DESIGN OF OPEN MICROWELL ARRAYS

FOR SINGLE CELL ANALYSIS

Tutor:

Prof. Roberto Guerrieri

Tutor:

Prof. Roberto Guerrieri

PhD Student:

Marta Lombardini

PhD Student:

Marta Lombardini

3rd year presentation

1. INTRODUCTION

2. MOTIVATION

3. OPEN MICROWELL ARCHITECTURE

OUTLINE

4. FABRICATION TECHNOLOGY

5. STRUCTURE OF THE SYSTEM

6. EXPERIMENTAL VALIDATION

7. CONCLUSIONS

INTRODUCTION

Lab-on-a-chip: miniaturization of a laboratory to a small device.

The 'laboratory' is created by means of channels, mixers, reservoirs, diffusion chambers, integrated

electrodes, pumps, valves and more.

GOALS:

• Automate standard laboratory processes.

Fast and cost efficient

Improved data quality

Increased separation abilityIncreased separation ability

• Improve Research in various areas of life sciences

(e.g. Immunotherapy, Stem cells, Cancer research, etc…)

Individually controlling single cells and particles

THE NEED:

-Analyze, manipulate and select one or few biological cells or artificial particles

- Recover the analyzed material (viable cells)

THE NEED:

1. Analyze, manipulate and select one or few biological cells

or artificial particles

– Controlled processes

– Efficient experiments

MOTIVATION OF OUR WORK

– Efficient experiments

2. Recover the analyzed material (viable cells)

To perform:

– Manipulation of single cells (Gene Therapy)

– Rare cells sorting (Immunology, Stem Cells)

OPEN MICROWELL ARRAY

MICROTITER PLATE OPEN MICROWELL ARRAY

Our platform is an OpenMicrowell array designed for High Throughput

recovery with integrated electrodes for dielectrophoretic manipulation with

single cell resolution

OUR SYSTEM:

1. Open on both sides: To permit

Top side:

Delivery-Processing-Recovery

DISPENSER

or RECOVERY

OPEN MICROWELL TECHNOLOGY

2. Micro size range: To handle a few or single cells (ø150 µm)

3. Active: By means of negative dielectrophoretic force

Top side:

→cells are dispensed and

recovered

Bottom side:

→channels provide suspension

buffer by capillarity.FLUID FLOW

SIDE

VIEW

CARRIER GLASS/ POLYCARBONATE

TECHNOLOGY REQUIREMENTS:1. High Parallelism

2. Cheap and disposable

3. Mechanical Compatibility with Standard Microtiter Plate

STANDARD

MICROPIPETTE

STANDARD

PITCH

FABRICATION TECHNOLOGY

OUR SOLUTION:

1. Printed Circuit Board technology

2. Suitable materials guarantee biocompatibility*

PITCH

BETWEEN WELLS

* Jung et al., Microsystem Technologies,2007

1. INTRODUCTION

2. MOTIVATION

3. OPEN MICROWELL ARCHITECTURE

4. F

OUTLINE

4. FABRICATION TECHNOLOGY

5. STRUCTURE OF THE SYSTEM

6. EXPERIMENTAL VALIDATION

7. CONCLUSIONS

DIELECTROPHORESIS (DEP)

A phenomenon in which a force is exerted on a dielectric particle when subjected to a non-uniform

electric field. This force does not require the particle to be charged.

All particles exhibit dielectrophoretic activity in the presence of electric fields.

V+ V-

Electric Field

------

----++++

++

--

--------------

----

--++

++++

++

++++++

++++++

V+ V-

Electric Field

----

--

--

--++++

--

--------------

----

--++

++

++

++

++++++

++++++

The dielectrophoretic force depends on:

• The medium and particles' electrical properties

• The particles' shape and size

• The frequency and magnitude of the electric field

V+ V-------++------

--------

--++++ ++++++++

pDEP

V+ V-------++--

----

--------

--++++ ++++++++

nDEP

εp > εm εp < εm

FUNCTIONING OF THE STRUCTURE:Trapping (nDEP) along z axis near boundaries.

1. Excitation: sinusoidal signals

- same Amplitude and Frequency

- opposition of phase

2. Pseudo-cylindrical nDEP cage

THE NEW IDEA OF OPEN MICROWELL (1)

(a)

x

y z

(b)

y

xz

V -V

-V V

E1 E2

E3 E4

z

x

LOCAL

MINIMA

105

1

0° 180°

180° 0°y

x

(c)

|E|2

REASONS FOR ALIGNMENT:

Measurement Efficiency is maximized.

1. Additional Electrodes to extend the processing phase :

- Impedance spectroscopy

- Electroporation

- Electrofusion

THE NEW IDEA OF OPEN MICROWELL (2)

x

y z

3D VIEW TOP VIEW

z

x

SIDE VIEW

y

z

REASONS FOR ALIGNMENT:

Measurement Efficiency is maximized.

1. Additional Electrodes to extend the processing phase :

- Impedance spectroscopy

- Electroporation

- Electrofusion

THE NEW IDEA OF OPEN MICROWELL (2)

x

y z

3D VIEW TOP VIEW

z

x

SIDE VIEW

y

z

FOUR ELECTRODES FOR NDEP TRAPPING:

1. Starting parameter: diameter D;

2. Other parameters: extracted from

Physical Simulations.

