function derives from the complexity afforded by protein building blocks. These highly efficient chemically powered biomotors rely on spontaneous reac-tions of energy-rich molecules, most commonly ATP, as a chemical fuel for the conversion of chemical energy into mechanical work. ATP serves as the primary chemical energy source in organisms and is present within cells at the millimolar concentration level. These motor protein classes, myosins, kinesins, and dyneins, use this highly efficient energy conversion to perform a wide variety of essential biological functions such as the long-distance intercellular transport of organelle and vesicle or the contraction of our muscles (Vale and Milligan, 2000). Motor proteins are thus considered as a basic building block for nanotransportation devices.

To overcome viscosity forces and Brownian motion, the muscle myosin and walking protein kinesin move (“walk”) in a linear fashion along actin or microtu-bule filaments (Figure 2.2). Microtubules are structurally polar cytoskeletal fila-ments that serve as tracks for kinesin and dynein motor proteins and provide mechanical integrity to the cell. Such microtubule filaments are hollow and stiff cylindrical polymers, made of α- and β-tubulin building blocks, with an internal diameter of approximately 18 nm and an external one of approximately 25 nm. The tubulin orientation within the microtubule defines the orientation at which kinesin binds and moves (Hess and Vogel, 2001; Hess et al., 2001). Microtubules have their own polarity, with one end being the plus (fast-growing) end and the other the minus (slow-growing) end. Kinesins move from the minus end to the plus end

Figure 2.2 Controlled motion of the ATP-consuming motor proteins kinesin (a) and myosin (b) along microtubule “tracks” and actin filaments, respectively, essential for the directed cellular transport of cargo. These biomotors move along their “tracks” by

using repeated cycles of coordinated binding and unbinding of their two heads, powered by energy derived from the ATP hydrolysis. (Reproduced with permission from Hess, Bachand, and Vogel, 2004.)


of the microtubule, whereas dyneins move from the plus end to the minus end. Kinesin-mediated transport is thus used to bring cargos toward the cell periphery.

The movement of motor proteins reflects the dramatic conformational changes of these biomotor proteins as they bind and hydrolyze ATP and release the prod-ucts in a cyclic process. Motor proteins possess a catalytic motor domain (the “head”), with two binding sites – one for the track and the other for ATP – and hence the binding and hydrolysis of the ATP fuel induce conformational change in the track-binding site. The similarities of the crystal structures of myosin and kinesin suggest that these biomotors have originated from a common ancestor (Kull et al., 1996; Vale and Milligan, 2000) and adopted a similar strategy for con-verting chemical energy into mechanical work. These biomotors thus move in steps related to the binding and hydrolysis (breaking down) of ATP. The energy stored in ATP is thus translated into movement by coupling the cycle of ATP hydrolysis to changes that result in the displacement of their motor protein domain in the direction of travel (power stroke) (Houdusse and Carter, 2009). This is fol-lowed by the dissociation of the motor from the filament track and reversal of the conformational change (recovery stroke). Such ability to convert chemical energy to mechanical work represents a remarkable example of the sophistication of biological motors. Motion thus derives from a mechanochemical cycle, during which the motor protein binds to successive sites along the substrate. These bio-motors are processive in that they can take hundreds of consecutive steps without detaching from their track.

2.2.3 Kinesins

2.2.3.1 Function and StructureKinesins represent a large family of proteins with diverse structures. There are over 250 kinesin-like proteins, and they are involved in diverse biological processes such as the movement of chromosomes, cell division, and the dynamics of cell membranes. The most studied kinesin, kinesin I, is referred to as conventional kinesin or simply kinesin.

Conventional kinesin is a dimer of two identical subunits, each consisting of 370 amino acids in length (Woehlke and Schliwa, 2000). Each subunit comprises three distinct domains: the N-terminal globular head (motor domain), the central 50-nm-long semiflexible coiled-coil stalk that holds the two chains together (facili-tates the dimerization) and the tail that is responsible for binding to cellular cargo (Figure 2.3) (Woehlke and Schliwa, 2000; Yang, Laymon, and Goldstein, 1989). The head is the signature of kinesin and its amino acid sequence is well conserved among the different kinesins. Each of the heads has two separate binding sites: one for the microtubule and the other for the ATP fuel. These heads are connected to the stalk through short (∼13 amino acids) “neck-linker,” a mechanical element that undergoes nucleotide-dependent conformational changes that facilitate the kinesin stepping.


