electro-physiological characterisation of cells for healthcare applications

Electro-physiological characterisation of cells for healthcare applications

Dr. Soumen DasAssociate Professor

School of medical Science & Technology

Indian Institute of Technology Kharagpur15th September 2016

Organisation of talk Introduction – Fusion of technologies at micro scale Reason for miniaturisation – Scaling effect Soft lithography Microfluidics Dielectrophoresis Flow cytometer Bioimpedance

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Photolithography and medicine were total strangers to one another. At present they are indispensable partners in biomedicine to envisage the scenarios of personalised medicine such as following:• Patient specific prevention and intervention strategies.• Early detection protocols to identify decease when it is easily subdued.•Technology allowing for long lives in the company of disease as good neighbour, without sacrificing the quality of life.

Development of miniaturized wearable/implantable BMW (BioMedical Wireless) sensor for personalized health care and beyond-hospital applications.

Decades of science and engineering knowledge are now converging to provide tools through micro and nanotechnologies that enable manipulation of biological systems at its length scales.

FUSION OF TECHNOLOGIES IN MICROSCALE For Biomedical Applications

3SMST, Indian Institute of Technology Kharagpur

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On Size and Scale !

Similarity in sizes and organization of common structures between the micro/nano scale devices and biological species makes this technology an obvious choice for creating advanced ultrasensitive clinical tools for direct detection of biological entities.

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MEMS vs. BioMEMSMEMS use micro-size components such as sensors, transducers, actuators, and electronic devices to sense (smell, feel, see, hear, taste) or to make something happen.Many of the MEMS used in consumer products and other areas (e.g., aerospace, agriculture, environmental) are also found in medical devices.MEMS pressure sensors are found in blood pressure monitors, infusion pumps, catheters, and intracranial probes.

For example, the MEMS inertial sensor used for airbag deployment in cars is also used in

rate responsive pacemakers.

Biosensors are ‘analytical devices that combine a biologically sensitive element with a physical or chemical transducer to selectively and quantitatively detect the presence of specific compounds in a given external environment’

SMST, Indian Institute of Technology Kharagpur

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Some of the MEMS used in the medical are unique in the sense that they incorporate biological molecules as an integral part of the device.

Microcantilever transducer coated with antibodies (green spheres) that capture a virus (red sphere) in a blood sample while ignoring the other components in the sample.

MEMS Cell Culture and analysis: creates a microenvironment for growing cells in vitro and in parallel, allowing for the analysis of multiple cell growth conditions.


Standard neuro probes

MiniMedParadigm®522 insulin pump

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In general, the use of micro and nano-scale detection technologies is justified byReducing the sensor element to the scale of the target species and hence providing a higher sensitivity; single entity/moleculeReduced reagent volumes and associated costs,Reduced time to result due to small volumes resulting in higher effective concentrations,Amenability of portability and miniaturization of the entire systemPoint-of-care diagnostic,Multi-agent detection capabilityPotential for use in vitro as well as in vivo

Reasons for Miniaturisation



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Precise control and manipulation of very small fluid flows

~ of the order of microliters or nanoliters ** a drop of water is approximately 25 μl

-Circulating and Respiratory System

-Arteria and venes in animals

- Capillaries in plants

• Historical Microfluidics: Glass capillary


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Biochemical assays: real-time PCR, immunoassay, dielectrophoresis for detecting cancer cells and bacteria, etc.

Chemical application: separating molecules from mixtures, chemical reactors, chemical detections. etc.

Biological application: Fundamental understanding of Bio-physical processes, cell co-culture, biosensor, drug screening, single-cell analysis, etc.


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Most sensing techniques scale poorly in the micro domain (-) Often large samples are required to get enough target species

collected (-) Short analysis time dictates small devices (+) Fast heating/cooling (e.g., for PCR) requires small samples (+) All flow is laminar (little turbulent mixing) (- for mixing) Surface tension becomes significant (+/-) No inertia effects (+/-) Apparent viscosity increases (+/-) Evaporation is very fast for small samples (-) Devices are almost always too large for Si to be a solution.


