BioMicroSystems: Labs and Fabs for Nanotechnology and ... · PDMS structure from ... most abundant biological compound; ... other crustaceans, insects, zooplankton and even the cell

Gary W. RubloffDirector, Maryland NanoCenter

Minta Martin Professor of Engineering

Department of Materials Science & Engineering, Institute for Systems Research, Institute for Research in Electronics and Applied Physics

University of Marylandwww.isr.umd.edu/gwrubloff , [email protected]

BioMicroSystemsBioMicroSystems: Labs and : Labs and FabsFabs for Nanotechnology for Nanotechnology and and NanomanufacturingNanomanufacturing

http://www.isr.umd.edu/gwrubloff

2Rubloff, IBM Research YKT 1/18/07

OutlineOutlineMotivation - biomicrosystemsChitosan as a bioreaction platformBioMEMS device and packaging technologyBiofunctionalization in bioMEMSMetabolic engineering drug discovery

Related workIntegrated sensing for diagnostics and controlBioMEMS as nanolabs and nanofactoriesCells as sensors and patientsRole of nanoparticlesNano-bio for non-bio applications Special thanks to

Reza Ghodssi (ECE/ISR)Greg Payne (UMBI-CBR/UMBC)Bill Bentley (BIOE/UMD/UMBI)


Why Why BioMicroSystemsBioMicroSystems??Fundamentals

Microfluidics versatile transportChemicals, particles, biomolecules, …

Biology super-selectivityPrecision assembly

FundamentalsMicrofluidics versatile transportChemicals, particles, biomolecules, …

Biology super-selectivityPrecision assembly

Enabling technology

Directed assembly

Biofabrication

Micro labs and factories

Enabling technologyEnabling technology

Directed assemblyDirected assembly

BiofabricationBiofabrication

Micro labs and factoriesMicro labs and factoriesApplicationsMetabolic engineering, drug

discovery(Bio)molecular synthesisChem/bio sensingCell-based sensing systems Cell clinics

ApplicationsMetabolic engineering, drug

discovery(Bio)molecular synthesisChem/bio sensingCell-based sensing systems Cell clinics

BioMEMS


Metabolic Engineering in a BiochipMetabolic Engineering in a Biochip

GOAL: Emulate multi-step, multi-site reaction sequences in artificial, controlled conditions to

Investigate biochemical reactionsConfirm or identify reaction pathwaysDetermine kinetics

Develop means to modify metabolic pathwaysIdentify candidate drugs which retard or enhance

reaction rates

Example: biosynthesis of quorum sensing molecule AI-2


BiofunctionalizationBiofunctionalization in in MicrofluidicsMicrofluidicsTARGETS:

Multi-step biochemical reaction pathways

Site-specific biofunctionalization to separate steps for diagnostics and control

Parallelism for rapid screening

CONSTRAINTS:Biology is delicate – cannot

withstand much processing for bioMEMS chip fabrication

Complexity of reaction sequences demands integrated sensing


SiteSite--Specific Specific BiofunctionalizationBiofunctionalizationStamping (µCP)

Press biomolecules onto surface

Mirkin (Northwestern)

Dip-pen nanolithography“write” biomolecules onto surface

Whitesides (Harvard)

Useful approaches to biofunctionalization, butNot easily compatible with enclosing microfluidics after attaching labile biological speciesNot readily scalable to complex bioreaction networksMay require sophisticated tools (AFM/DPN)


BioMEMSBioMEMS -- MicrofluidicsMicrofluidics

Whitesides, Anal Chem 2001Quake, Science 2002

Quake, Science 2000

Soft lithographyPDMS structure from

molding/casting

Multilayer PDMS microfluidic circuitsIntegrated valve controllers

Significant advances in bio-microfluidics, butSite-specific biofunctionalization not clearNot compatible with post-process characterization


ChitosanChitosan––Based Based BioMEMSBioMEMSMEMS

prefabricationbiofunctionalization

on demandbioMEMSoperation

1 2 3

reactants

products

surface biospeciesDNARNA

proteinenzyme

cell

Support multi-site bioreactionsIntroduce biology only when neededCompatible with post-process characterization and reuseSite-specific biofunctionalization through surface activation and surface-controlled reaction


ChitosanChitosan –– Biopolymer from NatureBiopolymer from Nature

http://www.ece.umd.edu/News/06_07_27_crab_detectors.html

Chitosan is derived from chitin. After cellulose, chitin is nature's most abundant biological compound; it makes up the shells of crabs and other crustaceans, insects, zooplankton and even the cell walls of mushrooms. Chitin is unusual in that it is a polymer (a large molecule composed of repeating units) produced by living things (biological). Chitin and its derivative chitosan are thus known as biopolymers.

