Bacteria Actuation, Sensing, and Transport (BAST) in

Micro/Nanoscale

Dr. MinJun Kim

Dept. of Mechanical Engineering & Mechanics

Drexel University

Layout of This Presentation

2. Microscale Bacterial Actuation - Chaotic Microfluidic Mixing System- Chemotactic Bacterial Sensing System- Self-sustained Microfluidic Pump- Autonomous Bacterial Transportation System

4. Microbial Risk Assessment

- Ultra-fast Bacteria Detection and Configuration- Rapid Bacteria Cell Lysis

1. Introduction of Flagellated Bacteria

5. Conclusions & Acknowledgements

3. Nanoscale Bacterial Actuation - Nanoscale Mechanical Actuator- Flagella-templated Nantube

Going Micro & Nano: Miniaturization Theory

Why do we need it?

- Reduced fabrication cost

- Reduced sample consumption

- High sample throughput

- Superior performance (speed / efficiency)

- MEMS and NEMS compatible

What are the applications?

- Molecular separations

- Chemical and biological synthesis

- Medical and clinical diagnostics

- Environmental monitoring

- DNA sequence analysis

- Process control

Why not use “nature”?

- Challenge to micron-nano scale actuation

- Intergration Engineering with Biology

Self-powered Bacterial Pump

Flagellated Bacteria (E.coli & Serratia marcescens)

Flagellated Bacteria:- Cell body & Flagella- Rod-shaped cell body : 2 m long, 1 m diameter- Flagella : rotary motor, hook, and filament

10 m

2 m

1m

A cell of E. coli, fluorescently labeled.(Turner, Ryu, and Berg 2000)

http://www.npn.jst.go.jp/ Namba

E.coli Rotary Motor, Hook, and Filament

25 nm

Filament – typically about 10 m and 20 nm in diameter. Helical shape in the unstressed state.

Hook – about 50 nm long and 20 nm in diameter. Plays the universal joint.

Motor – proton (H+) is the energy source. The typical rotation speed is about 100 Hz. The motor can rotate either direction.

Schematic diagram (Berg, 2003), electron micro-scopy image of the flagella motor (De Rosier, 1998), and http://www.npn.jst.go.jp/Namba

E.coli in Motion

Sequence of E. coli flagella bundling(Turner, Ryu, and Berg, 2000)

E. coli swim by rotating helical filaments. Filaments form a bundle and disperse the

bundle. Tumbles and runs change the swimming

directions.

Macro-scale Model of Bacterial Flagellar Bundling

Model Full-Scale

Fluid 10 5 cP 1cP

Flagella: 10 cm 10 um

Rotation: 0.3 Hz100 Hz

SetupTwo stepper motors.Epoxy-filled plastic tubes in helical shape.High viscosity silicone oil (100,000 cp).

Match geometryPitch, Aspect ratio, Number of turns.

Match flow characteristicsReynolds number 10-3 (Re 10-5 for Bacteria).

[ FRONT VIEW ] [ SIDE VIEW ]

Flow by Bundles Helices

- Flexible Helices Movie (Real time)

- Complex flow field induced by bundling

- Bundled state resembles single helix

flow

~

Bundled helices Double thickness helix

Test Geometry & Experimental Setup

y

x24 mm 20 mm 16 mm 12 mm 8 mm 4 mm 0.5 mm

Fluorescence

No Fluorescence

Width = 200 m

Depth = 40 m

(a)

(b)

• PDMS Microchannels Using Soft-Lithography Techniques

• E.coli: Tumbly (RP 1616), Wild type (HCB 33), and Immobile

• Concentrations of E. Coli: 0 ~ 109 /ml

• Flowrate: 0.5 ~ 1.25 l/min

• Velocity: 1 ~ 2 mm/s

(a) Buffer + 0.02% of FITC + Dextran (MW=77,000) 0.97 cp @ 24.3 C

(b) Buffer + 0.02% of Dextran (MW=68,800) 0.98 cp @ 24.3 C

Bacteria-Enhanced Diffusion

<Baseline> <Immobile E.coli>

<Tumbly E.coli> <Wild type E.coli>

Fixed at

U = 1.04 mm/s

x = 24 mm.

