Research Program

Project Summaries

2008

The Nanobiotechnology Center is a Science and Technology Center funded with the support of the National Science Foundation and the New York State Office of Science Technology and Academic Research (NYSTAR).

www.nbtc.cornell.edu

TABLE OF CONTENTS

Overview of the Nanobiotechnology Center.....................................................................................1 Biomolecular Devices and Analysis - Program Overview...............................................................7

Micro & Nanostructures for Molecular Detection, separation and analysis .............................................................. 8 On-Chip NanoPorous Membranes for Separation and Semipermeable Transport of Biomolecules ....................... 10 Microfabricated Rapid Fluid Mixers to Study Macromolecular Conformational Dynamics .................................. 12 Nanoscale Optofluidic Devices for Biomolecular Analysis .................................................................................... 14 Biosensor Based on Electrochemical/Gravimetric Detection of Intrinsic Antibody Catalysis................................ 16

Cell-Surface Interactions – Program Overview.............................................................................19 Cellular Responses to Topographic and Biochemical Clues ................................................................................... 20 Patterned Lipid Bilayers and Zero Mode Waveguides to Investigate Immune Cell Signaling ............................... 22 Colonization and Communication Among Plant-Associated Bacteria in Artificial Xylem Lumina ....................... 24 Divalent Helical Rigid Rods to Engage Receptors and Control Cellular Responses............................................... 26

Cellular Microdynamics – Program Overview..............................................................................29 Development of a Cell Biosensor............................................................................................................................ 30 Nanotechnological Assessment of Drug Toxicity ................................................................................................... 32 Isolation and Characterization of Immune Cells: Structure and Function............................................................... 34 Subcellular Molecular Distribution Analysis and Sorting ....................................................................................... 36 Bionanofabrication: In Situ Creation of Nanoscale Polymeric Features ................................................................. 38 Engineering Functional Microvascular Structure in vitro ....................................................................................... 40

Nanoscale Cell Biology - Program Overview .................................................................................43 Tracking Single Molecules and Transport Vesicles in Living Cells Using Fluorescent Nanoparticles .................. 44 A Scalable n x n Electrochemical Pixel Array with Integrated Electronics for Use as a Highly

Parallel Biosensor Device ................................................................................................................................ 46 Fabrication of Nanoscale Dendrimer-like DNA Structures as Multivalent Templates ........................................... 48 Nanotubes as Cellular Biosensors ........................................................................................................................... 50 Development of Novel Applications of Reverse Transfection for High Throughput Cell-Based

Functional Screening........................................................................................................................................ 52 Electrochemical Imaging of Exocytosis Using Microfabricated Devices ............................................................... 54 Dynamics of Viral Fusion Proteins ......................................................................................................................... 56 Chromatin Structure, Function, & Dynamics: From Mononucleosomes to Polytene Chromosomes ..................... 58

NBTC Shared Research Facilities ...................................................................................................60 NBTC Education Program ..............................................................................................................64

Overview of the Nanobiotechnology Center

1

2

NBTC Faculty Members Cornell University, Ithaca NY Harold Craighead Applied and Eng. Physics (NBTC Director) Harvey Hoch Plant Pathology, Geneva (Co-Director) Hector Abruña Chemistry & Chemical Biology Esther Angert Department of Microbiology Judith Appleton Department of Microbiology & Immunology Antje Baeumner Biological and Environmental Engineering Barbara Baird Chemistry & Chemical Biology Carl Batt Food Science Lawrence Bonassar Mechanical & Aerospace Engineering Theodore Clark Department of Microbiology & Immunology Geoffrey Coates Chemistry & Chemical Biology Brian Crane Chemistry & Chemical Biology David Erickson Mechanical & Aerospace Engineering Lara Estroff Materials Science & Engineering Claudia Fischbach-Teschl Biomedical Engineering Carl Franck Physics Emmanuel Giannelis Materials Science & Engineering Quan Hao Applied & Engineering Physics Ronald Hoy Dept. of Neurobiology & Behavior Brian Kirby Mechanical & Aerospace Engineering W. Lee Kraus Molecular Biology & Genetics Amit Lal Electrical & Computer Engineering David Lin Biomedical Sciences Manfred Lindau Applied and Eng. Physics John Lis Molecular Biology & Genetics Dan Luo Biology & Environmental Engineering Paul McEuen Physics Christopher Ober Materials Science & Engineering Lois Pollack Applied & Eng. Physics Michael Shuler Chemical & Biomolecular Engineering Dotsevi Sogah Chemistry & Chemical Biology Michael Spencer Electrical & Computer Engineering Tracy Stokol Department of Population Medicine

Chemical & Biomolecular Engineering arry Walker Bio. & Envir. Engineering

Abraham Stroock L

3

Michelle Wang Physics Watt Webb Applied and Eng. Physics Gary Whittaker Department of Microbiology & Immunology Ulrich Wiesner Materials Science & Engineering Mingming Wu Mechanical & Aerospace Engineering Weill Cornell Medical College

red Maxfield Biochemistry

ity, Washinglo

ones

Science Uners

cology

hitman College

e y

ter, NY State bany, NY aggana Core Facility rence

Tissue Culture Facility

Adele Boskey Biochemistry FTom Sato Cell and Development Biology Clark Atlanta University, Atlanta, GA Ishrat Khan Chemistry Howard Univers ton, DC Marmadou Dial Civil Engineering and ChemistryKimberly J Civil Engineering Oregon Health & iversity, Portland, OR Wolfhard Alm Vollum Institute Gary Banker Cellular & Molecular Neurobiology ToxiBruce Patton Cell and Developmental Biology Ginger Withers Department of Biology, W Princeton University, Princeton, NJ Bob Austin Department of Physics Edward Cox Department of Molecular Biology Saeed Tavazoi Department of Molecular Biolog Wadsworth Cen Health Department, AlMichele C Molecular Genetics DiagnosticDavid Law Environmental & Clinical Immunology William Shain Cell and

4

Biomolecular D lysis – Lois Pollack evices & Ana

. Micro & Nanofluidics for Molecular Detection, Separation and Analysis BDA2 Craighead, Luo, Coates

aration and Semipermeable Transport of Biomolecules BDA3

d Rapid Fluid Mixer s Exp iments BDA11

BDA13 Erickson, Baeumner, Lipson

. Biosensor Based on Electrochemical/Gravimetric Detection of Intrinsic Antibody Catalysis BDA14

etection of Genomic L Nanochannels BDA15 Austin, Craighead, Tavazoie . Cantilever Array Sensors BDA16

Production, Crys CD31, an Important xpress Protein BDA17

eractions – B

ses to Topographic CSI3 -Teschl

. Patterned Lipid Bilayers and Zero Mode Waveguides to Investigate Immune Cell Signaling CSI5 Baird, Craighead

sociated Bacteria in Artificial Xylem Lumina CSI6

ical Rigid Rods to Eng ses CSI10

. Imaging Axonal Growth and Axonal Organelle Transport Using Novel Devices that Integrate Microfluidics and Substrate Micropatterning CSI11

ity CSI12 , Boskey, Stroock

ee-Dimensional Cel CSI13 Fischbach-Teschl, Stroock, Bonassar

1

2. On-Chip Nanoporous Membranes for SepJones, Spencer

3. Microfabricate s for Protein Dynamic erPollack, Webb

4. Nanoscale Optofluidic Devices for Biomolecular Analysis

5 Baird, Abruña, Ober, Appleton, Clark 6. Electronic D ength DNA in 7 Lin, Craighead 8. A Novel P-gel for tallization and Structure Determination of But Difficult-to-e

Luo, Hao, Craighead

IntCell-Surface arbara Baird 1. Cellular Resp and Biochemical Clues on

Shain, Fischbach2

3. Colonization and Communication Among Plant-AsHoch, Wu,

4. Divalent Hel age Receptors and Control Cellular ResponKhan, Baird

5 Banker, Kirby 6. 3-D Matrices for Modeling the Bone-Cartilage Interface & Controlling Chondrocyte Activ

Estroff, Bonassar7. Recreating Thr l Niches with Microfluidic Tumor Cells

5

Cellular Microdynamics – David Lawrence

CM1 Spencer, Lawrence

tin, Lynes

nsport Vesicles in Living Cells Using Fluorescent Nanoparticles NCB1

. A Scalable n x n Electrochemical Pixel Array with Integrated Electronics for Use as a Highly Parallel Biosensor Device NCB2

. Fabrication of Nanoscale, Dendrimer-like DNA Structures as Multivalent Templates NCB3

NCB4

rs, Craighead, Baird, Ober NCB9

ctures, Function, and Dynamics: From Mononucleosomes to Polytene Chromosomes

1. Development of a Cell Biosensor

2. Nanotechnological Assessment of Drug Toxicity CM2 Shuler, Shain

3. Isolation and Characterization of Immune Cells: Structure and Function CM5 Lawrence, Aus

4. Subcellular Molecular Distribution Analysis and Sorting CM6 Austin, Lawrence, Cox

5. Bionanofabrication: In Situ Creation of Nanoscale Polymeric Features CM9 Batt, Coates, Ober Engineering Functional Microvascular Structure in vitro CM10 6. Stroock, Bonassar, Sato, Shuler, Stokol, Wu

Nanoscale Cell Biology – Manfred Lindau

ules and Tra 1. Tracking Single MolecLindau, Wiesner, Webb, Baird

2

Lindau, Ober 3

Luo, Baird 4. sensors Nanotubes as Cellular Bio

McEuen, Lindau, Craighead 5. ovel Applications of Reverse Transfection for High Throughput Cell-Based Development of N

Functional Screening NCB6 Maxfield

6. Electrochemical Imaging of Exocytosis Using Microfabricated Devices NCB8 Lindau, Alme

7. Dynamics of Viral Fusion Proteins Pollack, Whittaker, Crane

8. Chromatin StruKraus, Lis, Wang, Webb NCB10

6

Biomolecular Devices and Analysis - Program Overview

nalysis” (BDA) program is to use nanal systems to develop devices and m

s e specifically, the prpico- liter volu

apability to perform molecular svices for high throughput

strong commit

esearch Projects for 2008

paration and Analysis BDA2 Craighead, Luo, Coates

BDA11

Optofluidic Devices for Biomolecular Analysis BDA13

r Based on Electrochemical/Gravimetric Detection of Intrinsic Antibody Catalysis BDA14 lark

nomic Length DNA in Nanochannels BDA15

ation of CD31

Program Coordinator: Lois Pollack Program Objectives The overall goal of the “Biomolecular Devices and A ofabrication techniques, advanced material and advanced optic ethods for ob erving, manipulating, and quantifying the behavior of biomolecules. Mor ogram members are developing integrated techniques to work with nano- and mes, and to provide more quantitative data for use in genomics, proteomics, ecological, and other biological research act that by integrating our unique c eparation ivities. The expectation is

analyses at the nano-scaand le we can develop miniaturized "chip-like" dequantitative analysis of dynamic bioprocesses. In addition, there is a ment to the dev cation of nanostructures that detect individual molecules for early detection of elopment and applidisease or for high throughput analysis.

R

1. Micro & Nanofluidics for Molecular Detection, Se

2. On-Chip Nanoporous Membranes for Separation and Semipermeable Transport of Biomolecules BDA3 Jones, Spencer 3. Microfabricated Rapid Fluid Mixers for Protein Dynamics Experiments Pollack, Webb 4. Nanoscale Erickson, Baeumner, Lipson 5. Biosenso

Baird, Abruña, Ober, Appleton, C6. Electronic Detection of Ge

Austin, Craighead, Tavazoie 7. Cantilever Array Sensors BDA16

raighead Lin, C8. A Novel P-gel for Production, Crystallization and Structure Determin , an

tein BDA17 Important But Difficult-to-express Pro Luo, Hao, Craighead

7

Micro & Nanostructures for Molecular Detection, separation and analysis

: H. Craighead, G. Coates, D. Luo ocs: C. Reccius, S. Stavis, L. Bellan, K. Peretti, H. Liu

f biological systems relevant to biotechnology applications. anoscale optical and electrical devices have also been explored for detecting individual molecules and

tems such as lipid-bilayers and living cells.

Varied, nanoscale geometries were designed and tested for use in micro/nanofluidics. Channels with

mo cu king to integrate these channels with optics for molecular analysis.

spectroscopy and enable high throughput ethod to characterize tection l

he DNA strands opticr tropically driven processes of DNA

eate these struct

rming fibers of

es to be

in membranes and in living cells. The utility of these devices continues to be investigated.

Carbon nanotubes are exquisitely well-defined nanostructures that have been widely studied. We are working to integrate these into fluidic systems and explore their capabilities for observation of nanoscale molecular dynamics in biomolecular systems. Summary

Our group is constantly experimenting on new nanoscale fabrication techniques and we have shown to reliably create and utilize sub 100 nm structures for observation of biological systems. The work has been presented that past year in invited reviews in Nature and industrially-oriented international journals. The use of carbon nanotubes, nanofibers, nanofluidic systems, and zeromode waveguides has been demonstrated in numerous collaborative efforts and described in the listed publications.

Participating FacultyNBTC Students/PostdOther Students/Postdocs: J. Mannion, J. Cross, B. Cipriany Project # BDA2 Objectives

The goal of our research is to study new approaches for the creation and application of nanostructures for molecular detection, separation and analysis. Our interests focus on investigations of fundamental physical properties of nanoscale devices that can be utilized for detection, identification and manipulation of biomolecules. Detailed investigations of single DNA molecules and its polymer properties have been a significant part of our work, leading to increasingly sophisticated devices for single molecule studies of a variety oNinteracting with biological sysMethods

diameters between 100 and 500nm have permitted manipulation and detection capabilities on the single le le level. We are wor

Nanofluidic channels can also be used to drastically reduce the focal volume in fluorescence single molecule analysis. We used this m

among several other samples polymerase chain reaction (PCR) products with de evels far below rd standa gel techniques. We found that it is possible to track t ally with high

p e isi example, different kinds of enc on and to distinguish, for anochannels to mmolecules from n icrochannels. Our optical methods enabled us to distinguish folded

their retraction speed. molecules and investigate the influence on While electron beam and optical lithography is frequently used to cr ures, our group lso n lithographic techniques to create nanodevices, including the is a working on utilizing no

enca id nanofibers. Work included fopsulation of molecules in sol DNA-Au composites ed p. creat by the Luo grou

The use of simple sub-wavelength metallic apertures, or zero-mode waveguides, continuexploited for molecular investigations. These devices are useful for observing chemical reactions in free

as dynamic propertiessolution or surfaces as well

8

Accomplish• The mobility, compression, expansion, folding and

nalyzing molecules in

monstration of ori ositio of polymer fibers as vehicles for permanently elongating

ments stretching of individual DNA molecules in

nanochannels was observed and quantitatively analyzed. • Showed the utility of carbon nanotubes and zeromode waveguides for a

lipid layers • De ented dep n

molecules

Figure 1 – Experimental Schematic. (A) Fluorescently labeled DNA molecules are elongated by driving them electrophoretically from a nanoslit into a nanofluidic channel. The nanochannel is probed by two independent focused lasers. DNA molecules generate two similar fluorescent signals, shifted in

ese signals can be used to

time. Thdetermine single molecule properties such as speed, length and folding (B) Two photon count signals p resulting from the two focal volumes and a model fit to the data.

Figure 2. A nanotube under a supported lipid bilayer was used to detect the binding of a protein onto receptors im

up

bedded in the bilayer (upper inset). The nanotube was used as the semiconducting element of a field effect transistor. The conductance was measured as a function of the gating voltage. Shifts are observed

on formation of the bilayer and subsequent binding of the proteins.

Figure 3. Fluorescence micrograph of individual dye-labeled DNA molecules embedded in polymer nanofibers.

c

9

On-Chip NanoPorous Membranes for Separation and Semipermeable Transport of Biomolecules Participating Faculty: Kimberly Jones, Michael Spencer, James Turner NBTC Students/Postdocs: Jermey Matthews, Lori Lepak Other Students/Postdocs: Malaisamy Ramamoorthy, Xingqun JiangProject # BDA3

bjectives Complete the characterization of cellulose acetate membranes.

agen membranes. lness of alumina membranes for molecular separation, especially

mbranes for DNA-heme separation. ip CA membranes of different functionality. embranes using cellulose acetate or collagen spun on alumina.