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Photo lithography or etching

L-Edit, AutoCAD

Print

Soft lithography


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In soft lithography, an elastomeric stamp with patterned relief structures on its surface is used to generate patterns and structures with feature size ranging form 30 nm to 100 mm.

Elastomeric polydimethylsiloxane (PDMS) is most widely used. Other materials include polyurethanes, polyimides, and cross linked phenol formaldehyde polymers

Complete non-silicon based device – Flexible & biocompatible

• Micromolding • Microcontact printing• Replica Molding (REM)


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Proliferation of cancer disease occurs due to various physiological changes at the cellular level.

At present the detection techniques are limited to biochemical assay using labeling of diseased cells.

However, the mechanical, electrical and optical properties (non-biological parameters) of the cell also change during various stages of malignancy.

Thus, an alternative path is explored for label-free detection of the cancer cells by identifying and measuring those non-biological parameters of the cells to capture the signature of cancer disease.

In this aspect detection of electrical signals at a very low scale coming out of the cells can be possible by using ultrasensitive miniature bioMEMS sensors.

MEMS technology is indeed a boon as it provides a robust platform to meet such challenges in a very efficient way to meet the emerging needs of biosensing.


Hypothesis - Sensing non biological parameters

15Dept. of EE, Indian Institute of Technology Kharagpur

Alter Biochemical Composition Alter Dynamics•Membrane •Cytoplasm•Nucleus, etc.

•Cell division•Adhesion•Death, etc.

Conventional biological assay× Labeling × Efficiency of technicians× Time consuming× Expensive

Reflect in Mechanical, Electrical & Optical propertiesMicroscale detection system to probe cellular level information during cell cultureExploit advantage of scaling law -- Impedance measurement using microdevices

Disease causes

Schematicof a Cell

Microfluidics technology helps in electro-physical understanding of different disease cells (cancer) from its normal cells

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Dielectrophoretic Microfluidic Device for Separation of Cells

• Requirement of rapid diagnosis technique • Conventional Practice

Alternate Techniques

Bio-chemical staining & Microscopic observations× Labeling × Efficiency of technicians× Target cells lesser than large normal cells× Time consuming× Expensive

•Label-free and continuous separation utilizing Microfluidic technology

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Centrifugation

Particles of different densities or sizes

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Fluorescence-activated cell sorting (FACS) Provides a method for sorting a heterogeneous mixture of

biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell.


Break into individual droplets. Fluorescent characterization of interest cell By collecting light (scatter and

fluorescence) a computer determines which cells are to be separated and collected

An electrical charging ring is placed just at the point where the stream breaks into droplets.

The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge

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What is Dielectrophoresis (DEP)?


FDEP = 2 m a3 Re[ fCM (ω) ] E2

||εεpp** | < | | < | εεmm* |* |||εεp p *| > | *| > | εεmm*|*|

+++

__

_mB

Medium

Plate electrode

+

++

++ +

_ ____

mA

The translational motion of the neutral particles caused by the polarization effects in non-uniform electric field

Re[ Re[ ffcmcm ( (εε, , σσ, , ω)ω) ] ] > 0 : positive DEP> 0 : positive DEP

Re[ Re[ ffcmcm ( (εε, , σσ, , ω)ω) ] ] < 0 : negative DEP< 0 : negative DEP


Dielectric polarization : Change in the local distribution of bounded charge induced by an applied field.• Induced dipole formed • Charges of neighboring dipoles cancel, leaving behind a net induced polarization charge at surface


Since dipoles consist of positive and negative charges a distance apart, they generate their own electric field; this then warps the external electric field

Induced electric field is aligned counter to the external field, and the field is warped toward the surface of the particle and intersects the surface at near right angles

The dipole is oriented in the same direction as the external field and the field lines warp around the particle

Polarizability of the particle is a function of complex permittivity

Particle more polarizable (conducting ) than the medium

Particle less polarizable (insulating ) than the medium

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Lossy dielectric- suffer energy losses


ACd

1CZ

j C

~

1( ) ( )1 1

RCR d d j dz

jj RC A j A j j A

~j

Energy stored

Energy loss~ ( )jj

dRA

0

Permittivity dominated behaviour

Conductivity dominated behaviour

Between them, there is a transition in the dielectric behaviour from one type to another. This process is called dielectric dispersion