T

D

3D

VIEW

S

WTOP VIEW

D

Parameter Symbol Rule Value (µm)

DESIGN OF THE WELL

SIDE

VIEWH

T

4 c

m

Parameter Symbol Rule Value (µm)

Diameter D 150

Dielectric

Thickness

H H=D 150

Spacing

Electrodes

S S=D/3 50

Electrode

Thickness

T T=TMaxTech 25

Electrode

Width

W W=2D 300

1. INTRODUCTION

2. MOTIVATION

3. OPEN MICROWELL ARCHITECTURE

4. F

OUTLINE

4. FABRICATION TECHNOLOGY

5. STRUCTURE OF THE SYSTEM

6. EXPERIMENTAL VALIDATION

7. CONCLUSIONS

TRAPPING OF SINGLE BEADS OR

AGGREGATES ALONG THE Z AXIS.

Details of the Experiment:

1. Samples: 25 µm polystyrene bead

in DI water (nDEP);

(a) (d)

ONE MINIMUM TWO MINIMA

EXPERIMENTAL VALIDATION OF THE MODEL

in DI water (nDEP);

2. Excitation Signals: amplitude 6V,

frequency 600 KHz;

3. Device: ø150 µm well.

(b)

(c)

(e)

(f)

x

z

TRAPPING OF SINGLE BEADS OR

AGGREGATES ALONG THE Z AXIS.

Details of the Experiment:

1. Samples: 25 µm polystyrene bead

in DI water (nDEP);

(a) (d)

ONE MINIMUM TWO MINIMA

EXPERIMENTAL VALIDATION OF THE MODEL

in DI water (nDEP);

2. Excitation Signals: amplitude 6V,

frequency 600 KHz;

3. Device: ø150 µm well.

(b)

(c)

(e)

(f)

x

z

TRAPPING OF SINGLE BEADS OR

AGGREGATES ALONG THE Z AXIS.

Details of the Experiment:

1. Samples: 25 µm polystyrene bead

in DI water (nDEP);

(a) (d)

ONE MINIMUM TWO MINIMA

EXPERIMENTAL VALIDATION OF THE MODEL

in DI water (nDEP);

2. Excitation Signals: amplitude 6V,

frequency 600 KHz;

3. Device: ø150 µm well.

(b)

(c)

(e)

(f)

x

z

MISALIGNED STRUCTURES ALIGN PARTICLES ALONG A CURVED AXIS:

1. z direction : structure immune to misalignment (W>2D)

2. x direction : misalignments curve the alignment line

D s1234D

(a) (c)

MISALIGNMENTS CAUSED BY FABRICATION

4312D’D’

(a)

(b)

(c)

Cage Shape Misalignment

1 35 µm

2 40 µm

3 45 µm

4 50 µm

mis=35 µm

mis=35 µm

z

x

z

x

z

x

QUANTITATIVE CHARACTERIZATION OF THE STRUCTURE:

1. Particles trapped in y=yTRAP where DEPy=FG

2. yTRAP= fn(Voltage)

3. Experiments with 25 µm beads

4. Errors comparable with bead dimension

SIDE VIEW OF THE WELL

MICROSCOPE

MENISCUS

Voltage (V)

VERTICAL POSITION FUNCTION OF VOLTAGE

y=yTRAP

y=0DEPy

FG

CARRIER

FLUID FLOW

-60

-50

-40

-30

-20

-10

0

10

20

30

2 3 4 5 6

y (

µm

)

Voltage (V)

experiment

simulation

TRAPPING SINGLE CELLS OR SMALL

AGGREGATES ALONG THE Z AXIS.

Details of the Experiment:

1. Samples: K562 cells in

Physiological solution;

2. Excitation Signals: amplitude 4V,

EXPERIMENTS WITH K562 CELLS

K562 CELLS ARE TRAPPED

ALIGNED ALONG A PREDEFINED AXIS

2. Excitation Signals: amplitude 4V,

frequency 600 KHz;

3. Device: ø150 µm well;

4. Temperature: vital ranges.

5. Calcein viability test: no short

time effects (20 min)

1. NEW CONCEPT OF OPEN MICROWELLS:

- Trapping particles aligned along an axis

- Flex PCB technology

CONCLUSIONS

2. EXPERIMENTAL VALIDATION OF THE FUNCTIONING:

- 25 µm polystyrene beads in water

- K562 cells in physiological solution

PUBLICATIONS

Publications:

“Horizontal nDEP cages within open microwell arrays for precise positioning of cells and particles”

M. Lombardini, M. Bocchi, L. Rambelli, L. Giulianelli and R. Guerrieri. Biosensors (submitted Lab on Chip)

“Dielectrophoretic trapping in microwell for manipulation of single cells and small aggregates of particles”

M. Bocchi, M. Lombardini, A. Faenza, L. Rambelli, L. Giulianelli, N. Pecorari, R. Guerrieri. Biosensors (Vol. 24,

Issue 5), January 2009

Conferences:

Biosensor 2008

Shangai, China, 14-16 May 2008.

X National Biotechnology Congress

Perugia, Italy, 17-19 September 2008.

Nanotech 2009

Houston, Texas, 5-7 May 2009.

ECTC 2009 - Electronic Components and Technology Conference

San Diego, California USA, 26-29 May 2009.