M es were fabricated using differing initial polymer concentrations, n (w/v). Collagen thin films were spin cast onto commercially a polycarbonate (PC), and polyethyleneterephthalate (PET). CA m A (Electrokinetic Analysis), AFM, SEM, tangential flow dialysis, pressure-driven flux and rejection performance, and ATR-FTIR (surface chemistry). Pressure-driven salt rejection data was collected in addition to purification of DNA from heme by electrophoresis through the CA membranes. Summary: It is important to purify DNA before PCR to improve the sensitivity and efficiency of the amplification. For blood the major interferant is heme, and we have developed two methods for c separates DNA and heme. We have d is undamaged. However, it is important to understand the m ccurs and possibly expand the bio-separation applications of the m ch as surface charge, hydrophilicity, roughness and pore size can a -heme and other biomolecules.

olymer concentration of the polymer solution was varied in an e mbrane pore size, membrane thickness, and surface charge. In a anes were fabricated and characterized. CA and collagen were c atibility of those polymers, and alumina membranes were utilized as either stand-alone or support for the organic membranes. This year’s efforts were focused on linking specific membrane characteristics to separation mechanisms. That knowledge would allow a clear explanation for the observed DNA-heme separation and allow the mem rane properties to be controlled for a wider suite of separations.

We were successful in fabricating, characterizing and testing performance variables (flux, rejection) fo er concentration affects the permeability (pore size) and s embranes. Interestingly, the hydrophilicity and surface roughness were unaffected. The collagen membranes were fabricated on three different substrates (alumina, PC and PET). All three systems display visibly different film morphologies, typical pore sizes and pore size distributions. Furthermore, for any given substrate system, the deposition of additional layers of collagen decreases typical pore sizes. Alumina membranes were fabricated on silicon substrates and characterized in terms of diffusive flux for several biomolecules. The diffusivities were calculated to be on the order of 10-8 cm2 s-1 when the pore size was around 50 nm. The molecular flux was highly sensitive to the membrane thickness in the thin-film regime. It’s expected that the flux rate could be further enhanced by continuous reduction of alumina thickness.

O

Fabricate and characterize coll Complete evaluation of usefu

the purification of DNA. Compare the three types of me Complete development of on-ch Develop new class of on-chip m

ethods: Cellulose acetate membranamely 10%, 17.5%, 25% and 40%vailable porous substrates alumina, embranes were characterized by EK

ombining our CA membranes with electrophoresis that completely emonstrated that the purified DNAechanisms by which the separation oembranes. Membrane properties su

ffect separation efficiencies for DNA For the CA membranes, initial p

ffort to change and control the meddition, collagen and alumina membrhosen because of the biocomp

b

r the CA membranes. The initial polymurface charge (zeta potential) of the CA m

10

Accomplishments Linked surface morphology, surface functionality and pore size of CA membranes to •

mem anes with

A-he e separation

performance variables (flux and rejection) nt substrates, forming br • Fabricated collagen membranes on three differe

potential tailorable morphologies • Elucidated mechanisms for DN m

Fig.1: Anion rejection of CA membranes fabricated using differing initial polymer concentrations. Rejection is calculated from feed and permeate salt concentrations. Results, taken along with surface charge data, indicate that membrane surface charge affects rejection, and that Donnan interactions are important.

0.0

10.0

20.0

10 %CA 17.5 %CA 25 %CA

30.0

Rej

ectio

40.0 (%)

50.0

60.0

70.0

1 mM Cl- (0.195 nm)

1 mM SO42- (0.300 nm)

n

Fig.2: (A) Collagen film, 3 layers, on alumina. Magnification 50,000x. Note typical pore sizes 20-40 nm. (B) Collagen film, 3 layers, on polycarbonate. Magnification 25,000x. Collagen is present primarily in fibril form, with typical pore sizes (above holes in substrate) 20-100 nm. (C) Collagen film, 3 layers, on PET. Magnification 10,000x. Fibrils assume different arrangements when spanning holes than when in contact with substrate.

A B

Fig.3: (A) Bottom view of free-standing alumina membrane. (B) Zoom-in of (A). (C) Cross section of membrane type I, 1200 nm thick. (D) Cross section of membrane type II, 200 nm thick..

C

11

Microfabricated Rapid Fluid Mixers to Study Macromolecular Conformational Dynamics Participating Faculty: Lois Pollack, Watt Webb NBTC Students/Postdocs: Hye Yoon Park, Huimin Chen Other Students/Postdocs: Project # BDA 11 Objectives

The well-established connection between the structure and function of biological macromolecules provides strong motivation for probing the time-dependent structural changes of macromolecules that accom g or conformational dynamics. The long-t to monitor conformational changes on many time and length s because the motions of interest can occur in microseconds. Access to different length scales links local motions with large-scale changes in size and shape. This p application of microfabricated tools to probe conformational c ritical time and length scales.

Methods Microfabricated mixers are used to rapidly trigger conformational changes. These devices exploit the

unique properties of fluid flow on the micron length scale to achieve rapid mixing. As a result of design flexibility, we can interface these mixers with different probes to monitor either global or local structural changes. Optical probes, e.g. lasers, o n of a particular site that carries a fluorescent label ( ues probe changes in the overall size and shape of the m rical simulations are used to identify flow conditions that o ions of simulations are validated with experimental c using the strong signal from fluorescent dye flowing in the m ily on the use of NBTC fabrication and characterization f

SMicrofluidic mixers have been developed and applied to measure conformational dynamics of

proteins as they fold or function. This year, optically transparent mixers were used (a) to complete measurements of the time scale for conformational changes of the calcium binding protein calmodulin, and (b) to initiate studies of the conformational dynamics or fluctuations that accompany protein folding.

Calmodulin (CaM) is a ubiquitous calcium-binding protein that regulates numerous Ca2+ dependent processes. The microfluidic mixer was used in conjunction with conventional mixers to measure the i istinct conformational changes that occur as the protein binds u more rapid conformational change occurs on the m essible to the microfluidic mixer. These experiments identify a e state with only two of the four calciums bound which has a ~millisecond lifetime. When placed in the context of studies of CaM interaction with target proteins, these data suggest a biological, regulatory role for the half-calcium saturated protein.

This year, by coupling fluorescence correlation spectroscopy, FCS with microfluidic mixers, we initiated a project to monitor the conformational dynamics or fluctuations of proteins as they fold.

inary experiments have successfully detected rapid, conformational fluctuations of labeled roteins flowing within the microfabricated mixer.

pany biological activity, such as self-assembly/foldinerm goal of this project is to develop toolscales. Access to short times is essential

roject focuses on the development and hanges of proteins and nucleic acids on c

detect mlocal motions); x-ray scattering techniqacromolecules (global motions). Nume

ptimize uniform, rapid mixing; predictharacterization of the mixers, carried outicro-channels. This project relies heav

acilities.

ummary

tio

ntrinsic time constant for each of the two dp to four calcium ions, two at a time. The icrosecond time scale, which is only acc

n intermediat

Prelimp

12

Ac•

c ul n, suggesting an important

e ctuations within the microfluidic device by FCS

complishments Optically transparent microfluidic mixers have been applied to study microsecond-scale conformational dynamics of proteins

in mod i• Two discrete conformational changes are detected albiological role for an intermediate

ave initiated m• We h asurements of protein flu

Fig.1: The outline of the five-inlet port mixer is shown. The addition of sheathing flow from the diagonal channels (red fluid) allows us to separate hydrodynamic focusing of protein-containing solution (green) from the diffusive mixing into the thin jet (right) that triggers the reaction of interest.

Fig.2: An experiment designed to monitor the conformational change of calmodulin (CaM), a ubiquitous calcium binding protein, following the rapid addition of calcium ions, using the device shown in Figure 1. A fluorescent label is covalently attached to the CaM and reports on the conformational change. The dye emission is monitored as a function of position along the outlet channel, which corresponds to time after mixing.

Fig.3: Fluctuations of fluorescently labeled proteins, measured with fluorescence correlation spectroscopy (FCS), reveal their conformational dynamics. This year, fluctuations of labeled proteins flowing within the microfluidic device were detected. These measurements will enable the study of the fluctuations that accompany folding.

13

Nanoscale Optofluidic Devices for Biomolecular Analysis Participating Faculty: David Erickson, Antje Baeumner, Michal Lipson NBTC Students/Postdocs: Sudeep Mandal, Sam Nugen Other Students/Postdocs: Julie Goodard, Likun Chen, Peter Asiello, Brad Schmidt Project # BDA-13 Objectives

Recent interest in the development of new biosensors technology has been largely driven by the p ith disease states or pharmacological responses, and the need to r s. To capitalize on these applications successful next generation s ximize the total number of biomarker targets against which a s rrogated while being sufficiently sensitive and specific so as to e detection without the need for target labeling.

ram is the development of Nanoscale Optofluidic Sensor Arrays (NOSA) applied to the detection of Dengue virus RNA. The prototype along with a system schematic is shown in Fig. 1 and is based on our recently developed capability to: integrate nanoscale fluidics with optically resonant devices (Erickson and Lipson Labs) and create RNA based viral pathogen biosensors (Baeumner Lab). Within the first 8 months of this program our specific goals were to (1) develop, s ental proof of concept for the NOSA platform and (2) develop a D ntly orient DNA oligonucleotide probes in silicon substrates. M

hniques were used to simulate the devices and resulted in the unique 1D evanescently coupled optically resonant biosensor design which is the current focus of this p ricated in silicon-on-insulator using standard electron-beam l with PDMS based microfluidics as shown in Fig. 1c. When o istinct “dips” in the transmission spectrum (Fig. 2) where the e the resonant conditions of the resonator. When the local refractive index around cavity of the resonator changes (due to a RNA binding event) this resonant condition changes and its presence can be detected.

A variety of immobilization methods are being investigated and evaluated. All techniques involve an initial silanization of the silicon. Subsequently, DNA oligonucleotides are immobilized using glutardialdehyde as linker molecule, dendrimers as spacer molecule, or a direct immobilization of thiolated DNA. Immobilization success was evaluated using TAMRA-labeled DNA and monitored t obilization of DNA oligonucleotides via dendrimer spacers w immobilization methods.

tection, we used a 3 active resonator NOSA sensor functionalized w irus serotype 3, (R2) a control sequence and (R3) Dengue Virus s troduced a solution containing serotype 3 targets and observed the r e largest shift occurs at the resonator with the probes specific to the t ific Dengue-virus detection using the NOSA platform. Summary

The overall goal of this program is to develop a new approach to viral RNA detection that combines the exceptional sensitivity achievable with nano-devices with the parallelity offered by traditional microarray devices. In the first 8 months of this program we have constructed the prototype device, characterized the sensitivity, developed an oligonu leotide immobilization technique compatible with the device technology and demonstrated serotype specific detection of synthetic Dengue Virus RNA.

otential for associating biomarkers wapidly diagnose emerging viral threatensor platforms should be able to: maample or multiple samples can be intenable very low, sub-femtogram level

The overall objective of this prog

imulate, fabricate and provide experimNA immobilization method to efficie

ethods Finite difference time domain tec

rogram. These devices were fabithography techniques and integratedptically excited, these devices exhibit dxcitation wavelength matches

hrough a fluorescence microscope. Immas identified to be superior to the other

To demonstrate serotype specific deith probes specific to (R1) Dengue V

erotype 1. After functionalization we inesonators red-shifts shown in Fig. 3. Tharget serotype, suggesting serotype spec

c

14

Accomplishments • Development, simulation

nsi vity. chemistry

and fabrication of highly parallel “side resonator” biosensor devices• Fusion of new devices with microfluidics and characterization of the device se ti

f DNA onto silicon via dendrimer• Successful immobilization of high concentrations o

Fig. 1: Nanoscale Optofluidic Sensor Array. (a) Schematic of NOSA array during optical excitation. (b) Image of initial prototype as developed through NBTC program. (c) SEM of the device array comprising of 4 waveguides each with 5 evanescently coupled waveguides. White scale bar is approximately 1 µm.

Figure 2: Change in Resonant Dip location inrefractive i

response to change in local ndex. Blue spectrum shows

initial 5 resonator system each addressed with a separate microchannel filled with water (n = 1.33). Black spectrum shows red shift in of resonator #2 in response to the introduction of a ~1M CaCl2 solution. Overall sensitivity of device calculated to be 120 nm/RIU.

Figure 3: Initial results showing serotype specific detection of synthetic Dengue virus RNA sequences. Probes spe

(a) (b)

(c)

cific to Dengue virus serotype 3 were immobilized on the first resonator, a control (random) sequence on the second and serotype 1 on the third. When a sample containing the Dengue serotype 3 targets was introduced the largest shift was obtained at the complementdetection.

ary site, suggesting positive

Δλ

15

Biosensor Based on Electrochemical/Gravimetric Detection of Intrinsic Antibody Catalysis

isiting Scientists: R. Naal, Z. Naal BDA14

Objectives

loped for electrochemical/gravimetric detection of antibodies, in p e avian influenza virus (H5N1) hemagglutinin protein (H5). This a trinsic antibody-catalyzed water oxidation pathway (ACWOP) that w activity will be detected on a specifically modified electrode or w e (QCM hese detection methods, within microscale devices, are e re sensitive than current enzyme-linked-immuno-sorbant-assays ( Methods

Intrinsic ACWOP activity of antibodie confirmed by measuring direct production of hydrogen peroxide (H2O2) using a colorimetric (Amplex Red) assay that is also used for optimization. Measurements of antibodies bound to polymer brushes can be compared to electrochemical/gravime ic measurements of H2O2 produced by the same substrates in the development of the proposed device.

trochemical/gravimetric, devices includes determination of the s the assembly: polymer brushes, hapten and antibody, u mical (cyclic voltammetry), gravimetric (QCM), fluorescence and o OP is based on H2O2 reduction to water, using amperometry or g

oly(acrylic acid) are modified with haptenic groups and used to bind a and QCM surfaces. Atom transfer radical polymerization is used to g

or H5 are prepared in milligram amounts from cloned, secreting cell l g activity of purified antibodies is confirmed in hemagglutination inhibition and ELISA assays using killed H5N1 X PR8 viruses.

Preparation of H5 proteins from avian influenza virus is based on the full-length H5 gene, expressed in Tetrahymena. Other cell lines expressing several alternative versions of the recombinant H5 protein are being constructed to streamline protein purification and provide alternatives for conjugation to p S

une system are often the first signs of infectious diseases. Because antibodies recognize a vast repertoire of antigenic groups with exquisite specificity they are also v eutics, vaccine development, and research. The important need for d media is currently limited by sensitivity and specificity. We are c vimetric device to reach new detection limits, based on intrinsic catalytic activities of antibodies. Polymer brushes, modified with selected antigenic groups and other a surface of the detection device, such that antibodies bind specifically and the ACWOP activity can be measured sensitively. The device is initially being developed and optimized with a model antibody system that can then be generalized to antibodies of high medical importance. We are currently adapting the device for detection of antibodies specific for avian in uenza viruses (H5N1) that currently pose pandemic threat. The technological developments will be readily daptable to detection of other antibodies and also to other biosensors on the micro- and nanoscales.

Participating Faculty: B. Baird, H. Abruña, C. Ober, J. Appleton, T. Clark NBTC Students/Postdocs: N. Smith, S. Nad, A. Rastogi, Y. Bisharyan VProject #

Novel devices are being devearticular, antibodies specific for thpproach takes advantage of the inas previously characterized. Thisithin a quartz crystal microbalanc

xpected to be more specific and moELISAs).

). T

s is

tr

Assembly of general platform, elecurface coverage of the various components insing a combination of electrochether techniques. Detection of ACWravimetry as the analytical signal.

Polymer Brushes consisting of pntibodies specifically to electrode row brushes on gold or silicon oxide surfaces.

