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Engineering Applications of Kinesin Motor Proteins and Microtubule Filaments

Permanent Link: http://ufdc.ufl.edu/UFE0041059/00001

Material Information

Title: Engineering Applications of Kinesin Motor Proteins and Microtubule Filaments
Physical Description: 1 online resource (120 p.)
Language: english
Creator: Agarwal, Ashutosh
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: bionanotechnology, biosensors, biotin, devices, hybrid, kinesin, microtubule, shuttles, streptavidin
Materials Science and Engineering -- Dissertations, Academic -- UF
Genre: Materials Science and Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Hybrid bionanodevices utilize biological nanoscale components to provide critical functions in a synthetic environment. These devices merge the materials of biology and the techniques of nanotechnology in revolutionary combinations. Kinesin motor proteins have evolved in nature to attach to microtubule filaments, as well as to a variety of cargoes and transport them with high efficiency. These motors and filaments can be employed in microfabricated synthetic environments to develop engineering applications. Two such applications which utilize motor-filament attachment and motor powered active transport are described in this thesis. First, a novel quantification technique which measures protein non-fouling surfaces at extremely low protein coverages is reported. The test surfaces are initially exposed to kinesin motors (as probes). Subsequently, the measurement of the landing rate of fluorescently labeled microtubules (as markers) enables the determination of kinesin density. This characterization technique lowers the detection limit of established protein density measurement techniques (currently at ~ 1 ng/cm sub 2) by a hundred fold. Ultra-low limits of detection, dynamic range, ease of detection and availability of a ready-made kinesin microtubule kit makes this technique highly suitable for detecting protein adsorption below the detection limits of standard techniques. Secondly, utilization of molecular shuttles in the construction of smart dust devices is reported. Molecular shuttles achieve controlled transport of nanoscale cargo by patterning kinesin motors on a surface and employing functionalized microtubules, which are propelled by these motors, as cargo carrying elements. In this work, the challenge of cargo loading onto the shuttles is addressed. It is demonstrated that as a result of glue-like character of biotin-streptavidin bonds, the speed of streptavidin-coated microtubules has to be optimized to facilitate attachment of biotinylated cargo. The results from these investigations then enable the design of a functional smart dust biosensor powered exclusively by molecular shuttles. The device autonomically tags, transports, deposits and detects unlabeled analytes. Molecular shuttles capture antigens from the test solution, move until they bind fluorescent markers and accumulate at a distinct location for fluorescent read-out. The device does not need pressure-driven fluid flow or electroosmotic flow to drive the mass transport functions a critical impediment in nanoscale devices.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Ashutosh Agarwal.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Hess, Henry.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041059:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041059/00001

Material Information

Title: Engineering Applications of Kinesin Motor Proteins and Microtubule Filaments
Physical Description: 1 online resource (120 p.)
Language: english
Creator: Agarwal, Ashutosh
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: bionanotechnology, biosensors, biotin, devices, hybrid, kinesin, microtubule, shuttles, streptavidin
Materials Science and Engineering -- Dissertations, Academic -- UF
Genre: Materials Science and Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Hybrid bionanodevices utilize biological nanoscale components to provide critical functions in a synthetic environment. These devices merge the materials of biology and the techniques of nanotechnology in revolutionary combinations. Kinesin motor proteins have evolved in nature to attach to microtubule filaments, as well as to a variety of cargoes and transport them with high efficiency. These motors and filaments can be employed in microfabricated synthetic environments to develop engineering applications. Two such applications which utilize motor-filament attachment and motor powered active transport are described in this thesis. First, a novel quantification technique which measures protein non-fouling surfaces at extremely low protein coverages is reported. The test surfaces are initially exposed to kinesin motors (as probes). Subsequently, the measurement of the landing rate of fluorescently labeled microtubules (as markers) enables the determination of kinesin density. This characterization technique lowers the detection limit of established protein density measurement techniques (currently at ~ 1 ng/cm sub 2) by a hundred fold. Ultra-low limits of detection, dynamic range, ease of detection and availability of a ready-made kinesin microtubule kit makes this technique highly suitable for detecting protein adsorption below the detection limits of standard techniques. Secondly, utilization of molecular shuttles in the construction of smart dust devices is reported. Molecular shuttles achieve controlled transport of nanoscale cargo by patterning kinesin motors on a surface and employing functionalized microtubules, which are propelled by these motors, as cargo carrying elements. In this work, the challenge of cargo loading onto the shuttles is addressed. It is demonstrated that as a result of glue-like character of biotin-streptavidin bonds, the speed of streptavidin-coated microtubules has to be optimized to facilitate attachment of biotinylated cargo. The results from these investigations then enable the design of a functional smart dust biosensor powered exclusively by molecular shuttles. The device autonomically tags, transports, deposits and detects unlabeled analytes. Molecular shuttles capture antigens from the test solution, move until they bind fluorescent markers and accumulate at a distinct location for fluorescent read-out. The device does not need pressure-driven fluid flow or electroosmotic flow to drive the mass transport functions a critical impediment in nanoscale devices.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Ashutosh Agarwal.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Hess, Henry.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041059:00001


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1 ENGINEERING APPLICATIONS OF KINESIN MOTOR PROTEINS AND MICROTUBULE FILAMENTS By ASHUTOSH AGARWAL A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009

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2 2009 Ashutosh Agarwal

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3 To cows and flies, For their tubulin and kinesin

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4 ACKNOWLEDGMENTS First and foremost, I thank my mentor and guide for the past four years, Dr. Henry Hess for his brilliance, humility, and patience. Directly and indirectly, he has taught me far more things than what can be articulated in this note of acknowledgement. I would like to thank my committee members Dr. Laurie Gower, Dr. Brij Moudgil, Dr. Rajiv Singh and Dr. Kirk Ziegler for their time, guidance and constructive comments Appreciation is also extended to Al Ogden at the UF Nanofabrication Center staff at the UF International Center, Alice Holt at the MSE and Jo -Anne Standridge at the PERC for their help. I thank all the Hess group members; Thorsten Fischer for special mentorship, Rob and Parag for special moments in and out of lab, and Yoli, Inkook, Shruti, Kri shna, Ajoy and Isaac for friendship and companionship. I also thank my past and present roommates Amit, Abhinav and Vijay for all the stimulating and not so stimulating discussions which made Gainesville bearable. Finally, I thank my family members and m y girlfriend for their unwavering love. Though it was your support that made this entire thesis possible, it was your questions that especially inspired the first chapter!

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...................................................................................................... 4 LIST OF TABLES ................................................................................................................ 8 LIST OF FIGURES .............................................................................................................. 9 ABSTRACT ........................................................................................................................ 11 CHAPTER 1 INTRODUCTION ........................................................................................................ 13 Outline of the Thesis ................................................................................................... 13 Bionanomat erials ........................................................................................................ 15 Background and Inspiration ................................................................................. 15 Current Bionanomaterials .................................................................................... 17 2 THE KI NESIN MICROTUBULE SYSTEM AND NANOTECHNOLOGY .................. 21 Introduction ................................................................................................................. 21 Molecular Biology of Microtubule ............................................................................... 21 Molecular Biology of Kinesin ...................................................................................... 22 In v itro Utilization of Kinesin and Microtubules .......................................................... 24 Nanotechnological Applications derived from Microtubule Properties ............... 24 Nanoscale Motion derived from Kinesin.............................................................. 26 3 CHARACTERIZING NON-FOULING SURFACES USING KINESIN PROBES AND MICROTUBULE MARKERS .............................................................................. 32 Introduction ................................................................................................................. 32 Surface Modifications to Prevent Protein Adsorption ................................................ 32 Characterization Techniques to Measure Protein Adsorption .................................. 33 A Novel Technique to Measure Ultra low Protein Coverage .................................... 35 Landing Rate Model .................................................................................................... 36 Results ........................................................................................................................ 38 Fouling and Moderately Non-Fouling Surfaces .................................................. 38 Highly Non-Fouling Surfaces ............................................................................... 39 Conclusion .................................................................................................................. 40 Materials and Methods ............................................................................................... 42 Preparation of Surfaces ....................................................................................... 42 Assembly of Flowcells .......................................................................................... 44 Microscopy ........................................................................................................... 45 Determination of Landing Rates and Surface Density ........................................ 45

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6 4 THE MOLECULAR SHUTTLE SYSTEM AND DEVICE APPLICATIONS ............... 52 Introduction ................................................................................................................. 52 Nanoscale Transport System by Molecular Shuttles ................................................ 52 Guiding Molecular Shuttles ........................................................................................ 53 Loading and Unloading Cargo onto Shuttles ............................................................. 56 Controlling Shuttle Activity .......................................................................................... 58 Molecular Shuttle based Applications ........................................................................ 59 Manipulation of Single Molecules ........................................................................ 59 Self -Assembly ...................................................................................................... 60 Investigation of Surface Properties ..................................................................... 60 Biosensing ............................................................................................................ 61 Conclusions and Outlook ........................................................................................... 61 5 OPTIMUM VELOCITY FOR CARGO LOADING ONTO MOLECULAR SHUTTLES ................................................................................................................. 66 Intr oduction ................................................................................................................. 66 Experimental Design .................................................................................................. 67 Experimental Results .................................................................................................. 68 Attachme nt and Detachment Rate ...................................................................... 68 Attachment and Detachment Probability ............................................................. 69 Analytical Model .......................................................................................................... 73 Potential Energy Surface of Biotin Streptavidin .................................................. 73 Attachment Process ............................................................................................. 75 Detachment Process ............................................................................................ 76 Predicted Opposing Force ................................................................................... 76 Predicte d Loading ................................................................................................ 77 Conclusion .................................................................................................................. 77 Materials and Methods ............................................................................................... 78 Kinesin and Microtubules ..................................................................................... 78 Assembly of Flowcells .......................................................................................... 78 Microscopy ........................................................................................................... 79 6 A SMART DUST BIOSENSOR POWERED BY MOLECULAR SHUTTLES............ 86 Introduction ................................................................................................................. 86 Applications of Smar t Dust ......................................................................................... 86 Molecular Shuttle Technologies enable Smart Dust Biosensor Design ................... 87 Prototype of a Device ................................................................................................. 89 Results ........................................................................................................................ 92 Microscopy Images .............................................................................................. 92 Test for Specificity ................................................................................................ 93 Test for Transport Mechanism ............................................................................. 94 Conclusions ................................................................................................................ 96 Materials and Methods ............................................................................................... 97 SU8 Photolithography .......................................................................................... 97

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7 General Assembly of the Smart Dust Sensor ..................................................... 97 Assembly and Operation of the Streptavidin-Specific Sensor ............................ 98 Assembly and Operation of the Glutathione S-Transferase -Specific Sensor .... 99 General Read -Out of the Sensor ....................................................................... 101 7 CONCLUSION AND OUTLOOK .............................................................................. 106 LIST OF REFERENCES ................................................................................................. 109 BIOGRAPHICAL SKETCH .............................................................................................. 120

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8 LIST OF TABLES Table page 5 -1 Results from microtubule loading experiments. .................................................... 80

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9 LIST OF FIGURES Figure page 1 -1 Cellular components and the design of bionanomateri als .................................... 20 2 -1 Molecular biology of microtubule and kinesin ....................................................... 28 2 -2 Mechanochemical cycle of kinesin ........................................................................ 29 2 -3 Microtubule in nanotechnological applications. ..................................................... 30 2 -4 Kinesin in nanotechnological applications ............................................................. 31 3 -1 Landing rate methodology. .................................................................................... 48 3 -2 Landing rate for glass surface ............................................................................... 49 3 -3 Protein adsorption to fouling and moderately non-fouling surfaces. .................... 50 3 -4 Protein adsorption to highly non-fouling surfaces. ................................................ 51 4 -1 Guiding molecular shuttles. .................................................................................... 63 4 -2 Cargo loading onto molecular shuttles using the biotin-streptavidin chemistry .. 64 4 -3 Controlling shuttle activity. ..................................................................................... 65 4 -4 Molecular shuttle based applications. ................................................................... 65 5 -1 Sketch of principle. ................................................................................................. 81 5 -2 Expe rimental Results. ............................................................................................ 81 5 -3 Geometry of the microtubule-nanosphere collision ............................................. 82 5 -4 Sch ematic of the unbinding process ..................................................................... 83 5 -5 Biotin -streptavidin bond and binding and unbinding probabilities. ....................... 84 5 -6 Attachment and detachment rates as a function of microtubule velocity. ............ 84 5 -7 Predicted mi crotubule loading ............................................................................... 85 6 -1 Concept and device layout. .................................................................................. 102 6 -2 Active transport of optical tags into the detection zone. ..................................... 103 6 -3 Accumulation of microtubules at the detection zone over time. ......................... 103

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10 6 -4 Experimental results for streptavidin as analyte. ................................................ 104 6 -5 Experimental results for GST as analyte. ............................................................ 105

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11 Abstract o f Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ENGINEERING APPLICATIONS OF KINESIN MOTOR PROTEINS AND MICROTUBULE FILAMENTS By Ashutosh Agarwal December 2009 Chair: Henry Hess Major: Materials Science and Engineering Hybrid bionanodevices utilize biological nanoscale components to provide critical functions in a synthetic environment. These devices merge the materials of biol ogy and the techniques of nanotechnology in revolutionary combinations. Kinesin motor proteins have evolved in nature to attach to microtubule filaments, as w ell as to a variety of cargoes and transport them with high efficiency. These motors and filament s can be employed in microfabricated synthetic environments to develop engineering applications. Two such applications which utilize motor -filament attachment and motor powered active transport are described in this thesis. First, a novel quantification t echnique which measures protein non-fouling surfaces at extremely low protein coverage s is reported. The test surfaces are initially exposed to kinesin motors (as probes). Subsequently, the measurement of the landing rate of fl uorescently labeled microtubu les (as markers) enables the determination of kinesin density. This characterization technique lowers the detection limit of established protein density measurement techniques (currently at ~ 1 ng/cm2) by a hundred fold. Ultra low limits of detection, dyna mic range, ease of detection and availability of a

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12 ready made kinesin microtubule kit makes this technique highly suitable for detecting protein adsorption below the detection limits of standard techniques Secondly, utilization of molecular shuttles in t he construction of smart dust devices is reported. Molecular shuttles achieve controlled transport of nanoscale cargo by patterning kinesin motors on a surface and employing functionalized microtubules, which are propelled by these motors, as cargo carryin g elements. In this work, the challenge of cargo loading onto the shuttles is addressed. It is demonstrated that as a result of glue -like character of biotin-streptavidin bonds, the speed of streptavidincoated microtubules has to be optimized to facilitat e attachment of biotinylated cargo. The results from these investigations then enable the design of a functional smart dust biosensor powered exclusively by molecular shuttles. The device autonomically tags, transports, deposits and detects unlabeled analy tes. M olecular shuttles capture antigens from the test solution, move until they bind fluorescent markers and accumulate at a distinct location for fluorescent read out. The device does not need pressure-driven fluid flow or electroosmotic flow to drive th e mass transport functions a critical impediment in nanoscale devices.

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13 CHAPTER 1 INTRODUCTION Outline of the T hesis People interested in me often inquire about my area of research. If I want to avoid follow up questions, I say that I am developing engineering applications of biomolecular motors. However, the shorter answer, Bionanomaterials is almost always followed b y the questions: "What are bionanomaterials?" and "What is the scope of the field?" These impulsive questions have turned out to be surprisingly tricky, and this introductory chapter seeks to answer those. The concept of bionanomaterials, its inspiration, and its most promising applications are described. In Chapter 2 t he biological motor of interest, kinesin, along with its associated filament, microtubule is introduced. Some of the nanotechnological applications derived from the remarkable properties o f these motors and filaments are also reviewed. In Chapter 3 one such application based on the strong attachment between kinesin and microtubule is reported. A novel quantification technique for detecting extremely low protein coverages on protein -resisti ng surfaces has been developed. It utilizes the measurement of landing rates of fluorescently labeled microtubule filaments on kinesin proteins adsorbed to a surface. The technique is applied in evaluating the performance of standard surfaces, surface poly mer coatings by poly(ethylene oxide) (PEO) and a new type of polymer coating for preventing protein adsorption. Ultra low limits of detection, dynamic range, ease of detection and availability of a ready ma de kinesin microtubule kit make this techniq ue highly suitable for quantifying protein adsorption.