Each Concentration of E.coli = 1.05 109/ml. channel wall

Flow

Chemotactic Bacteria-Sensors

1

7

6

5

4

3

2

y

x

1.

3.

5.

7.

1

2

3

Bacteria’s Chemotatic Receptors

Sudden Change

Rotary Motor Performance Affected

Global Microfluidic Effects

Monitoring or Detecting

Bio-Sensor

Controlled-Mixing in Microfluidics

Formation of Bacterial Carpets

• Concentration of Serratia Marcescens

: 2 ~ 5 109/ml

• Time : ~ 1 hour

• Flow rate : 0.06 l/min

On : 10 seconds

Off : 5 minutes repeatedly

Flow

…etc…

…etc…

Glass wall

PDMS wall

15 m

Fill

fa

cto

r [%

]

Time [s]10

010

110

210

310

410

0

101

102

PDMSGlass

17 6 5 4 3 2

y

x

2

1

3

1

23

MJ Kim and KS Breuer, PNAS, 2007

Cell Orientation on Bacterial Carpets

-100 -50 0 50 1000

0.002

0.004

0.006

0.008

0.01

0.012

PD

FCell Orientation [Degree]

-30 < degree < 30 : 54.9013%

-40 < degree < 40 : 64.3485%

-50 < degree < 50 : 74.5086%

Single Cell : 80.5192 %

Group Cell : 19.4808 %

Bacterial Carpet: 50 m x 50 m

Chaotic Mixing with Bacterial Carpet

Baseline Dead Bacterial Carpet Live Bacterial Carpet

Depth: 15 mWidth: 200 m

0 5 10 15 20 250

2

4

6

8

10x 10- 7

D [

cm

2/s

]

Concentration of Bacteria [ x 108/ml]

BaselineDeadTumblyWild- type (1)Wild- type (2)Wild- type (3)

Baseline

Dead Bacterial Carpet

Live Bacterial Carpet

MJ Kim and KS Breuer, JFE, 2007

Active bacterial carpet in the microchannel (1 micron dia. fluorescence bead motion)

Autonomous Bacterial Pumping System

• Coat surface of racetrack with Serratia marcesens using flow-deposited carpet

• Seed with 500 nm fluorescent particles

• Pumping velocities ~ 10 m/sec in the racetrack microchannel

PD

F-10 -5 0 5 10 15 20 250

0.02

0.04

0.06

0.08

0.1

0.12

0.14

2 mm

200 m

50 m

1.6 mm

Channel Blockedby Glue (RTV)

Streamwise Drift Velocity [ m/s]

MJ Kim and KS Breuer, APS DFD Meeting. 2004

Fully Developed Pumping Velocity

Pumping Enhancements in the Open System:

1) Glucose (Food) Effects

2) Geometric Effects

Flagellar Motor Acitivities

Large-scale Self-coordinations

Various Effects on Bacterial Microfluidic Pumps

MJ Kim and KS Breuer, PNAS, In review. 2007

Autonomous Bacterial Transportation System

Micro-Barges:

- Fill Factor: 90 ~ 95 %

- Typical Velocity : ~ 5 m/s

- Chemotaxis, Phototaxis, and Aerotaxis

PDMS barge

Glass substrate

Microbarges in Motion

Engineered Bacterial Systems

Cell Patterning Using Colloidal Lithography

DK Yi, MJ Kim, et al. Biotech. Lett, 2006

Conclusions

ACKNOWLEDGEMENTS:

E. Steager (Ph.D), R. Mulero (Ph.D), C.-B Kim (PostDoc), C. Naik (UG), J. Patel (UG), L. Reber (UG), S. Bith (UG).

Kenny Breuer, Tom Powers (Brown), Howard Berg, Linda Turner (Harvard), Nick Darnton (U.Mass), MunJu Kim (U.Pittsburgh).