Monoclonal antibodies specific fines. Bindin

olymer brushes.

ummary Antibodies produced by the imm

aluable reagents for medical therapetection of antibodies in diverse onstructing an electrochemical/gra

gents, provide the interface on the

fl

a

16

Ac• Confirmed ACWOP activity of various antibodies, including anti-DNP and anti-H5 antibodies

crobalance ombina specific anti-H5 monoclonal antibodies

complishments

• Prepared polymer brushes that specifically bind anti-DNP • Measured H202 activities by cyclic voltametry and quartz crystal mi• Prepared rec nt H5 proteins and

Fig.1: Schematic for proposed biosensor based on electrochemical/gravimetric detection of intrinsic antibody catalysis (ACWOP)

Fig.2: Cyclic voltammograms of an HRP modified gold electrode displaying the enhanced catalytic current for the reduction of phendione bis-phenanthrolinosm

e ium (III) (used as a redox mediator)

due to the activity of the immobilized HRP enzyme in the presence of hydrogen peroxide

Fig.3: Synthesis of DNP modified poly(acrylic acid) brushes NO2

NO2

HN

COOH

Br

S

DPTS, DIPC, DMF

H2NO

OH

Br

S

O

NH

OO

H

DPTS, DIPC, DMF

Br

S

O

NH

OO

HN

O2N

NO2

O

OH

O

ΠACWOP rxn: 1O2 + H2O ⇑ H2O2 + O2

a) polymer brushes

1O2 is generated by photosensitizer; H2O2 is detected by redox chemistry

hapten

antibody

20 nm

-0.2 -0.1 0.0 0.1 0.2

-0.14

-0.12

-0.06

-0.10

-0.08

-0.04

-0.02

0.00

0.02

WE : Au + SAM-DTSP + HRPA

RE : Ag/AgCl

I / μ

CE : PtScan Rate : 10 mV/s

0 mM H2O2 0.1 mM H2O2 0.4 mM H

2O

2 0.7 mM H2O2

Voltage (V)

17

BLANK PAGE

18

Cell-Surface Interactions – Program Overview Program Coordinator: Barbara Baird Program Objectives How cells interact via their surface membranes with environmental signals or substrata is the thrust of this program. The roles of the dimensions, chemistry, and topography of interacting components in regulating cellular behavior are explored. A wide range of cell-types, including mammalian cells, higher plant cells, fungal cells, and bacteria are studied. In addition to synthesis, the Program utilizes techniques that pattern topography and receptor-specific ligands or delicate biomolecules that are of greatest biological relevance, and also develops me ods for documenting their impact on cell growth and polarity, differentiation, and function. Microcontact printing, lithographic and parylene lift-off patterning of chemical domains, patterning on strep fabrication of pillared arrays, and incorporation of sensing junctions are some of the techniques used as part of the research efforts of this program. The discoveries made for the cellular processes examined in this program are provide valuable insight and perspective for the other programs within the NBTC. Research Projects for 2008

1. Cellular Responses to Topographic and Biochemical Clues CSI3 Shain, Fischbach-Teschl 2. Patterned Lipid Bilayers and Zero Mode Wa tigate Immune Cell Signaling CSI5 Baird, Craighead 3. Colonization and Communication Among Plant-Asso lem Lumina CSI6 Hoch, Wu, 4. Divalent Helical Rigid Rods to Engage Receptors and Control Cellular Responses CSI10 Khan, Baird 5. Imaging Axonal Growth and Axonal Organelle Transport Using Novel Devices that Integrate

Microfluidics and Substrate Micropatterning CSI11 Banker, Kirby

6. 3-D Matrices for Modeling the Bone-Cartilage Interface & Controlling Chondrocyte Activity Estroff, Bonassar, Boskey, Stroock CSI12

7. Recreating Three-Dimensional Cell Niches with Microfluidic Tumor Cells CSI13 Fischbach-Teschl, Stroock, Bonassar

th

tavidin-conjugated hydrogels,

veguides to Inves

ciated Bacteria in Artificial Xy

19

Cellular Responses to Topographic and Biochemical Clues

: William Shain, James Turner ocs: Matthew Hynd (Postdoc), John Frampton (Graduate student)

rned neural networks. Patterned hydrogel were used as templates for the development of neural-based biosensors.

pounds from hydrogels will be used in the development of neural-based biosensors.

lution consisting of 10% (w/v) poly (ethylene) glycol diacrylate,

tota streptavidin-conjugated acrylamide was added to 8 μL of hydrogel solution.

Sila ss coverslip was placed on top of the solution. The hydrogel solution was photo-

peptide, biotin-GIKVAVY was patterned onto hydrogel surfaces using the NanoEnabler molecular rd

coit #7740 substrate. An additional 10 nm titanium layer was deposited on the gold

HF (3:1) solution for gold. A triple stack insulating

stress 512) mask was used for reactive ion etching of electrode

nd contact sites. The upper titanium layer was etched in the same cycle. MEAs were designed to have 2 electrodes (in a 4X8 array) with 200-μm site spacing. The exposed area of each electrode site was 00 μm2. The mean measured impedance was 1.91 MΩ in phosphate buffered solution at 1 kHz. In rder to decrease electrode impedance, all sites were electroplated with H2PtCl6. For neural networks, rimary hippocampal neurons were plated at a density of 200 cells/mm2 on neurotrophin-containing ydrogel-coated MEAs. ummary

The ability to control the spatial localization of molecules at micrometer scales on biologically compatible materials is a key requirement for such technologies. In the present study the biotin-streptavidin system has been adapted to create novel biomimetic hydrogel surfaces functionalised with the streptavidin protein. Modified surfaces were patterned with the laminin protein epitope, GIKVAVY to enhance and direct neural cell growth and adhesion using the automated NanoEnabler protein arrayer. Hippocampal neurons were found to organize along hydrogel patterned regions only and resulted in the formation of functional neural networks on MEAs. Release of bioactive neurotrophins from hydrogels was shown to modulate network function in vitro. These results demonstrate the use of automated molecular bioprinting for the rapid construction of neural-based biosensors.

Participating FacultyNBTC Students/PostdOther Students/Postdocs: Project # CSI3 Objectives

1. Development of novel biomimetic hydrogel surfaces. Synthetic hydrogels composed of poly(ethylene) glycol diacrylate were used as templates for protein attachment and cell growth.

2. Construction of low-density neural networks in vitro. Biomolecular printing was used to print defined geometric patterns of peptides onto hydrogel surfaces using a robotic arrayer (NanoEnabler). These surfaces will be used to direct attachment and growth of neural cells on microelectrode arrays.

3. Hydrogel-coated microelectrode arrays for stimulation and recording of patte

Release of bioactive com

Methods A hydrogel pre-polymer so

(PEGDA, Mw = 3400 Da), 0.1% Irgacure 2959 and in HEPES-buffered Hanks saline was prepared. A l of 150 pmol (2 μL) of

The mixture (10 μL) was applied to a quartz slide pre-treated with PlusOne Repel-Silane ES. A Bind-ne treated 18mm gla

polymerized by exposure to UV light (9 mW/cm2) for 5 min on a Spectrolinker XL-1000 at 365 nm. The

bioprinter (Bioforce Nanosystems). Microelectrode arrays (MEAs) were fabricated using standasemi nductor processes. Briefly, a 30nm titanium adhesion layer and a 300 nm gold layer were depos ed on a Pyrex layer to provide for adhesion to the insulation layer. Metal layers were patterned by wet etching with (1%) for titanium and HCl, and a HNO layer 3(SiO2/Si N /SiO ) was deposited by plasma enhanced chemical vapor with compensated 3 4 2thicknesses of each layer. A photoresist (AZ1a31ophS

20

Accomplishments • Development of novel biomimetic hydrogel surfaces. Synthetic hydrogels composed of

s tem lates for protein attachment and

- etworks in vitro. The spatial patterning of cells was based the molecular b of olecules onto hydrogel surfaces. The laminin protein

epitope, GIKVAVY, was nanopatterned onto functionalized hydrogel surfaces. Cell cultures of ary hippocampal neurons were used to characterize adhesion to peptide-modified

al

w neuronal networks form and for the development of neural-based

poly(ethylene) glycol diacrylate were developed a pgrowth of neural cells in both 2-D and 3-D (Figure 1).

• Construction of low density neural non ioprinting m

primhydrogels (Figure 2).

• Hydrogel-coated microelectrode arrays for stimulation and recording of patterned neurnetworks. Patterned hydrogel surfaces were used as templates for the development of neural-based biosensors. Primary hippocampal neurons were cultured at a density of 200 cells/mm2 on patterned microelectrode arrays (Figure 3). Results from these studies will allow understanding of hobiosensors.

Fig.1: Hydrogel-coated MEA patterned with the laminin peptide, biotin-GIKVAVY. The laminin peptide biotin-GIKVAVY was printed onto using the automated NanoEnabler bioprinter. Deposited peptide was arranged in a pattern consisting of orthogonal 2 μm-wide lines connecting 10 μm diameter node. Scale bar = 200 μm.

Fig.2: Patterned neuronal network at 4 weeks in vitro. Neurons were stained with microtubule associated protein 2 (MAP2; red). Patterned neurons were shown to develop functionally active synapses (green; synaptophysin). Cell nuclei were stained with DAPI (blue). Scale bar = 25 μm.

Fig.3: Fluorescent image 3-D hydrogel

r neural cell growth. LRM55 constructs foglioma cells (green, actin-phalloidin; arrowheads) were photo-encapsulated in biomimetic poly(ethylene) glycol diacrylate hydrogels. Primary hippocampal neurons (red, βIII-tubulin) were seeded on top of the hydrogels. Cell nuclei were stained with DAPI (blue). Neurons showed extensive outgrowth by 48 hr in vitro. Images were taken using a Nikon TE2000 inverted microscope and image stacks deconvolved. Scale bar = 200 μm.

21

Patterned Lipid Bilayers and Zero Mode Waveguides to Investigate Immune Cell Signaling

a

ponses to external stimuli, leading to targeted responses. The

f biomolecular distributions and dynamics on ~100 nm length scale. With IgE ceptors on mast cells as our prototypic system we seek to elucidate mechanisms controlling the spatial

distribution of signaling events that are initiated by immune receptors. M

photolithography for parylene lift-off or ZMWs; patterned l encoded fluorescent constructs and immunofluorescence d nd electron microscopic imaging; fluorescence confocal m orescence microscopy; fluorescence correlation spectroscopy ( Summary

Surfaces patterned with antigens on the micron scale are utilized for examination of spatially localized events in receptor-mediated signal transduction and cellular responses. Our previous studies revealed the participation of the cytoskeleton in co-redistribution of membrane and signaling c ptors. Adhesion to the substrate also involves cytoskeletal i d by focal adhesion complexes. Patterned bilayers containing a these interactions based on integrin preference for the silicon o ors were clustered by the antigen. These studies revealed r n proteins, independent of integrins. Complementary b hat these interactions are involved in negative regulation of signaling.

The ZMWs that confine excitation of fluorescence to scale of 100nm proved to be very effective for examining dynamics of cell membranes at this high level of resolution. With a combination of fluorescence and electron microscopy we demonstrated that plasma membranes from live cells penetrate t r exploration of the nanoapertures depends heavily on actin f sting that cells extend filopodia-like membranous extensions t n filaments. The presence of actin filaments within ZMWs w ence from genetically encoded GFP-actin as it polymerized w ted volume. This positioning of the plasma membrane into the a diffusion processes through spectroscopy of fluorescently l red the diffusion of fluorescently labeled lipid analogs DiIC12 a found distinctive diffusive behaviors, suggestive of different e s show that the use of optical nanostructures enables the m ngle molecule resolution in sub-diffraction volumes.

Participating Faculty: Barbara Baird, Harold Craighead NBTC Students/Postdocs: Pangshun Zhu, Prabuddha SenguptOther Students/Postdocs: Jose Moran-Mirabal, Alexis Torres Project # CSI5 Objectives

Cells spatially control their resimportance of the spatial localization is reflected by the sophisticated structures employed by cells to restrict and compartmentalize many biomolecular interactions. The focus of this project is to visualizeboth 2D compartmentalization (domains or interaction zones within plasma membrane) and molecular-scale dynamics and interactions. Patterned surfaces allow fluorescence visualization on the micron scale, whereas electron microscopy, zero mode waveguides (ZMW), and new optical systems to be developed enable examination ore

ethods Micro- and nanofabrication including

ipid bilayers and proteins; genetically etection within living cells; optical aicroscopy; total internal reflection flu

FCS).

omponents with antigen-clustered recenteractions with integrins that are mediatentigens were used to spatially separate xide surface at the same time that recepteceptor interactions with focal adhesioiochemical studies indicate t

hese nanostructures. We found that celluilaments but not on microtubules, suggehat elongate by the polymerization of actias confirmed by monitoring the fluorescithin the structures and entered the exci

pertures, allows the study of membraneabeled molecules. With FCS, we monitond DiIC16 in plasma membranes and nvironmental interactions These resulteasurement of membrane events with si

la

22

Ac•

te ins ar exclu d. tin-dependent fashion.

Ws enabled single ed lipid analogs.

complishments Determined with patterned bilayers that focal adhesion proteins vinculin and paxillin co-redistribute with clustered IgE-receptors, whereas α5-in gr e de

• Plasma membranes shown to invaginate into ZMWs in an ac• Localization of plasma membranes within the excitation volume of ZM

molecule spectroscopy of fluorescently-label

Fig.1: Confocal micrographs of RBL-2H3 mast cells labeled with a GFP fusion of paxillin and α5-integrin interacting with the antigen containing patterned lipid bilayers. Paxillin, a well studied focal adhesion protein, shows selective recruitment towards the clustered IgE receptors while α-5 integrin shows exclusion. These data suggest possible involvement of focal adhesion proteins in FcεRI signaling,

t of integrin interactions. independen

Fig.2: Scanning electron microscope image of n nanostructured substrates show fixed cells o

that cells explore and enter the ZMWs. The cell is outlined in orange and the position and periodicity of the structures is shown with yellow circles. This micrograph was featured on the cover of Nanotechnology, Vol. 18, No. 19, 16 May 2007.

Fig.3: Comparison of the FCS autocorrelation curves taken from ZMWs filled with AlexaFluor488-dUTP in solution, POPC-DHPE-LR bilayers, or cell membranes labeled with DiIC12 and DiIC18, shows that cell membrane dyes can be distinguished from model systems and between themselves. Larger residence times reveal slower diffusion through the illuminated volume.