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14 In Chapter 4 the "molecular shuttle" concept in the design of hybrid bionanodevices is discussed Molecular shuttles integrate biological motors and associated filaments with synthetic structures to achieve autonomous transport and actuation at the nanoscale. T hree main streams of research have emerged in this field: guiding the movement of shuttles, loading and unloading of cargo onto shuttles and control of motor activity. A summary of the recently reported applications, a brief outlook into the future, and review of the major roadblocks to its widespread implementation is presented. In C hapter 5 efforts in addressing an engineering challenge behind cargo loading onto mole cular shuttles are presented. Ca rgo is attached to microtubules via conventional bioconjugation techniques, for example by utilizing the receptor ligand pair streptavidin -biotin. It is demonstrated that the speed of streptavidin-coated microtubules has to be optimized to facilitate attac hment of biotinylated cargo. E xperimental loading data and the detailed model describing the dynamic process of loading of cargo onto shuttles are presented. The biotin-streptavidin bond gains its ultimate strength on a timescale of milliseconds due to exi stence of metastable binding states. The modeling of the attachment and detachment processes reveal the glue -like behavior of biotin streptavidin linkages. Glue -like bonds are much weaker if subjected to pulling forces immediately after formation and need a certain curing time to achieve their ultimate strength. Finally, in Chapter 6 t he first major device application of t he molecular shuttle technology a kinesin powered smart dust biosensor is demonstrated. The sensor autonomically tags, transports, deposits and detects unlabeled analytes in a complex

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15 solution. The principal challenge in designing miniaturized biosensors is integrating transport functions with energy supply into the device. Our device is powered by ATP and relies on antibody -functionalized microtubules and kinesin motors to transport the target analyte into a detection region. The transport step repla ces the wash step intrinsic to traditional double antibody sandwich assays. Bionanomaterial s Background and Inspiration Materials scientists often take pride in the fact that entire periods in the timeline of human existence are named after discoveries made in materials science. The prehistoric bronz e and iron ages represented efforts in masteri ng new alloys and metals. The se efforts paid off by equipping men with increasingly improved devices, and in turn benefitting almost every form of human endeavor; hunting, agriculture, transportation and war. However, societies have an insatiable demand fo r ev er increasing functionality in their materials. This demand is usually met in gradual increments, driven by technological advancements to manipulate the material of that age. This process continues until there is an abrupt discovery of an entirely new class of functional materials and c onsequently, the most unrealistic dreams of superior functionality are rapidly realized. Nanomaterials and nanotechnology might very well be the adventure of the age we live in. While the field has always received ample h yperbole, distinct areas have now emerged where both technology and science stand to benefit from it. Much of technology stimulus is being provided by the area of electronics, specifically integr ated chip design and manufacturing1. Miniaturization with t he current photolithography based technologies is destined to hit fundamental challenges and limits in the next decade2.

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16 Nanotechnologies based on the functional nanostruct ures of nanorods3 or nanotubes4 are expected to emerge as a practical alternative. M uch of the science stimulus, however, is being provided by the field of bionanotechnology. It is a highly interdisciplinary endeavor which operates at the fringes of biology and nanotechnology, and in the end, serves both. Biology has been aided by nanotec hnological advancements which include probes (such as nanoparticles5 and nanopores6), designer surfaces (such as self assembled monolayers7 and protein microarrays8), and advanced experimental techniques (such as optical tweezers9 and single molecule imagi ng10). Furthermore, developments in the area of nanomedicine, such as drug delivery systems11 and engineered architectures for tissue regeneration12 hold g reat promise to prevent and cure diseases. The second thrust area of bionanotechnology, and the focus of this thesis, is the advancement of nanotechnology by our greater understanding of biology. At the heart of each biological system are molecular building blocks (such as phospholipid membranes and DNA genetic material) and machineries (such as antibody proteins and motor proteins) The molecular building blocks and machines work in tandem to produce living entities. In a modern reductionist view, these nano -components are active nanomaterials -capable of storing information, assembling, sensing, adapting, r epairing, and transporting. This arsenal of molecular building blocks and machines has piqued the imagination of nanotechnologists for a long time. The broad objective of the field of bionanomaterials is to c ombine the active organic nanomaterials of t he biological world with the functional inorganic nanostructures synthesize d with modern nanotechnologies.

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17 This fresh approach towards materials design could enable unforeseen superior functionality in hybrid bionanodevices Current Bionanomaterials The f ield of bionanomaterials is currently driven by r esearchers who are challenging the conventional viewpoint of materials being static load bearing structures. They are creating devices and materials which have the ability to change form, store information, self clean, repair damage, produce motion, and act as sensors. T he components of a cell are being explored to tailor materials at the nanoscale. In the following, we briefly look at those cellular components and their application in the construction of bionanomaterials. Cell m embrane based materials. Lipid bilayers, most commonly found in cell membranes, have been used to synthesize liposomes13 (Figure 11 a) These v esicles can be synthesized in sizes varying from 20 nm to 10 m and make ideal nanocontainers for drug delivery applications. They retain their relative s tability and low permeability for biomolecules. Also, their surfaces can be decorated by various molecules such as polyethylene glycol (for longer circulatory life of the vesicle) or with antibodies (for specific targeting). S -layer s another kind o f biological membrane, assemble into a crystalline layer with spacing of assembly units in the range of 330 nm. These layers can be deposited on various substrates and are now routinely used to create regular nanopatterns of nanoparticles, enzymes and antibodi es14. DNA based materials. DNA serve s as a very effective genetic material owing to its structural properties. DNA structures self assemble from two complementary strands in which the guanine moieties interact with cytosine and adenine interact with thym ine. These structural properties also enable the use of DNA as a building material for

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18 tailored nanostructures (Figure 1-1 b)15. DNA oligomers that have internal complementary recognition interfaces spontaneously self assemble into these structures. Today researchers are combining well established motifs (such as "junctions", "tiles" and "crossovers") and designing new ones to synthesize elaborate 3D structures. The structure based assembly of DNA molecules also provides a handle for computation to be incorporated into the assembly pathways of DNA arrays. This has given rise to the field of DNA based computing where large parallelism could provide advantages over conventional electronic computing for certain problems. Antibody based sensors. Antibodies are a key component of the immune system. They can distinguish between extremely similar ligands and attach to their complementary ligand with high specificity and high affinity. Diagnostic immunoassays have been utilized in commercial products such as pregnancy kits which detect traces of a human hormone. However, ultra sensitive nanotechnological devices which utilize molecular recognition by antibodies are now being developed (Figure 1-1 c)16. T hese devices utilize nanowire field effect transistors. The nanowires are functionalized by various antibodies and upon the highly selective binding of biomolecules, discr ete conductance changes provide real -time and parallel detection. These multiplexed nanowire sensor arrays can simultaneously detect viruses and prot eins at femtomolar concentrations. Apart from electrical detection, antibodies are widely used by nanotechnologists to develop other sensing platforms, locate target cells and direct drug delivery vehicles. Biomolecular motors in hybrid bionanodevices. Ge nerating controlled nanoscale motion has been a long term goal of nanotechnology. It can be traced back

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19 to the famous 1959 lecture by Feynman17 in which he articulated, I want to build a billion tiny factories, models of each other, which are manufacturin g simultaneously, drilling holes, stamping parts, and so on. Indeed, nanotechnological advancements such as the atomic force microscope and the scanning tunneling microscope have made it possible to manipulate individual atoms However, these techniques a re prohibitively slow sophisticated and expensive. Biomolecular motors, which convert the chemical energy stored in ATP molecules directly into motion, can provide the solution (Figure 11 d) There are two basic types of biomolecular motors: the rotary m otors (such as the F1-ATPase) and the linear motors (such as kinesin and myosin). Rotary motors, properly immobilized on a surface, have been used to rotate biological filaments18 and inorganic rods19. While the technological applications of such rotary mo tors still remain to be demonstrated, linear motors have gained much more attention of the nanotechnologists20. These motors, along with their complementary filaments, have been either directly utilized in nanotechnolog ical applications (reviewed in Chapte r 2 ) or interfaced into synthetic environments for the control led transport o f nanoscale cargo (reviewed in Chapter 4).

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20 Figure 11 Cellular components and the design of bionanomaterials. a ) Lipid bilayers are used to synthesize liposomes which carry drugs and genetic material. Their surface can be decorated with homing peptides and non-fouling PEG moieties. b) DNA strands are designed to self assemble into complex nanostructures15. c) Antibody functionalized nanowires detect the binding of correspondi ng ligands with very high sensitivity16. d) Rotary motor protein, ATP synthase and linear motor protein, kinesin can be immobilized on the surface to rotate18 or propel20 biofilaments.

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21 CHAPTER 2 THE KINESIN MICROTUBULE SYSTEM AND NANOTECHNOLOGY Introduc tion Cells are the fundamental building blocks of life. Within almost every cell there exists complex nanoscale machinery, capable of repairing damage, communicating, and creating entirely n ew cells with high fidelity21. Specifically, the cytoskeleton of t he cell is the protein scaffold that assists the cell in organizing its cytoplasm, responding to external mechanical s timuli, and generating motility22. The cytoskeleton consists of a network of actin filaments, microtubules and intermediate filaments22. T he actin and microtubule filaments are also associated with the myosin and the kinesin family of motor proteins respectively. Actin and myosin constitutes the primary component of muscles. Microtubules along with their complimentary motor, kinesin are util ized by cells for intracellular transport of cargoes such as organelles and chromosomes. In this chapter, the properties of microtubule filaments, kinesin motors and their interactions are described. Some of the nanotechnological applications based on thei r physical properties are also reviewed. Molecular Biology of Microtubules Microtubules21,22 are long, stiff, and hollow biopolymers composed -tubulin heterodimers They span the entire cytoplasm and provide str uctural support to the cell T hese remarkable constructs are involved in a variety of cellular functions such as intracellular transport, cell motility, and mitosis More importantly, microtubules serve as tracks for the kinesin motor proteins. Tubulin dimers bind headto -tail to form linear protofilaments with an 8 nm repeat distance and a variable number (10 -18, often 13) of protofilaments and assemble into a

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22 cylindrical structure with an outer diameter of about 25 nm and a length of many micro meters (Figure 21 a) Since the protofilaments bind to each other in the same tubulin is slow growing and called the minus end, and the fast tubulin is named the plus end. However, protofilaments bind to each other with an offset of 0.92 nm, which results in an accumulated offset of 12 nm from 13 protofilaments. This exactly equals the length of three monomers and hence, for a 13 protofilament microtub ule, a discontinuity in the structure or a linear seam exists. The structural properties of microtubules are critical to their cellular functions as well as their nanotechnological applications. During polymerization, microtubules can display a phenomenon termed dynamic instability: stochastic switching between growing and shrinking phase s on a timescale of minutes When microtubules are in a growth phase, the tubulin dimers joining the plus end have a Guanosine triphosphate (GTP) molecule bound to them, which they su bsequently hydrolyze to Guanosine di phosphate (GDP) If polymerization proceeds faster than hydrolysis, a cap of GTP -tubulins is formed at the microtubule tip, which stabilizes the microtubule. If hydrolysis overtakes polymerization, the GT P cap is destroyed and rapid depolymerization sets in (a phenomenon known as catastrophe). The depolymerization of the microtubule stops when a surviving GTP tubulin is encountered in the microtubule lattice, which arrests the depolymerization and initiates a new growth phase (the process is termed rescue) Molecular Biology of Kinesin Kinesin 1 motors21,22 are involved in intracellular trans port of vesicles and organelles, cell division and the organiz ation of cilia and flagella. Their role becomes

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23 p articularly important in neurons because vital cargo has to be trans ported over large distances Kinesin 1 is a twoheaded motor which walks towards the plus end of the microtubules. It is a tetramer of two identical heavy chains, and two associated light chains, which in vivo are responsible for cargo binding. The heavy chains fold into two globular heads at one end, a stalk with a hinge in the middle, and a tail domain at the other end (Figure 2 -1 b). The key to the generation of motion is a conformati onal change in the globular head domain of the motor as a result from adenosine triphopsphate (ATP) hydrolysis while the head is attached to the associated filament. The conformational change is amplified and results in movement of the load bearing tail region of the motor in a specific direction along the filament. The amplification is provided by the lever arm neck domain and depends directly on the length of t his lever arm After this power stroke, the head detaches, advances and rebinds at the n ext binding site on the filament. The fraction of time that each head spends in its attached state is called the duty ratio of the motor. Differences in the duty ratio are important in determining if the motors are processive or not Kinesin 1 is a highly processive motor and hence utilized individually by cells for long distance intracellular transport The heads step on the binding sites along the microtubule protofilaments. These sites are spaced 8 nm apart. Each head of the kinesin molecule takes 16 nm s teps, thus moving the entire molecule 8 nm in each step in a handover hand mechanism. The heads pass each other on alternating sides to keep the stalk from getting twisted during the walk. For every step, kinesin consumes one ATP molecule. The two heads are tightly coordinated so that one head does not detach before the other is securely

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24 attached. This makes kinesin a highly processive motor, allowing it to walk for several micrometers before detaching. The exact sequence of events in the mechanochemical c ycle of kinesin is still debated but one possible mechanism is depicted in Figure 2 -2. In vitro kinesin motors walk along microtubules at speeds of 1 m/s (saturating ATP concentrations). The force at which the mean velocity drops to zero, the stall forc e, is about 8 pN and is independent of ATP concentration. Because of its high degree of processivity, even a single kinesin molecule is capable of propelling microtubules along a surface. In vitro Utilization of Kinesin and Microtubules Advances in biotech nology have enabled the use of biological components outside their natural environments. For example, tubulin is routinely purified from bovine brains and can be commercially purchased. The kinesin motor proteins can be purified from cells or expressed in recombinant bacterial systems and harvested in large quantities. This has spurred a spate of nanotechnological applications based on these two remarkable constructs. Nanotechnological Applications derived from Microtubule Properties Regulated assembly and disassembly of microtubules can generate pulling and pushing forces23, and is used by cells to position the mitotic spindle and separate the two sets of chromosomes into the two daughter cells Dynamic instability also allows cells to recycle the tubulin b uilding blocks. From a materials perspective, constantly turning over the subunits of a structural element might also allow cells to ensure that these co mponents remain defect free24. The diversity of tasks that cells are able to fulfill by controlling dynamic instability has piqued the imagination of nanotechnologists too. Simulations have shown that the

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25 seemingly unreliable stochastic process of dynamic assembly can be employed to bui ld a variety of nanostructures25. Strategies to sort, pattern, harvest, and deliver nanopartic les have been evaluated (Figure 2-3 a). Reproducible control over the actions of these nanosystems can be exercised only at the swarm level, where the stochasti c nature of individual elements is averaged25,26. The initial steps towards an experimental demonstration of these concepts have been taken by assembling threedimensional polar oriented synthetic mic rotubule organizing centers27. The reversibility of tubulin polymerization is also employed for the preparation and purification of proteins functionalized with fluorophores or biotin, for example28,29. It is essential that the ability of the protein to polymerize is not compromised after functionalization. Hence, microtubules are labeled in the polymer form, excess label is removed and functional protein is selected by repeated cycles of polymerization, centrifugation and depolymerization. Fluorescently labeled tubulin and biotinylated tubulin have been prepared and are commercially available in lyophilized form in ready to -use aliquots (Cytoskeleton Inc., Denver, CO). Frequently, a static microtubule structure is desirable. To this end, depolymerization can be suppressed for days by the addition of paclitaxel ( taxol) to the buffer solution30. Microtubules can also be chemically cross -linked using glutaraldehyde. This extends their lifetime to weeks and makes them stable at temperatures as high as 90C and against large variations in pH31,32. Due to their nanosc ale cross -section, very high aspect ratio, and well ordered surface functionality, stabilized microtubules can be used as templates for the production of inorganic nanowires3335 and nanoparticle arrays (Figure 23 b) 35,36. Iron

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26 oxide -coated microtubules3 3 have been produced by a biomimetic mineralization process with a fair amount of control over the oxide thickness and crystallinity. Tubulincarbon nanotube hybrids, including microtubule encapsulatednanotubes have also been reported37. Nanoscale Motion derived from Kinesin Biophysicists have for a long time utilized in vitro assays to study motor proteins and their associated cytoskeletal filaments in a synthetic environment38. Motility assays39,40 have been conducted in the bead geometry (filaments st ationary, motors moving) and in the gliding geometry (filaments moving, motors stationary). Both approaches have been adopted by engineers aiming to mimic the biological applications of motor proteins, and in particular their function as nanoscale transp ort systems. Bead geometry. In the bead geometry, the microtubule is immobilized by adsorption to a surface while the motor is attached to a polystyrene microsphere (the bead) and walks along the filament (Figure 2 4 a) Optical tweezers can be used to exert precisely calibrated opposing forces on the bead and consequently on the motor. The distance over which the bead is transported depends on the number of motors connecting the f ilament and the bead41, but can only exceed the length of the filament if the immobilized filaments are overlapping42. Gliding geometry. The gliding geometry (or inverted geometry) utilizes immobilized kinesin motors and moving microtubule filament s (Figure 2 -4 b) This is advantageous as the filament can move for centimeters without interruption, provided a sufficient area has been covered with motors. Gliding assays are typically performed in simple flow cells43 assembled from a glass slide and a glass coverslip separ ated by spacers of roughly 100 m height (Figure 24 b)

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27 The interaction between the kinesin motors and the internal surface of the flow cell in a gliding assay has been shown to be critical44. The direct binding of a motor to the glass surface of the coverslip typically leads to denaturation of the motor protein with a complete (neither filament binding nor transport) or partial (binding but not transport) loss of function of the motor. Coating the surface with a blocking protein such as casein has been shown to dramatically enhance the motor activity after nonspecific adsorption43. In addition, strategies to specifically adsorb motors to coatings on the surface have been explored. To mimic the biological transport system, the functionality of the bead or gliding assay would have to be significantly expanded. By integrating directed transport along predefined paths, loading and unloading of specific cargoes and user -controlled activation of the motors a fully functional biomimetic nanoscale transport system could be created. While efforts to create such a molecular shuttle45,46 system are reviewed in Chapter 4 an application which primarily takes advantage of kinesinmicrotubule binding is reported in t he Chapter 3.