Kinesin I is responsible for the movement of cargo within the cell. It is one of the most widely studied motor proteins and serves as a model protein for under-standing the molecular basis of intracellular transport. Within living cells, diffu-sion is the primary way of transport. Kinesin motor proteins address the need for fast and directed movement of certain cargo, such as vesicles filled with neuro-transmitters in neurons, by cooperatively transporting cargoes along the protein microtubule filamentous structures (Figure 2.2a). These biomotors are thus able to pick up vesicles at the right place, transport them in a timely manner along a microtubule track substrate, and deliver them to the right location.

2.2.3.2 Kinesin MovementSubstantial efforts have been devoted toward understanding the movement mech-anisms of kinesin (Asbury, Fehr, and Block, 2003; Okada and Hirokawa, 1999). Kinesin utilizes the chemical energy stored in ATP in order to produce directed force along microtubule filaments (Figure 2.2a). Key for the movement of kinesin is a conformational change associated with the ATP hydrolysis. The ATP binding and hydrolysis, and the corresponding ADP release, change the conformation of the microtubule-binding domains of kinesin and the orientation of the neck linker with respect to the head, leading to the protein movement (Hirokawa et al., 1989). Single molecule studies have revealed that the heads move parallel to the micro-tubule at an amazing speed of 1.8 μm/s. Kinesin thus moves rapidly in discrete 8-nm steps, related to the binding and hydrolysis of ATP, and corresponds to the distance between adjacent tubulin heterodimers within the microtubule track (Svoboda et al., 1993). Each of these steps corresponds to the hydrolysis of a single ATP molecule. The step size is independent of the ATP concentration or the force. The movement of kinesin is thus closely coupled to the cycle of conformational changes. Yildiz et al. (2004) suggested that kinesin is bound to the microtubule by both heads while it waits for ATP in between steps. Kinesin binds to microtubule track and walks on these tracks for many enzymatic cycles. Such movement of kinesin along the microtubule filaments involves numerous steps without disso-ciating itself and provides the directionality necessary for the cellular transport. Kinesin thus performs about 100 steps before detaching from the microtubule. Such cellular transport greatly benefits from the high force of kinesin motors. For example, a single kinesin motor can exert a force up to 7 pN from the hydrolysis

Figure 2.3 Structure of kinesin 1 (conventional kinesin), including the motor domain (head), the central coiled-coil stalk domain, and the C-terminal fan-shaped tail, which binds to cellular cargo.

Coiled-coil stalk Tail

Head domain


of a single ATP molecule, implying an energy conversion efficiency higher than 50%.

2.2.4 Myosins

Myosins constitute a large family of ATP-driven motor proteins, which has been shown to interact with actin, hydrolyze cellular ATP, and produce force and move-ment (Sellers, 1999). Myosin-based molecular machines are involved in the process of force generation during muscle contraction, wherein thin actin filaments and thick myosin filaments slide past each other. In addition to muscle contraction, they are also involved in transporting cargoes along actin filaments, cell move-ment, or endocytosis and exocytosis. The muscle myosin has served as a model system for understanding motility for decades. This motor protein uses the energy from ATP hydrolysis and related conformational changes (described later) to bind and move along actin filaments and generate force (Figure 2.2b). Myosin biomo-tors are thus involved in powering our voluntary motions (walking, running, lifting,) and in involuntary muscles (such as the heart beating) (Balzani et al., 2000).

Myosin II, known as conventional myosin, is the myosin motor responsible for producing muscle contraction. Muscle contraction is a sophisticated process involving actin and myosin and is a result of actin filaments sliding on myosin heads. The muscle fiber consists of repeated sections of actins and myosins allow-ing for the movement of large muscles. The thick filaments of muscle consist of several hundred myosin molecules, associated in a parallel staggered array by interactions between their tails. During muscle contraction, the ATP hydrolysis leads to conformational changes in the myosin motor, which induce net move-ment of the two filaments relative to each other.