23

Colonization and Communication Among Plant-Associated Bacteria in Artificial Xylem Lumina Participating Faculty: Harvey Hoch, Mingming Wu, (Chris Smart and Thomas Burr—non-

ther Students/Postdocs: Luciana Cursino dos Santos (postdoc with Hoch/Burr), Donald Lee (graduate student with Wu)

Project # CSI6 O

veloped as ‘artificial’ plant xylem vessels to study bacterial-p accomplished in planta. The fabricated artificial vessels a poral and spatial activities of plant pathogenic bacteria, p monas campestris pv. campestris. Specific objectives were t terial pili and a substratum. These studies used wild-type s ith defects in pili genes, 2) evaluate signaling factors on p osa, 3) evaluate influence of pathogenicity factors e.g. DSF m biofilm development in X. fastidiosa and X. campestris, and 4) assess type IV pili-mediated ‘twitching’ motility of various X. fastidiosa pilus-mutants. Methods

Photolithography and either deep RIE or SU-8 processes in were used to pattern silicon wafers, from which PDMS replicas were made and sandwiched between a glass microscope slide and coverslip to create ‘artificial’ xylem vessels. Fluid flow was achieved through in and out ports with high p acterial activities was accomplished principally through time-l software. Mutants of Xylella fastidiosa were generated by r transposome system. S

um, is limited to colonizing xylem vessels (water conduits) of p iseases by developing cell aggregates and biofilms that block x Through NBTC support, we previously discovered that X. fastidiosa migrates via type IV pili-mediated twitching motility at speeds up to 5 μm min-1 against a rapidly flowing medium (20,000 μm min-1). There are two length classes of pili, long type IV pili (1.0-5.8 μm) and short type I pili (0.4-1.0 μm). Using ramped flow through a microfluidic flow chamber to create drag force on bacteria, in conjunction with pili-defective mutants it was determined that the average adhesion force necessary to detach wild-type X. fastidiosa cells was 147±11 pN, while mutant cells possessing only type I pili r l, and cells possessing only type IV pili required 119±8 pN to d speed of twitching movement against the flow direction of m s 0.86 µm min-1. Mutants with only type IV pili moved six t ith an adhesion defect (pilY1 protein) in the type IV pilus tip m ). These and other results are summarized in movies at: http://www.nysaes.cornell.edu/pp/faculty/hoch/movies/

NBTC faculty) NBTC Students/Postdocs: Leonardo De La Fuente (Hoch) Tanya Taylor (Hoch), Daniel Rhoads

(Wu) O

bjective In vitro microfluidic systems were de

lant relationships in ways that can not bellowed optical assessment of both temarticularily Xylella fastidiosa and Xanthoo: 1) assess adhesion forces between bactrains and mutant strains X. fastidiosa wromotion of autoaggregation in X. fastidiolecules on cell migration and

recision syringe pumps. Assessment of bapse light microscopy and image analysisandom mutagenesis using an EZ::TN Tn5ummary

Xylella fastidiosa, a pathogenic bacterilants. It causes economically important dylem sap flow.

equired a force of 204±22 pN for removaislodge these cells. In a related study, theedia by wild type cells was measured a

imes faster (4.85 µm min-1) and mutants woved three times slower (0.28 µm min-1

lagellar motility; however, little is known as to when the

flagellar genes are deactivated and biofilm formation begins within the confines of xylem vessels. Using a GFP flagella promoter and a GFP type II secretion system (involved in various aspects of cell function, including extracellular polysaccharide matrix—EPS formation) promoter, the ctivation/deactivation of these genes was determined in artificial xylem vessels. GFP fluorescence iminished as the cells attached to the microfluidic chamber walls indicating loss of flagella; EPS

related GFP expression began subsequently.

Xanthomonas campestris relies on f

ad

24

Accomplishments Item 1. Used microfluidic devices to show that short type I pili slow type IV pili twitching motility •

• ted th iosa

secretion system t pathogenic bacteria. bly

o

of plant pathogenic bacteria. Item 2. Demonstra at pilY1-adhesins are important in pilus attachment of Xylella fastid

• Item 3. Developed a microfluidic system to visualize GFP expression of flagella and the type IIs plan

• Item 4. Demonstrated that a diffusible signal factor slowed Xylella fastidiosa motility, probavia regulation f the pilQ gene.

Fig. 1. Diagrammatic representation of the dual-

icrofluidic device used to examine

es of this figure are available as

channeled mtwitching motility in Xylella fastidiosa. Comparative paths of type IV pilus-mediated twitching movement of wild type (WT) cells, and pilY1 and fimA mutants. Colored traces correspond to migratory paths (R to L) against medium flow (L to R) of three representative cells for each cell type over same time period. Each line within the graphs (right) indicates distance displaced for each 30 sec interval by twitching movement for one representative cell of each cell type. fimA mutants moved greater distances than the length of the field of view during the time interval shown. Total time, 150 min. Movisupplemental material at http://www.nysaes.cornell.edu/pp/faculty/hoch/movies/.

Fig. 2: Microfluidic devices were used to show that Xanthomonas campestris pv. campestris with GFP driven by the flagellin promoter (top channels) and GFP driven by the type II secretion system (bottom channels) were switched off/on depending on the promoter function. Flagellar function is lost in close confines, while extracellular polysaccharide production is switched on. Left images are fluorescence; Right images DIC. Cells in the upper chamber were initially bright (not shown), but with loss of flagellar function are now non-fluorescent; Cells in lower chamber were initially not GFP-fluorescent, but are as the type II secretion system is activated.

Fig. 3: Five-channeled microfluidic device used to examine diffusible signal factor (DSF, a bacterial pheromone), associated with cell-cell signaling and gene regulation. All channels contained a nutrient medium, PD2, with either DSF or MeOH added. DSF reportedly down regulates pilQ gene expression involved in type IV pili activities. Preliminary observations indicate X. fastidiosa bacteria in DSF-amended streams twitch-migrated less than bacteria in all other channels.

25

Divalent Helical Rigid Rods to Engage Receptors and Control Cellular Responses

Partic , Barbara Baird, Harold Craighead and Larry Wang (Clark

Othe ss, Brandy Jones, Omotunde Olubi

bjectives: Synthesis and characterization of DNP functionalized polymers (both conductive and non-

and immune receptors are being carried out. These polymers a eceptor binding and cell activation, with eventual application i diagnostics. During the current reporting cycle we have developed a conductive IgE functionalized polypyrrole and have been successful is preparing fibers by e hydrofuran solution). Also, during the current period, our g had to do with preparing polymers with appropriate s quired for electrospinning. Our intentions were also to d initrophenyl caproic][poly(ethylene oxide)-b-poly(2-m CDNP-PEO-P2MS-PEO-CDNP), capable of selectively i L mast cells. M he polymers are carried out by established methods. In a lized electronic polymers, detailed determination of the morphological properties were carried out by differential scanning calorimetry to correlate the observed g perature (Tm) of the polymers with the ease of f e product fibers. Capacity of the ligands to stimulate IgE-F ted with a number of assays used routinely in the Baird l osphorylation western blots, microscopic imaging of f he electrosprayed fibers are prepared by methods well eS eriod we made a significant observation in that water soluble D shown in Figure 1) is a powerful inhibitor of degranulation r be noted that a high degree of inhibition remains with p his is very interesting and suggests the possibility of d ese polymers. We are in the process of starting a study to u of inhibition by these polymers. The synthetic methods used to prepare these polymers have been completely developed and preparing controlled polymers of any structure is possible. Also, we have completed the study with our functional polymers, α,ω-bi[2,4-dinitrophenyl c oxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-P ese ligands are capable of selectively interacting with anti-D binding affinity for these polymers at this stage of d ited by the solubility of the functional polymer in water. W r solubility of these functional polymers will lead to the d or allergy treatment. Thus, we are very excited with our w uring the current reporting period, we have developed c spun into fibers. Electrospun fibers are shown in figure 3. These fibers are a blend of the DNP functionalized conductive polymers (75% by weight) and polystyrene sulfonate (25%) by weight. During the current period, we solved a series of challenges which had to do with developing polymers with appropriate solubility, viscosity and rheological properties required for electrospinning. We are currently working on the problem to solve the specificity of interaction between the DNP decorating the electrospun fiber and solution IgE.

ipating Faculty: Ishrat M Khan

Atlanta and CREST Member) NBTC Students/Postdocs: Biswajit Sannigrahi and Dwaipayan Sil

r Students/Postdocs: Darkeyah Reuven, Jereme DoProject # CSI 10

Oconductive) that are specific to antibodiesre expected to be useful for controlling rn molecular (nanoscale) therapeutics and bio

lectrospinning the polymers from a THF (tetraoals were to solve some challenges whicholubility, viscosity and rheological properties reevelop a totally water soluble α,ω-bi[2,4-dethoxystyrene)-b-poly(ethylene oxide)] (

nteracting with anti-DNP IgE receptors on RBethods: Synthesis and characterization of t

ddition to basic characterization of the functiona

lass transition temperature (Tg) and melting temabrication of the fibers and the quality of thcεRI mediated cellular responses are tes

aboratory: degranulation, Ca2+ mobilization, phluorescent IgE and other cellular components. Tstablished in the Craighead laboratory. ummary: During the current reporting pNP functionalized polymer (see structure

esponses in mast cells (see figure 2). It is to olymer concentrations as low as 1 μg/ml. Teveloping nanoscale therapeutics based on thnderstand the mechanism

aproic][poly(ethylene oxide)-b-poly(2-methEO-CDNP) and have demonstrated that thNP IgE receptors on RBL mast cells. The evelopment is in the range of 33 μM and is lime expect that further improvement in the wate

evelopment of functional synthetic polymers fater soluble ligand systems. Furthermore, d

onductive DNP functionalized polymers capable of being electro

26

Accom• Preparation of divalent DNP functionaized water soluble ligand based in poly(2-

Observation of the e granulation of mast cell by water soluble

plishments

methoxystyrene)ffectiveness of inhibition of de•

ligands • Success in preparing fibers by electrospinning DNP functionalized conductive polymers

Fig.1: Structure of water soluble difunctional sulfonated DNP-poly(2-methoxy styrene) based ligands

R

R

OMe OMe

MeO MeO

n

nO

Om

m NH

HN

O

ONO2

O2NNO2

O2NSO3Na

NaO3S SO3Na

NaO3S

Water Soluble Polymer

Fig.2: The water soluble polymer is an effective inhibitor of degranulation in mast cells (see Figure 1).

Fig.3: Blend of FPPY: PSSO3,Na (75:25 w/w ratio) electrosprayed fibers on to silicon surface and exposed to Tyrodes Buffer w/ 5% gelatin

Inhibition with bs5-25

50

60

70

80

90

100

400.001 0.01 0.1 1 10

[bs5-25] (mg/ml)

% Inhibition

27

BLANK PAGE

28

Cellular Microdynamics – Program Overview Program Coordinator: David Lawrence Program Objectives The overall program objectives are to utilize nano- and micro-meter fabricated devices to quantify certain types of cells and their response to the envi onment in which they are exposed. The cell types under investigation range from bacteria to gastrointestinal endothelial cells. The parameters being evaluated are physical properties of cells (such as deformability, rigidity, and size), differentiation and growth, viability, release of products, trafficking, and cell communication. The devices are designed to simulate in vivo conditions allowing high visibility and conservation of reagents and cells. New projects in this program have added an additional dimension to cell analysis with inclusion of new substrates for cell interactions and 3-D vascularized patterns to assess cellular functions. Research Projects for 2008

1. Development of a Cell Biosensor CM1 Spencer, Lawrence 2. Nanotechnological Assessment of Drug Toxicity CM2 Shuler, Shain 3. Isolation and Characterization of Immune Cells: Structure and Function CM5 Lawrence, Austin, Lynes 4. Subcellular Molecular Distribution Analysis and Sorting CM6 Austin, Lawrence, Cox 5. Bionanofabrication: In Situ Creation of Nanoscale Polymeric Features CM9 Batt, Coates, Ober 6. Engineering Functional Microvascular Structure in vitro CM10

Stroock, Bonassar, Sato, Shuler, Stokol, Wu

r

29

Development of a Cell Biosensor

Michael Spencer, David Lawrence ocs: Xingqun Jiang, Ling Lu

capture of CD4+ monocytes and non-CD4+ cells and still allow capture of CD4+ T cells.

A three-electrode system was fabricated on a 4-inch precleaned glass wafer by depositing 200 nm-nm-thick Ti. The Au was patterned by lift-off to produce a

wo 300 µm, a counter electrode of 2 mm × 2 mm, together with a reference to 5 mm × 5 mm. The reference electro

opt hloroplatinic acid to increase its surface area. A microfluidic channel with a bout severa

long, able to cover 8 working electrodes in a row. The distance between two consecutive working annel was designed for th

flushing reference and counter electrodes. The pattern of channels were made on a silicon wafer first in a d the PDMS

cleaned for 1 min, and then combined together to produce an encapsulated electrode

o the electrode surface by Au-thiol affinity binding. econd, N-hydroxysuccinimide (NHS) is linked to MPA through a dehydration reaction. Last, CD4+ ntibody is bound to MPA, replacing NHS. CD4+ cell sample was flown through the microfluidic hannel and bound to the antibody-modified working electrodes. The impedance spectrum of the

biosensor was measured in PBS solution by applying 10 mV a.c. bias across the working and the counter electrodes. The frequency ranged from 0.2 to 100 kHz. The impedance spectrum was displayed as either Bode or Nyquist plot. Summary

Accurate quantitation of CD4+ cells in blood requires the measurement of at least 200 cells with a resolution of a few cells. This requires a large working (cell capture) electrode. Traditionally biosensors require a small working electrode for consistent stable operation. We developed a stable biosensor with a very large working electrode (300 µm × 300 µm) and a dynamic range exceeding 100 by fabricating a large platinized reference electrode with an extremely large surface area. We also implemented a covalent antibody linking method to produce a high density of capture antibodies on the cell capturing electrode. This was important to maximize the amount of the captured CD4+ cells and their stability during experimental operations. EIS measurements showed the impedance increased by a factor of 2 after CD4+ cells were attached to the antibody-activated electrode surface. Since capture efficiency and high binding energy are essential, we have developed a new biosensor by incorporating the three-electrode system into a microfluidic channel device. Compared to other open-surface biosensors, this encapsulated three-electrode system was advantageous of having a higher capturing efficiency due to the constrained flow of CD4+ cells over the working electrode in very close vicinity (<50 µm), as well as providing the possibility of circulating the cell sample over the working electrode surface for multiple times to further improve the capture efficiency.

Participating Faculty: NBTC Students/PostdOther Students/Postdocs: Tadahiro Kaburaki, Thomas Zieziulewicz Project # CM1 Objectives

• Develop an on-chip biosensor for quantification of CD4+ cells in human blood sample • Develop an chemically modified electrode surface for capturing antibodies with high efficiency • Quantitate the capture of CD4+ cells • Develop microfluidics to increase shear forces to lower non-specific

Methods

thick Au with an adhesion layer of 20 rking electrode of 300 µm ×

electrode of different sizes, ranging from 2 mm × 2 mm de was ionally platinized in c

width of 2 mm and depth of 50 µm was patterned on PDMS. The channel is a l centimeters

electrodes was 5 mm. Two more channels parallel to the main ch e purpose of

negative tone and then transferred to PDMS. The three-electrode system an cover were in a plasma cleaner

system. CD4+ antibody is immobilized on the surface of the working electrode through three steps: First, 3-mercaptopropionic acid (MPA) is attached tSac

30

Accomplishments • Covalent bonding of CD4+ antibodies to Au working electrode

ody difi d electrode • Impedance change upon attachment of CD4+ cells to the antib -mo ee for higher capture efficiency • Biosensor integrated with a microfluidic devic

Fig.1: Fluorescence Mouse

f antibody (le

images (FITCIgG labeled) of covalent coating of 5 ng CD4+ antibody on chemically modified Au electrode (right) versus physical

ion of the same adsorptamount o

ft)

Fig.2: Impedance spectrum before (red) and after (dark blue) CD4+ cell attachment, and the corresponding phase angle curves (light blue and green). Impedance at low frequency increased by a factor of 2 after CD4+ cells attached.

Fig.3 Schematics of the three-electrode

edded in a system embmicrofluidic channel. The depth of the channel is 50, about 5 times of the diameter of CD4+ cell. The working electrode is 300X300 µm, and the reference electrode is from 2X2 mm to 5X5 mm.

31

Nanotechnological Assessment of Drug Tox

icity

ther Students/Postdocs: G. McAuliffe, J. Sung Project # CM2 O

ple micro and nanofabrication techniques with cell cultures to predict toxicology a aceuticals. Our cell culture analogs (CCA) containing interconnected cell cultures a ic pharmacokinetic models of human. Together the Shain-Shuler group have worked on blood-brain-barrier (BBB) models, and Shuler has worked with R.P. Glahn (USDA-ARS) on G ier models are to be coupled with systemic or “body” models (e.g. liver-lung-fat-other tissue) to provide a detailed prediction of dynamic response of humans to exposure to drugs. Associated studies have focused on using the CCA system to evaluate potential combination therapies for cancer. Methods

design and operation of the systemic CCA model has been published by us in B

Participating Faculty: Michael Shuler, William Shain, R. Glahn, W. Lee Kraus NBTC Students/Postdocs: Hui Xu, J. Frampton O

bjectives Our goal is to cou

nd efficacy of pharmre constructed to mim

I tract models. These barr

The basic approach toiotechnol. Prog. 20:316-323; 338-345; 590-597 (all in 2004). These devices were all fabricated in

s icularly interested in coupling our systemic CCA with “barrier” modules; in particular, the gastro-intestinal (GI) tract and the blood brain barrier (BBB). We are using hydrogel ( -cultures to build BBB mimics and membrane based supports for co-cultures to m . We perform experiments to determine adsorption-distribution-metabolism-e aracteristics of drug or chemical mixtures. New studies for the toxicity of nanoparticles has been initiated using fluorescent carboxylated polystyrene particles (50 to 200nm) in s sing physiologically-based pharmacokinetic models to design appropriate CCA experiments and as a basis for comparison to animal/human response. We are also developing optical techniques to monitor the response of multiple CCA’s. Tissue impedance microscopy is used to characterize 3-D cell-hydrogel mimics of the brain. S

thelial, goblet, and M cells that we have developed may be the most realistic b e GI tract available. We have used this model to show that both 50nm and 200nm carboxylated polystyrene particles interfere with iron transport across the GI tract, but by different m udies are among the first to examine potential chronic side-effects of oral exposure t are also completing initial studies on integration of the GI tract model with a body m irculation.

model using hydrogels (modified alginates) and astroglial, neuronal, and endothelial cells have progressed towards more realistic models of brain tissue and blood brain barrier. The 3-D hydrogel cultures of astroglial cells has been used successfully as a model to evaluate m abricated neural probes.