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28 Figure 21. Molecular biology of microtubule and kinesin. a) Schematic representati on of a microtubule with 13 protofilaments. Protofilaments are made from tubulin dimers which bind to each other in a head -to -tail fashion. Protofilaments bind to each other with an offset which results in a seam in the microtubule structure. The ribbon di agram (bottom) shows th e GTP and taxol binding sites. b) Schematic representation of kinesin showing the cargo binding tail domain, the coiled coil stalk domain, the neck linker, and the head domain of the molecule. The ribbon diagram (bottom) shows the AT P binding site in the head domain.

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29 Figure 22. M echanochemical cycle of kinesin. The hand over -hand mechanism is elucidated by Block47. a) ATP binding to the leading head induces a conformational change in the molecule. This power stroke results in the movement of the trailing head towards th e plus end of the microtubule. b) The trailing head reaches the next binding site, 16 nm away on the microtubule lattice, after a diffusional substep. c) Binding to the microtubule catalyzes ADP release from the new leading head. This strains the neck linker region which gates the two head domains in different states. d) ATP hydrolysis and release of phosphate from the now trailing head relieves the strain, freeing the leading head to bind to an ATP molecule to repeat the cycle.

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30 Figure 23. Microtubule in n anotechnological applications a) Snapsh ots from a simulation study25 which illustrates the harvesting of nanoparticles by microtubules and the delivery of harvested particles to defined locations. The harvesting process and formation of stable tracks to the destination location employs dynamic instability. b) Microtubules have been used as templates to nucleate and grow nanoparticles from metal ion solutions in presence of reducing agents35. The transmission elect ron microscopy image shows a microtubule densely covered by gold nanoparticles; th e scale bar represents 100 nm.

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31 Figure 24. Kinesin in nanotechnological applications. a) Bead geometry. This is most often employed in an optical tweezer setup utilized by motor biophysicists. Filaments adsorbed to a substrate serve as tracks for motors. Microsphere cargo is bound to the tail region of the motors and held in place by a laser trap. b) Gliding or inverted geometry. Motors are adsorbed to a substrate through t heir tail domains. The heads project out into the solution and bind to filaments. The experiments are usually carried out in a flow cell which enables solution exchanges (but does not utilize continuous flow) and investigation under a microscope.

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32 CHA PTER 3 CHARACTERIZING NON-FOULING SURFACES USING KINESIN PROBES AND MICROTUBULE MARK ERS Introduction Proteins are known to adsorb on almost all surfaces, hydrophilic or hydrophobic. This is largely because proteins can interact with surfaces by any one or more of the following general force classes48: 1) Electrostatic forces due to the presence of two or more charged groups near the interface, 2) H ydrophobic interactions in the presence of water molecules at th e protein-surface interface and 3) H ydrogen bo nding due to dipole dipole interactions. It is widely believed that protein adsorption on surfaces dictates the response of other biological species such as other proteins protein agglomerates and cells towards the surface49. Hence, extensive developments in the fields of biotechnology, medicine and food processing have given rise to a large variety of applications which require controlled protein -surface interactions In many cases, this implies comp lete prevention of protein adsorption to the surface. In this chapter, the surface techniques to prevent protein adsorption and the established techniques to measure protein adsorption with a special emphasis on their detection limits are first outlined. Then, a novel quantification technique which measures the performance of protein-resisting surfaces at extremely low protein coverage s is reported. Surface Modifications to Prevent Protein Adsorption The most widely used system to render a surface protein resistant (non-fouling) is surface immobilization of poly (ethylene oxide) (PEO) chains of varying lengths (number of EO units varying from 3 to 50). The methods to attach these polymers include physiosorption of PEO -PPO -PEO triblock co -polymers onto hydrophobic surfaces50,

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33 formation of self assembled mono layers (SAM) of Oligo(ethylene oxide) (OEO) or PEO modified alkane-thiols on gold surfaces51, and utilization of surface initiated atom transfer radical polymerization (SIATRP)52,53. Surfaces grafted with oligosaccharide surfactant polymers have also been shown to resist protein adsorption by the formation of a glycocalix like structure54. Apart from using PEO or PEO -like polymers to coat the surface, polymeric structures such as tetraethlyeneglycol dimethylether (tetraglyme)55 and formation of stable phospholipi d bilayers on surfaces have also been shown to prevent adsorption of most proteins56. Characterization Techniques to Measure Protein Adsorption With advances in non-fouling surface technology it becomes important to characterize these surfaces for protein adsorption. A large number of different techniques to qualitatively and quantitatively measure protein adsorption have been developed over the years. Examples include Radiolabeling, Surface Plasmon Resonance (SPR) Spectrometry, Ellipsometry, Quartz Crystal Microbalance (QCM) X ray Photoelectron Spectroscopy (XPS), Time of Flight -Secondary Ion Mass Spectroscopy (TOF -SIMS), Enzyme Linked Immuno Sorbent Assay (ELISA) and Single Molecule Detection techniques Of these examples, XPS, TOF -SIMS, and ELISA are ma inly used to qualitatively characterize the adsorbed protein layer and are at best only semi quantitative. The common quantitative techniques are Ellipsometry SPR QCM and Radiolabeling. Ellipsometry and SPR are optical techniques and provide information regarding the adsorbed protein layer in terms of refractive index and thickness. The thickness values are used to obtain information regarding the mass of the protein layer and the

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34 density of adsorbed proteins on the surface. The detection limit of for measuring protein density using ellipsometry57 is about 20 ng/cm2. SPR requir es a metal -dielectric interface and is well suited to quantify protein adsorption on gold or silver surfaces coated with self assembled-monolayers (SAMs ) of polymeric chains. SPR is also used to measure protein adsorption on metallic nanoparticles as well as in high sensitivity biosensors to measure antibody binding to adsorbed proteins on metallic nano-pa rticles and structures. The detection limit for quantifying the amount of adsorbed protein using SPR52 is 0.5 ng/cm2. Quartz crystal microbalance (QCM) is a highly sensitive technique for measuring the adsorbed mass of protein layer on a surface. Using recent developments in QCM known as QCM -D, information regarding the rigid ity of the adsorbed protein layers can also be obtained. The detection limit for adsorbed protein layer on surfaces using QCM58 is 3.5 ng/cm2. The method of radiolabeling to measure protein density involves conjugation of proteins with common radio labels such as 3H, 32P, 35S, 14C, 125I and 131I and measuring the radioactive emissions using a Geiger Muller counter The detection limit for measuring the amount of protein adsorbed using radioactivity of labeled protein molecules59 is 5 ng/cm2. Techniques that can detect single protein molecules adsorbed on a surface would theoretically be the most sensitive techniques to quantify protein adsorption. If the protein coverage is extremely small, one should be able to directly count the total number of protein mol ecules to arrive at the density of adsorbed proteins The techniques include optical methods such as Near Field Scanning Optical Microscopy

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35 (NSOM ), Total Internal Reflection Fluorescence (TIRF) Confocal Microscopy and non optical methods such as At omic Force Microscopy (AFM) and Scanning Tunneling Microscopy (STM). While single molecule detection techniques are well suited to detect the presence or absence of an individual protein at a specific spot scanning a large area (say 100X100 m2) for every single protein molecule adsorbed is practically impossible. Furthermore, single molecule detection requires extremely high optical sensitivity which can only be accomplished with sophisticated and expensive instrumentation. While observing single molecules has b een a long term goal of biologists, no examples of the use of such methods to measure protein density have been reported. A Novel Technique to Measure Ultra -low Protein Coverage Development in the quality of polymer coatings on surfaces to further improve their protein resistance needs to be supplemented with quantification techniques that can acurately characterize these surfaces. The limits of detection of current standard techniques such as SPR and Radiolabeling are close to 1 ng/cm2 (close to the resi d ual protein adsorption on stan dard PEO coated surfaces). Novel surfaces that perform better than the current standards cannot be characterized accurately by these techniques. Therefore more sensitive techniques for the measurement of protein adsorption are needed. The binding of intra -cellular filaments, such as microtubules filaments, is readily observed by fluorescence microscopy in vitro60, since these filaments are composed of thousands of protein subunits and carry typically at least a thousand covalen tly linked fluorophores Furthermore, it has been demonstrated that observing the attachment of microtubules from solution t o surface adhered kinesin motor proteins enables the

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36 determination of motor protein 2 by measur ing the rate of microtubule attachment 40,43. Attachment rate measurements have subsequently been adapted to the determination of relative kinesin motor activity on different surfaces61 and to the evaluation of guiding structures for microtubule transport6 2. Since kinesin s long tail domain evolved to efficiently connect to cargo within cells, one can hypothesize that it serve s as a particularly efficient probe for attachment points on the surface in vitro. Measuring the attachment rate of microtubules as m arkers to the surface adhered kinesins, termed as landing rate, enable s the estimation of the absolute coverage of functional kinesins in the range of 0.004 1 ng/cm2, thus enabling to differentiate the performance of even the best non -fouling surfaces. While landing rate measurements in effect count individual proteins, their complexity is low compared to single molecule fluorescence measurements due to the availability of a kinesin/microtubule kit (Cytoskeleton Inc.) and the high brightness of fluores cent microtubules which can be imaged with a standard fluorescence microscope. In the following, it is demonstrated that the quantification of ultra low kinesin coverages by landing rate measurements is a valuable tool in determining the performance of coatings with outstanding resistance to protein adsorption. S pecifically, the adsorption model underlying the method i s described, experiments which demonstrate the determination of kinesin coverages on fouling and non-fouling surfaces are presented, and the advantages and limitations of the proposed method are discussed Landing Rate Model Microtubule attachment rate measurements are interpreted in the context of a two stage adsorption model (Figure 31) : First, kinesin probe molecules adsorb from

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37 solution to the surface, filling a fraction of the available binding sites. Second, the kinesin solution is replaced by a m icrotubule marker solution and microtubules bind specifically to the kinesin motors bound to the surface Both processes, kinesin adsorption and microtubule binding, are assumed to be irreversible on the timescale of the experiment (< 1 hr), since the solu tion exchange removes weakly bound motors, and the use of AMP -PNP (a nonhydrolyzable ATP analogue) prevents detachment of microtubules from motors. The surface concentration of ads orbed kinesin motors obtained for a given dosage of kinesin solution is the n quantified by measuring the initial landing rate R of microtubules on the coated surfaces, and the maximal, diffusionlimited landing rate Z on a control surface dens ely coated with motor proteins. The initial landing rate is related to the diffusion lim ited landing rate as given by Equation 3-1. The probability of microtubule landing and binding to the surface, P, is the probability that there is atleast one kinesin motor in the area A of interaction between the microtubule and the surface adsorbed kines in. Here, A=Lw is also known as the footprint of the microtubule, where the average length L of the microtubule is measured and the width is assumed to be w=25 nm which is the diameter of the microtubule. PZ R (3 -1) The probabil ity distribution of kinesin molecules in an area on the surface can be atleast on e kinesin is within the footprint of the microtubule is given by Equation 32. [1exp()] PA (3 -2)

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38 From Equation 31 and Equation 3-2 one can express the initial landing rate of microtubules R as a function of the kinesin surface dens -3. *[1exp()] RZA (3 -3) The initial landing rate R begins to substantially deviate from the diffusionlimited maximal landing rate if A<1, where A~0.1 m2 for typical microtubule lengths. Since the minimal measurable landing rate is on the order of 1 mm2s1, and the diffusion limited landing rate Z is on the order of 100 mm2s1, microtubule landing rate measurements are sensitive to motor densities between 0.1 m2 and 30 m2. Results Fouling and Moderately NonFouling Surfaces It is well established that direct adsorption of kinesin to fouling (glass) and moderately non-fouling (Pluronic F108 coating) surfaces leads to denaturation and loss of its mic rotubulebinding ability43. However, if the surface is covered by either denatured kinesin motors or a blocking protein (e.g. albumin or casein) interstitial binding of kinesin tails to the surface results in high motor functionality. These observations ar e reproduced in our measurements (Fig ure 32), which show that direct absorption of kinesin to glass results in a nonlinear increase of the landing rate as the amount of available kinesin is increased. In contrast, pre-coating of the glass, PU and Pluroni c surfaces with casein results in an initially linear dependence of microtubule landing rate and surface density of active motors (Figure 3-3 ) on the amount of available kinesin. On these surfaces, the diffusion-limited landing rate is reached for moderat e (20 fold) dilutions of the kinesin stock solution whose concentration can be calculated as 175 nM (see Methods). This value was used to determine dosage values in Figure 3 -3

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39 In all subsequent experiments, the diffusionlimited landing rate Z for a given microtubule preparation is assumed to be equal to the observed landing rate on a casein -coated glass surfaces exposed to a 17.5 nM kinesin solution (10-fold dilution of stock) for 5 min. Since physi o sorbed Pluronic F -108 reduces protein adsorption by ~ 80% one can interpret the observed four -fold lower density of microtubule binding motors as a reflection of the reduced adsorption of casein (Figure 3-3) Kinesin contact to the bare surface will always lead to denaturation, whereas kinesin contact to ads orbed casein will result in a functional motor. Similarly, the density of active motors is 40% lower on PU surfaces compared to glass surfaces at equal kinesin dosages. Highly Non-Fouling Surfaces With the fundamental principle of the kinesin/microtubul e based assay established, the focus was shifted to three types of highly non -fouling surfaces: (EG)3OH -terminated SAMs 4 arm Star PEO, and polyethylene glycol methacrylate (PEGMA) coated surfaces. To prepare star PEO and PEG MA coatings, functional groups were first introduced onto the substrates using chemical vapour deposition (CVD) polymerization of 4 amino paracyclophane. Direct adsorption of kinesin to these highly non-fouling surfaces led to low landing rates which did not significantly increase wit h increasing kinesin concentration in solution (Figure 3 -4 ). Precoating PEG -SAM surfaces with casein followed by exposure to 17.5 nM of kinesin for 5 min reduced the observed microtubule landing rate from ~ 31 mm2s1 to ~ 2 mm2s1, corresponding to a 15-fold reduction in kinesin surface density Our interpretation of these observations is that the highly non-fouling surface helps conserve kinesin motor function after adsorption, and that casein acts as a competitor

PAGE 40

40 for a very limited number of adsorption sites. Furthermore, since the surface density of adsorbed kinesin does not linearly increase with kinesin dosage, the residual kinesin adsorption is not a consequence of kinesins slowly penetrating the coating63. Instead, the surface density of adsorbed ki nesins is equal to the density of defects in the coating (Figure 34) EG3OH -terminated SAM surfaces, a widely studied model system adsorbed an 2 (0.17 0.03 ng/cm2). This average is derived from three sets of identical surfaces prepared on different days (SAM1, SAM2 and SAM3). In comparison, the values measured using SPR for the adsorption of fibrinogen at hundredto thousand-fold higher dosages are 0.35 1.75 ng/cm2 (1 mg/ml adsorbed for 3 min)64 and 2.8 1.05 ng/cm2 (1mg/ml adsorbed for 30 min)65. PEGMA surfaces had no non-specific adsorption of microtubules and had exceptionally low l anding rates The average motor density on these surfaces is 0.16 2 (0.0064 0.0008 ng/cm2; average of PEGMA1 and PEGMA2). These d ata suggest that PEGMA surfaces represent a significant improvement over (EG)nOH terminated SAM surfaces. Conclusion Kinesin protein adsorption followed by microtubule landing rate measurements enables the determination of active protein coverages between 2 (0.004 1 ng/cm2). This detection range extends the lower end of the detection range of established methods. In essence, microtubule landing rate measurements afford single molecule sensitivity by exploiting the thousand -fold amplification of a fluorescence signal provided by labeled microtubules. The detection limit can be further reduced by increasing the observable number of microtubule landing events (increasing the field of

PAGE 41

41 view, observation time, or microtubule solution concentration) It is possible, that the ability of kinesin to bind microtubules is reduced after adsorption to the highly nonfouling surfaces. However, this reduced activity affects primarily the absolute protein coverage calculated from the data, and not the relative performance of tw o highly nonfouling surfaces. While the method does not determine the performance of non-fouling surfaces in blood serum or solutions of blood proteins, the reduced detection limit enables the quantification of adsorption events which would be invisible to established techniques. As a result, the performance of highly non-fouling surfaces can be determined and optimized. For example, the adsorption of kinesin to PEGMA surfaces is twenty -fold reduced compared to EG3OH -terminated SAM surfac es. It is hope d that the low technical requirements (fluorescence microscope with camera) and the commercial availability of a kinesin motility kit (Cytoskeleton Inc.) make this method widely accessible. In the context of hybrid devices integrating motor proteins, the measurements demonstrate that the newly developed coatings can achieve the extreme degree of adsorption resistance desirable for the reliable placement of kinesin motors. Similar to such hybrid devices, biosensors utilizing nanowires or other nanostructures as transducing elements require highly adsorption resistant surfaces to maintain their performance advantages on the system level. These are only two examples of the diverse applications of high performance protein-resistant coatings in bi onanotechnology.