Chesapeake Bay60 million pounds (2004)

Blue crab


Nucleic acids- DNA/RNASandwich assay

Proteins, enzymesEnzyme catalysis

VirusesCells

20μm

Yi, Langmuir (2005)

Proteins

Pederzolli, IRST, Italy

Cells

Yi, Analytical Chem 2003

Nucleic acids

Viruses

Yi, Nano Letters 2005

ChitosanChitosan as as BioreactionBioreaction PlatformPlatform

Review: Yi et al, Biomacromolecules 6 (6), 2881-2894 (2005)


ChitosanChitosan ElectrodepositionElectrodeposition

High pH region at negative electrode due to hydrogen evolutionChitosan molecules deprotonated, immobilized at electrode surface

Cat

hode

-------

pH gradient

NH3+

NH3+

NH3+

pH>6.3

2H+ + 2e- H2

H2

Low pH, soluble High pH, insoluble

OO

OHO

NH3+

OH

OH

*

OH

*

NH3+

n OO

OHO

NH2OH

OH

*

OH

*

NH2

n H+

2n+

Unusual polysaccharide

Chitosan electrodeposition


Opticalmicroscopy

Fluorescence microscopy

before deposition

Magnification 20×

after deposition

Wu et al., Langmuir, 18, 8620-8625 (2002)Wu et al, Langmuir 19 (3), 519-524 (2003)Fernandes et al., Langmuir, 19, 4058-4062 (2003)

ElectrodepositionElectrodeposition of Fluorescent of Fluorescent ChitosanChitosan


Deposition Process MonitoringDeposition Process Monitoring

0 50 100 150 200 250 300

0

1000

2000

3000

4000

3 A/m2 constant current

Res

ista

nce

[ohm

]Deposition time [sec]

Electrical (resistance)

0 50 100 150 200 250 300

0.0

0.2

0.4

0.6

0.8

1.0 3 A/m2, constant current

Thic

knes

s [μ

m]

Deposition time [sec]

10nm/min10nm/min

Physical (thickness)

Optical (reflectance)

Real-time, in-situ

Post-process


PostPost--Process CharacterizationProcess Characterization

Chemical & compositional (XPS)

2 x 2 2 x 2 μμmm

Roughness Δt / t = 3 nm / 1.1 μm ~ 0.3 %

Structural (AFM)Biological (cells)


Film micro/nano structure can be manipulated by deposition conditionsCompact film to hydrogel

Mechanism (pH gradient) and permeability demonstrated

Biofabrication possible

Composites possible by incorporation

Materials science challengingBiofunctionality works well

ChitosanChitosan Materials and ProcessingMaterials and Processing

Fernandes et al, Langmuir, 19, 4058-4062 (2003)

Zangmeister et al, Electrochemica Acta 51, 5324-5333 (2006).

Fernandes et al, Langmuir 20 (3), 906-913 (2004)

Payne et al; Chen; Zhitomirsky

Roughness Δt / t = 3 nm / 1.1 μm ~ 0.3 %


Spatial ResolutionSpatial ResolutionChitosan patterned and biofunctionalized to 1-2μm or smaller

50X10μm patterned lines1-10μm spacings

MicroRaman Spectroscopy (E. Dreyer et al)Fluorescence Microscopy (S. Beatty et al)


ActivatedSurface

Probe ssDNA

Target ssDNA

DNA Hybridization on DNA Hybridization on ChitosanChitosan

Chitosan

glutaraldehyde


Reversible DNA Hybridization on Reversible DNA Hybridization on ChitosanChitosan


Yi et al, Anal. Chem. 76 (2), 365-372 (2004)

Reversible DNA Hybridization on Reversible DNA Hybridization on ChitosanChitosan


Sandwich Assay:Sandwich Assay:Purified Total RNA from Purified Total RNA from E coliE coli

Electrode ← Chitosan ← Glutaraldehyde ← ssDNA (probe)

mRNA (analyte) Hybridization

F F

HybridizationssDNA (sandwich probe)

Yi et al, Anal. Chem. 76 (2), 365-372 (2004)


Protein Assembly on Protein Assembly on ChitosanChitosan

DsRed GFP

Yi et al, Langmuir 21 (6) 2104-2107 (2005)


- ssRNA virus- 17 kD coat protein- Self-assembles into nanotubes- 4 nm inner diamater- 18 nm outer diameter- Length of 300 nm - Engineered to