The CCA body modules are under development for application to colon cancer treatment with drug combinations and for evaluation of response to endocrine disruptors. Optical techniques to monitor cells

multiple chambers on multiple devices have been developed. Potential synergistic interactions of rugs to treat multidrug resistant cancer have been discovered.

ilicon. We are part

modified alginate) coimic the GI tract

limination-toxicity ch

ize. We are also u

ummary The triculture of epi

iological model of th

echanisms. These sto nanoparticles. We odule and systemic c

Our neural tissue

icrof

ind

32

Accomplishments • Item 1 The micro CCA using rea ction of two multidrug

lectively

m 2 Improved I trac el with epithelial, goblet, and M cells shows that both reduce iron transport from the gut to the

emic circulation. The 50 nm particles interfere with diffusional iron transport and the 200

listic doses predicts synergistic interaresistance (MDR) suppressing compounds (cyclosporine and nicardipine) on se

esence of the chemotherapeutic doxcrubicin reducing growth of MDR resistant cancer in the prwithout increased toxicity to other tissues.

• Ite G t modcarboxylated polystyrene particles at 50 and 200 nmsystnm interfere with vesicle-mediated iron transport.

• Item 3 An alginate-RGD hydrogel using various concentrations of astroglial cells was shown to be a good model for evaluating potential reactive cell responses to microprobes in the brain through measurement of electrochemical impedence.

Fig.1: Redesign chip to test combination drug therapy for r. Cell lines representative of each tissue type

Ma

colon canceis culture in the appropriate compartment in 3-D using

trigel for liver and colon and alginate for the bone marrow. Fluid is recirculated through this chip using an off-chip pump and reservoir.

Fig.2: A uCCA device filled with red dye for visualization of fluidics pathways. This uCCA is designed to study environmental endocrine disruptors. Included in this device are cell culture compartments for the liver cells, mammary cells, endometrial cells, and fat-

mimic cells.

Fig.3: Design of custom culture systems for real time impedance measurements from the Neuronexus neural probe using an alginate-astroglial model of brain cortex. The alginate is modified with RGD-peptides.

33

Isolation and Characterization of Immune Cells: Structure and Function

cellular response). Two distinct devices were

ze distribution histograms comparable to that of forward light scatter in a conventional Flow eter but in a much less expensive, on-chip, microfluidic device. An in vitro capillary flow model

was designed to assess alterations in immune cell function. Methods: Leukocyte differential size histograms were produced from a drop of human whole blood o s run through the “bump array” device under constant a and fluidity get bumped further from plasma and subsequently end up in exit channels that are farther away from smaller cell types. The channels in which the immune cells exited the device were evaluated using a fluorescence camera mounted on an i rder. The type of immune cell and its corresponding exit channel infor deo recording and plotted in a histogram format which was compared to the histogram of the same sample produced by the flow cytometer. The next step is to evaluate leukocyte subset distribution or separation directly within the device in real time. The “bump array” will be used as a front end device to a GCSPRI chip, similar to the one shown in Figure 4 of the continued funding proposal. The advantage of this setup is that leukocyte subset distribution or changes in “hydrodynamic” cell size can be determine within the device, in real time and without the use o ithout the use of camera’s, video recorders and the laborious t evice. The GCSPRI chip will have regions of interest ( ture antibodies for each leukocyte subset in each of the “ xit the device they will be captured by their respective capture antibody based on their subset. Since this device will be run with the GCSPRI chip, the cells exiting the bump array can be characterized and quantified by their capture on the ROIs. The change in the degree of the plasmonic refraction from each ROI indicates the number and type of cells evaluated in real-time. Summary: We have shown that the “bump array” microfluidic device can effectively separate different leukocyte types in blood as well as bacterial toxin-activated from non-activated lymphocytes. The device differentially separates leukocytes whose diameter size may only differ by a few microns or less; however, additional chemical or physical parameters, such as membrane rigidity, may influence the t e assayed. The microfluidic device, which separates c e distribution histograms for a mixed cell sample that w ram of the same sample by Flow Cytometry (Fig. 2). This m tinct leukocyte cell types (i.e. CD4+, CD14+ and J45 T-Cells), which were known to have different cellular diameters. This device also showed that its sensitivity is good enough to detect minor increases in cell size due to exposure to staphylococcal entertoxin B (SEB). SEB induces some lymphocytes to become activated and become lymphoblast, which are larger in size compared to normal lymphocytes. This device showed that it could detect this minor change in lymphocyte volume upon activation (Fig. 3).

Participating Faculty: David Lawrence, Robert Austin, Michael Lynes NBTC Students/Postdocs: TJ Zieziulewicz Other Students/Postdocs: David Inglis Project # CM5

Objectives: Microfabricated devices were used to evaluate alterations of immune cell structure (i.e. size, shape, deformability) and function (adhesion, chemotaxis, designed and fabricated in order to individually evaluate each of these cellular parameters (Fig. 1).Alterations of immune cell structure were evaluated using a microfluidic “bump array”, which wasdesigned to separate cells based on changes in “hydrodynamic” cell size. This device would produce siCytom

btained from a finger prick. The whole blood wand repeatable pressure. Cells of increasing size

nverted microscope which is connected to a recomation was extracted from the vi

f florescent stains or markers as well as wask of counting leukocyte subsets exiting the dROI, spotted antibodies in a set grating) with capbump array” exit channels. Therefore, as cells e

rafficking through the device and thus need to bells mainly by “hydrodynamic” size, produced sizas comparable to the forward light scatter histog

ixed sample contained three dis

34

Accomplish• Design and Fabrication of the “Capillary F

nd iapedesis in an “in

e differential requirements to perform the adhesion, emotaxis and diape under flow conditions

namic” cell sizing on a microfluidic chip that

ments low Model” and “Bump Array” Devices

• Successful demonstration of leukocyte rolling, adhesion, chemotaxis a dal fluid flow conditions vitro” capillary model under physiologic

v• Determined that leukocyte subsets hach desis Successfully separated leukocytes by “hydrody• has sensitivity and resolution comparable to that of a commercial flow cytometer

Fig.1: (Left) Microfabricated capillary to assess cellular diapedesis. Device has four input channels and two output channels to control flow. The center barrier wall consists of 3 or 5 micron gaps which cells chemotax through. Test area is 1cm in length and the center wall separates the device into two separate distinct regions except for where the gaps are located. (Right) Top view image of etched silicon device showing central bump array, high fluidic resistance channels, whole blood injection channel, and sand-blasted holes for backside fluid connections.

CAPILLARY DEVICE

Barrier Wall•Pillars

20μM Long5μM Wide

•Gaps3 or 5μM Wide

Waste Ports

25μM

Inpu

t Por

ts

75μM

Inpu

t Por

ts

Test Area

100μM wide

100μM wide

Test Area

Test Area-1cm in Length-100μM Wide on either side of barrier wall-Device is separated into two compartments by a barrier wall- Chemokine can be introduced in soluble form or imbedded in an extracellular matrix gel (“Matrigel”)

Fig.2: Comparison of size measurements for three cell types from the experimental “bump array” device (A) and conventional flow cytometry (B). CD4+ (blue) and CD14+ (black) cells are from whole blood, CD4+ labeled J45 T-lymphocytes (red) from cell culture. (A) The error bars express the standard deviation observed between six independent tests. (B) The forward scatter value for only those cells whose PE fluorescence value was over 200 (of 1024) are included in the plot.

Fig. 3: Comparison of size measurements for blood incubated with the activating toxin SEB using the microfluidic device and conventional flow cytometry. For each method, the SEB sample (red) has a higher proportion of moderately larger cells compared to the control (black). In (A) the number of cells beyond the down-sloping crossover point increased by 2.6 times in the SEB stimulated sample. For the sample measured by flow cytometry (B) the increase was 2.7 times.

35

Subcellular Molecular Distribution Analysis and Sorting Participating Faculty: Robert Austin, David Lawrence, Ted Cox NBTC Students/Postdocs: Chih-kuan Tung Other Students/Postdocs: David Inglis, John Davis, Keith Morton, Peter Galajda Project # CM6 Objectives

Our basic mission for this project is to develop innovative, new ways to sort the contents of single cells using a deep understanding of hydrodynamics at the sub-micron scale. As our sorting technology f , we will use this technique to track adaptation and evolution in cells. M

learn when you cross over into real biological systems is that rarely are two biological objects identical, they are quite often different. Any attempt to find patterns within a b gned to look at a large number of supposedly identical molecules which m

is from the start, and our original design used photolithography to make a s, and standard photolithographic techniques remain a core technology in our work. However, photolithography cannot be used to make sub-micron width objects easily, and electron-beam lithography is tedious and an expensive way to make nano hannels over large (cm x ) areas. One of our collaborators, Professor Steven Chou at Princeton University (Electrical Engineering), has developed ways to transfer patterns of nanochannels into substrates that be used as etching masks, P Morton has developed ways narrow the channels and create arrays with feature sizes approaching 10 nm.

in interfacing biological objects with cellular components is the i ts with the materials used in microlithography. Since interrogation of p e using various spectroscopic methods, it is necessary that the materials o indows on the microfabricated flow cell are made are transparent to the desired wavelengths. At the same time it is important that the chosen materials can be m at allow appropriate fluid manipulations such as fast mixing required f ding kinetics. While silicon still remains the material of choice for high aspect ratio feature etching, materials traditionally used spectrograph cuvettes such as fused silica (also called quartz or amorphous quartz) or calcium fluoride are most desirable as the observation windows d rescence and hi transmittance in the UV, visible and IR range. This creates c ar materials or to fabricate the structures in optical materials for which high a readily available.

nanofabrication area one meets further problems: sealing to nanostructures n t the molecular level, surface properties become ever more important as the s pressures needed to maintain flow rates of interest rise to the kilobar level r Also, surface defects in materials become more important as sizes shrink these defects can act as blockages in nanodevices. Much or our recent work involves dealing with these q

or subcellular objects develops

ethods One of the early lessons you

iological system must be desiay have a large variance.

We have been aware of thrrays of obstacles and channel

c cm

rof. Chou’s grad student keith

Another substantial problemnteraction of cell componenroteins and nucleic acids is donut of which the observation w

icromachined into structures thor measurements of protein fol

ue to their low fluohallenges to bond dissimilspect ratio etching is not so

As one moves into the ow must be truly hermetic aurface/volume ratio rises, the equiring very strong bonds.

uestions.

gh

36

Accomplishments • Theoretical models and exper

ays.

ling of array and els wn to sub-100 nm sizes for true molecular fractionation.

imental tests made for the critical particle size of fractionation in deterministic arr

and evolution using nano/microtechnology coupled • New technology for probing adaptationwith genomics and ecology.

• Sca nanochann do

Fig.1: Experimental poi

array, versus the row ion.. Open

poi

str

nts of the particle diameter divided by the gap size in a deterministic bump

shift fractnts represent

bumping mode where particles move at an angle to the flow, solid points represent

aight motion with the flow. The solid line is our theoretical prediction of the change in motion.

Fig.2: True color view of two competing strains of bacteria (one green the other red( battling in a microhabitat array.

Fig.3 SEM of a complex patterned array used to separate cellular components after lysis of the cell.

37

Bionanofabrication: In Situ Creation of Nanoscale Polymeric Features

Coates, and Christopher K. Ober

athew (undergraduate student)

echanism of enzymatic surface-initiated polymerization (ESIP) of ate (PHB) and to enhance the growth of PHB on the solid surfaces interaction between various mammalian cell lines and the ESIP PHB derivatized

e their performance for 2D patterning of mammalian cells. e of ESIP PHB as a surface modification for 2D patterning of mammalian cells.

M ctionalization of ESIP PHB on the solid surfaces. PHB is a biodegradable a phatic polyester produced by a variety of microorganisms as a reserve energy s cific attachment of the key catalytic enzyme, PHA synthase, on lithographic-f sequent addition of 3-hydroxybutyryl-CoA substrates (3HB-CoA), allowed us to create spatially ord eric micro-/nano-structures on the patterned surfaces via in situ E albumin (BSA) proteins were added during the immobilization as well as the p n order to enhance PHB surface polymerization forming thick PHB-BSA films up t full surface coverage (Figure 1A). By using biotinylated BSA conjugates, we w rporate biotin groups into the PHB polymer matrix, thus generating a bioactive surface that can be used for displaying other functional biomolecules through streptavidin-biotin interaction on t igure 1B). I y studies of mouse embryonic fibroblasts and embryonic stem cells on ESIP PHB and ESIP PHB-BSA fabricated surfaces. Various gold patterned substrates used for our surface p studies were fabricated using standard lithography techniques housed within N We also collaborated with Professor Jun-Lin Guan (College of Veterinary Medicine, Cornell University) to study the interaction between mouse embryonic fibroblasts (MEFs) and the fabricated ESIP PHB patterned surfaces and with Professor Tom Sato’s lab (Weill Medical College of Cornell University) to study mouse embryonic stem (ES) cells. Summary: Cell studies performed with MEFs showed a substantially increased cell attachment and proliferation on the fabricated PHB surface as compared to the unmodified surface. Moreover, we demonstrated that micropatterning of PHB structures created through a combination of parylene lift-off and in situ biofabrication of PHB could be used for mammalian cell patterning (Figure 2). Besides MEF cells, we also evaluated the biocompatibility assessments of the fabricated PHB surface with mouse ES cells. Our preliminary results suggested that the in situ fabricated PHB polymer on a planar gold surface could provide a favorable 2D matrix for ES cells maintenance and propagation. More embryonic bodies ( two days of cultivation on this PHB surface in comparison to the unmodified gold surface. In addition, these resulting EBs were found to remain in an undifferentiated state for over a week without the need for fibroblast feeder cells (Figure 3A-B). Besides the PHB planar surface, we also evaluated the effect of a simple 2D geometrical shape of the ESIP-PHB and found that the mES cells localized onto the PHB stripes, especially during and after EB formation (Figure 3C-D). Our current goal is to use the fabricated PHB surfaces as a promising biomaterial for culturing and differentiating ES cells into other cells, such as epithelial cells, nerve cells, and bone cells. In addition, we will evaluate the effects of different geometric patterned surfaces fabricated with PHB, which we hope to find out information that will offer us new levels of control over ES cell behavior.

Participating Faculty: Carl A. Batt, Geoffrey W. NBTC Students/Postdocs: Nuttawee Niamsiri (PhD graduate student) Other Students/Postdocs: Esha MProject # CM9 Objectives: • To understand the m

polyhydroxybutyr• To investigate the

surfaces and evaluat• To evaluate the us

ethods: Growth and funnd biocompatible aliource. Here, site-speabricated surfaces and sub

ered PHB polymSIP. Bovine serum olymerization steps io 1 μm in height with ere able to inco

he PHB structures (Fn vitro biocompatibilit

olymerization and cells BTC & CNF at Duffield Hall.