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42 Materials and Methods Preparation of Surfaces For glass surfaces, coverslips (FisherfinestTM, Premium Cover Glass, no 1, Fisher Scientific, Pittsburgh, PA) were cleaned with ethanol and dried. For polyurethane (PU) surfaces, UV curable PU precursor NOA 73 (Norland Products Cranbury, NJ) was spincoated onto glass coverslips at 3500 rpm for 40 s and cured for at least 2 hrs using a 365 nm UV lamp (Spectroline EN -280L, Spectronics, Westbury, NY). Physiosorbed Pluronic surfaces were prepared according to. Glass surfaces were cleaned twice by batch sonication for 15 min in 5% (v/v) Contrad 70 soap (Fisher Scientific, Pittsburgh, PA). Prior to the second batch sonication, all samples were sonicated in deionized distilled water for 15 min. After a final rinse with water and drying at 200C for 15 min, the coverslips were treated with 5% dimethyldichlorosilane in toluene (v/v, Cylon CT, Supelco Inc., Bellefonte, PA) and rinsed twice with toluene and thri ce with methanol. These visibly hydrophobic surfaces (contact angle = 86 measured using a Rame-Hart goniometer, Model 100-00, Rame-Hart, Mountain Lakes, NJ) were treated overnight with 2 mg/ml Pluronic F108 (BASF, Mount Olive, NJ) solution in water. Unbou nd Pluronic was removed from the surface by rinsing twice with water and once with BRB80 buffer (80 mM PIPES, 1 mM MgCl, 1 mM EGTA, pH 6.9). After the Pluronic treatment, the contact angle changed to 71 which matches the reported contact angles for physio sorbed Pluronic Self assemb led monolayers terminated in EG3OH were prepared by sputter coating cleaned glass coverslips with a transparent gold layer (25 -18 Multi Target Sputter Deposition machine (Kurt Lesker, Clai rton, PA), and immersing them overnight in 1mM solution of 1Mercapto11 -undecyl

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43 tri(ethylene glycol) (Asemblon Inc., Redmond, WA) prepared in 99.9% methanol (Sigma -Aldrich, Saint Louis, MO) according to. PEGMA coatings: Unless otherwise specified, all ch emicals were purchased from Aldrich. 2bromoisobutyryl bromide (98%), triethylamine (TEA, 99.5%), Cu(I)Br (99.999%), Cu(II)Br2 (Fluka, 99%) were used as received. Hexanes were distilled over calcium hydride. 2,2 -dipyridyl (bpy, 99%) was sublimed. Poly (et hylene glycol) methyl ether methacrylate (PEGMA, average Mn ~475) was pas sed through a 20 cm column of in hibitor remover and stored at 20 C. Glass slides were initially modified via CVD polymerization of 4 amino [2.2]paracyclophane using a custom -built installation 50 mg of the paracyclophane were sublimed at 90 C and 0.12 torr, thermally activated, and transferred into a deposition chamber, where the polymer film was deposited at 15 C. Films made under these conditions had a thickness of 50 70 nm as determined by ellipsometry (EP3, Nanofilm AG). The resulting amino -functionalized glass slides were immersed into an anhydrous hexane solution containing 200 L 2 bromoisobut yryl bromide and 300 L TEA was added subsequently. After incubated for three minutes at room temperature, the surface modified glass slides were removed from the solution, washed sequentially with water and ethanol, and dried under a stream of nitrogen. Next, PEGMA (40 mL), deinoized w ater (20 mL), bpy (304 mg), and Cu(II)Br2 (40 mg) were charged into a Schlenk flask, stirred until homogeneous at room temperature, and degassed using three freeze-pump thaw cycles. CuBr (86 mg) was then added under nitrogen purge with the contents in the flask being frozen. The flask was then evacuated and backfilled with nitrogen five times, and finally backfilled with nitrogen and warmed up to room

PAGE 44

44 temperature. The mixture in the flask was stirred until the formation of a homogeneous dark brown solution was observed. Finally, surface-modified glass slides were incubated with this solution in a nitrogen purged glove bag at room temperature for three hours. The substrates were then removed from PEGMA solution, washed sequentially with water and ethanol, an d dried under a stream of nitrogen. Assembly of Flowcells assembled from two coverslips and double-stick tape. In all flow cells, the bottom surface and top cover surface had identical surface chemistry. Solutions were exchanged within a few seconds by pipetting the new solution to one side of the cell and removing the old solution using filter paper from the other side. A kinesin construct consisting of the wild -type, full -length Drosophila melanogaster kinesin heavy chain and a C -terminal His -tag was expressed in Escherichia coli and purified using a Ni -NTA column. The density of functional motors in the eluent was estimated by this technique as described later. Microtubules were prepa red by -tubulin (Cytoskeleton Inc., Denver CO) in 6.5 L of growth solution containing 4 mM MgCl2, 1 mM GTP and 5% DMSO (v/v) in were 100In one set of experiments (glass, PU and Pluronic surfaces), 0.5 mg/mL casein (technical grade, Sigma, Saint Louis, MO) dissolved in BRB80 buffer was adsorbed for 5 min to reduce denaturation of kinesin. Next, diluted kinesin solution (kinesin stock solution, BRB80 buffer, 0.5 mg/ml casein, 1 mM AMP PNP from Sigma) was flowed in and after five minutes, exchanged against a 4g/ml microtubule solution (sheared thrice

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45 by passing through 30G 1 needles from Becton Dickinson, Franklin Lakes, NJ) in BRB80 buffer containing 1 mM AMP -PNP (Sigma -Aldrich, Saint Louis, MO), 0.2 mg/mL casein, stem to reduce photobleaching (20 mM D glucose, 0.02 mg/mL glucose oxid ase, 0.008 mg/mL catalase, 10mM DTT) In another set of experiments (all surfaces except PU and Pluronic), casein adsorption was omitted and kinesin dilutions were made directly in BRB80 containing 1mM AMP -PNP but no casein. The microtubule solution was u nchanged. Time elapsed since microtubule injection was measured using a digital stopwatch with one second accuracy. Microscopy An Eclipse TE2000 -U fluorescence microscope (Nikon, Melville, NY) with a 40X oil objective (N.A. 1.30), an X -cite 120 lamp (EXFO, Ontario, Canada), a rhodamine filter cube (#48002, Chroma Technologies, Rockingham, VT) and an iXon EMCCD camera (ANDOR, South Windsor, CT) and were used to image microtubules on the bottom surface of flow cells. Images were collected every 10 seconds wi th an exposure time of 0.5 s. Determination of Landing Rates and Surface Density Microtubule landing events were manually counted within a field of view using UTHSCSA ImageTool version 3.0 and plotted against time elapsed after microtubule solution injecti on. The N(t) plots were fitted (error weighted least squares) with N = Nmax[1 exp( -Rt/Nmax)] where N is the number of landed microtubules (in a field of view of 0.04 mm2), t is the elapsed time and R is the initial landing rate specific to that motor densi ty. However, for EG3OH terminated SAM surfaces significant non -specific adsorption of microtubules was observed. This necessitated modification of the

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46 expression to N = Ninit + Nmax[1 exp( -Rt/Nmax)] in order to account for the non-specifically adsorbed MTs on the surface. The parameter Ninit is independent of time, since it was observed that in the absence of kinesin the number of non-specifically adsorbed microtubules does not change within the observation time beginning 100 s after microtubule injection. In the second PEGMA experiment, a small amount of non-specific adsorption was observed, and the data points reflect a fit with Ninit, R, Nmax as parameters, while the error bars are extended to include the coverages derived from a fit with only R and Nmax as parameters. The average microtubule length L was measured by taking the arithmetic mean of at least 250 landed microtubules for every new microtubule preparation to calculate the area, A=Lw assuming a width w of 25 nm. The diffusion limited landing rate Z is assumed to be equal to the landing rate observed on casein-coated glass surface at a very high kinesin solution concentration (> 10 nM, 10-fold dilution from stock solution), since a dilution series shows a saturation of landing rates reached for 40-fold dilution of stock solution ( ~5nM kinesin concentration) For gold-coated coverslips (used for EG3OH -terminated SAM surfaces), it was observed that the maximum landing rate differed significantly from the rate measured on glass. Hence, the value of Z for EG3OH terminated SAM surfaces was taken to be equal to the landing rate observed on casein coated gold surfaces at a very high kinesin solution concentration (10 -fold dilution from stock solution). Using A, Z (both measured on each day of experiment s) and the landing -[ln(1-R/Z)]/A. The dilution series data for casein-coated glass surfaces also enable an exact determination of the concentration of active kinesin motors in the stoc k solution under

PAGE 47

47 the experimentally validated assumption that all kinesin in the solution adsorbs uniformly to the casein -coated glass surface within 5 min The stock kinesin 00 can be obtained by fitting the equation R=Z[1 0coated glass surfaces as

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48 Figure 31 Landing rate methodology. a) I n the first step, the test surface is exposed to a known dosage of kinesin Kinesin adsorbs to a fraction of defect sites. b) Kinesin is then exchanged by a microtubule solution of known concentration. At low kinesin coverages, the landing rate of microtubules is directly proportional to the functional kinesin density on the surface.

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49 Figure 32. Landing rate for glass surface. Direct adsorption of kinesin to glass (open circles) results leads to denaturation and a non linear increase of the landing rate Secondary adsorption of kinesin to casein-coated glass (open squares) results in high motor functionality and an initial linear dependence of microtubule landing rate. 1E-3 0.01 0.1 10 100 Glass Landing rate R ( mm-2s-1)Dilution of kinesin stock solution Casein-coated Glass

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50 Figure 33 Protein adsorption to fouling and moderately non -fouling surfaces. The polyurethane (PU) and Pluronic surfaces are precoated with casein. The lower kinesin activity on these surfaces compared to casein coated glass is a reflection of reduced casein adsorption on these surfaces. 0.02 1 10 Casein coated Pluronic Active kinesin surface density ( ng/cm2)0.4 0.04Active kinesin surface density ( m-2)Initial kinesin concentration (nM) Casein coated Glass Casein coated PU 0.2 2 Glass PU Pluronic

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51 Figure 34 Protein adsorption to highly non -fouling surfaces. These surfaces help conserve motor functionalit y after adsorption and do need a layer of blocking protein such as casein. The measured kinesin densities are well below the detection limit of established characterization techniques. 0.1 1 10 SAM31000 100 10StarPEOPEGMASAM2 Kinesin surface density ( ng/cm2)0.4 0.04Kinesin surface density ( m-2)Kinesin dosage (nM*min) 0.004SAM1

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52 CHAPTER 4 THE MOLECULAR SHUTTL E SYSTEM AND DEVICE APPLICATIONS Introduction The basic capability of motors to generate directed nanoscale motion can be integrated in vitro with static synthetic components to enable nanoscale transport systems. However, t o mimic the fully functional biological transport system, the functionality of the bead or gliding assay s would hav e to be significantly expanded. Three basic topics that most of the experimental contributions have sought to address are: the guiding of shuttle movement along predefined tracks the loading and unloading of specific cargo onto the shuttles, and the user -controlled activation of motor s. Such a system, termed molecular shuttle system45,46, was envisioned to serve as a component of miniaturized devices and functional materials. These molecular shuttles have generated significant scientific interest over the past decade, resulting in over 200 publications. This chapter focuses on the efforts to construct kinesin and microtubule based molecular shuttles and their use in device applications. Nanoscale Transport System by Molecular Shuttles Molecular shuttle designs can be based on either the bead or the gliding assay. However, the gliding assay offers distinct advantages. Control over direction of motion is most elegantly achieved in the gliding assay. Due to the intrinsic structural polarity of the filaments, the resulting motion in a gliding assay is already always towards one end of the filament. Hence, t he gliding filaments can be guided in a specific direction by a variety of techniques, which are disc ussed in the next section. For directional control in bead assays, oriented filaments have to be immobilized on the surface in high densities.

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53 Furthermore, diverse cargoes can be attached in high density to filaments by convention al bioconjugation techniqu es29. T ubulin subunits are typically first functionalized and purified, and then reassembled into filaments with the desired properties. In a bead assay, however, apart from generating dense tracks of oriented filament, functional motors need to be adsorbed in high density to the cargo surface41. Guiding Molecular Shuttles On a planar surface uniformly coated with motors, shuttles perform persistent random walks66. These paths result from the Brownian motion of the filament tip, which searches for the next motor as the advancing filament is anchored to the preceding motors. While some device designs can take advantage of the random movement of shuttles, in most cases it is essential to guide molecular shuttles along predetermined paths. In addition, it is de sirable to define a specific direction of transport along the path as well as to have the ability to switch between alternative paths. Guiding using surface topography. Surface topography provides physical confinement by imposing barriers to filament motil ity. When the shuttles collide with the walls of the barriers, the propelling force of motor proteins is transformed into bending forces for the filaments. This results in guiding along the direction of channel walls (Figure 41 a)67. The first physical guiding design45 was simple ridges and grooves along the shear axis of a mechanically deposited PTFE film on glass. Since then, rapid design improvements have taken place which include replica molding of polyurethane channel46, arrowhead -shaped direction rectifiers68, undercuts at the bottom of the channel wall to provide greater confinement69 (Figure 41 b), combination of undercuts with rectifier designs42,70, completely enclosed channels71, combination of enclosed

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54 channels with direction rectifiers72 and the use of electrical fields in closed channels for dynamic control of the filament path73. Guiding using surface chemistry. P atterns in motor density and/or functionality bias the Brownian motion of the filament tip in the dir ection of the track (Fi gure 41 c)67. When a filament moving on a track reaches the boundary of a region which is either free of motors or contains inactive motors, an overhanging filament tip not bound to any motors emerges. This tip either keeps growing in length until it deta ches completely from the surface or it bends back to the motor -rich area as a result of its Brownian motion. It has an increased chance of returning to the motor -rich area when it is held only by the very last motor, because it can sw ivel around the motor axis67. Contrasts in kinesin surface density have been produced either by the suppression of non -specific binding of kinesin by non -fouling coatings67, or through directing the specific binding of biotinylated kinesin74. Guiding using surface topography a nd chemistry. In topographical only confinement, filaments can climb the sidewall (due to motors being present there) and escape. When only chemically confined, filaments crossing the boundary may detach from the surface, because their motion is not redire cted by a wall. The logical solution to these problems is to combine both approaches and confine motor adsorption/functionality only to the bottom surface of the guiding channel (Figure 4-1 d). For the kinesin -microtubule system, the combined guiding has been achieved by various combinations of non-fouling topographical features such as polymer channels coated with Triton X -10068, or Pluronic F10862, SU -8 walls coated with Pluronic F10867,

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55 and gold patterns etched into a SiO2 surface which were later funct ionalized with poly(ethylene glycol) chains75. Guiding using flow fields. Shear flow exerts drag forces on the leading tip of the gliding filament. The random tip fluctuations develop a bias towards finding a motor in the direction of the shear flow which ultimately leads to polar alignment of the entire filament in that direction76. However, if the pressure-driven shear flow is halted, the direction of filament movement becomes randomized again. Oriented filament arrays which support directed transport in the bead geometry are created by immobilizing the filaments immediately after alignment. Flow oriented microtubule arrays have served as tracks to transport micrometer sized kinesin -coated cargoes on flat surfaces77 and in microchannels78. Guiding using electrical fields. Electric fields exert electrophoretic forces on the negatively charged microtubules, which bend the cantilevered tip of the gliding filament towards the cathode and over time, reorient the entire structure79. Kinesin surface density and microtubule translocation speed along with electric field strength affect the rate of redirection of microtubules80. However, the electrodes used to generate the electric fields have to be placed far apart from the filaments to alleviate the effects of he ating and the generation of oxygen, which interferes with fluorescence imaging81. Active control over the motion of individual microtubules has been achieved with electric fields73 generated by direct current. In the presence of alternating electric fields, dielectrophoretic forces on moving filaments align them towards the highest field gradient. Although AC generated force fields are of short range compared to static DC