Permittivity is frequency dependent


Force on the particle

( ) ( )F Q E r d Q E r


•Force on the particle

If E is uniform => F=0

( ) ( )F Q E r d Q E r

The translational motion of the neutral particles caused by the polarization effects in non-uniform electric field

2302 Re[ ( , , )].DEP m cmF a f E

We have to produce non-uniform electric field

* *

* *

( )( 2 )

P Mcm

P M

f

* j

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2302 Re[ ( , , )].DEP m cmF r f E

Re[ ( , , )] 0cmf

Re[ ( , , )] 0cmf

p-DEP

n-DEP

•CM factor varies with applied frequency, properties of particle and medium

SMST, Indian Institute of Technology, Kharagpur

•DEP is a non-linear phenomena due to dependence on the electrical field (E2 )•DEP force is present only when the electric field is non-uniform•DEP force does not depend on the polarity of the electric field=>works both DC and AC•DEP force is proportional to particle volume=> can separate size wise•DEP force is proportional to electrical properties of the particle and the medium•DEP force depends upon the sign and the magnitude of the Clausius-Mossotti factor, fCM

27SMST, Indian Institute of Technology, Kharagpur

FDEP = 2 0 m r3 Re[ fcm (ω) ] E2

||εεpp** | < | | < | εεmm* |* |

||εεp p *| > | *| > | εεmm*|*|

++ +

_ __B

Medium

Plate electrode

+

++

++ +

_ ____

A

Re[ Re[ ffcmcm ( (εε, , σσ, , ω)ω) ] ] > 0 : positive DEP> 0 : positive DEP

Re[ Re[ ffcmcm ( (εε, , σσ, , ω)ω) ] ] < 0 : negative DEP< 0 : negative DEP

-

28 SMST, Indian Institute of Technology Kharagpur

* *

* *

( )( 2 )

P Mcm

P M

f

* j

( ) ( )

( 2 ) ( 2 )

p m p mcm

p m p m

jf

j

2

2 22

1( )( 2 ) ( )( 2 )Re[ ] 1( 2 ) ( 2 )

p m p m p m p m

cmp m p m

f

0

( )lim Re[ ]

( 2 )p m

CMp m

f

( )lim Re[ ]

( 2 )p m

CMp m

f

Sign is determined by conductivity

Sign is determined by permittivity

Re[ ] 0CMf FDEP=0


p m p mand p m p mand

Re[ ] 0CMf FDEP=0 => cross over frequency

Frequency where n-DEP switches to p-DEP


• and of normal and cancerous cells are different

•Separation- Find a particular frequency at which one group of cells will experience positive DEP whereas other group of cells will fill negative DEP

•Manipulation- Vary Re[fcm(w)] with frequency

Based on sizeParticle’s dielectric property

( , ) ( )a

2302 Re[ ( , , )].DEP m cmF a f E

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Cells exhibit polarizability in non-uniform fieldHow does cell attain higher polarizability? Major portion of cell is water Polar molecules-proteins, sugar, DNA etc are dissolved in

intercellular regions Lipid membrane acts as capacitive region


The cell membrane consists of a very thin lipid bilayer, which is highly insulating with a conductivity of about 10-7 S/m. The conductivity of the cytoplasm (interior part of a cell) can be as high as 1 S/m, since cells contain many ions and charged particulates. Upon cell death, the membrane becomes permeable and its conductivity can dramatically increase by a factor of 10 4.