EBs) were formed after

38

Accomplishm• Obtained a better understanding about the mechanism of ESIP PHB on the solid surfaces and could

he Eorable scaffolds for 2D

surf es are biocompatible for maintaining the culture of mouse

embryonic stem cells in an undifferentiating state

ents

further functionalize t SIP PHB matrix via biotinyated BSA • Demonstrated that the fabricated ESIP PHB surfaces could be used as fav

patterning of mammalian cells • Proved that the fabricated ESIP PHB ac

Fig.1: (A) AFM images of the unmodified planar gold surface and the modified planar gold surface with either PHB or PHB-BSA via ESIP (scan size 20 x 20 μm). (B) Biotin functionalized PHB patterned surfaces through the incorporation of biotinylated BSA into the PHB polymer matrix. Fluorescence analysis of biotin-derivatized PHB patterned surfaces after the binding of Alexa Fluor® 488-conjugated streptavidin.

Fig.2: Fluorescence images of surface synthesized PHB on patterned surfaces after staining with Nile blue dye, and mouse embryonic fibroblasts (MEFs) attachment on fabricated PHB surfaces after 24 hr of cell culture (Scale bars = 100 μm)

Figure 3. (A) EBs formation on Day 2 from initial cell seeding onto a planar PHB surface (no pattern). (B) EB clusters on the PHB surface stained for Oct4 (green) to indicate the undifferentiated state of ES cells. (C) EB formation on Day 4 from initial cell seeding onto the PHB patterned surface. (D) Oct4 staining on the PHB patterned surface on Day 7 (green), indicating undifferentiated cells.

100 μm

PHB directs the attachment of MEF cellsPHB Micropatterned Surfaces

100 μm

100 μm

PHB directs the attachment of MEF cellsPHB Micropatterned Surfaces PHB directs the attachment of MEF cellsPHB Micropatterned Surfaces

100 μm

10 μm

BSAPHA synthase

Biotinylated BSAPHB polymer

Au Au BSAPHA synthase

Biotinylated BSAPHB polymer

Au Au

ESIP PHB ESIP PHB-BSA

0 10.0 20.0 μm

0 10.0 20.0 μm

0 00 10.0 20.0

μm

0

Au alone20.0 20.0 20.0

10.0 10.0 10.0

ESIP PHB ESIP PHB-BSA

0 10.0 20.0 μm

0 10.0 20.0 μm

00 000 10.0 20.0

μm

00

B

Au alone20.020.0 20.020.0 20.020.0

A

10.010.0 10.010.0 10.010.0

10 μm10 μm10 μm

BSAPHA synthase

Biotinylated BSAPHB polymer

Au Au BSAPHA synthase

Biotinylated BSAPHB polymer

Au Au

ESIP PHB ESIP PHB-BSA

0 10.0 20.0 μm

0 10.0 20.0 μm

0 00 10.0 20.0

μm

0

Au alone20.0 20.0 20.0

10.0 10.0 10.0

ESIP PHB ESIP PHB-BSA

0 10.0 20.0 μm

0 10.0 20.0 μm

00 000 10.0 20.0

μm

00

B

Au alone20.020.0 20.020.0 20.020.0

A

10.010.0 10.010.0 10.010.0

39

Engineering Functional Microvascular Structure in vitro

Larry Bonassar (co-PI – BME and ME and

) Tracy Stokol (co-PI – Vet. Med); Mingming Wu (co-PI – CBE). erie

ther Students/Postdocs: Kevin Wong (CBE), Dr. Eugene Kalinin (CBE), Karla Comacho (CBE), Ankur Chaudhury (BEE), Rosa Rosales (CBE), Dr. Daniel Rhodes (CBE), Christopher Lee (MSE), Inna Lipchina (Weill).

Project # O

uidic and micromechanical methods to study and control single cell and m mensional (3-D) cultures in vitro. 2) Develop optical and biochemical t lar dynamics with sub-cellular resolution within 3-D cultures.

rs and dynamics of vasculogenesis and angiogenesis. 2) Develop an a l barrier as it serves to mediate metastasis and pharmacokinetics. 3) E tructure within 3-D scaffolds for tissues in the context of regenerative m Methods

1) Living lithography (Stroock, Bonassar). We have adapted soft lithographic methods to transfer photolithographically defined features into cell compatible hydrogels such as calcium alginate, agarose, and collagen with cells in the bulk and on surfaces. Living cells can be present in the bulk of the material and plated onto the surfaces. 2) Endothelializable microfluidic networks (Stokol, Shuler). We h lti-scale networks of semi-cylindrical microchannels in silicon (xenon f s have been transferred into polystyrene for casting into PDMS. We have p ial cells in these channels. 3) Microfluidic migration assay mammalian c We have developed a microfluidic platform in agarose that allows for i iple soluble factors on mammalian cells. The fluid in the cell chamber can be either stationary or flowing. Summary

Our team has made significant advances toward uniting microfabrication and microfluidics with complex, 3-D cell culture. We have used hydrogel-based microfluidic devices to allow for the control of the soluble chemistry in the microenvironment of cells via convective mass transfer. As illustrated in Fig. 1, channels embedded with a hydrogel act as a vascular system that can control the chemical state of t temporally (Fig. 1a) and spatially (Fig. 1b and 1c). In order to form p tes for vascular structure, we have developed new fabrication processes i ational networks of channels with round cross-sections (Fig. 2). In an i tanding the physical cues that control the initial stages of vascular d the effects of geometrical and mechanical characteristics of a 3-D culture on the behavior of vascular endothelial cells (Fig. 3). For example, we find that mechanical c ensity have dramatic impacts on cellular self organization. These studies have allowed us to identify conditions that lead to the formation of extensive vascular networks in vitro (Fig. 3c). These advances represent exciting steps for advanced cell culture and form basis engineering microstructure with which to study and control the development of microphysiological structure in vitro.

Participating Faculty: Abraham Stroock (PI – CBE);

MAE);Thomas Sato (co-PI – Weill); Michael Shuler (co-PI – BCBE

NBTC Students/Postdocs: Brittany Held (Chem), Jong Sung (CBE), James Camp (BME), ValCross (BME)

O

CM-10

bjectives Tools: 1) Develop microfl

ulticellular dynamics in three-diechniques to characterize cellu

Studies: 1) Elucidate driveccurate model of the endotheliangineer functional vascular sedicine.

ave designed and fabricated muluoride etch). These structureerformed cultures of endothelell micrgration assays (Wu). mposition of gradients of mult

he bulk of a 3-D culture bothhysiologically relevant templan silicon to create multi-genermportant step toward undersevelopment, we have mapped

onfinement and cell-seeding d

40

Accomplishments • Demonstration of microfluidic scaffolds for spatial and temporal control of soluble chemistry

res• Map of effects of geo• Endothelialized netw

in 3-D tissue cultu . metrical parameters on vasculogenesis in vitro. orks of microchannels for studies of cancer metastasis.

Fig.1: Microfluidic tissue scaffolds. (a) microfluidic e points during

reen)

Fluorescence micrographs ofscaffold in calcium alginate at tim

livery of solutes (fluoine B (red)) to scaf embedded microchannels. (b) micrograph of chondrocyte-seeded

mi

sequential de rescein (gand rhodam fold via single network ofFluorescence

crofluidic scaffold in calcium alginate after delivery green vital stain to left network and red stain to right network. (c) Cross-section of top layer of microfluidic scaffold in (b). Scale same in (b) and (c).

Fig.2: Model networks for the study of cancer (a) Network architectures with

diametastasis.

meters for each level of structure. (b) Scanning electron micrograph of a junction in network etched into silicon via an isotropic etch with xenon difluoride. (c) Fluorescence micrograph showing cross-sectional view of human endothelial cells grown on a fibronectin-coated silicon channel. Scale bars = 30 μm.

Fig.3: In vitro vasculogenesis as a function of eters. (a) 3-D cell culture in

en gel of thickness, H, and with ini

geometrical paramconfined collag

tial cell-cell spacing, λcell. (b) Diagram of cellular structures formed as a function of λcell and H. (c) Composite micrograph of 2-D percolating state formed in 400 μm-thick culture with λcell = 100 μm. (d) Flourescence confocal micrograph of the vertical cross-section of a culture of VECs as in (c). The walls of capillaries are visible (bright) due to the staining of cytoskeletal proteins in the cells.

41

BLANK PAGE

42

Nanoscale Cell Biology - Program Overview Program Coordinator: Manfred Lindau Program Objectives The Nanoscale Cell Biology Program of the NBTC is aimed at approaching a mechanistic understanding of cellular function with nanoscale precision. To achieve this aim new nano- and microscale tools are developed and applied. Microfabricated devices are also developed to probe cellular function in highly parallel manner for proteomics and drug testing. The approaches include nanoparticles, molecular force measurements, small angle x-aray diffraction, carbon nanotubes, integrated chip design, nanofabricated devices and molecular design. All projects involve interdisciplinary teams with PIs, post-docs and students from many different disciplines supplemented by outside collaborations on the national and international level.

Research Projects for 2008

1. Tracking Single Molecules and Transport Ve icles in Living Cells Using Fluorescent Nanoparticles NCB1

Lindau, Wiesner, Webb, Baird 2. A Scalable n x n Electrochemical Pixel Array with Integrated Electronics for Use as a Highly

Parallel Biosensor Device NCB2 Lindau, Ober

3. Fabrication of Nanoscale, Dendrimer-like plates NCB3 Luo, Baird

4. Nanotubes as Cellular Biosensors NCB4 McEuen, Lindau, Craighead

5. Development of Novel Applications of Reve e Transfection for High Throughput Cell-Based Functional Screening NCB6

Maxfield 6. Electrochemical Imaging of Exocytosis Using Microfabricated Devices NCB8

Lindau, Almers, Craighead, Baird, Ober 7. Dynamics of Viral Fusion Proteins NCB9

Pollack, Whittaker, Crane 8. Chromatin Structures, Function, and Dynam s: From Mononucleosomes to Polytene

Chromosomes NCB10 Kraus, Lis, Wang, Webb

s

DNA Structures as Multivalent Tem

rs

ic

43

Tracking Single Molecules and Transport Vesicles in Living Cells Using Fluorescent Nanoparticles

: Manfred Lindau, Uli Wiesner, Watt Webb, Barabara Baird

ostsynaptic densities, neurotransmitter receptors, ion channels and specific mRNAs distal dendritic locations, as well as components of presynaptic terminals, adhesion molecules, and itochondria to axonal locations. These cargoes are carried by molecular motors that tread along an

bules. In this project we focus on the use of fluorescent nanoparticles to vestigate the motion of the vesicles along microtubules, of secretory vesicle recycling in neurons as

vestigated the understanding of synaptic targeting, synaptic plasticity, vesicle

cell membrane. er-

ethods [1, 2]. The average particle size was significanl-dye) sensors with hydrodynamic diameters below 15 nm, while maintaining

cle access to confined vesicular and intracellular locations, such as synaptic vesicles articles. Poly(ethylene glycol) (PEG) coated particles

nginee stability in buffers, due to their steric (rather than electrostatic) le

-clogging by the particles. Prototyperat dots were synthesized including a number of multifunctional particle architectures,

porosity, reporters capable of localized targeting, stimulation,

intracellular vesicle tracks, which have intrinsic structural polarity. ratiometric

eters (Fig. 1), which can more easily access the confineter), and other intracellular locales. As well, we have developed

ynthetic protocols for attaching oligomeric chains of hydrophilic polymers such as poly(ethylene nhance their stability against agglomeration. These particles were

successfully introduced into single cells using single cell electroporaton (Fig. 2). A microscopy setup was built that can collect both the forward- and backward-directed fluorescence

and second harmonic emissions. Using this setup, we imaged microtubules in individual neurites within acute brain slices. To address the question whether microtubule polarity is different for various neurite types in vivo, we prepared acute slices from Thy1-YFP line-H transgenic mice, which labeled a subset of neurons in the hippocampus and neocortex. Using SHG and multiphoton microscopy (Fig. 3), we found, in addition to axons, large stretches of apical dendrites that contain uniform polarity microtubules. These arrays can extend for more than 270 µm with an estimated polarity of 83% and are presumably mediating the rapid transport of AMPA receptors in dendrites that we observed using fluorescent nanoparticles [4].

Participating FacultyNBTC Students/Postdocs: Andrew Burns , Raymond Molloy, Alex Kwan Other Students/Postdocs: Project # NCB1 Objectives: The ultimate goal of understanding cellular function on the nanoscale requires to imaging of the movement of molecules and organelles and monitor their function with nanometer precision and high time resolution. Produced in the soma, anterograde vesicular transport through neurites delivers cargoes including ptomextensive network of microtuinwell as movements of membrane proteins in the cell plasma membrane. The questions to be inhere are particularly relevant torecycling and receptor dynamics in the Methods: Calcium- and pH-sensitive fluorescent silica nanoparticles were synthesized by both Stöbbased and reverse micelle-based m tly decreased, yielding ratiometric (duathe enhanced optical properties that the core-shell silica architecture is known for. This decrease in parti size facilitateswhich would be inaccessible to larger p were e red, which exhibit enhancedstabilization as well as minimal protein adsorption. These could be introduced into single cells by singcell electroporation (SCE) [3] avoiding excessive tip s for the next gene ion of C incorporating e.g. multiple sensing capabilities, magnetic materials and towards the development of multifunctional single particle reporting and recovery. Second harmonic generation (SHG) microscopy is a nonlinear optical technique employed to image microtubules, the Summary: We have adapted the C dot sensor synthesis techniques to create core-shell sensors with sub-15nm diam ed volumes of synaptic vesicles (~30nm diamsglycol) to these particles to e

44

Accom•

of sensing pH or Ca+2 concentrationc adso ption

n in uniform polarity microtubules that can extend for more than 270 µm f 83%

plishments Synthesized Ratiometric core-shell nanoparticles with hydrodynamic radii of <15 nm, capable

in solution. • PEG-coated particles with enhanced particle stability and decreased non-specifi r

were introduced into cells using single cell electroporation • Apical dendrites co ta

ing an estimatedhav polarity o .

Fig.1: a. A schematic representation of the core-shell silica nanoparticle sensor architectures used in this work, with the key attributes of the particles highlighted. b. A scanning electron microscopy image of 8-10 nm core-shell Cy5/Fluo-4 Ca+2-sensor particles. c-d. Plots of calibration data for the Cy5/Fluo-4 particles from spectrofluorometry

Fig.2: Single Cell electroporation of bovine chromaffin cells was used to load cells with FITC/Cy5 Sensor Dots (green).

Fig.3: Uniform polarity microtubule arrays in the neocortex of adult Thy1-YFP mice. This is a composite image (left panel), of multiphoton-excited YFP fluorescence (green) and SHG (red), in layer V cortex of a 11-month old mouse. The same region, shown with SHG only, is shown in black and white (right panel) for better contrast. Uniform polarity microtubule arrays co-localize with apical dendrites of layer V pyramidal neurons (arrowheads). Scale bar = 30μm

45

A Scalable n x n Electrochemical Pixel Array with Integrated Electronics for Use as a Highly

Quantitative measurements of neurotransmitters have wide applications in basic sciences as well as

medical treatments. The goal of the project is to design, fabricate, and characterize active miniature electrochemical pixel array devices to assay quantal exocytosis from thousands of cells in a day. Using appropriate chemical modification with charged polymer brushes, such devices are also able to detect m zable with high sensitivity and with reduced influence by interfering s dy of glutamate release and general biosensing applications. M

echniques and deep-submicrometer CMOS foundry processes are e ay that measures amperometric currents with on-chip amplifiers and r ifier (RCA) potentiostat was designed and fabricated at MOSIS with 0 noise comparable to that of patch clamp amplifiers [1]. In post-processing, the focused ion beam is used to remove native oxide on the aluminum electrodes and then deposit platinum (Pt) (Fig. 1a). Electrochemical detection is tested applying dopamine at various concentrations using a Picospritzer and measuring catecholmines released from chromaffin cells. The potentistat circuit maintains the voltage applied to the electrode at 0.7 V.