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56 fields, they have been reported to be more effective than electrophoretic forces for guiding microtubules82. Guiding using magnetic fields. Guiding using magnet ic fields is made possible by functionalizing filaments with ferritic particles. Microtubules functionalized with ferritic particles have been aligned83,84 and guided85 by externa lly applied weak magnetic fields. In summary, the field of guiding shuttles has now reached a matured state. The trajectory persistence length of gliding filaments has been determined experimentally66,86 and the mechanistic details behind dispersion in shuttle motion have been understood66. These developments have made it possible to simulate the motion of shuttles within the structures87,88. This in silico approach together with the wealth of information generated by the experimental studies enables the rational design of devices powered by biomolecular shuttles. Loading and Unloading Cargo onto Shuttles In nature, kinesin -1 motors serve as motile cargo transporters moving along the stationary microtubules. Molecular shuttles, which often utilize the gliding g eometry require different, engineered approaches to connect with and disconnect from various types of cargo. Ideally, the cargo attachment is strong, durable, specific, reversible, adaptable to diffe rent types of cargo, and does not interfere with gliding motion of filaments46. Biotinylated microtubules, sometimes coated with streptavidin, have carried a wide variety of streptavidin-functionalized or biotinylated cargoes (Figure 4 -2), including quantu m dots89, micro and nanospheres46,90, ferritic particles83,85, DNA9194 and carbon na notubes95. It was discovered early that conjugating biotinylated microtubules with

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57 cargoes such as avidin83, strept avidin-coated quantum dots90 and ferritic particles84 b efore introduction into the flow cell precludes subsequent binding of these filaments to the motors on the surface. To circumvent this problem, filaments can be biotinylated in segments85,90 to define cargo binding re gions and motor binding regions Altern atively, cargo can be connected to filaments already bound to the surface, although, this too interferes with m otor -filament interactions96. A major advance in loading cargo onto shuttles has been the loading of unfunctionalized cargo onto gliding microtub ules (Figure 42 c). Analytes, such as myoglobin proteins97 and virus particles98 were directly captured by antibodies attached to the shuttle. Furthermore, an optical tag was loaded onto this construction by employing a double antibody -sandwich. This technology has recently been expanded to multi analyte assays99, opening avenues for the design of multiplex biosensors. Other creative cargo-loading schemes have provided shuttles systems with cargo unloading capability100,101 and with an in situ supply of fuel102. Spatial control over cargo binding has been achieved by defining surface regions with high cargo dens ities101,103. This design spatially separates cargo pick up by shuttles and its subsequent utilization. In summary, our ability to load cargo onto molecular shuttles has rapidly advanced. Loading of DNA molecules91 and assembly of double antibody -sandwich assays onto moving shuttles97 have inspired studies of single molecule manipulation93 and biosensors104 respectively. In addition, controlled unloading of cargo would be beneficial for various molecular shuttle designs. Shuttles would be able to transfer cargoes between different stations, and could also be used multiple times.

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58 Controlling Shuttle Activity Biomolecular motors are enzymes of the ATPase family and can be described by the Michaelis Menten model for enzyme kinetics C ontrolling ATP concentration is a direct route to controlling shuttle velocity. In addition, the dependence of motor activity on a variety of other factors such as divalent ion concentrations, pH, temperature and motor density has been in vestigated for kinesin105. Motor activity can be reversibly controlled over time by changing the concentration of ATP106, localized temperature changes107, application of an electric al field108, by reversibly inhibitin g motors with local anesthetics109, or by engineering kinesin mutants with activity depending s witch like on the presence of calcium110 or zinc111 ions Photolysis of caged ATP by UV light releases ATP for motor consump tion However, the high diffusivity of ATP and the low consumption rate of ATP by motors impose fundamental limitations with respect to temporal and spatial control of activation, which can only be partially reduced by enzy matic sequestration of ATP26 (Fig ure 4-3 a) The speed of microtubules gliding over a kinesin-coated surface has also been directly controlled by the application of an electric field. Although electric fields have been primarily used to guide the direction of filament motion, it was shown108 that the microtubule gliding speeds can increase five-fold by application of assisting fields or decrease t o zero for opposing fields (Figure 4-3 b) Another area of activation control focuses on smart surfaces which are responsive to an electrical112 or thermal stimulus113. While electrically switchable surface affects kinesin activity112, thermally responsive PNIPAM chains affect kinesin -microtubule binding113.

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59 In summary, f or most control approaches, the rapid diffusion of molecules or heat at the nanoscale limits spatial resolution. To prevent diffusion of control molecules beyond the desired zone of activation, a sequestration system can be employed. However, the increased spatial control comes at the expense of a reduced efficiency in the utilizati on of the control molecules26. The utilization of electric pulses or light as stimuli seems generally preferable to an exchange of the buffer solution. However, the design of integrated traffic signals for molecular shutt les possibly utilizing stimuli r esponsive polymers, has not yet been achieved. Molecular Shuttle based Applications G eneral application concepts which incorporate the technical approaches outlined in the preceding sections have now emerged. Manipulation of Single Molecules T he reproduci ble and well -characterized nanoscale motion generated by motors and the ability to attach cargo via bioconjugation techniques onto filaments has inspired several single molecule manipulation studies. Molecular shuttles have been used to transport and stret ch indivi -phage DNA molecules. DNA stretching was achieved when a moving microtub ule picked up the end of a DNA molecule already attached to the substrate at its other end via biotin/ streptavidin linkage91, thiol/gold linkages93 or antibody antigen in teractions94 (Figure 4 -4 a). Interactions between a microtubule carrying a streptavidin-coated microsphere and a clamped microtubule have been used to develop a versatile forcemeter114. This forcemeter can m easur e rupture forces of single receptor/ligand pairs in the pN range.

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60 Self Assembly Self assembled strained structures can be produced by kinesindriven biotinylated microtubules by partially covering the microtubule surf ace with streptavidin115,116 or streptavidin -coated quantum dots90. The gliding m icrotubules first assemble into nanowires, which in turn form nanospools115 (Figure 4 -4 b). It has been argued that distinct advantages can be conferred to the assembly process if the building blocks are actively transported by molecular shuttles117. B esides accelerating the process for larger structures, it facilitates the utilization of stronger bonds. Instead of relying on thermal activation to dissociate mismatched parts, force generated by motors can be used to rectify errors in assembly. Most impo rtantly, it permits the assembly of strained, nonequilibrium structures. Investigation of Surface Properties The parallel process of autonomous, self -propelled shuttles provides a conceptual alternative to the deterministically controlled macroscopic cant ilever probes of the scanning force microscopes. When fluorescent microtubules are randomly propelled on a topographically structured surface they do visit the elevated por tions of the surface. This shows up as dark spots in an image created by overlaying hundreds of fluorescence microscopy images acquired successively from the same surface region118 (Figure 44 c) Now, t his concept has been expanded to include local height information with the use of fluorescence interference contrast (FLIC) wide-field m icroscopy119. The strong interaction between kinesin and microtubules has also been explored as a technique for quantifying the surface coverage of functional kinesin at extremely low densities This is already detailed in Chapter 3.

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61 Biosensing The active transport of proteins and viruses enabled by molecular shuttles is a valuable tool in the context of lab ona -chip devices, since it can be exploited to concentrate analytes104 and to replace mass transport by pressure driven fluid flow or electroosmotic f low66. While concentration of analytes confers higher sensitivity in sensors, active transport provides a particular benefit for smart dust devices. The capture and concentration of labeled streptavidin (serving as analyte) with biotinylated microtubule s moving on a kinesin-coated surface has already been shown104. Furthermore, shuttles have been loaded with unfunctionalized cargoes through the construction of a double antibody sandwich97. These developments have enabled the construction and working of a smart dust b iosensor, which is reported in Chapter 6. Conclusions and Outlook Cells have evolved sophisticated molecular machinery that drives movement and active transport. Driven by the direct conversion of chemical into mechanical energy, through hydr olysis of the biological fuel ATP, biomolecular motors enable cells to transport organelles and molecules to designated locations. The engineering of nanosystems with biomolecular motors as functional components is a part of the molecular shuttle design. Within the last decade, it has emerged as an established subfield of bionanotechnology. Successful approaches to the key challenges of guiding shuttle motion, loading cargo and controlling activity, are continuously refined in laboratories around the world. A key consideration for all hybrid devices is of course the degradation of the biological components prior and post activation. This issue was studied in some detail for kinesin/microtubule shuttles. Taxol -st abilized microtubules were found to limit the

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62 lifetime of the device to less th an a day81, while partial chemical cross -linking of microtubules can extend the lifetime to a week without gliding32. Freezing, freeze drying, and critical point drying are appr oaches to prepare kinesin/microtubule s ystems for extended storage120122. Surprisingly, kinesin is not very susceptible to chemicals used in the processing steps of microfabrication, such as removers/solvents or developers, which may enable a close integr ation of biomolecular self assembly processes and topdown fabricati on123. The central challenge at this stage is the development of convincing application concepts, which integrate the above discussed elements into a complex device delivering significant performance enhancements.

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63 Figure 41 Guiding molecular shuttles. a) Guiding using surface topography. The normal force provided by the walls to the motor propelled filaments bends them along the wall. However, a fraction of the filaments, depending on the approach angle, are able to climb the walls and escape due the availability of motors on the walls. b) Guiding in the presence of an undercut just below the wall. Filaments that climb the wall are redirected back into the region between the channels. c) Guiding using surface chemistry by generating sufficient contrasts in motor functionality between two regions. When filaments cross the track edge an overhanging tip emerges, which fluctuates under the influence of thermal forces until it binds to a motor in the motor -rich region. A filament bound to a last motor can rotate more freely because motors can swivel around their axis. d) Guiding using surface topography and chemistry. The surface chemistry of the walls is designed to interfere with either bi nding or functionality of motors. Filaments are redirected along the walls for steep and small approach angles, resulting in superior guiding as compared to surface topography alone or surface chemistry alone approaches.

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64 Figure 42 Cargo loading onto m olecular shuttles using the biotin -streptavidin chemistry. a) Biotinylated nanospheres can be attached to streptavidin-coated biotinylated microtubules b) Specific capture of fluorophore linked target DNA by microtubules functionalized with appropriate pr obe DNA. T he biotinylated probe strands w ere attached to biotinylated microtubules using streptavidin as a crosslinker. c) A double antibody -sandwich assay by linking a biotinylated antibody to a biotinylated microtubule with streptavidin, selectively capt uring analytes from solution, and detecting the captured analytes via binding of antibody -functionalized optical tags

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65 Figure 43 Controlling shuttle activity. a) Light activation and control of molecular shuttles26. A cylindrical cone of UV light locally photolyzes caged -ATP to produce ATP. As the ATP diffuses outward, it is hydrolyzed by kinesin and consumed by hexokinase. Addition of hexokinase increases the gradient of the ATP concentration profile at the expens e of the maximum shuttle speed. b) Manipulation of microtu bule speeds with electric field108. Negatively charged microtubules move faster in the presence of assisting fields by a factor of 5 and can be completely stalled in the presence of opposing fields. Figure 44 Molecular shuttl e based applications. a) Manipulation of single molecules93. One end of DNA molecules is stuck to surface through thiol -gold interactions while the other end is loaded onto microtubules through biotin-streptavidin. Subsequent transport results in unwinding and stretching of the DNA strands b) Non equilibrium self assembly of sticky microtubules into nanowires and nanospools. c) Investigation of surface properties using molecular shuttles118. Information about surface topography can be revealed in a time-in tegrated image of microtubules movi ng randomly on the surface a Stretched DNA Biotin Streptavidin and Thiol functionalized DNA Biotinylated microtubule Kinesin DNA loading Gold pad 0 min 8 min 18 min Cylindrical posts b c

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66 CHAPTER 5 OPTIMUM VELOCITY FOR CARGO LOADING ONTO M OLECULAR SHUTTLES Introduction Controlled transport of nanosized cargoes has been a long term of nanotechnology. The ability to address and transport individual molecules would open new opportunities for actuation, sequential assembly and disassembly, biosensing repair and design of reaction pathways. Cells, for example, have sophisticated molecular machinery that drives nanoscale movement and active transport of cargo While kinesin tail binds to a variety of cargoes, microtubules serve as tracks for long distance intracellular transport. In the molecular shuttle design, kinesin motors are anchored to the track surface through their tails. C onsequently, chemical modifications of tubulin are needed to specifically bind cargo. The strong and selective binding between biotin and streptavidin has been successfully utilized as a coupling element to connect cargo to the shuttles46. The strong interaction of biotin and streptavidin is widely used to separate proteins, thus making the key components (biotinylated tubulin, streptavidin, biotinylated cargo) commercially available. Biotin -streptavidin linkages remain the most popular chemistry to load cargo onto molecular shuttles and have been utilized by others to load quantum dots89, micro and nanospheres46,90, ferritic particles83,85, DNA91 94 and carbon na notubes95. In this chapter, the effect of shuttle velocity on cargo attachment via biotin streptavidin linkages is investigated and an unexpected optimum in the shuttle velocity discovered. Similar to dynamic effects introduced by other complex intermolecular bonds such as the catch bond bet ween FimH proteins and mannose124 or P -selectin a nd P -selectin glycoprotein ligand1125 this dynamic effect originates from the glue -like

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67 character of the biotin -streptavidin bond. The bond gains its ultimate strength on a timescale of milliseconds, due to existence of metastable binding states. The at tachment process can only be understood by combining rigorous mechanical engineering analysis with detailed physico -chemical models. Experimental Design The experiments are performed in the usual gliding assay format. (Figure 5 -1) Kinesin motor proteins ar e adsorbed to a surface precoated with casein; biotinylated microtubules adhere to the kinesin and are subsequently coated with rhodamine-labeled streptavidin. The microtubule gliding velocity was controlled via the concentration of the kinesin substrate A TP and varied between 50 nm/s and 450 nm/s. Finally, biotinylated fluorescein labeled nanospheres were added in concentrations ranging from 25 pM to 100 pM, which resulted in surface densities of 250 to 2000 nanospheres within a field of view of the fluore kinesin -coated surfaces, but not to surfaces coated only with casein. Nanospheres attached to microtubules only as a result of collisions between gliding microtubules and nanospheres adhering to t he surface. Binding of nanospheres from solution to moving microtubules was never observed, and stationary microtubules (in the absence of ATP) did not capture nanospheres from solution. The attachment and subsequent transport of fluoresceinlabeled, bioti nylated nanospheres by microtubules coated with rhodamine-labeled streptavidin can be visualized by fluorescence microscopy, since loaded nanospheres appear as a distinct group of moving nanospheres colocalized with a moving microtubule. Control experiment s were carried out in which 10% rhodamine and 90% biotin-labeled microtubules were used but the streptavidin coating step was omitted. These

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68 microtubules did not bind any nanosphere, which proves that the binding of nanospheres occurred specifically throu gh the streptavidin-biotin chemistry. A striking dependence of nanosphere attachment on microtubule velocity was observed. The loading initially increases with velocity and reaches a maximum at a velocity ~200 nm/s before decreasing again. Experimental Re sults Attachment and Detachment Rate To gain a quantitative understanding of this unexpected observation, the number, N, of nanospheres loaded per microtubule was recorded as a function of time, t, after bead injection for different nanosphere concentrati ons and microtubule velocities. These datasets are represented in Figure 52. The data were modeled under the assumptions that cargo loading onto microtubules is reversible, that cargo attachment is a zero order reaction (because of large excess of vacant binding sites on microtubules and negligible decline in surface density of cargo), and that detachment is a first order reaction (rate proportional to sites loaded per microtubule). The dynamic process can be written as Equation 51. N k k dt dNoff on (5 1) where kon is the attachment rate and koff is the detachment rate. Upon solving, we get the time dependent loading, N (t) from the Equation 5-2. t k k k Noff off on exp 1 (5 -2)

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69 The dataset for each experiment was fitted by the equation 5-2 to generate the values of kon and koff for each dataset. The fits are shown in Figure 52 and the fit values are tabulated in Table 51. Attachment and Detachment Probability In the attachment process, since loading occurs only via s urface collisions, the attachment rate, kon can be expressed as a product of rate of collision between a microtubule and nanospheres, kMT NS, and the sticking probability for each collision, SMT NS: ) ( ) ( v vS g v S k kNS MT MT NS NS MT NS MT on (5 -3) wh NS and gliding velocity v are directly measured. The grasp gMT of the microtubule is estimated to be 80 nm based on the geometry of the kinesin-bound microtubule and nanosphere explained as follows (Figure 53 a): The mi crotubule is elevated 17 nm above the substrate surface126 and the nanosphere sits on a 2 nm casein layer127. The radius of a microtubule increases from 12 to 18.7 nm upon biotin-streptavidin functionalization and the radius of a nanosphere increases from 20 to 21.7 nm upon biotinylation. Hence the distance between the two centers is 40.4 nm (18.7+21.7). Furthermore, the center of the microtubule is at a vertical position of 29 nm (17+12) while the center of the nanosphere is at a vertical position of 23.7 nm (2+21.7). Hence, the vertical separation between the two centers is 5.3 nm (2923.7). Now the horizontal separation between the two centers (=gMT/2) can be simply calculated to be 40 nm. The resulting values of SMT for each dataset are also tabulated in Table 5-1.