- assemble w/o RNA- assemble to greater lengths- bind metals (metalization)

J.N. Culver, UMBI

Tobacco Mosaic Virus (TMV):Tobacco Mosaic Virus (TMV):Template for NanostructuresTemplate for Nanostructures


Virus Assembly on Virus Assembly on ChitosanChitosan

Yi et al, Nano Lett 5 (10) 1931-1936 (2005)

300 nm


BioMEMSBioMEMS Device and PackagingDevice and PackagingDevelop platform for multi-step,

multi-site biochemical reaction pathways metabolic engineeringTranslate chitosan biofunctionalization to

microfluidic biochipIntegrate sensing into platform


MicrofluidicMicrofluidic SealingSealing

SU8 knife edge / PDMS gasket sealing

Uses only local mechanical forces to sealRobust, reusableEnables post-process analysis

Current technology• Thermal bonding• Anodic bonding• UV adhesive

Elevated temperatures, …Ultraviolet exposureNo post operation, ex-situ analysisNot easily reusable


MicrofluidicMicrofluidic SealingSealingCompression ensures leak-tight

sealingSealing componentsSU8 micro-knife-edge (400 μm wide, 150 μm deep) PDMS gasket (~300 μm).

Flow conditionsFlow rate : 5 μl/minWater with coloring agent

With compression Without compression


Device TechnologyDevice Technology

Pyrex substrateThermal stability at process temperature (~95°C)Resistance to process chemicalsOptical transparency and high electrical resistivity

SU-8 microfluidic channelsChemical, and thermal stabilityVertical sidewalls and high aspect ratios

Park et al, Lab on a Chip 6 (10) 1315-1321 (2006)


Packaging TechnologyPackaging Technology

device

package



Packaging & ControlPackaging & Controlcontrolsystem


NHSNHS--FluoresceinFluorescein -- ChitosanChitosan

(a) chitosanelectrodeposition

(b) duringNHS-fluoresceinflow

(d) control(no voltage)

(c) after NHS-fluoresceinflow

post-process profilometry



Plot profile

Image J analysis of fluorescence image

GFP Assembly on GFP Assembly on ChitosanChitosan



Green Fluorescent Protein (GFP)Green Fluorescent Protein (GFP)

GFP shows green fluorescence when structure maintained

Conventional conjugation of GFP to chitosan through glutaraldehyde, or

Enzyme-activated conjugation of GFP to chitosan

Lewandowski et al, Biotech and Bioeng (2006)


EnzymeEnzyme--Activated GFP AssemblyActivated GFP Assembly

Tyrosinase activation of GPF in bioMEMSHomogeneous reaction (liquid)Possible heterogeneous reaction

(nearby activated chitosan)

GFP flow with tyrosinase assembly onto chitosan scaffold

GFP flow without tyrosinase – no assembly onto chitosan scaffold

Lewandowski et al, Lab on Chip (in revision)


BiomolecularBiomolecular Conjugation to Individual Conjugation to Individual ChitosanChitosan Scaffold Sites in Scaffold Sites in BioMEMSBioMEMS

Demonstrated forNHS-fluoresceinGFP using glutaraldehydeGFP with Tyr tag using tyrosinase enzymePfs enzyme

Preliminary results forTMV virusDNA hybridization

Chitosan electrodeposited at local sites in bioMEMSprovides a versatile platform for metabolic engineering


BiomolecularBiomolecular CommunicationCommunication

Quorum sensing: bacterial attack on host is triggered by elevated AI-2 concentration due to sufficient bacterial population

Example: biosynthesis of quorum sensing molecule AI-2

Adenine Homocysteine

?

O

OH OH

Adenine S

NH 3

+OOC

-

O

OH OH

Adenine S

NH 3

+OOC

-

O

OH OH

Adenine S

NH 3

+OOC

-NH 3

O

OH OH

OH S

+OOC

-NH 3

O

OH OH

OH S

+OOC

-NH 3

O

OH OH

OH S

+OOC

-

OH

OH O

OOH

OH O

OOH

OH O

O

?