For chemical modification, surface initiated atom transfer radical polymerization (SI-ATRP) is used to grow poly(sodium methacrylate)-b-poly (2-hydroxyethyl methacrylate) block copolymer brushes on gold electrode surfaces. Fourier transform infrared spectroscopy (FTIR) was used to characterize the structure of the polymer brushes (Fig. 3a). Cyclic voltammetry (CV) and amperometry were used in combination to detect ascorbic acid on both bare gold electrodes and modified gold electrodes. F o study the protein immobilization on the brushes. S

On-chip detection of dopamine was demonstrated recording currents in response to ejection of 7uM dopamine the pipette onto a single platinum electrode on the chip (Fig. 1b). This demonstrates for the first time electrochemical detection on a CMOS chip. To measure cellular exocytosis using our amplifier we connected an external carbon fiber electrode to the potentiostat on a chip without post-processing. F composed of amperometric spikes (Fig. 2b,c). with amplitudes of ~ pical values for exocytotic events from chromaffin cells. The circuit i affin cells and is furthermore flexible such that i on-chip electrodes or alternatively connected to external electrode a ssed strong interest in marketing the technology.

d that block copolymer brushes were successfully grown on gold e e carboxylic groups are ionized at pH 7.4 CV results using block c ctrodes showed that ascorbic acid oxidation peak was shifted to a m odified electrodes (Fig. 3b) whereas CV of dopamine was not significantly affected. The hydroxyl groups on poly(2-Hydroxyethyl methacrylate) can be activated by t lize proteins at high density (Fig. 3c). Suggesting that this method m me-modified electrochemical detectors.

Parallel Biosensor Device Participating Faculty: Manfred Lindau, Christopher Ober, Bradley Minch NBTC Students/Postdocs: Sunitha Bandla, Rong Dong Other Students/Postdocs: Khajak Berberian, Kassandra Kisler, Project # NCB2 Objectives

olecules that are not per se oxidipecies. Such devices enable the stu

ethods Ultra-low-level circuit design t

mployed in developing a pixel arread-out. A regulated cascade ampl.5 µm CMOS technology with a current

luorescence microscopy was used t

ummary

ig 2a shows amperometric currents100 pA and half width of ~5ms, ty

s thus capable to detect single release events from chromt can either be fully integrated withrrays. The company ALA has expre

FTIR experiments demonstratelectrode surfaces and most of thopolymer brush-modified gold eleuch higher voltage compared to unm

osyl chloride and then used to immobiay be used to engineer enzy

46

47

A• First electrochemical CMOS • Measurement of cellular exocytosis using our potentiostat circuit

rface and used to

ccomplishments chip detection of dopamine

• PMAA-b-PHEMA block copolymer brush grown on gold electrode suimmobilize proteins

Fig.1: (a) Pt deposition on Al pads ng focused ion beam. A haze of

Pt around the electrode can make it difficult to pattern Pt on dense arrays with this method. (b) On chip detection of 7uM dopamine ejection

usi

Fig.2: (a) Amperometric recordings using a carbon fiber electrode (inset) connected to our amplifier. (b). A single event with an amplitude of 120pA and half width of ~5ms. (c) Another event with an amplitude of 60pA and half width of ~5ms.

Fig.3: (a) Cyclic voltammetry of ascorbic acid on bare gold electrode

and polymeelectrode su

r brush modified gold

of rface. (b) FTIR spectra

PMAA brushes and PMAA-b-PHEMA brushes on gold surface after rinsing with PBS buffer. Most of the carbonyl groups show the peak at around 1580 cm-1, which corresponds to ionized carboxylic acid groups (c) Image of fluorescently labeled BSA immobilized on PHEMA brushes activated by tosyl chloride.

18161412108.06.04.02.020

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60

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)

7uM Dopamine Injection (b) (a)

7.1 7.2 7.3 7.4 7.520

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Fabrication of Nanoscale Dendrimer-like DNA Structures as Multivalent Templates Participating Faculty: Dan Luo, Barbara Baird NBTC Students/Postdocs: Dwaipayan Sil, Jong Bum Lee Other Students/Postdocs: Chikako Torigoe

roject # NCB3 Objectives

er-like DNA structures that can be modified with specific groups on 5’ t tructures serve as multivalent templates to investigate how antigenic a e clustering of cell-surface IgE-receptors (FcεRI) and consequent s lls. The purpose is to identify structural constraints within the signaling m lated by the structural properties of antigens..

Methods

Nucleic acid engineering: designing, synthesizing, and annealing Y-shape DNA with selected structural features; Chemical modification of DNA ends with fluorescent and antigenic groups (e.g., DNP); fluorescence resonance energy transfer to measure distances between pairs of 5’ ends; Fluorescence spectrometry to measure binding of DNP-DNA ligands to IgE in solution and on cells; Gel p analyze formation of DNP-DNA-IgE oligomers; Immunoprecipitation a stimulated tyrosine phosphorylation of signaling proteins; Assays for s ding Ca2+ mobilization and degranulation; Fluorescence microscopy t cs of DNP-DNA crosslinked IgE- FcεRI on cells. S

igen-mediated crosslinking of IgE bound to its receptor, FcεRI, stimulates degranulation and other responses in mast cells and basophils to initiate allergic responses. To investigate possible roles for structural constraints of crosslinked receptors on signaling, we utilized double-stranded DNA spacers to synthesize ligands with tunable lengths and valencies. We produced and characterized a series of symmetric trivalent ligands with rigid spacing between 2,4-dinitrophenyl (DNP) haptenic groups in r ds all bind to anti-DNP IgE on RBL mast cells with similar avidity, and they all crosslink IgE receptor complexes effectively. They initiate length-dependent tyrosine p γ subunits and the adaptor protein LAT that are ~5-10-fold less for the l ortest. Receptor-mediated Ca+2 responses exhibit different kinetics and m d length, and degranulation responses parallel differences in these Ca+2 r hosphorylation. In contrast, tyrosine phosphorylation of phospholipase C s are relatively length independent, indicating differences in signaling p lux and release from stores. The length dependence of FcεRI p onsistent with a transphosphorylation model of receptor activation leading to degranulation, whereas length-independent activation of PLCγ1 reveals unanticipated d ional signaling pathways for this process.

P

This project utilizes dendrimermni. These rigid, branched srchitecture determines effectivignaling events in RBL mast ceechanism and the particular steps that are regu

ermeation chromatography to nd western blotting to examine timulated cellular signaling, incluo assess distribution and dynami

ummary Ant

ange of 4.5-13.5 nm. These lig

hosphorylation of FcεRI β and ongest ligand compared to the shagnitudes as a function of ligan

esponses and in FcεRI tyrosine pγ1 and Ca2+ release from storeathways important for Ca+2 infhosphorylation that we observe is c

ivergence from convent

an

48

Accomplish

-sha f dif rent lengths that bind with same affinity to

Y-DNP-DNA ligands and revealed gth-based discrim ong gnaling pathways in RBL mast cells.

• d DNA templates with fluorescence resonance gy transfer

ments • Prepared rigid Y ped DNP-DNA ligands o fe

IgE and cluster multiple IgE-receptors on cells t ar• Assessed several cellular activities tha e stimulated by

len ination am siConfirmed rigidity and assess shape of Y-shapeener

RBL mast cells that leads to

kin

Fig.1: Schematic of IgE-FcεRI crosslinked by the rigid Yn-DNP3 on the surface of transmembrane coupling with signaling

ases Lyn and Syk.

Fig.2: Using fluorescence resonance energy transfer (FRET), we have

determined the molecular sstingly, it is no

A

hape of Y-t entirely flat. DNA. Intere

model based on our calculation is built as shown.

Fig.3: Degranulation of RBL-2H3 mast cells stimulated by Yn-DNP3 ligands β-Hexosaminidase release from cells after stimulation with varying doses of specified Yn-DNP3 ligand in the presence of 2 μM cytochalasin D. Data shown are from a single, comparative experiment that is representative of 5-10 different experiments with each ligand. Error bars represent range of triplicate samples. Degranulation is quantified as the percentage of total β- hexosaminidase released in TX-100 cell lysates. 0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2 0

4 0

6 0Y 1 6 -D N P 3Y 2 6 -D N P 3Y 3 6 -D N P 3Y 4 6 -D N P 3

% D

egra

nula

tion

[D N A ] (n M )

49

Nanotubes as Cellular Biosensors

bjectives Our principal objective is to develop a new class of carbon nanotube devices to probe the properties

of both cell membranes and supported lipid membranes and cell membranes. Of particular interest is the use of nanotubes for the detection of chemical, optical, and electrical signals at the surface of cell m poral resolution. Also of interest is the physical perturbation that t ane and its effect on the structure and function of the membrane. Finally, we are interested in nanotube/supported lipid bilayer devices as platform to detect and sort m oteins. Methods

In previous work, we demonstrated the formation of continuous supported lipid bilayers over nanotube transistors (see upper inset in Figure 1). In the last year, we fabricated improved device geometries using aluminum oxide to isolate the electrodes from the electrolyte solution. Electrical m e formation of the SLB dramatically shifts the threshold voltage of the transistor, as shown in Figure 1 (dashed red curve). Furthermore, if functionalized lipids are incorporated in the membrane, the binding of proteins to the membrane can also b curve).

e interaction of cells with two different kinds of nanotube transistor devices was investigated. The first type were the same as those discussed above, with the nanotube resting on a fused silica substrate. In the second, a micron-sized trench was etched in the substrate, leaving a portion of the nanotube suspended. A micropipette was used to press a cell onto the nanotube, a l was seen. If, on the other hand, a portion of the tube was suspended, a large change in the conductance of the transistor was observed ( hift in the threshold voltage of the transistor (Fig. 3 right). This i the cell membrane and the nanotube is required. S

anotube transistor can be used to electrically sense both cell m rs. In supported lipid bilayer devices, we further demonstrated that t membrane could be easily detected. In the case of cell membranes, w nd the carbon nanotube in order to obtain a signal. These results d are a powerful new tool for exploring the local electrostatic and physical properties of lipid membranes.

Participating Faculty: Paul McEuen, Manfred Lindau, Harold Craighead NBTC Students/Postdocs: Xinjian Zhou, Sunitha Bandla (1/2) Other Students/Postdocs: Lisa Larrimore, Jose Miran-Mirabal, Kassandra Kisler, Samantha Roberts,

Yaqiong Xu Project # NCB4 O

embranes with high spatial and temhe nanotube produces on the membr

embrane-bound pr

easurements of these devices demonstrate that th

e electrically detected (dotted green In a second set of experiments, th

s shown in Figure 2. If the nanotube rested on the substrate, no signa

Figure 3 left) corresponding to a sndicates that intimate contact between

ummary Our results demonstrate that a n

embranes and supported lipid bilayehe specific binding of proteins to the e found that it was critical to suspeemonstrate that nanotube transistors

50

Accomplishments • Created improved carbon nanotube

u ported ipid b ayer e evices with c lls.

devices for membrane sensing • Used nanotube as electronic sensor to detect binding of streptavidin to s p l il

nded nanotub d e• Performed first experiments electrical interaction of suspe

Figure 1: Detection of tin/streptavidin binding with

SWNT FET. Conductance G of nanotube transistor as a function of electrolyte voltage before (black) and after (dashed red) formation of biotinylated lipid bilayer on surface. The dotted green curve was measured after streptavidin was bound t

bio

o the biotin. Lower inset: Optical

f the device used for micrograph osensing. Upper inset: Schematic of nanotube device with functionalized lipid bilayer.

After lipidbilayerformed

After Strept.binding

Figure 2: Setup for probing interaction between suspended nanotube transistors and cells. Left: optical micrograph (top view) of a cell held by a micropipette pressed onto a nanotube transistor. Right: Schematic cross-section showing cell and nanotube transistor.

Figure 3: Electrical response of nanotube transistor to chromaffin cell. Left: Conductance G versus time as a cell is lowered onto the nanotube. Right: Conguctance versus electrolyte gate voltage showing that the interaction with the cell shifts the threshold voltage of the transistor.

51

Development of Novel Applications of Reverse Transfection for High Throughput Cell-Based

BTC Students/Postdocs: Nina Pipalia, Ph.D.

Project # NCB6 Objectives

ct is to develop assays to enable high throughput screening using reverse transfection technology. The major effort has been in the adaptation of the reverse transfection technology to a high throughput format. Considerable progress in overcoming technical obstacles has b ing specific examples of problems in which this technology may be u en the expre everal different src family kinases in arrayed g f a 96-well pla e effects of potential chemical inhibitors of these k is way, effect h compound on many different kinases could be dM

previously has been shown to allow fabrication of reverse transfection m elatin-based printing ink. This is important because viscous protein-containing inks can potentially lead to clogging of the printing pin and irreproducible array printing. A modified dextran surface has been shown to give good performance for reverse transfection using a printing ink containing only buffer. Further surface modifications are being developed and evaluated to optimize assay performance (i.e., increased transfection efficiency, decreased cell toxicity, etc.).

We have adapted the reverse transfection assay from the conventional slide format to a high t reverse transfection assay has essentially three steps: printing the m lls to the array surface, and imaging the resulting transfected m eps required modification for a high throughput setting. To this end, p gned to print DNA constructs in 96-well microplates. Protocols for a ll addition, washing and fixing steps, have also been developed.

on methodology for reverse transfection applications, enabling the screening and quantification of non-epitope tagged constructs. This co-transfection method was used to study in parallel the structure/function of multiple versions of the v-Src protein. The wild-type v-Src protein and four mutants having insertions or deletions in the SH2 or SH3 domains displayed high levels of tyrosine kinase activity in HEK293T cells. Three other mutated v-Src proteins, including a kinase-dead version, were shown to be defective for tyrosine kinase activity.

family tyrosine kinases in arrayed clusters in a single well and t e level of phosphotyrosine in the cells. S

ction method using reverse transfection technology that allows us to e proteins even if they do not have a fluorescent tag or an epitope tag. This can be used in a multi-well format for high throughput screening of chemical libraries against m in different patches of cells in a single well. We are now developing this technology for testing effects of chemical inhibitors on tyrosine kinases.

Functional Screening Participating Faculty: Frederick Maxfield, Fawad Faruqi (Corning) NOther Students/Postdocs:

The overall objective of this proje

een made, and we are now testseful. One such example has beroups of cells within each well oinases could then be tested. In thetermined simultaneously. ethods

A surface chemistry identified icroarrays without requiring a g

ssion of ste. Ths of eac

hroughput microplate format. The icroarrays, adding mammalian ceicrospots. Each of these three st

rinting programs have been desiutomated cell culture, including ce

We developed a co-transfecti

We have now expressed several src ested the effects of inhibitors on thummary

We have developed a co-transfexamine the effects of transfected

ultiple target proteins expressed

52

A• Demonstration of Tyr kinase act forms • Development of high throughput immunofluorescence assay for phosphotyrosine

ccomplishments ivities of multiple src iso

• Testing chemical inhibitors of tyrosine kinases

Fig.1: Parallel functional analysis of multiple mutant versions of t everse transfectionhe v-Src protein using r . Plasmids for eight different versions of the v-Src protein

seven mutant proteins) were co-printed

tran

, and SPX1), while osine staining (KD,

was

(wild-type andwith the phMGFP “marker” plasmid on reverse

sfection cell arrays. (A) A schematic showing the location of each printed spot is shown. Following the addition of HEK293T cells, a cell array was produced showing clusters of cells expressing GFP protein. To monitor the tyrosine kinase activity of the co-expressed v-Src proteins, the cell arrays were subjected to phosphotyrosine immunostaining. Representative images taken using a 2.5X objective are shown. Five of the v-Src proteins displayed elevated phosphotyrosine staining (wild-type, ΔSH2, ΔSH3, ΔSH2ΔSH3

ts showed no phosphotyrthree mutanSHX10, SHX20). The level of v-Src protein expression

also determined by immunostaining a replicate well with an anti-Src antibody. (B) The level of phosphotyrosine staining of each expressed v-Src protein in GFP-positive cells was quantified using MetaMorph image analysis software. The plot shows the total integrated phosphotyrosine intensities (arbitrary units) per GFP-positive cell.

Fig.2: Cells in 96-well plates were transfected with GFP alone (control) or GFP plus cSrc, vSrc, bcr-abl, Fyn, Yes, Lyn, Lck, or Syk. Wells were incubated with various trial inhibitors and stained for anti-phospho-tyrosine as in Figure 1. The level of phosphotyrosine in the GFP-labeled cells was determined using automated microscopy.