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70 Sticking is a result of the repeated attempts of streptavidins attached to the microtubule to grasp the biotinylated nanosphere, and SMT NS is thus given by SMT NS( v ) 1 1 Pon( v ) n (5 4) where Pon(v) is the velocity dependent probability that a linkage is successfully formed during an individual biotin-streptavidin encounter, and n is the number of encounters. The number of encounters n is given by the product of the number of streptavidins on the microtubule simultaneously within reach of the biotin linkers on the nanosphere, the number of tubulin dimers along the length, and the probability that a tubulin dimer carries a streptavidin To estimate the number of simultaneous streptavidin biotin contacts, the details of the interface between microtubule and nanosphere (Figure 53 a,b) are examined. Both are covered with a layer of biotin whose thickness is equal to the most probable end-to end distance of the flexible linker (1.7 nm, biotinXX). The microtubule is further coated with streptavidin, adding another 5 nm to its diameter. The most probable radius of the microtubule biotin-streptavidin (MBS) structure and the biotinylated nano sphere are represented by the dashed lines. Due to Brownian motion, the biotin linkers can extend up to their full contour length (3 nm) and increase the diameter of the MBS structure as well as the diameter of the biotinylated nanospheres to the dotted li ne. Contact between a MBS structure and the biotinylated nanosphere is established at the most probable configuration (contact of dashed lines). It can be concluded that streptavidins on neighboring protofilaments (cross -section, Figure 53 a) cannot be si multaneously in contact with the biotinylated nanosphere even when the biotin linkers are fully extended.

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71 Along the same protofilament (axial direction, Figure 5 -3 b), the two neighboring streptavidins can be simultaneously in contact. However, the biotins on both, the microtubule and the nanosphere, would have to be extended to 90% of their contour lengths for these additional contacts to be established. The probability of this is small, and thus the contribution of these streptavidins to the overall attac hment of the biotinylated nanosphere can be neglected. Hence, approximately one streptavidin on the microtubule interacts with the biotinylated nanosphere at a given instant. By measuring the average length of the ation ratio of the tubulin (0.60.1 biotins per tubulin dimer with a length of 8 nm) it can be determined that in average, n=34090 attempts to form a linkage were made during each collision between a microtubule and a nanosphere. In detachment process, th e detachment rate can similarly be modeled as a product of the rate of collision between the loaded nanosphere and nanosphere binding sites on the surface, and the probability of unbinding in each collision. The unbinding probability per collision is twice the rupture probability of a biotin/streptavidin bond at the specified force, since two biotin/streptavidin bonds connect the nanosphere to the microtubule. The nanosphere binding sites in our case are the kinesin motors. Because nanospheres bind only t o kinesins on the surface, a collision occurs if the loaded kin 2, kinesin density measurement details in Chapter 3) or defective motors (present at a density66 kin 2) The detachment probability in the collision with a functional

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72 and with a defective motor are denoted Poff and Poff respectively. Active and defective motors thus make independent contributions to the detachment rate: off kin off kin NS offP v pN P g v k ) 53 ( 2 (5 5) The calculation of the distance gNS over which a nanosphere loaded onto a microtubule can grasp a kinesin is complicated by the fact that the gliding microtubule likely rotates around its axis128 placin g the nanosphere out of reach for roughly half the time. A detailed calculation yields equal to 33 nm (Figure 53 a) a s follows : A microtubule rotates about its longitudinal axis as it is propelled along its length by kinesin. Hence a nanosphere load ed onto a microtubule can be present anywhere along NS is -3 a) and averaged over all angles: d d g gNS NS) ( (5 6) The calculation of gNS the surface and the grasp is zero. provides: nm gNS 2 2)^ sin 4 40 29 ( )^ 7 38 ( 2 ) ( (5 7) casein layer, yielding a grasp of 61 nm. It is assumed that the microtubule has to deform while it is being rotated and propelled to allow the nanosphere cargo to remain attached to it.

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73 Combined, the average value of the grasp of a nanosphere for a kinesin is determined to be 33 nm. Analytical Model It is proposed that the veloci ty dependence of Pon which decreases from 35 x 104 to 7 x 104 as SMT NS decreases from 0.7 at 50 nm/s to 0.2 at 400 nm/s can be understood by considering the kinetics of the attachment process. The duration of each biotin-streptavidin contact, tc, can be calculated from the microtubule velocity and the distance d = 5 nm (Figure 5-3 c) that the MBS structure moves from the initial contact with the stationary nanosphere until the biotin linkers have extended up to their full contour length and either rupture of the connection or attachment of the nanosphere have to occur. Potential Energy Surface of Biotin-Streptavidin In the attachment process, the newly formed biotin-streptavidin bond competes with the non -specific attachment of the nanosphere to th e surface. The force exerted by the moving microtubule makes unbinding favorable for both bonds, and the outcome of the process (rupture of the biotin/streptavidin bond or the nanosphere/surface connection) is determined by the relative height of the two energy barriers to unbinding (Figure 54). The small probability of attachment (Pon<0.01) implies that the newly formed biotin-streptavidin connection is weaker, or in other words that its activation energy to unbinding is 57 kT smaller compared to the nan osphere -surface connection. If both linkages would be adequately represented by potentials with a single energy minimum (Figure 5 4), it would be possible to specify potentials which result in a desired value of Pon, e.g. 35 x 104. However, a reduction in microtubule velocity, which increases the time available to complete the attachment process, would provide more

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74 time for the weaker of the two bonds to overcome the barrier to rupture. Thus, a decreasing velocity would not result in the observed increas e in the attachment probability. Instead, it is proposed here that the velocity dependence of Pon (and hence of SMT NS) is a result of the unusual shape of the biotin -streptavidin potential energy surface together with the varying time of contact tc betwe en biotin and streptavidin, which depends inversely on the microtubule velocity and is on the order of milliseconds. Biotin -streptavidin linkages which are allowed to form over long periods of time are stronger than the linkages which are subjected to pull ing for ces immediately after formation129. This results from the shape of the biotin-streptavidin energy landscape, which displays two metastable states in addition to the energy minimum (Figure 5 5 a)130. Contact between biotin and streptavidin initially populates the outermost state (state 3), but a fraction of the population can overcome the barriers and reach the more stable equilibrium state (state 1). It is assume d that only these stronger bonds can overcome the non-specific adhesion of the nanosp here to the surface and contribute to nanosphere attachment to the microtubule, meaning that the attachment probability Pon is equal to the probability to transition from state 3 to state 1. Conversely, for the detachment process, the detachment probab ility Poff is equal to the probability of escaping from state 1. To calculate the fraction of the encounters leading to the formation of a strong biotin-streptavidin bond (and attachment), the timedependent master equation governing the population of st ates 1 2 and 3 using the potential energy surface

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75 parameters provided by P incet et al129 is solved. The occupancies of the states can be calculated using the following relations: 1 1 1 1 1 1 ii i ii i i i i i i i ik P k P k P k P dt dP (5 -8) )} ) ( ) ( ( exp{ 21T k F E F E kB mi bi bi mi ii (5 -9) )} ) ( ) ( ( exp{ 21 1 1T k F E F E kB mi bi bi mi i i (5 10) Where ki i+1 is the transfer rate from state i to i+1, the subscripts mi and bi refer to the ith metastable state and the ith barrier respectively, K is the curvature of the local la obtained from Pincet et al.129 Attachment Process T he transfer rates depend on the shape of the potential energy surface which itself is altered in the presence of the force. Hence, Pon, the probability of filling up of state 1, was solved for as a function of force, F and time of contact, tc. Theoretical Pon values in the presence of an opposing force of 53 pN are plotted in Fi gure 5-5 b. Theoretical SMT NS values were fitted to the experimental SMT NS values after converting microtubule velocities to corresponding times of contact. A n opposing force of 535 pN yields an excellent fit of the calculated velocity dependence of the sticking probability SMT NS to the experimental data (Figure 56 a). This constant opposing force is provided by the kinesin motors and balanced by the adhesion of the nanosphere to the surface

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76 Detachment Process The velocity -dependent detachment probability for encounters with functional motors Poff (the probability of emptying state 1) was calculated in the presence of an opposing force of 53 pN. These values are plotted in Figure 55 b. Defective motors are able to bind to microtubules very tightly, since the binding of a gliding microtubule to a defective motor results in buckling of the microtubule131 and spiraling132. It is assume d that the binding of nanospheres to defective motors is at least three-fold stronger than the binding to functional motors, causing the unbinding probability Poff to be close to unity. The calculated unbinding rate koff combining the velocity -independent contribution from encounters with active motors and the linearly increasing contribution from encounters with defec tive motors fits the observed data effortlessly (Figure 56 b). Predicted Opposing F orce Each microtubule has in average 11 motors attached to it, based on the measured average length of 2 (determi ned b y landing rate measurements; details in Chapter 3). These 11 motors can provide a force of ~50 pN at a stepping velocity which is reduced by only one third relative to the unloaded velocity133, if a linear scaling of force generation with motor number is a ssumed41. The non-specific adhesion between the nanosphere and the surface can also readily provide a counteracting force of 50 pN for a few milliseconds, considering the force tolerance s of typical non -covalent bonds134. Thus, it can be concluded that the velocity dependence of the sticking probabilities is indeed a result of the time dependent strength of the biotin-streptavidin interaction.

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77 Of course, a constant force does not accurately reflect the complex variation of the force exerted on th e newly formed biotin-streptavidin bond over time. However, the calculated linear dependence of the population of state 1 (and the attachment probability Pon) on the time of contact tc is simply a result of the small probability of overcoming the two bar riers and not a result of the assumed force profile In other words, changing the force profile affects the value of the force providing the best fit, but not the shape of the calculated dependence of SMT NS on velocity Predicted Loading Our attachment an d detachment rate model allows the predict ion of the loading behavior for microtubules which use biotin-streptavidin chemistry and surface attachment of cargo as t he loading mechanism (Figure 5-7 ). The predicted loading rises linearly from zero with increasing velocity, reaches a maximum at a velocity of ~200 nm/s, and falls to 75% of the maximum at the top speed of the molecular shuttle. The predicted loading behavior matches the experimental data scaled to a constant nanosphere density. Conclusion The ex istence of an optimal velocity for cargo attachment to molecular shuttles is a consequence of the complex binding energy landscape of the biotin-streptavidin linkages. The behavior of biotin -streptavidin linkages is best captured by the glue metaphor, which emphasizes that a certain curing time is required to achieve the ultimate strength of the linkage. In contrast, lectin-selectin or mannose -FimH catch bonds function as hooks which gain strength under load, DNA -DNA linkages act like adhesive tape, a nd Nickel/His -tag linkages resemble magnets. Translating the description of molecular building blocks and their behavior into appropriate metaphors is

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78 important for the design and understanding of nanosystems, since it enables the correct application of our engineering intuition honed by decades of experience with macrosystems. Materials and Methods Kinesin and Microtubules A kinesin construct consisting of the wild -type, full -length Drosophila melanogaster kinesin heavy chain and a C -terminal His -tag was expressed in Escherichia coli and purified using a Ni -NTA col umn. biotin labeled -tubulin (Cytoskeleton Inc., Denver CO) in 6.5 L of growth solution containing 4 mM MgCl2, 1 mM GTP and 5% DMSO (v/v) in B RB80 buffer for 30 min at -fold diluted and (Invitrogen Inc.) was used to determine that there are 0.60.1 mol es of biotin per mole of tubulin. Assembly of Flowcells flow cells assembled from two coverslips and double -stick tape .30 First, a solution of casein (0.5 mg/mL, Sigma) dissolved in BRB80 (80 mM PIPES, 1 mM MgCl2, 1 mM EGTA, pH 6.9) was injected into the flow cell. After 5 minutes, it was exchanged with a kinesin solution (~10 nM in BRB80 with 0.5 mg/mL casein and varying ATP concentration). 5 minutes later, this was exchanged agai nst a motility solution (an antifade system made up of 20 mM D allowed for

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79 microtubule attachment after which 20 nM Alexa568-labeled streptavidin (Molecular Probes) in motility solution was perfused into the flow cell and incubated for five minutes to cover all the biotin sites on the microtubules .16 Finally, after th ree washes with motility solution, biotinlabeled 40 nm nanospheres loaded with fluorescein dye (Molecular Probes Inc.) at different concentrations (25-100 pM) in motility solution were introduced into the flow cell. Microscopy The flow cell was mounted on the microscope stage and the time elapsed since nanosphere introduction was recorded. An Eclipse TE2000-U fluorescence microscope (Nikon, Melville, NY) equipped with a 100X oil objective (N.A. 1.45), an X -cite 120 lamp (EXFO, Ontario, Canada), a FITC fi lter cube (#48001), a TRITC filter cube (#48002, Chroma Technologies, Rockingham, VT) and an iXon EMCCD camera (ANDOR, South Windsor, CT) was used to image microtubules and nanospheres on the bottom surface of flow cells. The exposure time was 0.5 s, while the time between exposures was varied from 4 to 8 seconds depending on microtubule speeds. Uninhibited transport of nanosphere loaded microtubules at nanosphere concentrations as high as 1 nM was observed if the excitation light intensity was reduced from 2.1 mWcm2 to 0.1 mWcm2, indicating tha t the previously described limit90 of 10 pM is a result of photodamage.

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80 Table 5 1. Results from microtubule loading experiments. v (nm/s) (1600 1 m 2 ) kon (min 1 ) koff (min 1 ) SMT NS 50 2 1364 37 0.132 0.019 0.087 0.016 0.685 0.064 175 9 1900 43 0.357 0.036 0.061 0.019 0.377 0.067 325 11 1375 37 0.229 0.032 0.178 0.029 0.180 0.028 452 20 800 28 0.143 0.098 0.208 0.166 0.139 0.012 85 3 675 26 0.069 0.007 0.046 0.007 0.423 0.133 140 9 550 23 0.105 0.007 0.026 0.006 0.479 0.120 296 10 510 23 0.113 0.014 0.089 0.014 0.259 0.096 442 22 432 21 0.114 0.021 0.165 0.035 0.209 0.032 100 4 272 16 0.052 0.005 0.029 0.006 0.603 0.086 297 10 288 17 0.109 0.012 0.091 0.014 0.446 0.051 420 23 238 15 0.047 0.011 0.108 0.030 0.154 0.023

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81 Figure 51. Sketch of principle. Biotinylated microtubules are coated with rhodamine labeled streptavidin. Biotinylated fluorescein -labeled polystyrene nanospheres (40 nm diameter) adhere to the surface and attach to the streptavidin -coated microtubules as they move on the kinesin-coated surface. Figure 52. Experimental Results. Observed nanosphere load ing for various microtubule speeds and nanosphere densities as a function time elapsed since nanosphere injection. The data points are fitted to E quation 5 -2 to determine attachment and detachment rates. Biotinylated Nanosphere Streptavidin coated microtubule Surface adsorbed nanosphere 0 10 20 30 40 50 60 0 1 2 3 4 50 nm/s, 0.8 m-2 175 nm/s, 1.2 m-2 325 nm/s, 0.9 m-2 450 nm/s, 0.5 m-2Ntime (min) 0 10 20 30 40 50 60 0 1 2 110 nm/s, 0.2 m-2 300 nm/s, 0.2 m-2 450 nm/s, 0.1 m-2Ntime (min) 0 10 20 30 40 50 60 0 1 2 3 4 85 nm/s, 0.4 m-2 140 nm/s, 0.4 m-2 300 nm/s, 0.3 m-2 440 nm/s, 0.3 m-2Ntime (min)