O

OOB- OHHO

HO

HO

CH 3O

OOB- OHHO

HO

HO

CH 3O

OOB- OHHO

HO

HO

CH 3

S-adenosylhomocysteine S-ribosylhomocysteine 4,5-dihydroxy-2,3-pentadedione (DPD)

SAH SRHAI-2

Pfs LuxS

Cell signaling: bacteria manufacture and sense a small “signaling”molecule (e.g., AI-2)

AI-2

cell

cell


Emulate reaction sequences in artificial, controlled conditionsInvestigate biochemical reactionsIdentify or confirm reaction pathwaysDetermine kinetics

Introduce candidate drugs to modify metabolic pathways Suppress AI-2 synthesisCreate new antimicrobial strategy which avoids bacterial resistance

BioMEMSBioMEMS Laboratories for Drug DiscoveryLaboratories for Drug DiscoveryBioMEMS

Supported by Robert W. Deutsch Foundation Bentley, Ghodssi, Rubloff (UMCP)Payne (UMBI), Ghandehari (UMB)

AI-2 cell signaling (quorum sensing)


Building Building pfspfs Reactive Sites in Reactive Sites in BioMEMSBioMEMSUse tyrosinase activation to bond pfs-chitosan

Bond, then deposit

Bond while depositing


Downstream Product AnalysisDownstream Product Analysis

100 % conversion SAH SRH + Adenine at 5µL/minResidues 20% conversion for negative control (no electrodeposition voltage)

HPLCsignal

5 10 15 20

Minutes

-29

0

50

100

150

200

250

mV olts

0.49

9

1.05

8

1.48

21.

845

4.33

7

5.15

4

14.1

45

14.7

16

15.1

8615

.532

16.5

64

18.0

08

18.9

47

II+II- WI:8II+ II- II+WI:16II- II+

Adenineproduct

Adenineproduct

SAHreactant

controlreaction

Lewandowski et al (submitted to Lab on Chip)

Pfs-chitosan reactive sites SAH catalytic conversion to SRH & adenine

HPLC analysis


Enzymatic Reaction in Enzymatic Reaction in BioMEMSBioMEMSCatalytic activity of pfs demonstrated for

1st step in AI-2 synthesisPfs-chitosan conjugate, with expected

flow rate dependenceSimultaneous flow of pfs and tyrosinase

activatorRobust with substrate re-use, extended time in

flowSome nonspecific binding Bond, then deposit

Bond while depositing


Drug Development PathDrug Development PathCostly avg $403M million

Lengthy 5-8 yrs preclinical 6 yrs clinical trials18 mo’s approval

DiMasi et al, 2003


Integrated Sensing in Integrated Sensing in ChitosanChitosan--BioMEMSBioMEMS

Powers et al, Lab on a Chip (2005)

biochemical optical biochemical mechanicalKoev et al, Lab on a Chip (2006)

5x5, 10x10, 20x20 μm2

biochemical electricalBuckout-White et al (in preparation)


Integrated Fluorescence SensingIntegrated Fluorescence Sensing

Powers et al, Lab on a Chip (2005)

fluid channel

SU8 waveguideSU8 ridge

NHS-fluorescein, DNA on chitosan


DNA Detection using DNA Detection using ChitosanChitosan MicrocantileversMicrocantilevers

-0.2

0.8

1.8

2.8

3.8

4.8

0 25 50 75 100Position along cantilever (μm)

Hei

gh t

( μ m

) Before hybridization

After hybridization After denaturation

Frequency (kHz)57 58 59 60 61 62 63 64

Ampl

itude

(A. U

.)

0.6

0.8

1.0

1.2Before hybridizationAfter hybridizationAfter denaturation

static deflection (in vitro) dynamic oscillation (dry)

Koev et al Lab on Chip 2007


ChitosanChitosan––Based Based BioMEMSBioMEMSMEMS

prefabricationbiofunctionalization

on demandbioMEMSoperation

1 2 3

reactants

products

surface biospeciesDNARNA

proteinenzyme

cell

Support multi-site bioreactionsIntroduce biology last Compatible with post-process characterization and reuseSite-specific biofunctionalization through surface activation and surface-controlled reaction


cf. Conventional Biotechnologycf. Conventional Biotechnology

similarities in bioprocesses, BUT actually VERY DIFFERENT …isolate individual reaction steps spatially and temporallyidentify and control individual reaction stepsminimize material requiredexploit parallel arrays

controlled throughputcombinatorial discovery

Estimate from pfs catalysis of SAH:∼100X less reactant needed in bioMEMS to make same amount of product


cf. Conventional Microelectronicscf. Conventional Microelectronics

similarities in “manufacturing”, BUT actually VERY DIFFERENT …build factory (bioMEMS) before needed – novel supply chaininstall tools (biology) through biomicrofluidicsmake products (synthesize), provide services (analysis, factory=product)sensing & advanced process control – implement integrated schemes

make productsbuild the factory install the tools


MicroMicro--Clinics for CellsClinics for Cells

Cells as sensors and patients:BioMEMS environments to maintain

and monitor cellsTechniques to measure response of

cells to stimulantsCells as intelligent chem-bio

sensorsCells as indicator of nanoparticle

risk

Smela, Abshire, Shapiro et al


Designer NanostructuresDesigner NanostructuresSelf-assembled

nanostructures (AAO)Self-aligned

nanofabrication (ALD, ECD, …)