Myosin consists of a globular head, which binds to the actin on a given substrate, and a coiled tail, which reacts with ATP and attaches myosin to a cell. All myosins share a motor domain on their heavy protein chains at the amino-terminus of their “head” domain (at the amino-terminus), but they differ greatly at their “tail” domain (at the carboxy-terminus). Myosin II transports cargo using actin filaments as a track. Myosin V is thought to be involved in several types of intracellular transport, especially the movement of vesicles from the center of the cell to the periphery. All myosins thus bind to actin filaments via a globular “head” domain located at the end of the heavy chains, and such actin binding increases the ATPase activity of all myosins (reviewed in Hwang and Matthew, 2009).

Myosin produces mechanical force from ATP hydrolysis by cyclically interacting with actin filaments in a four-step cycle known as the Lymn–Taylor cycle (Figure 2.4) (Hugel and Lumme, 2010; Kühner and Fischer, 2011). The principle underly-ing each of the four Lymn–Taylor steps is that structural change in one region of the myosin is coupled closely to a change in another domain of the protein. Similar to kinesin, myosin cannot lift up both legs at the same time from the actin fila-ments or it will fall off the filament and diffuse away (Figure 2.4). Myosin II works


by converting small structural rearrangements at the catalytic site within the motor domain into a large swing or power stroke of the light-chain binding domain. This serves as a flexible lever arm, transferring force to the object that is being moved. In this model, the presence of nucleotide (ATP or ADP and inorganic phosphate) at the catalytic site is tightly coupled to the affinity of myosin for actin as well as to the lever-arm position. The myosin remains tightly bound to actin until the release of ADP, at which point ATP binds rapidly, causing the release of the myosin from the filament. On rebinding to actin and releasing phosphate, the ADP-bound myosin head undergoes a transition from a weak to a strong actin-binding state, which is accompanied by a reverse conformational change to a poststroke state, leading to a sliding motion at the myosin–actin interface (Kühner and Fischer, 2011; Spudich and Sivaramakrishnan, 2010). The fraction of the ATPase cycle time that a myosin spends strongly bound to an actin filament is known as the duty ratio. Bryant’s team (Chen et al., 2012) demonstrated that myosin motors can be engineered to change their direction of motion reversibly in response to a calcium input. Adjusting the local concentration of calcium ions in the surrounding solution thus allowed myosin to walk in either direction along an actin track.

Muscle tissues represent an elegant natural example of a system that effectively combines the output of many molecular motors. The actin–myosin interactions

Figure 2.4 Processive stepping (movement) of Myosin V (in green) on an action filament (in red) during the various stages of its conformational cycle. a) Both heads contain ADP and are bound to the track, (b) triggered by intramolecular tension the rear head releases ADP, (c) the rear head binds ATP and detaches from the filament, (d) the

rear head hydrolyzes ATP and makes a forward stroke, (e) the rear head becomes the front head and binds to the actin filament. D, ADP; Pi, inorganic phosphate; T, ATP. Myosin V thus utilizes one ATP per approximately 36 nm step. (Reproduced with permission from Hugel and Lumme, 2010.)

Coiled-coil

(a) (b)

(c)

(d)

(e)

Lever arm

Motor domainD D*

D*

D*

D D+Pi

D

Current opinion in biotechnology

D+Pi

T

– +


play a central role in cell biology. Such ATP-fueled cyclical interactions are respon-sible not only for muscle contraction but also for a variety of movements of non-muscle cells, including cell division. Multiple myosin II molecules generate force in skeletal muscle through a “power stroke” mechanism. Such complete “power stroke” cycle involves ATP-binding, hydrolysis, and phosphate release. This cycle is fueled by energy released from ATP hydrolysis and occurs at the release of phosphate from the myosin molecule after the ATP hydrolysis while myosin is attached to actin. Such phosphate release leads to a conformational change in the myosin molecule that moves the myosin along actin filaments.

2.2.5 Dyneins

Dyneins are also microtubule-associated protein motors that convert the energy stored in ATP into movement to power a wide variety of cellular activities. The dynein family of motors is the largest of the three linear motor proteins (1–2 MDa), and resembles kinesin in several ways although it is nearly 10 times larger (Burgess and Knight, 2004; King, 2000; Porter and Johnson, 1989; Samso et al., 1998). There are two types of dyneins classified by their structure and function: cytoplasmic and axonemal dyneins. Cytoplasmic dyneins, found in all animal cells, have a variety of cellular responsibilities that include intracellular transport, mitotic chromo-some separation, and dictating cellular movement. The microtubule motor cyto-plasmic dynein has been implicated in a variety of intracellular transport processes (e.g., organelle transport), by moving processively along the microtubules. Axone-mal dyneins are involved primarily in cilia and eukaryotic flagella movement, where they cause sliding of microtubules in the axonemes and produce the bending motion.