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82 Figure 53. Geometry of the microtubule nanosphere collision. a) Cross section. The microtubule is elevated 17 nm126 above the substrate surface and the nanosphere sits on a 2 nm127 casein layer. The radius of a microtubule increases from 12 to 18.7 nm upon biotin-streptavidin functionalization and the radius of a nanosphere increases from 20 to 21.7 nm upon biotinylation. The flexible biotin linker confers an average boundary (dashed lines) and a limiting boundary (dotted lines) on the supramolecular structures. b) Top view. A second biotin-stre ptavidin -biotin connection can only be established from a neighboring tubulin dimer if the biotins are almost fully extended, which is a rare event. c) A biotin-streptavidinbiotin connection formed upon initial contact can strengthen while the biotin link ers are extended to their full contour length as the microtubule moves a distance d = 5 nm. 20 6.7 8 10 23 21.7 12 2 18.7 20 gMT/2 20 21.7 23 17 gNS/2 v d 1.7 1.7 3 3a c b Nanosphere Nanosphere Nanosphere Tubulin monomer KinesinCasein Micro tubule

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83 Figure 54. Schematic of the unbinding process. (Left) The formation of a microtubule/nanosphere linkage via a simple bond with a single potential energy minimum can reproduce the frequency of attachment but not the dependence on microtubule velocity, which determines the time of contact. (Right) The formation of a microtubule/nanosphere linkage via a biotin/streptavidin bond, which strengthens over time by transi tioning into more tightly bound states, causes the increase in binding probability with increasing time of contact. Microtubule Nanosphere Surface Initial Contact F = 0 Increasing tension F > 0 Rupture Pick up (rare) No attachment (frequent) E Microtubule Nanosphere Surface Initial Contact F = 0 Increasing tension F > 0 Rupture Pick up (rare) No attachment (frequent) Pick upBiotin/Streptavidin Simple bond 1 2 3 1 2 3 1 2 3

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84 Figure 55. Biotin-streptavidin bond and binding and unbinding probabilities. a) The potential energy landscape of the biotin-strepta vidin bond129 in the absence of a force (dotted black curve) and in the presence of a 53 pN opposing force (solid green curve). b) Pon. Probability for a biotin -streptavidin bond that is initially in the outermost binding state 3 to transition to the equil ibrium binding state 1 during the time of contact in the presence of a 53 pN opposing force (green solid curve). Poff. Probability to transition out from the equilibrium binding state 1 in the presence of a 53 pN opposing force (red dashdot). Figure 56. Attachment and detachment rates as a function of microtubule velocity. a) kon/kMT NS as a function of microtubule velocity. b) Detachment rates koff as function of microtubule velocity. The data points represent the fit parameters obtained from experi mental data points depicted in Figure 52 while the curves represent the theoretical model developed for the attachment and detachment process. 0.0 0.4 0.8 1.2-40 -30 -20 -10 0 state 3 state 2 state 1 Distance (nm) Energy (kBT) 0 pN 53 pN 0 20 40 60 80 1000.000 0.005 v (nm/s) t c (ms) Pon (53 pN) Poff ( 53 pN )Pon, Poff50 100 500 a b 0 100 200 300 4000.0 0.1 0.2 0.3 koff (min-1 )v (nm/s) 0 100 200 300 4000.0 0.2 0.4 0.6 kon/ kMT-NSv (nm/s) a b

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85 Figure 57. Predicted microtubule loading. The attachment and detachment model are used to predict loading as function of microtubule velocity at different times after bead injection. The onand off -rates were calculated assuming a 2. Experimental values at 10 min after nanosphere injection (green squares) were also normalized to a nanosphere 2. 0 250 500 750 1000 0 2 4 10 min N at equilibrium 30 min 10 min 5 min Nv (nm/s)

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86 CHAPTER 6 A SMART DUST BIOSENSOR POWERED BY MOLECULAR SHUTTLES Introduction Biosensors can be miniaturized by either injecting smaller volumes into the micro and nanofluidic devices or immersing increasingly sophisticat ed particles in the sample. The 'smart dust' concept reflects these competing paradigms in miniaturization; it originally described cubic -millimetre wireless semiconducting sensor devices that could invisibly monitor the environment in buildings and public spaces135, but later it also came to include functional micrometer -sized porous silicon particles used to monitor yet smaller environments136. The principal challenge in designing 'smart dust' biosensors that can be immersed into the sample is integrating transport functions with energy supply into the device. A molecular shuttle-powered smart dust biosensor is reported. It autonomically tags, transports, deposits and detects unlabeled an alytes in a complex solution. T he device does not need pressure-drive n fluid flow or electroosmotic flow to drive the mass transport functions a critical impediment in nanoscale devices. Furthermore, the active transport is generated by direct conversion of chemical energy (stored in ATP molecules and packed within the device) into motion, eliminating the need for an external power supply. Due to their small size and autonomous function, it is envision ed that large numbers of smart dust biosensors can be inserted into organisms or distributed into the environment and read out by remote sensing. Applications of Smart Dust Miniaturized biosensors (i.e. all outer dimensions < 1 mm) would have unique applications in biodefense and biomedicine. For biodefense, these devices would

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87 enable remote sensing in high-risk areas such as detection of biological and chemical warfare directly on the battle field. For biomedicine, these devices could locally sample biomarkers in locations which are difficult to access such as the gastrointestinal tract. The initial impetus towards fabricatin g microscale analytical devices was provided by electrical engineers when they formulated the concept of smart dust135. It was conceived as a network of distributed electronic devices with millimetre dimensions, which are capable of sensing their environment and communicating with each other and the user. Sailor et al.136 expanded the concept to include engineered particles capable of changing optical properties in response to an environmental stimulus (e.g., the presence of volatile organic compounds). The optical response of such particles to the stimulus can then be remotely detected, e.g., by light detection and ranging (LIDAR)137. Molecular Shuttle Technologies enable Smart Dust Biosensor Design A significant constraint in developing microscale analy tical devices is the packaging of energy sources within the devices to perform work. Miniaturization of electrical energy sources such as batteries to the size of the devices has proven unsuccessful. Furthermore, the use of pumps in microfluidic devices suffers from conceptual roadblocks because t he transport velocities scale down with the square of the tube diameters In a generalized context, the need for such external power supplies and batteries in these devices may be removed by constructing an integrated device powered exclusively by molecular shuttles20. The energy required to power biomolecular motor powered components will be supplied by the photo-induced release of caged ATP), which in turn will activate the device. A nalytes will be detected by spe cific capture with receptor molecules and subsequent tagging with optical tags. A common strategy, especially for protein and virus analytes, is to employ a doubleantibody -sandwich

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88 assay. In traditional assays, excess sample solution has to be removed aft er the capture process and unbound optical tags have to be removed before the detection process. These solution exchanges are usually driven by external pumps or manual pipetting. Molecular shuttles provide the possibility of replacing the capture wash tag wash detect sequence in these assays with a capture transport tag transport detect sequence. Several key developments have enabled such a design. Successful assembly and transport of double antibody -sandwi ch assays by molecular shuttles97 has been demonst rated for a variety of antigen types and now, for different analytes in the same assay99. Spatial control over pickup and delivery of cargoes has been achieved by designing loading stations103. This is critical because in the shuttle-based biosensor design functionalized shuttles would capture antigens and then move into a tagging zone to pick up the optical tags. Shuttles loaded with optical tags can also be collected into a centralized small location to enhance the signal from the tags. This has been de m onstrated104 by concentrating biotinylated microtubules which captured labeled streptavidin at the center of a spiral pattern. Challenges regarding cont rolled activation of motors26, m aximal loading of shuttles (explained in t he Chapter 5), temperature stability of motors138, and long term storage and operation of the overall hybrid device121 have also been investigated in detail. Based on these enabling technologies and the overall size of the device, the proposed device aptly fits into smart dust tec hnology for remote sensor applications. The proposed devices may be fabricated in a highly parallel manner, combining the batch fabrication technology of the semiconductor industry with

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89 self -assembly methods of biotechnology, which will be essential for deploying large numbers of these devices over target areas. Prototype of a Device Several of the above described concepts have been integrated into a prototype of a molecular shuttle powered smart dust biosensor. The m icrodevice replaces the capture wash-tag wash de tect sequence in traditional double antibody sandwich assays (Figure 61 a) with a capture-transport tag-transport detect sequence rel ying on molecular shuttles (Figure 6 1 b ). (1) Antibodies immobilized on microtubules capture antigens from soluti on. (2) Kinesin motors are activated and move the antigenloaded microtubules. (3) Collisions of antigen -loaded microtubules with fluorescent particles functionalized with a second antibody leads to pick -up. (4) Fluorescent particles are transported from t heir initial location to a deposition zone. (5) The detection of particle fluorescence at the deposition zone is indicative of the antigen presence. Streptavidin and Gluthathione-Stransferase are captured from solution with functionalized microtubules, t he target -coated microtubules are moved until they bind fluorescent markers, and the microtubules carrying both target an d marker are further moved to a separate location. To reduce the detection time, a balance between the time required for the analyte to slowly diffuse to a surface-bound microtubule139 and the time required for the microtubule to move quickly on a diffusive trajectory87 to the boundary of the cell is desirable, suggesting that a flat device is desirable (Figure 61 c). After additional consideration of assembly and imaging processes, height and diameter of the device surface is formed by a coverslip to enable the observation of the internal processes by

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90 fluo photolithographically patterned SU8 photoresist, which exhibits low autofluorescence. It comprises a three zone design: functionalized microtubules are adsorbed into a capture zone, while functionalized fluorescent tags are adsorbed into an overlapping tagging zone. The chosen SU8 processing conditions result in an extended overhang at the sidewall and immobilization of gliding microtubules as they contact the sidewall. This enables t he third detection zone. It is created under the photoresist overhang at the wall of the circular device, because the overhang effectively prevents microtubules and tags from reaching the surface in the vicinity of the wall. Controlled activation of m otor -driven transport within the device relies on photolysis of 1-(4,5-dimethoxy -2 nitrophenyl)ethyl) -caged ATP (DMNPE-caged ATP), which is here provided by a UV lamp. However, sunlight can effectively serve the same purpose140. Selective recognition of the analyte by both, molecular shuttles and optical tags, is required to realize the DAS assay. In a first set of experiments, a simplified system has been employed where biotinylated microtubules act as molecular shuttles, streptavidin as analyte, and bioti nylated fluorescent nanospheres as the optical tags. The photoresist structure is fabricated on a glass coverslip and placed in a humidified chamber. The internal surfaces are coated with casein and kinesin before a solution containing biotinylated, rhodam ine -labeled microtubules and a solution of biotinylated 40 nm polystyrene nanospheres is added. The large overhang at the wall prevents the exchanges, streptavidin is added t o a final concentration of 1.0 nM, and subsequently

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91 the caged ATP in the solution is photolyzed by a 90 s pulse of UV light. Microtubules commence moving, collide with nanospheres on the surface, pick -up the nanospheres if streptavidin is present on either nanosphere or microtubule, and eventually reach the wall (Figure 62) where their accumulation can be imaged (Figure 63). In a second set of experiments, a commercially available pair of antibodies for Glutathione-S-transferase (GST) is utilized to creat e a true DAS. In this system, the biotinylated GST antibody is linked via streptavidin to biotinylated microtubules, GST serves as the analyte, and quantum dots conjugated to the second GST antibody serve as the optical tag. Since the antibody -labeled quan tum dots did not adhere to the surface, they were dispersed together with the caged ATP in the solution. In solution, quantum dots are invisible to our imaging system because they are out of focus and rapidly diffusing. In both experiments the distinction between the zones for analyte pick -up and tagging has been abolished for three reasons: (1) It is permissible for the analyte to bind to the antibody on the tag first and then to the antibody on the gliding shuttle, (2) a sufficient number of encounters b etween shuttles and tags still occurs, and (3) the device assembly process is dramatically simplified. The experiments shown here are the results of an initial round of parameter optimization. In order to achieve efficient pick up of nanospheres, the speed of microtubules was adjusted to 100-150 nm/s by controlling the caged ATP concentration and light dosage. The biotinylation ratio of microtubules was reduced to 6%, since higher ratios of biotin tubulin to unmodified tubulin resulted in impaired motilit y. The signal from GST capture was improved when the GST analyte was present in large

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92 excess of the antibody -labeled quantum dots (35 -fold excess is better than 3-fold excess). The concentration of solutions, the incubation times and the number of wash ste ps between assembly steps were optimized. The microfabrication process was optimized to prevent delamination of the thick SU8 resist and create the overhang at the wall. Results Microscopy Images The experimental results for the streptavidin system are shown in Figure 64. Within hours (Figure 6-3), the microtubules together with their cargo of nanospheres are deposited at the wall, creating a distinct, new band of fluorescent particles indicative of the presence of streptavidin in the solution (Figure 64 b,e; images false -colored green). The overlays in (Figure 6-4 c,f,i) demonstrate the colocalization between nanospheres and microtubules at the wall. In a second set of experiments, Glutathione -S -transferase (4 nM) was captured and detected by a commercially available combination of a biotinylated antibody and a quantum dot -conjugated antibody (Figure 6 5). The observed analyte-dependent deposition of quantum dots (Figure 6 -5 b,e; image false -co lored green) as microtubules accumulate at the wall after the activation of the device (Figure 65 a,d; images false colored red) confirms the formation and active transport of a DAS of antibody functionalized microtubules, Glutathione-Stransferase and an tibody -functionalized quantum dots. Again, Figure 6 -5 c,f demonstrate the colocalization between quantum dots and microtubules at the wall. The autofluorescence of the photoresist is absolutely and relatively stronger in these images, due to the short exci tation wavelength and the low brightness of the quantum dots, respectively.

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93 Signal from the device was quantified as the surface density of optical tags in the detection zone 3 hours after analyte introduction. All measured surface density values repres ent mean standard deviation. Optical tags in the presence of analyte can accumulate in t he dete ction zone of the biosensor well by a combination of three different routes: (1) Active transport of tags into the detection zone after getting loaded onto mic rotubules through specific interactions, (2) Active transport of tags into the detection zone after getting non-specifically loaded onto microtubules and (3) Diffusion of tags into the detection zone. To quantify the device performance, control experiments were carried out to separate the effect of the first mechanism from the other two in the accumulation of the signal. Test for S pecificity In the streptavidin system, the number of transported nanospheres is proportional to the number of microtubule nanosphere collisions; hence the collected signal needs to be normalized with the surface density of nanosphere in the tagging zone of the respec tive sensors. In the experiment with streptavidin present, nanospheres were 2 from 2 different locations within the same well, nanospheres counted within an area of m2) while the density of nanospheres in the tagging zone was 0.45 0.02 2 2). In the control experiment (streptavidin absent), nanospheres were deposited in densities of 2 within the 2) while the density of nanospheres in the tagging zone was 0.49 0 .02 2 (791 nanospheres counted within an area of 2). The ratio of densities in the detection zone and tagging zone is 0.89 0.09

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94 (streptavidin present) and 0.17 0.02 (streptavidin absent). A student t -test to evaluate the statistical significance between these two values yielded the two-tailed probabi lity to be 0.05 (t value 11.4 and 1 degree of freedom) and confirms that the increase of nanosphere density in the detection region is due to the presence of streptavidin. In the GST system, quantum dots were deposited in the presence of GST in a density of 0.12 0.01 2 2). In the absence of GST, the quantum dot density in the detection zone was 0.0016 0.0011 2 (average from 5 different locations within the same well, nanospheres 2). Since quantum dots do not adsorb to the surface and since equal concentrations and amounts are introduced, a normalization of the signals is unnec essary. A student t test between these two values yielded the two-tailed probability to be 0.04 (t value 17.4 and 1 degree of freedom) and confirms that the increase in quantum dot density in the detection zone as a result of the addition of GST as analyte is statistically significant. Test for Transport Mechanism In the streptavidin system, the optical tags (biotinylated nanospheres) are immobilized onto the sensor surface during device fabrication. Excess nanospheres in the solution are washed out and the non-specifically bound nanospheres remain tightly adhered to the surface throughout the operation of the device. Hence, after the removal of optical tags from solution during device fabrication, no new tags reach the detection zone by diffusion. Immediately after device assembly, a nanosphere density of 0.074 2 2) while

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95 the density of nanospheres in the tagging zone (indicative of the nanosphere dosage) was 0.49 0 .02 2 2). After device operation in presence of streptavidin, the measured signal was 2 (average from 2 different lo cations within the same well, nanospheres counted within an 2) while the density of nanospheres in the tagging zone was 0.45 0.02 2 2). The ratio of densities in the detection zone and tagging zone is 0.89 0.09 (optical tags delivered by active transport) and 0.15 0.02 (optical tags delivered by diffusion). A student t -test to evaluate the statistical significance between these two values yielded the two-tailed probability to be 0.05 (t value 11.6 and 1 degree of freedom) and confirms that the nanospheres are actively transported into the detection zone. In the GST system, it was critical to test the diffusion based contribution to the signal because quantum dots are always present in solution. For this control, microtubules were accumulated at the sensor walls until no microtubules remained in the tagging zone of the sensor. The GST analyte was then introduced and the signal was measured 3 hours after analyte introduction. Quantum d ots were deposited in a density of 2 2) in this control experiment. In contrast, the signal measured in the GST detection experiment (GST present, quantum dots actively transported) was 0.12 0.01 2 (average from 2 different locations within the same well, quantum dots counted within 2). A paired t -test yielded the two tail ed pvalue as 0.05 (t value