Nanostructures in templates Energy (supercap, solar)Display (electrochrom)

Nanostructures released Targeted drug deliveryEnergy

MaterialsToday Dec 2006

100 nm

Anodic aluminum oxide (AAO)

Atomic layer deposition (ALD)

Shape-differentiated

Selectively functionalized


Center for Center for NanoparticleNanoparticle Risk,Risk,Impact, & AssessmentImpact, & Assessment

NanoparticlesSynthesis, characterizationBiofunctionalization

Cellular response in bioMEMSEarly indication of clinical

response

Clinical assessment

Benign nanoparticletechnologies & manufacturing

www.nanocenter.umd.edu/centers/UMD/Nanoparticle_Risk/center_nanoparticle_risk.php

NIST $1.5M

http://www.nanocenter.umd.edu/centers/UMD/Nanoparticle_Risk/center_nanoparticle_risk.php


Microfluidics to transport nanocomponents from source to targetBiological decoration and selectivity to achieve desired configuration

of nanocomponents into a system

BioBio--Assisted Assisted NanoassemblyNanoassembly

Self-assembled nanostructuresregistered to nanotemplate

Free biodecoratednanostructures

Biodecorated nanostructures

Microfluidic transport

Biodecoratednanoelectrode

target

Bioselectiveattachment

BioMEMS-assistednanoassembly


ConclusionsConclusionsBioMEMS promising for nanobiotechnology

Metabolic engineering, drug discoveryCellular response, cell-based sensingNon-bio applications

Chitosan – programmable biofunctionalization (time, space)Robust, reusable science and development vehicle

Chitosan-based bioMEMS enableMulti-step, multi-site biomolecular reaction processes

New paradigm: manufacturing at the micro/nano scale MEMS on the shelfBiology on demandOperation for the application


AcknowledgementsAcknowledgementsStudents

Jung Jin Park, Susan Beatty, XiaolongLuo, Erin Dreyer, Israel Perez

Michael Powers, Stephane Koev

FacultyReza Ghodssi, Greg Payne, Bill

BentleyHyunmin Yi, Li-Qun Wu, Jim Culver

Other collaboratorsElisabeth Smela, Pamela AbshireMariano Anderle group (IRST-Italy)Sang Bok Lee, Michael FuhrerRebecca Zangmeister, Mike Tarlov

(NIST)

Support


BioMicroSystemsBioMicroSystems: Labs and : Labs and FabsFabs for for Nanotechnology and Nanotechnology and NanomanufacturingNanomanufacturing

While most of today’s nanotechnology resides in electronic systems constructed using complex manufacturing tools in enormous fabs, a broader scope of microsystemspromises a wider variety of functions and applications in nanotechnology and nanomanufacturing. Biological and microfluidic microsystems are particularly intriguing in providing new strategies to manipulate nano- and micro- scale components for applications in biotechnology and bio-assisted nanotechnology. We have demonstrated a unique platform for executing biomolecular reaction processes and sequences. Chitosan, a naturally-occurring, amine-rich biopolymer, is electrodeposited at predefined, programmable sites in a polymer-based microfluidic system. Such activated electrode sites support conjugation of proteins, enzymes, nucleic acids, and viruses while retaining biofunctionality such as DNA hybridization or enzyme catalysis. We have also developed a reusable microfluidics device and packaging technology to support multi-site bioreaction steps, employing fluidic, electrical, and optical networks to provide a platform for metabolic engineering and its applications. A primary target is quorum sensing, the enzyme-catalyzed small-molecule reaction sequence by which bacteria signal and sense their proximity to each other: a biomicrosystem which simulates the metabolic pathway is an attractive testbed for discovery of drugs that can impede bacterial attack. We are also pursuing other biological applications, including cell-based microsystems as intelligent chem/bio sensors and as models to assess health and environmental risk of nanoparticles. Finally, we are exploring the application of biological decoration and microfluidic transport as a means for directed assembly of nanocomponents into larger systems.

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BioMicroSystems: Labs and Fabs for Nanotechnology and ... · PDMS structure from ... most abundant biological compound; ... other crustaceans, insects, zooplankton and even the cell