The structure and force-generating mechanisms of dyneins are quite different from those of kinesins and myosins (Oiwa and Sakakibara, 2005). The dynein structure consists of one to three heavy polypeptide chains of >500 kDa in the form of globular heads (responsible for the movement along the microtubules through their flexible stalk structures), and several intermediate chains (for anchoring cargo) and light chains (Burgess et al., 2003). The heavy chains contain the sites of ATP hydrolysis and microtubule binding, and hence constitute the “motor” domain. Cytoplasmic dynein has two heavy chains with globular “heads” that “walk” along the microtubule, to which they are bound by the “stalks.” The move-ment of dynein combines cycles of track binding and release with cycles of force-generating nucleotide hydrolysis.

2.2.6 Biomotor-based Active Nanoscale Transport in Microchip Devices

Inspired by cellular transport processes, there have been considerable efforts to exploit the use of protein motors for motion-driven active transport in microchip devices (Goel and Vogel, 2008; van den Heuvel and Dekker, 2007). Several groups


have thus demonstrated the use of kinesin biomotors for developing on-chip microsystems powered by autonomous transport (Bachand et al., 2009; Hess and Vogel, 2001; Hess et al., 2001, Hess , Bachand, and Vogel, 2004; Schmidt and Vogel, 2010). Particular attention has been given to the pick-up and guided trans-port of selected cargo by kinesin/MT-based molecular shuttles within microchan-nel networks. Such kinesin–MT system has served as a model system for integrating biomotor-driven transport into microengineered devices. Actin/myosin-based molecular shuttles can also be used in connection to actin filaments (AFs).

The development of kinesin/microtubule-based molecular shuttle requires proper attention to key issues of nanoscale transport: guidance, loading, and discrete movement. Accordingly, and similar to intracellular transport systems, kinesin-based active-transport microchip devices also require a positioning of a microtubule “track” within the channels. Alternately, kinesin can be bound to the surface and used to glide the microtubules, which serve as the microtrans-porters that carry nanoscale cargos to destinations (Figure 2.5). Modern micro-fabrication techniques coupled to chemical patterning have been used to create well-defined tracks for filament shuttles necessary for the spatially controlled movement. So thus far, the motion of protein motors has been confined mainly along individual tracks. Clemmens et al. (2004) demonstrated the feasibility of using kinesin-coated tracks to actively transport microtubule shuttles through engineered microchannel networks and characterized various track junctions and directional sorters for such molecular shuttles. Hancock’s team used lithographic patterning for orienting and guiding microtubules traveling over kinesin-coated surfaces (Moorjani et al., 2003). Functionalization of the biomotors or microtu-bules with fluorescent dyes or nanocrystals provides an attractive approach for long-term imaging of these molecular shuttles. For example, Muthukrishnan et al. (2006) demonstrated also the conjugation of fluorescent quantum dots to kinesin biomotors through a neutravidin bridging molecule. Such use of biotin

Figure 2.5 Kinesin/microtubule-based molecular shuttle. Multiple kinesin motor proteins, adhered to surfaces, support the movement of microtubules functionalized

with selective linkers for cargo attachment and transport. (Reproduced with permission from Hess and Bachand, 2005.)

Cargo

Microtubule

Kinesinmotor

Track surface

Shuttledetail


linkers permits the selective loading and functionalization of molecular shuttles. This study indicates also the potential for biomotor-driven nanoparticle transport and assembly.

The guided and controlled directional motion of kinesin/MT-based molecular shuttle should be combined with high degree of speed regulation. Various param-eters affect the activity of these motor proteins and hence their speed, including the concentration of the ATP fuel, or the presence of ATP-regenerating or hydro-lyzing enzymes, divalent cations, or inhibitors. Appropriate (bio)chemical stimuli can thus been used to modulate the motion of biomotors. For example, Hess et al. described the use of light for controlling the movement of kinesin (Hess and Vogel, 2001; Hess et al., 2001). Such light-regulated motion of kinesin was accom-plished by exploiting a UV-induced release of caged ATP combined with enzymatic ATP degradation by hexokinase to turn the molecular shuttles “On” and “Off” sequentially. Repetitive light “On/Off” cycles thus resulted in corresponding increase and decrease in the velocity. Similar “On/Off” switching of the movement have been achieved by changing the concentration of inhibitors.