Tyrosine Kinase Inhibitor Treatment

1.00

1.20

1.40

1.60

1.80

0.00

0.20

0.40

0.60

0.80

GFP cSrc vSrc bcr-abl Fyn Yes Lyn Lck SykcDNA

PP1_1.7 uM PP1_3.6 uM PP2_15.8 nM PP2_31.6 nM

AG957_9.25 nM AG957_18.5 nM HS_4.35 uM HS_8.7 uM

53

Electrochemical Imaging of Exocytosis Using Microfabricated Devices

. Torres, R. Dong, K. Berberian

ther Students/Postdocs: Q.Fang, A.Ngatchou, B.Kim, R.Molloy P O hormones by neuronal cell types as well as histamine and o brane bound vesicles by the mechanism of exocytosis via a een the intravesicular and the extracellular space. We i ase of single vesicles in neuronal cell types and mast cells c icrofabricated devices, fluorescence imaging and FRET c anges in the SNARE complex. M e properties and transmitter release is studied by patch a iber electrode inserted into a patch pipette is used as e eleased from single vesicles in chromaffin cells while the f ance analysis [1, 2]. For electrochemical imaging of single v ell as transparent ultrathin gold or indium tin oxide (ITO) e microscope cover glass. These devices detect the opening o fluorescent markers are observed simultaneously by total i oscopy [5]. Fluorescent tags are attached to proteins i s in their interactions with fusion pore dynamics. Electrochemical imaging of exocytosis is also applied to study the spatio-temporal dynamics of stimulus secretion easuring release of preloaded serotonin. For chemical modification allowing detection of molecules that are not per se oxidizable such as glutamte, surface initiated atom t ation (SI-ATRP) is used to grow poly(sodium methacrylate)-b-poly (2-h ) block copolymer brushes on gold electrode surfaces. S perometry and patch amperometry experiments showed that in contrast to c ines through the early fusion pore is not associated with cation flux through channels in the vesicle membrane but with Na+ influx through the fusion pore following electrodiffusion [2]. It was shown that transparent 5 nm thin electrodes are capable of detecting a ic foot signals, and thus single fusion pore openings, while fluorescence changes can be

aged through the electrode (Fig.1). Block copolymer brushes were successfully grown on gold lectrode surfaces. The hydroxyl groups on poly(2-Hydroxyethyl methacrylate) can be activated by syl chloride and then used to immobilize proteins t images showed that high density

f proteins can be immobilized onto brushes by this method. The protein SNAP-25 plays a key role in exocytosis [8]. To investigate its structural changes in the

NARE complex the CSNAC construct with a FRET donor (CFP) at the N terminal of the first SNARE motif and a FRET acceptor at the N terminal of the second (using a tetracysteine (C4) - motif that was post-translationally labeled with the biarsenical dye FlAsH) [9]. The endogenous cysteines of SNAP-25 were replaced with alanines. A large FRET increase was observed when it forms a binary complex with the SNARE syntaxin, or a tertiary complex with syntaxin and the SNARE VAMP. The effect is not seen when CSNAC2 lacks either of the two SNARE motifs, nor when CSNAC2 is incubated with VAMP alone (Fig.2).

Mast cell exocytosis was detected with ECD arrays using poly-D-lysine (PDL) stimulation surface patterned employing parylene dry lift-off. Placing individual mast cells on ECDs with PDL (but not without PDL) produced amperometric serotonin signals at multiple electrodes (Fig.3).

Participating Faculty: M. Lindau, W. Almers, H. Craighead, B. Baird, C. K. Ober NBTC Students/Postdocs: K. Kisler, O. Varmalov, AO

roject # NCB8

bjectives: Release of neurotransmitter and ther mediators by mast cells occurs from mem fusion pore that forms a connection betwnvestigate the mechanisms of fusion and releombining electrochemical measurements, monstructs indicating protein conformational ch

ethods: The relation between fusion pormperometry, a technique where a carbon flectrochemical detector of catecholamines rusion pore properties are measured by admittesicle exocytosis platinum electrodes [3] as wlectrodes [4] are patterned on the surface of a f singe fusion porees electrochemically whilenternal reflection fluorescence (TIRF) micrmplicated in fusion to correlate change

coupling in mast cells m

ransfer radical polymerizydroxyethyl methacrylate

ummary: Combined amurrent belief [6, 7], release of catecholam

mperometrimeto . Initial fluoresceno

S

54

Accomplishm• Amperometric detection of exocytic rele

onfor ation

ikes by planar elec ays

ents ase with transparent 5 nm gold electrodes

• Improved SNAP-25 FRET construct using FlAsh as acceptor probes SNAP-25 c mchange during complex formation with syntaxin.

tosis detected as amperometric • Surface patterned poly-D-lysine stimulates mast cell exocysp trode arr

Fig.1: Amperometric detection of fusion pore openings with transparent 5 nm gold electrodes in a chromaffin cell. (A) Fluorescently labeled granules are visible between as well as through the electrodes (red outlines). (B) Amperometric signals with low

noise show clear foot signals indicating fusion pore expansion. (C) Amperometric charge detected per vesicle with gold electrodes is about twice that detected by ITO electrodes.

A B C

Fig.2: CSNAC and its mutants. A. The SNARE motifs SN1 (green) and SN2 (red) are connected by a linker (white) that targets SNAP25 to the plasma membrane. The donor fluorescent protein Cerulean (cyan) is at its N terminal and the tetracysteine motif C4 (yellow) at different positions along the linker (at amino acids R142, G132, E125 and A99 in CSNAC 1-4. B. The SNARE domain of syntaxin was added to purified CSNAC mutants for 30 min at the concentrations given on the abscissa, and the fluorescence was measured. FRET ratio was calculated as the emission at 530 nm divided by that at 475 nm.

Fig.3: Electrochemical detection of serotonin release from a rat peritoneal mast cell placed on top of a surface patterned poly-D-lysine stimulus locared between the ECD electrodes. (A) Bright field microscope image. (B) Integrated amperometric charge detected by the 4 individual electrodes imdicating time course and position of release in a single fusion event. The measured amperometric spike is shown in the inset.

0.60.81

1.21.41.61.8

pC

Gold, n=6

ITO, n=5

00.20.4

Average Quantal Size

55

Dynamics of Viral Fusion Proteins Participating Faculty: Lois Pollack, Gary Whittaker, Brian Crane NBTC Students: Jessica Lamb, Xiangjie Sun, Ken Gee Others: Ikenna Madu, Suzette Pabit, Shoshannah Roth, Brian Zoltowski, Sandrine

Belouzard Project # NCB 9 O

to invade cells to deliver genomic material. We propose to unravel critical steps in this attack by studying the conformational dynamics of proteins r d host cell membranes. Three research groups will collaborate to m es upon activation of fusion proteins from viruses, including i echniques will reveal the pre and post-fusion structures of these p e dynamics that accompany this process. This work may enable engineering of novel nano-scale devices or reagents for delivery of macromolecules into living cells. M

rovide structural information about macromolecules. While x-ray c and relative position of each atom in a macromolecule, small angle x nformation about the dynamics that accompany conformational c mployed to determine the structure of inactive and active viral fusion p tor the kinetics associated with these transitions.

ly a new method for measuring the dynamics of viral fusion protein a ll be used to initiate the pH jump that triggers the conformational c be monitored by small angle x-ray scattering (SAXS). SAXS s l states will be compared with structures determined by x-ray c

or preparing the ample quantities of viral fusion proteins required for t e prepared either from the intact virus, or with the assistance of an expression system S

above (static and time-resolved SAXS in addition to x-ray crystallography), we expect to contribute to structural and dynamic characterization of viral fusion p in viral infectivity of a healthy organism, we are bringing a new a oblem in human health.

derstand these important biological reactions and to contribute to the future engineering of novel reagents for delivery of macromolecules into living cells.

bjectives Viruses are the ultimate nano-machines, programmed

esponsible for fusion of viral aneasure the conformational chang

nfluenza, VSV and SARS. X-ray troteins, and, for the first time, th

ethods X-ray scattering techniques p

rystallography reveals the location-ray scattering (SAXS) provides ihanges. Both techniques will be eroteins. SAXS will be used to moni

We propose to develop and appctivation. A microfluidic cell wihange. Structural dynamics will tructures of the initial and finarystallography.

Two methods will be explored fhese experiments. Proteins will b

.

ummary Using the methods outlined

roteins. Since fusion is the first steppproach to bear on an important pr

Our goal is to more thoroughly un

56

Accomplishments • Purification of HA protein and construction of baculovirus expression system • SAXS profiles and reconstructions of HA and SARS proteins

. • Successful testing of laser trigger of conformational changes

Fig.1: Cart events oon diagram of theoccurring during influenza entry into

ral spike or fusion protein nizes the host cell (via

bin

cells. The vi(HA), recog

ding to sialic acid) and enters the endosome of the cell, where low pH triggers a conformational change in HA to expose the viral fusion peptide (shown in red), which inserts into the host cell membrane to initiate the process of membrane fusion and the delivery of the viral genome into the cytoplasm.

Fig.2: The three different viral fusion pro

ind

teins we plan to study. The bold letters indicate conformations that have confirmed (e.g. pre and post-fusion state of HA and G). Open letters indicate states that have not been identified, but are the targets of this proposal. Arrows

icate conformational dynamics to be explored by SAXS. None of the conformational dynamics has been previously measured.

Fig.3: ctural reconstructions of the avirus spike (fusion) protein

ect

trim

StruSARS coron

odomain at pH 7 (left) and pH 5 (right). These structures were derived from small angle x-ray scattering (SAXS) profiles of protein in solution. The SAXS data indicate the formation of

ers (right) from monomers (left) as the pH is decreased. (The size of the reconstructed protein is not to scale.)

57

Chromatin Structure, Function, & Dynamics: From Mononucleosomes to Polytene Chromosomes

Participating Faculty: W. Lee Kraus, John Lis, Michelle Wang,

BTC Students/Postdocs: Nasun Hah, Jing Jin, Katherine Kieckhafer Watt Webb

ther Students/Postdocs: Joanna Berrocal, Chris Denfel, Scott Forth, Matthew Gamble, Mike Hall, Chris Fecko, Raga Krishnakumar, Donald Ruhl, Jie Yao, Katie Zobek

P O

to develop and use a set of complementary approaches in n tin, a biomolecule critical in maintaining gene structure and f using approaches from nanobiotechnology to study chromatin s ith a focus on histone variants and nucleosome-binding proteins, a methodologies in nanobiotechnology to the study of chromatin s ies progress from nano-resolution single-molecule methods that e ts under defined conditions to multi-photon microscopy methods that examine molecular dynamics and interactions, also at nano-resolution, in living cells. The integration of inform these diverse measurements should provide a molecularly detailed and biologically r matin. M

together three approaches from nanobiotechnology to explore the s (1) single molecule optical trapping, (2) atomic force microscopy, a he integration of these approaches allows us to examine chromatin and associated factors in an unprecedented way both under highly defined conditions with single m environment of living cells, all at nano-scale resolution. Single m raps are devices capable of manipulating and detecting sub-n ments for sub-micron dielectric particles, making them very useful for the m nipulation and study of single molecules of DNA or protein attached to those dielectric particles. This technique can be used to probe dynamic events directly and quantitatively under physiological conditions, without the averaging and smearing effects associated with measurements taken from populations of molecules. Atomic force microscopy: AFM provides three-dimensional views of biological molecules with nanometer resolution. AFM can also be extended to the determination of the molecular identities of specific components within complexes or mixtures. Multi-photon microscopy: MPM provides real-time views of gene regulation in living tissue that are of unprecedented clarity and resolution. The deep-penetrating and low background MPM is ideal for imaging tissues and cells. S

f nucleosomes (DNA wrapped around a protein core of histone p portant and specific roles in determining the structure and function of chromatin in vivo. The dynamic properties of chromatin include: (1) s osome (e.g., removal of H2A/H2B dimers), (2) mobilization of n A (e.g., translational repositioning), and (3) inter-nucleosome interactions leadi paction of nucleosomes into higher-order structures (e.g., chromatin condensation). Our group is using the set of complementary approaches described above to study the nano-structure and dynamics of chromatin. Specificially, we are determining (1) the mobility of canonical and variant histones within chromatin, (2) the effect of nucleosome-binding proteins on the

ructure of chromatin, and (3) the mechanisms by which chromatin remodeling complexes alter e structure and stability. Our integrative approach should provide a molecularly detailed and

biologically relevant view of the dynamics of chromatin and its molecular partners.

NO

roject # NCB10

bjectives The goal of our research is

anobiotechnology to study chromaunction. Specifically, we are: (1)tructure, function, and dynamics, wnd (2) developing and applying newtructure and dynamics. Our studxamine purified componen

ation fromelevant view of the dynamics of chro

ethods The studies from our group bring

tructure and dynamics of chromatin:nd (3) multi-photon microscopy. T

olecules and in the complex native olecule optical trapping: Optical t

anometer displacea

ummary Chromatin is a dynamic polymer o

roteins) whose biochemical and biophysical properties play im

tructural alterations within a nucleucleosomes along a length of DN

ng to the com

stnucleosom

58

Accomplishments

• We performed single molecule experiments using several distinct optical trapping tecelucidate the mechanism of nucleosome remodeling by the SWI/SNF and ACF c

hniques to hromatin remodeling

was used to determine that both

them while maintaining used AFM to defi uctural features of PARP-1 that underlie its ability to promote the

localized compaction of nucleosomes. Our results indicate that the DNA binding domain and the lytic domain function together to form the minimal functional module of PARP-1 required for

enzymes. The DNA unzipping technique, developed by the Wang lab, ACF and SWI/SNF are capable of displacing single nucleosomes along a DNA template, repositioning

their canonical structure. (Lead group = Wang) • We ne the str

catachromatin compaction. (Lead group = Kraus)

• We developed the use of photo-activateable GFP (paGFP) and other photochromic proteins for use with MPM to determine the fate of histone proteins that package a gene that is inactive and which subsequently becomes transcriptionally activated. For example, we have tested fly lines that produce fusions of canonical histone H2B or variant histone H2Av with photoactivatable GFP (paGFP). (Lead groups = Lis and Webb)

Fig. 1: ACF remodeling activity on a mononucleosome is detected by holding DNA tether at constant low force (<3pN). (A) Cartoon illustration of experimental configuration.

(brown cylinder) was assembled on Nucleosome601 nucleosomal positioning element flanked by

ed DNA (red wavy line). ACF is indicated by pink ovals. (B) DNA extension vs. time under three different conditions indicated by different colors. Cross-correlation with step function clearly showed that without ACF or after removal of ACF and ATP, extension had no change over experimental time (60s); however, in the presence of ACF and ATP, DNA extension fluctuated between two states.

nak

A

B

Time(s)

Time (s)

DN

A le

ngth

(bp)

Time (s)

No ACF

25 nM ACF+ 1mMATP

Fig. 2: The DNA binding domain (DBD) and catalytic domain are necessary and sufficient for chromatin compaction by PARP-1. (A) Schematic diagram of the PARP-1 deletion mutants used in these studies. (B) AFM images of in vitro-assembled chromatin with a staurating amount of wild-type (Wt) PARP-1 or the PARP-1 deletion mutants indicated. (Top) Scan probe oscillation amplitude images. The length scale is indicated by the white baThe

r. (Bottom) Topographical images. height scale is shown along the bottom of each

image.

Fig. 3: Photo-activation of H2B-paGFP in an optical section of a salivary gland nucleus in a transgenic Drosophila line expressing this tagged core histone. The H2B-paGFP is strikingly photo-activated.

No PARP-1 Wt PARP-1 DBD-NBD DBD CAT

100 nm 100 nm 100 nm 100 nm100 nm

A

B

Wt PARP-1 1014

DBD BRCT

Catalytic Domain

NAD+ Binding

DBD

DBD-NBD

AMD

CAT

0 nm 8 0 nm 25 0 nm 25 0 nm 8 0 nm 8

Pre-activation 2-photon activation (800nm)

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