Paired micro tips electrode

Our Aim : Design and fabrication of a microfluidic device by micromachining technology for rapid and continuous separation of cervical cancer cells

Composed of the micro-channel and planar electrodes The cell mixture is injected through inlet AC signals applied to electrodes generate the DEP force to move

the cells in the mixture


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2302 Re[ ( , , )].DEP m cmF a f E

Cross-section view

•D.Das, et al., Medical Engineering & Physics, 2014

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PhotolithographyUV

Mask 1

Growth of SiO2 Si

Cr/ Au layerdeposition

Coating ofpositive Photoresist

Patterned electrode

Coating ofSU-8 Photoresist

SU-8 Open Channel

PhotolithographyUVMask 2

PDMSCovering

Si

SiO2

Cr/ Au

+VePhotoresist

Mask

SU-8 PDMS

Process steps for fabrication

Fabricated Device & Measurement Setup

Microscopic view of the electrode and micro-channel

Microphotograph of the fabricated DEP-microfluidic device

Schematic of Experimental setup for continuous cell manipulation

Fabricated DEP-microfluidic deviceafter covering and punching

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Experimental Observation: Vpp =10V, Flow: 2 μl/minParticle diameter:10 μm

At both100 kHz and 1 MHz frequency 10 µm

Micro-beads move through centre electrode

Experienced n-DEP force

Theoretical Comparison: In the frequency range of 100 Hz to 100 MHz Re [fCM] factor is always negative

Particles will always experience n-DEP in this frequency range

Variation of CM factor with frequency for polystyrene beads

Beads


Micro-beads movement

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Variation of CM factor with frequency for HaCaT cells

Microscopic observation of movement of HaCaT cells (a) @ 100 kHz, (b) 1 MHz

Experimental Observation:@ 100 - 600 kHz frequency HaCaT cells move through centre experience n-DEP effect @ 800 - 1 MHz maximum HaCaT cells move towards side electrode experience p-DEP

Theoretical Comparison:There is a crossover frequency @ 736 kHz

Frequency <736 kHz the Re [fCM] factor --negative

> 736 kHz value of Re[fCM] is positive

Cell

(a) (b)

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736 kHz

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Variation of CM factor with frequency for HaCaT cells and

beads Microscopic observation of movement of

Beads and HaCaT cells

Experimental Observation:

•@ 100 kHz frequency both HaCaT cells and beads experienced n-DEP effect

•@1 MHz frequency HaCaT cells moves along the side, whereas beads move over central electrode

Theoretical Comparison:

Cell

Bead

@ 100 kHz

@ 1MHz


•D.Das, et al., ICST, 2015


Shashank Shekhar, Paul Stokes, and Saiful I. Khondaker, ACS Nano, 2011, 5 (3), pp 1739–1746

• How to align the nanotubes direction?• How to bridge the nanotubes between source and drain

electrodes?

Manipulation of nanotubes

Developing sensitive Sensors- gas, humidity,Molecule sensor etc.


Journal of Colloid and Interface Science, vol. 355, pp 486-493, 2011.

(a) Randomly distributed hepatic cells are loaded into the microfluidic chamber.

(b) The hepatic cells are captured and patterned onto the 1st DEP patterning electrodes

(c) The endothelial cells are, then, loaded, guided and positioned in-between the patterned hepatic cells on the 2nd DEP patterning electrode.

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Microflow Cytometer

Counting number of cells/ particles in a fixed volume of sample


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Microflow CytometerImportance of cell/particle counting in Healthcare•Determining the health condition of a patient•Live/dead cells under drug treatment•Researching the behavior of infectious viruses, bacteria and other pathogensTechniques•Hemocytometer- counting under microscope

•Spectrophotometry- based on turbidity and light absorbed by cells

•FACS- count depending on optical scattering of fluorescent labeled cells.

Alternate Techniques • Counting in microchannel based on the electrical property —portable, cost effective, integrated in lab-on-a-chip system.

Issues:×Time consuming× Skilled person× Labeling× Expensive× Complex analysis


When particles passed overall resistance between electrode pairs changed. The change of resistance causes a pulse.Total number of electrical pulses ~ number of particlesVoltage variation due to change of Rch is amplified and detected in a data acquisition (DAQ) system.

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Schematic representation of the impedance flow cytometer

Analogous Model


Fabricated microfluidic device after covering and punching

Microscopic view of the electrode and microchannel

Common electrode Microchannel

Sensing electrode


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Flow rate - 5 µl/min Each pulse corresponds to change of

resistance due to a particle. Width of pulse proportional to the

cell size Magnitude proportional to electrical

property of particles.

160 180 200 220 240 260 280

6.4

6.5

6.6

6.7

6.8

6.9

7

Time(seconds)

Out

put(v

olts

)

a

b

d

cfe

g hl m

k

jino p

rq

s

Time trace output captured by Agilent DAS.