The kinesin/MT-based active-transport microchip systems enable selective loading directional transport and release of selected cargo within microchannel networks. Vogel and colleagues discussed the challenge of attaching cargo to protein transport systems without compromising their transport performance remains (Schmidt et al., 2012). The capture and transport of a wide range of target analytes including proteins, nucleic acids, virus particles, and liposome by kinesin-driven molecular shuttles have thus been demonstrated (Hess and Vogel, 2001). In particular, the cargo unloading (on-demand release) capability was dem-onstrated for using the kinesin motor protein connection to various triggers, such as light (Hess and Vogel, 2001; Hess et al., 2001), chemical (Hirabayashi et al., 2006), or temperature (Hiyama et al., 2010) stimuli. DNA hybridization reactions have also been attractive for releasing of captured nucleic-acid cargo from kinesin microtransporters without external stimuli. For example, Hiyama et al. (2010) described an autonomous system that selectively loads, transports, and unloads reactor-liposomes using biomolecular motor-based motility and DNA hybridiza-tion using ss-DNA-labeled microtubules gliding on kinesin-coated surfaces (Figure 2.6).

The selective loading of kinesin-powered molecular shuttles with protein cargo has enabled “smart dust” biosensing applications, discussed in Chapter 6 (Fischer, Agarwal, and Hess, 2009). Such use of antibody-functionalized microtubules and kinesin motor proteins as molecular shuttles can selectively capture analytes from solution and deliver the analytes to a sensor patch, and overcome the mass transfer limitations for nanoscale sensors (Katira and Hess, 2010). Although protein motors are capable of complex functions and operations, a major limitation to their ex-vivo microchip applications lies in their inherent instability and restric-tions in the environmental conditions they operate in within such engineered paths (van den Heuvel and Dekker, 2007). Prolonging the lifetime of protein motors in their functional states is thus critical for many applications where such biomotors are integrated into synthetic materials or devices.


Active transport by motor proteins can also be used for self-assembly processes. Hess et al. demonstrated that active transport powered by biomolecular motors can be utilized to drive the self-assembly of extended linear and circular mes-oscopic ordered structures that would not form without such active transport (Hess and Bachand, 2005; Hess et al., 2005).

Related biosensing applications of such biomotor-based microchip systems are described in detail in Section 6.2.

2.3 Rotary Biomotors

The enzyme ATP synthase is the most important and amazing natural rotary motor in view of its remarkable design and performance (Boyer, 1997; Weber and Senior, 2003). According to Boyer (who shared the 1997 Nobel Prize in Chemistry), “Among all enzymes, ATP synthase is one of most beautiful as well as one of the most unusual and important” (Boyer, 1997). Such beauty and uniqueness reflect the three-dimensional structure of the F1-ATPase component and the structural com-plexity and reaction mechanism of the enzyme, respectively. The large amount of ATP synthesized per day indicates the importance of ATP synthase (Boyer, 1997). This highly efficient nanopropellor, which manufactures ATP from ADP and phosphate at rates exceeding 100 ATP molecules per second, provides about 80% of the cellular ATP in most living organisms.

The structure and mechanochemistry of ATP synthase have been studied exten-sively (Boyer, 1993; Noji et al., 1997; Weber and Senior, 2003; Yasuda et al., 2001). ATP synthase is an assembly of proteins anchored in the cell lipid bilayer. As illustrated in Figure 2.7, this multisubunit enzyme consists of two rotary molecu-lar motors, F1 and F0, attached to a common shaft, each powered by a different

Figure 2.6 Kinesin-based microchip delivery of liposomes. Loading of the liposome is accomplished by DNA hybridization using single-strand (ss) DNA-functionalized

microtubules and liposome functionalized with the complementary ss DNA. (Reproduced with permission from Hiyama etal., 2010.)

