Capacitance-Based Real Time Monitoring of Endocytosis

Kyung-Hwa Yoo

Dept. of Physics

Yonsei University

Contents

1. Introduction- What is cell?- Capacitive cell sensors

2. Monitoring proliferation and cell death

3. Monitoring receptor-mediate endocytosis

4. Monitoring pinocytosis & phagocytosis

5. Summary

Introduction

What is cell?

structural and functional unit of all living organisms.

Image of HELA cell

Cell membraneCell membrane ( plasma membrane ): lipid by layer

a finite membrane potential due to different ionic concentrations between inside and outside of the cell

Capacitive Cell Sensors

The biological cells may be regarded as dielectric particles The capacitance depends on their morphology, number, etc.

Can we monitor cell activities in real time using the capacitance measurements ?

Sensor (I)

Fluidic channel

Silicon well

out

in

(c)

50nm100nm

Cell Death

Apoptosis

1 min 70min 90min

10μm

Trail apoptosis

170min 310min

TE2

0 200 400

0.85

0.90

0.95

1.00

C/C

o

Time(min)

50 ng/ml

100 ng/ml

150 ng/ml

cell-free medium

Ri Mi Lee, et al, Biosensors & Bioelectronics, 24, 2586 (2009)

Necrosis

0 200 400

0.80

0.85

0.90

0.95

1.00

C/C

o

Time(min)

200 mM

300 mM

500 mM

1 min 60min 200min

150 ng/ml ethanol necrosis

290mim 330min

TE2

Sensor (II)

0 10 20 30 40

1.2

1.5

1.8 NIH3T3

HeLa

TE2

G/G

o

Time(hr)

TRAIL

NIH3T3: mouse fibroblast cell line (noarmal cell)

HeLa: human cervical cancer cell line

TE2: human esophageal cancer cell line

50nm100nm

Endocytosis

Endocytosis

Receptor –mediated endocytosis

Replication-competent adenovirus

Hep1

Ri Mi Lee, et al, Biosensors & Bioelectronics, 25, 1325 (2010)

Replication-incompetent adenovirus

The capacitance peak is due to internalization of adenovirus

0 200 400

1.00

1.02

1.04

C

/C0

Time(min)

CAR Abs+Ad

Ad-E1/E3

The capacitance peak was ascribed to receptor-mediated endocytosis

Car fiber receptor

Blocked by Car-Abs

HEP1 cell

X

Replication-incompetent Ad tagged with GFP TIRF Image

Until 30 min. after 30 min.cell cell

adenovirus

TIRF: Total Internal Reflectance Fluorescence

Treated with CAR-specific Abs & replication-incompetent Ad tagged with GFP

TIRF Image

cell

X

X

Car-Abs

X

Receptor-mediated endocytosis of Antibody

Pinocytosis

internalization

Lysosomedisruption

PS particles(100 nm)

AUNPs(10 nm)

Observed by capacitance sensor

Pinocytosis

Pinocytosis

PS particles

cell

Phagocytosis

internalization

Phagosomedisruption

Observed by capacitance sensor

E-coli (1~2 μm)

Macrophage(RAW264.7)

Cell eating

Phagocytosis

RAW cells treated with live bacteria

RAW cells treated with dead bacteria

Phagocytosis:Confocal Images

21 min 22 min

23 min 24 min

Cell: GFP infection(green)

e. coli: mcherry(red)

Engulfing e.coli yellow

Array chip for high throughput screening

Computer

LCR meter

CO2 incubator (37oC, CO2: 5%)

co

scanner

connecting

Summary

• In the future, the capacitance sensor array will be a valuable

tool for high-throughput screening to identify antibodies or

ligands with high affinities to specific cell types.

• Three different kinds of endocytosis can be distinguishable

by capacitance sensor

Acknowledgements

Ri Mi Lee, Yoon Chung Park

( Department of Physics, Yonsei University)

Chae-Ok Yun, Pyung Hwan Kim, Joung-Woo Choi , Jun-Soo Shin

(College of Medicine, Yonsei University)

Donghyun Kim, Kyujung Kim

(School of Electrical and Electronic Engineering, Yonsei University)