204.75 204.8 204.85 204.9 204.95

6.38

6.4

6.42

6.44

6.46

6.48

6.5

6.52

6.54

X: 204.7Y: 6.377

Time(Milisecond)

Out

put(v

olts

)

X: 204.8Y: 6.422

X: 204.8Y: 6.406

X: 204.9Y: 6.423

X: 204.9Y: 6.543

X: 205Y: 6.38

e

size

electrical property LimitationRequires offline data processingDoes not provide real time counting


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Overview of counting-instrumentation

IA- amplifies voltage variation due to this change of impedanceNotch Filter- suppress power frequency harmonicsAD 843-shapes signal to square wave


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•The value of TIMER 1 of the microcontroller is processed based on following equation to get count number:

Count = (integer value × 2562 ) + (timer1H value × 256) + (timer1L value).

Limitation-• A chance of 10 count error in output value if the frequency of pulse is greater than 25 kHz.

Particle/cell concentration

Flow rate Flow time Real-time Count

Count by DAQ

108 beads in 2ml PBS

2 µl / min 40 sec 64090 64150

60 sec 96140 95650

106 cells in 2ml PBS

2 µl / min 40 sec 650 664

60 sec 980 991


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Bio-Impedance Mechanical, electrical & optical properties of cell also

change along with bio-chemical property during disease process

Label-free characterization of biological cells using change in electrical properties


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Impedimetric characterization mode

Frequency sweep at fixed time Static characteristics

Real-time at fixed freq Dynamic property

•Electrical features•Membrane capacitance •Cytoplasm resistance

•Real time cell growth•Attachment, Spreading,

Death

Modeling & Analysis

Comprehensive understanding of cancer behavior from Electrical point of view:

R, C, ε, σ

Suspended/Adherent cell colony

Combine static & dynamic features infer complete pathological stage of cell


Aspire: Simple but sensitive sensor in the

dimension of cells Cell culture compatible Modeling and analysis of

impedance data

Device Fabrication Process:

Final fabricated device

3D schematic of the device

50

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Impedance of media

Impedance of media +Cell

Seed Cell

Allow to grow

Optimum freq: • 100 Hz- 10 MHz with 10 mV from COMSOL Simulation

Impedance response

Measurement steps: High freq currentLow freq current

Impedance Analyzer: Agilent 4294A


Electrode Medium Cells

Proposed a novel fragmental frequency analysis techniqueMore flexibilityDetail understanding of system

Parameter extraction

Fitting softwareCompulsory initial guess No of data point

Alternate

Cross sectional view of one microwell

Equivalent circuit

•D. Das et al. IEEE Transactions on Instrumentation & Measurement, 2014.


Impedance measured at 40 kHz, 5 min interval with Agilent 4294A Impedance Analyzer

Healthy cells- HaCaT cell line Breast Cancer cells- MCF-7 & MDA-MB-231 cell lines

HaCaT (Normal) MCF-7 (Cancer) MDA-MB-231 (Cancer)

Both cancer cells have higher raising slope faster growth rateAny further insight ??Uninterrupted Impedance fluctuations– correlated with cellular micromotions

Fluctuations

slope slopeslope

Less fluctuations

#NZ- Normalized Impedance

•D. Das, et al., Physical Review E, 2015.


Understanding effect of drug to enhance success of chemotherapy

Drug dose selection

Dynamic cell-drug interaction To develop a toxicity index

Two breast cancer cell lines treated with Paclitaxel drug MCF-7 MDA-MB-231

Slopeincrease

# NZ Normalized Impedance


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Microfluidics promises miniaturization of liquid-manipulation Processes

Scaling effects at micro-scale help in-vitro experiments; accurate and sensitive detection

Easy pattering of PDMS polymer avoid costly fabrication process Dielectrophoretic microfluidic device for separation of cells have

been demonstrated Micro-chips enable to capture and characterize cancer cells in

terms of electrical properties in a non-invasive manner Microfluidics can solve many complicated bio-chemical-physical

process with fundamental understanding

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