Loading and transport of reactor-liposomes

Calcein

Liposome

15-basessDNA 23-base

ssDNA

Kinesins

Glass substrate

Microtubule (MT)

2.3RotaryBiomotors 25

fuel attempting to rotate in the opposite direction. The hydrophilic (water-soluble) F1 portion is a chemical motor, powered by ATP.

ATP synthase is a remarkable reversible coupling device. It uses the free energy of ATP hydrolysis for rotating in one direction. In this operation, the enzyme hydrolyses the ATP to ADP while turning left-handed 120°, with each 120° step consuming a single ATP molecule. The F1 motor can also rotate right-handed upon providing it with ATP. In this reverse operation, the F1 head rotates right-handed in 120° steps, every time an ATP molecule is synthesized. The mechanical energy of this rotation thus drives the ATP synthesis. Such rotary catalysis repre-sents a remarkable feature of ATP synthase. In contrast, the second motor – the membrane-bound hydrophobic F0 portion – uses the energy stored in a transmem-brane electrochemical gradient to turn in the opposite direction. This F0 motor is responsible for transporting protons across the membrane via its ring of c-subunits. Such transport of protons through F0 drives the release of ATP product. The proton translocation through F0 (during the ATP synthesis) causes the rotation of the γ subunit within F1. The mechanical force of subunit rotation is transmitted to the catalytic sites to drive ATP synthesis from ADP and inorganic phosphate (Pi). Three ATP molecules are produced per 12 protons that pass through the motor.

The first suggestion that rotation of internal subunits was part of the catalytic mechanism of the F1 portion was published by Paul Boyer in the early 1980s (for review see Boyer, 1993). Subsequently, in 1997, Noji et al. (1997) directly observed

Figure 2.7 Structure of F0F1-ATP synthase: two rotary molecular motors attached to a common shaft, each attempting to rotate in the opposite direction The catalytic region consists of the subunits a, b, g, d, and e. The

proton channels lie at the interface between subunits a and c; dashed lines indicate the putative inlet and outlet channels. (Reproduced from Balzani, Credi, and Venturi, 2008.)

F1H+

δ α α

α

β

β β

γ ε

H+a c

b

F0Membrane

ATP

ADP + Pi


the rotation of the F1 motor. Kinosita and co-workers (Adachi et al., 2007) further elucidated how the rotation of the ATP synthase subunits is coupled to the chemi-cal reactions that generate ATP.

Montenegro’s team at Cornell University (Soong et al., 2000) demonstrated in 2000 the integration of biomolecular motors and nanoscale inorganic systems, in a hybrid nanomechanical device powered by a biomolecular motor. The device consisted of three components: an engineered substrate, an F1-ATPase biomolecu-lar motor, and fabricated nickel rod nanopropellers (of ∼1 μm length) connected to the motor through biotin–streptavidin bonds. Addition of ATP caused rotation of the propeller. On addition of the ATP fuel, the nickel nanorod propellors remained attached and spun at eight revolutions a second. The reversible rotation of the nanopropeller was accomplished in the presence of 2 mM adenosine tri-phosphate (“On”) and the sodium azide inhibitor (“Off”).

The same team described also the rational design, preparation, and characteriza-tion of a mutant F1-ATPase motor containing a metal-binding site that functions as a reversible zinc-dependent “On/Off” switch (Liu et al., 2002). Repeated cycles of zinc addition and removal (chelation) resulted in inhibition and restoration, respectively, of both ATP hydrolysis and motor rotation of the mutant, but not of the control wild-type F1 fragment. Such ability to engineer chemical regulation offers considerable promise to control (switch) F1-ATPase-powered integrated nanobiomechanical hybrid devices at the single-molecule level.

2.4 Swimming Microorganisms

Natural microorganisms inhabit a world of low Reynolds number regime, from flagellated bacteria (Re∼10−5) to spermatozoa (Re∼10−2) (Figure 2.1). The absence of inertia means that the conventional knowledge of the hydrodynamics of mac-roscale swimmers cannot be applied to the propulsion of microorganisms. Back in 1951, Geoffrey Taylor (Taylor, 1951) first showed that propulsion is possible in the absence of inertia by propagating a sinusoidal traveling wave along the body. He computed asymptotically the leading order propulsion speed as a2k2c/2, where a, k, and c are the amplitude, wavenumber, and phase speed of the wave, respec-tively (assuming the wave amplitude is smaller than the wavelength).