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96 13.5 and 1 degree of freedom), which indicates that the number of quantum dots in the detection zone increases significantly as a result of active transport by microtubules. Conclusions While the signal strength of the device ha s to be significantly improved to enable remote detection141, it is the first time that capture of unlabeled analyte by molecular shuttles has been integrated with tagging, transport and localized deposition. As a result, the demonstrated device concept represents a significant advance with respect to previous demonstrations of conceptual building blocks, such as analyte capture by antibody -coated microtubules97, selective pick -up of tags103, or capture and concentration of labeled streptavidin104. The mo lecular shuttle -powered smart dust biosensor is a significant step towards mimicking the autonomous, micron-sized, multifunctional sensors engineered by nature. In order to apply such smart dust biosensors in a practical setting, the design requires furthe r iterations to improve specific processes as well as their integration. For example, opaque material covering the tagging zone will ensure that only fluorescent tags transported to the peripheral detection zone and a negligible fraction of the freely diff using tags contribute to the sensor signal. In addition, strategies t o manufacture, package and store120,121 such devices at high yield and low cost have to be developed and taken into consideration in the design process. The performance has to be optimiz ed and matched to specific application scenarios. Multiplexing can be achieved by preparing devices with different antibody pairs and s pectrally separable optical tags99 and mixing the devices in the desired combinations. In summary, while centimeter -size radio -frequency identification (RFID) devices begin to penetrate the market, the design of submillimeter -size distributed sensors for biomedical and biodefense applications is a

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97 new frontier in engineering to which biomimetic and hybrid systems offer inte resting solutions. Materials and Methods SU8 Photolithography The layout of the photo mask was drawn using progeCAD LT 2006 (progeSOFT, Como, Italy) and transferred to a precision laser photoplot (resolution 1/40.64 mil, Photoplot Store, Colorado Springs CO).Round cover slips ( 25 mm, No. 2, Fisher brand, Fisher Scientific, Hampton, NH) pretreated by rinsing with acetone, methanol and water serve as substrate. To dehydrate the surface, the substrate was baked for 10 min at 200 C on a contact hot plate. Approximately 1 mL of SU8 -2015 (MicroChem, Newton, MA) was spin-coated onto the substrate (static dispense; spread cycle: ramp to 500 rpm at 100 rpm/s acceleration; spin cycle: ramp to 2000 rpm at 300 rpm/s acceleration and hold for 30 s). The resist was soft -baked for 1 min at 65 C and for 3 min at 95 C, exposed (i Line, 5 mW/cm2) for 30 s in a MJB3 mask aligner (Suss MicroTec, Garching, Germany) and post baked for 1 min at 65 C and for 2 min at 95 C on a contact hot plate. The structures were develop ed for 10 min in a Branson 200 ultrasonic bath (Branson, Danbury, CT) using SU8 developer (MicroChem, Newton, MA). Following development, the substrate was rinsed briefly with isopropyl alcohol and then dried with a gentle stream of nitrogen. The thickness of the resist was measured using a Dektak II (Sloan now Veeco, Woodbury, NY). General A ss embly of the Smart Dust S ensor The whole assembly and operation of the smart dust sensor was carried out under minimal illumination to prevent accidental release of c aged ATP. To prevent drying, degradation and photobleaching, the sensor was also kept under an atmosphere of

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98 humidified nitrogen except for the solution exchange steps. Prior to assembly, the sensor surface was cleaned by rinsing with ethanol and water. So lution exchange on the sensor surface was carried out manually using two 100 L pistondriven air displacement pipettes. All solutions were applied as droplets onto the sensor surface and exchanged by suctioning the existing droplet and deposition of a new droplet. Assembly and Operation of the Streptavidin -Specific Sensor Rhodamine -labeled microtubules and biotinlabeled microtubules were prepared by polymerizing 20 g of rhodamine -labeled and biotinylated tubulin (both Cytoskeleton, Denver, CO), respectively, in 6.25 L of growth solution (4 mM MgCl2, 1 mM GTP and 5% DMSO (v/v) in BRB80 buffer (80 mM PIPES, 1 mM EGTA, 1 mM MgCl2, pH 6.9) for 30 min at 37 C.30 5 L of each microtubule suspension was subsequently diluted 100fold into BRB80 contain The surfaces of the sensor were wetted for 5 min with BRB80 (100 L), coated for 5 min with casein (0.5 mg/mL in BRB80, 100 L) and for 10 min with kinesin (~10 nM in BRB80 with 0.5 mg/mL casein and 0.1 mM DMNPE -caged ATP, 100 L). Th e surface was then washed once with antifade solution (0.2 mg/mL casein, 0.1 mM DMNPE caged ATP, 20 mM D -glucose, 20 g/mL glucose oxidase, 8 g/mL catalase and 10mM DTT in BRB80, 100 L). The surface was incubated for 5 min with a motility solution cont aining biotinylated and rhodamine-labeled microtubules (ratio 10:1, 16.0 g/mL biotinylated microtubule s, 1.6 g/mL rhodamine-labeled microtubule s, 0.1 mg/mL casein, 0.1 mM DMNPE -caged ATP, 20 mM D glucose, 20 g/mL glucose oxidase, 8 g/mL catalase and 1 0 mM DTT in BRB80, 100 L). The surface was washed again with antifade solution (100 L) and incubated for 5 min with a suspension (100 L) containing biotinylated fluorescent microspheres ( 40 nm, FluoSpheres F8766,

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99 Molecular Probes, Eugene, OR). The m icrosphere suspension was prepared by suspending 1 L of the microsphere stock suspension in 49 L BlockAid Blocking Solution (Molecular Probes, Eugene, OR), sonicating for 5 min in an ultrasonic bath and diluting 20-fold in antifade solution to give the f inal concentration of 0.5 nM. After washing the surface twice with antifade solution (2x 100 L) and incubating it with antifade solution (100 L) the sensor was ready for operation. An analyte solution of AlexaFluor568-labeled streptavidin (Invitrogen, Carlsbad, CA) was loaded by solution exchange (1.0 nM streptavidin in antifade solution, 100 L). For the control experiment, this step was omitted. Caged ATP was subsequently released by illuminating the sensor for 90 s with UV light from a LB LS/30 ligh t source (Sutter Instrument Company, Novato, CA) equipped with a UV band pass filter (D350/50x, Chroma Technologies, Rockingham, VT). The intensity at the sample was measured to be 1.4 mW/cm2 using a UV light meter (UV -340, Mannix, New York, NY). Assembly and Operation of the Glutathione -S-T ransferaseSpecific S ensor Mixed microtubules (13% rhodamine, 6 % biotin) were prepared by polymerizing a mixture of 40 g of unlabeled, 6.4 g rhodamine labeled and 3.2 g biotinylated tubulin (all Cytoskeleton, Denver CO) respectively in 16.5 L of growth solution (4 mM MgCl2, 1 mM GTP and 5% DMSO (v/v) in BRB80 buffer (80 mM PIPES, 1 mM EGTA, 1 mM MgCl2, pH 6.9)) for 30 min at 37 C. 5 L of the microtubule suspension was subsequently diluted 100-fold into BRB80 cont Rhodamine microtubules were prepared by polymerizing 20 g of rhodamine labeled tubulin (Cytoskeleton, Denver, CO) in 6.25 L of growth solution for 30 min at 37 C and 100 -fold diluting 5 L of the microtubule suspension into BRB80 containing

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100 L), coated with casein (0.5 mg/mL in BRB80, 100 L, 10 min) and kinesin (~10 nM in BRB80 with 0.5 mg/mL casein and 50 M ATP, 100 L, 10 min). The surface was then washed thrice with 100 L antifade solution (0.2 mg/mL casein, 0.1 mM DMNPE caged ATP, 20 mM D -glucose, 20 g/mL glucose oxidase, 8 g/mL catalase and 10 mM DTT in BRB80). The surface was incubated for 10 min with a motility solution containing mixed and rhodamine-labeled microtubules (ratio 50:1, 15.0 g/mL biotinylated microtubule s, 0.3 g/mL rhodamine-labeled microtubule s, 0.1 mg/mL casein, 50 M ATP, 20 mM D glucose, 20 g/mL glucose oxidase, 8 g/mL catalase and 10 mM DTT in BRB80, 100 L). The surface was again washed thrice with 100 L antifade solution and incubated for 10 min with a solution of unlabeled streptavidin (Invitrogen, Carlsbad, CA) (8.0 nM streptavidin in antifade solution, 100 L). Thereafter the surface was washed thrice with 100 L antifade solution, and incubated for 10 min with a solution of biotinylated ant i -GST (Goat, Rockland, Gilbertsville, PA) (32 nM biotinylated anti -GST in antifade solution, 100 L). Finally, the surface was washed thrice with antifade solution (3x 100 L) and incubated with a suspension of anti -GST conjugated Qdot 655 (Goat, Invit rogen, Carlsbad, CA) (0.2 nM Qdots in antifade solution, 50 L) for a final quantum dot concentration of 0.1 nM. GST analyte was loaded at a final concentration of 4 nM by introducing 50 L of GST (Schistosoma japonicum GenScript, Piscataway, NJ, 7.7 nM GST in antifade solution) without mixing. For the control experiment, this step was omitted. Caged ATP was released after 25 min by illuminating the sensor for 240 s with UV light. The light source and intensity was the same as in the previous experiments (see above).

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101 General R e ad Out of the Sensor Readout was performed with an Eclipse TE2000U fluorescence microscope (Nikon, Melville, NY) equipped with a 100x oil objective (N.A. 1.4) and a 10x air objective (N.A, 0.3), a X -cite 120 light source (EXFO, O ntario, Canada), and an iXon EMCCD camera (ANDOR, South Windsor, CT). For imaging microtubules and AlexaFluor568 -labeled streptavidin a rhodamine cube (#48002, Ex 535 nm, Em 610 nm), for FluoSpheres a fluorescein filter cube (#48001, Ex 480 nm, Em 535 nm) and for Qdots655 a modified Qdot cube (#32104a modified with D655/40 EM, Ex 420 nm, Em 655 nm) was used (all cubes Chroma Technologies, Rockingham, VT).

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102 Figure 61. Concept and device layout. a) The capture wash tag wash detect sequence of a traditional doubleantibody sandwich (DAS) assay. b) The capture transport tag transport detect sequence of the smart dust device, in which antibodies on microtubules capture antigens from solution. c) A smart dust device utilizing active transport by molecular shuttles to conduct a series of steps analogous to the DAS assays. A basic device layout is a circular well created in photoresist on a coverslip. Analyte harvesting, tagging, and detection is performed in different radial zones. The photoresist overhang at the wall of the well defines the detection zone by preventing optical tags from reaching the wall during the tag deposition step of the assembly procedure. cLayout of molecular shuttle powered sensor 800 m 20 m Analyte Capture Analyte Detection Analyte Tagging Analyte Tag Overhang

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103 Figure 62. Active transport of optical tags into the detection zone. Biotinylated and rhodamine -labeled microtubules loaded with biotinylated nanospheres travel from the tagging zone into the detection zone and get immobilized there. Other immobilized microtubules with optical tags attached to them can be seen in the detection zone. Im ages captured with the rhodamine filter cube were false colored red and images captured with fluorescein filter cube were false colored green to generate these overlays. Figure 6 3. Accumulation of microtubules at the detection zone over time. The biotinylated and rhodamine labeled microtubules are imaged using a rhodamine filter cube. The sequence of images shows that microtubules take a few hours to concentrate within the detection zone of the sensor.

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104 Figure 6 4. Experimental results for strepta vidin as analyte. a) Biotinylated m icrotubules (imaging with the rhodamine cube and false colored red) have aggregated at the wall 6 h ours into the experiment. b ) A ring of deposited nanospheres (imaging with the fluorescein cube and false colored green) is faintly visible against the autofluorescence of the photoresist c) A false -color overlay of the images shown in Panel a and b. d), e), f) Imaging of the perip heral region of a different well 3 hours after the start of the experiment shows the distinct transition between the capture/tag region (visible from the cross -talk of nanospheres) and the deposition region (visible from the accumulation of microtubules). g), h), i) Results from the control experiment (streptavidin absent). After 3 hours, microtubules have accumulated at the wall but the region remains free of nanospheres.

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105 Figure 6 5. Experimental results for GST as analyte. a) (I mag ing with rhodamine fi lter cube and false colored red) Microtubules functionalized with anti -GST antibodies and rhodamine have aggregated at the wall 3 h ours into the experiment. b ) (I maging with quantum dot filter cube and false colored green) Microtubules captured GST and ant i -GST antibody conjugated quantum dots from the solution, and transported the double antibody -sandwich to the wall. c) A false -color overlay of the images shown in Panel a and b. d) In the absence of GST, microtubules have accumulated at the wall. e) In th e absence of GST, the region remains free of quantum dots. f) A falsecolor overlay of the images shown in Panel d and e.

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106 CHAPTER 7 CONCLUSION AND OUTLO OK Nanoscale engineering with molecular shuttles has advanced significantly in the past decade, but it still remains a scientific frontier142. While most technologies proceed in a series of incremental steps, where each new device generation resembles the previous generation in its design and functi on, the field of molecular shuttle engineering represents a fresh approach to the challenge of purposefully converting chemical energy into mechanical work143. As a result, the field has to develop an ecosystem of application ideas, which give direction to its future development. Two such applications which employ the functionality of the kinesinmicrotubule system to fulfill a unique need are reported. While both these applications represent a significant advancement in the state of art, they also pave a clear path forward for future improvements. First144, a technique for quantifying protein adsorption with a 100-fold lower limit of detection than established characterization techniques was reported. The technique is applied to measure kinesin density on novel non -fouling coatings, which already perform at a per formance level invisible to established techniques145. While the technique is demonstrated for the adsorption of kinesin probes and with the use of microtubule markers, it can be developed for other proteins as well. One example is determin ing the density of fibrinogen by measuring the landing rate of micron sized fluorescent beads functionalized with anti -fibrinogen molecules, so that they land specifically on adsorbed fibrinogen. The simplicity of design and ease of detection make this technique useful fo r characterization of a large variety of protein resistant surface s. Second146, the integration of several molecular shuttle design elements into the development of a smart dust biosensor was discussed. For purposes of loading cargo

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107 onto shuttles, the disc overy of an unexpected optimum in shuttle velocity was reported. The optimum is a consequence of the complex binding energy landscape of the biotinstreptavidin linkages, which gain their ultimate strength on a time scale of milliseconds. The study helps i n t ranslating the description of biotin -streptavidin, one of the most popular molecular building blocks in bionanotechnology into appropriate metaphors These metaphors will enable the correct application of our engineering intuition, honed by decades of e xperience with macrosystems, in the design of nanosystems. In the prototype of the smart dust device147, molecular shuttles capture antigens from the test solution move until they bind fluorescent markers and accumulate at a distinct location for fluoresc ent readout. It is envisioned that this autonomous microdevice can be manufactured in large numbers, stored in an inactivated state, reconstituted, distributed in an aqueous solution, activated by light and read out by standoff fluorescence detection. In order to apply such smart dust biosensors in a practical setting, the design requires further iterations to package these biocomponents into useful devices. While the prospect of utilizing molecular shuttles to directly construct active nanobiomaterials is an exciting one, it is fraught with considerable challenges Amongst the most significant are the shuttles limited lifetime and the need to be sustained in aqueous solutions of precise ionic composition and temperature. These challenges are fundamental in nature and common to many fields within bionano technology. I t may take decades to resolve them or to develop synthetic components as repla cements of the biological parts which are more robust and as functional. However, molecular shuttles might find pr actical applications in devices that do not require long service

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108 lifetimes (such as surface characterization techniques) or in microdevices implanted into physiological systems and can operate by scavenging energy from biological hosts (such as smart dust biosensors).

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120 BIOGRAPHICAL SKETCH Ashutosh Agarwal was born in 1983 in Narnaul, Haryana, India. His parents ensured that he received first rate education throughout his career. He attended the Delhi Public School during his formative years where he was awarded a gold medal for his academic achievements. Ashutosh earned his Ba chelor of Technology in Metallurgical and Materials Engineering from the Indian Institute of Technology, Roorkee. After a brief stint as a process engineer in a mining and metallurgical company in India, he came to the United States to pursue doctoral educ ation. With an aim to shift from shop floor engineering to engaging in good science, he joined the group of Dr. Henry Hess in the Materials Science and Engineering department at the University of Florida. Ashutosh now looks forward to his days of some mo re good science as a postdoctoral researcher.