As was discovered in the following three decades, natural microorganisms use a variety of techniques for overcoming the viscous drag forces. These techniques are largely different from those used by macroscale swimmers (Zhang, Peyer, and Nelson, 2010; Childress, 1981; Purcell, 1977; Lauga, 2011). Microorganisms are technologically well ahead of our synthetic nanoswimmers in achieving small-scale propulsion. Indeed, the remarkable underlying locomotion principles of these natural microorganisms serve as general guidelines for designing efficient small-scale artificial magnetic swimmers.

The movement of microorganisms is usually accomplished by breaking the time-reversibility, and hence escaping from the scallop theorem (Purcell, 1977).

2.4SwimmingMicroorganisms 27

They move in a nonreciprocal manner, such as screw-like and flexible oar-like movements. The fluid mechanical processes involved in the propulsion of such microorganisms were discussed extensively since the 1970s (Berg and Anderson, 1973; Brennen and Winet, 1977). Considerable progress has been made toward understanding the underlying locomotion principles of small natural swimmers in a variety of complex environments and under various practical constraints (Lauga and Powers, 2009). The efficiency of a swimming microorganism was defined by Lighthill as the power that would be needed to drag an object of the same size with the same speed through viscous fluid, divided by the total viscous dissipation in the fluid (Lighthill, 1952).

2.4.1 Bacterial Flagella – Escherichia coli

Many bacterial species are motile by means of flagella. Swimming bacteria rotate their helical rigid flagella using rotary motors embedded in the cell walls (Berg, 2003; Berg and Anderson, 1973;) or via whole-body wave deformation propelled by flagella beneath the cell’s outer membrane (Goldstein and Charon, 1990). For example, Berg and Anderson (1973) discovered in 1973 that bacteria, such as Escherichia coli (E. coli), swim at Reynolds numbers as low as 10−4 by rotating their flagella filaments in a screw-like motion (Figure 2.1a), with each filament propagat-ing a helical wave. The same team demonstrated in 2000 the real-time imaging of the fluorescent flagellar filaments (Turner, Ryu, and Berg, 2000).

Bacterial flagella are helical filaments – of several micrometers long and about 20 nm in diameter – organized in a bundle of four or five. The filaments can exist in different polymorphic forms, having distinct values of curvature and twist. It has been shown recently that the form used during forward swimming runs (the normal form) is the most hydrodynamically efficient (Spagnolie and Lauga, 2011). Each flagellar filament is driven at its base by a reversible molecular rotary engine of about 45 nm in diameter. Such engine is assembled from about 20 different kinds of parts that are similar to those found in mechanical motors. The rotary motor is powered by an ion flux (protons moving down an electrochemical gradi-ent), and not by ATP. The flow of ions through peptidoglycan-bound stator com-plexes, down an electrochemical gradient into the cytoplasm, is thus responsible for the rotor rotation via electrostatic interactions at the rotor–stator interface. The rotational speed of flagella thus varies in response to the intensity of the proton motive force. This reversible and powerful rotary motor can turn clockwise or counter-clockwise at a high frequency, leading to flagella kinematics akin to that of traveling helical waves, and thus to a propulsion. The rotary biomotor generates a torque of up to 4500 pN nm at 100 revolutions per second (Figure 2.8), which is 200 times than the torque of an F1-ATPase.

Bacterial swimming occurs at very low Reynolds numbers (Re∼10−4) and hence the fluid motion is governed by Stokes flow. The propulsion matrix proposed by Purcell (1997), which relates the translational and angular velocity of the flagellum to the torques and forces propelling the bacterium, was shown to give an adequate


description of the bacterial swimming over a physiological range of velocities (Chattopadhyay et al., 2006). For such freely swimming bacterium, the viscous drag of the cell body is balanced by that acting on the flagellar bundle. Unlike the viscous drag of the cell body, the contributions of the flagellar bundle to the total drag are more difficult to calculate. Such flagellar mechanism thus generates the nonreciprocating motion necessary for generating a net motion, thrusting the bacterium forward and swimming at the low Reynolds number regime (Berg, 2003). The natural design and motion of bacterial flagella have inspired the fabrica-tion of magnet

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