electro-physiological characterisation of cells for healthcare applications
TRANSCRIPT
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
2
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
4
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.
5
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
6
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.
SMST, Indian Institute of Technology Kharagpur
Standard neuro probes
MiniMedParadigm®522 insulin pump
7
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
SMST, Indian Institute of Technology Kharagpur
SMST, Indian Institute of Technology Kharagpur
8
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
SMST, Indian Institute of Technology Kharagpur
9
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.
SMST, Indian Institute of Technology Kharagpur
10
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.
SMST, Indian Institute of Technology Kharagpur
11
Photo lithography or etching
L-Edit, AutoCAD
Soft lithography
SMST, Indian Institute of Technology Kharagpur
12
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)
SMST, Indian Institute of Technology Kharagpur
13
14
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.
SMST, Indian Institute of Technology Kharagpur
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
16
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
17
Centrifugation
Particles of different densities or sizes
18
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.
SMST, Indian Institute of Technology Kharagpur
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
19
What is Dielectrophoresis (DEP)?
SMST, Indian Institute of Technology Kharagpur
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
20SMST, Indian Institute of Technology Kharagpur
21SMST, Indian Institute of Technology Kharagpur
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
22SMST, Indian Institute of Technology Kharagpur
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
23
Lossy dielectric- suffer energy losses
SMST, Indian Institute of Technology Kharagpur
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
24SMST, Indian Institute of Technology Kharagpur
Force on the particle
( ) ( )F Q E r d Q E r
25SMST, Indian Institute of Technology Kharagpur
•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
26
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
29 SMST, Indian Institute of Technology Kharagpur
p m p mand p m p mand
Re[ ] 0CMf FDEP=0 => cross over frequency
Frequency where n-DEP switches to p-DEP
30 SMST, Indian Institute of Technology Kharagpur
• 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
31
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
SMST, Indian Institute of Technology Kharagpur
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.
32 SMST, Indian Institute of Technology Kharagpur
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
SMST, Indian Institute of Technology Kharagpur
33
2302 Re[ ( , , )].DEP m cmF a f E
Cross-section view
•D.Das, et al., Medical Engineering & Physics, 2014
34
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
35
SMST, Indian Institute of Technology Kharagpur
36
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
SMST, Indian Institute of Technology Kharagpur
Micro-beads movement
37
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)
37
SMST, Indian Institute of Technology Kharagpur
736 kHz
38
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
SMST, Indian Institute of Technology Kharagpur
•D.Das, et al., ICST, 2015
39 SMST, Indian Institute of Technology Kharagpur
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.
40 SMST, Indian Institute of Technology Kharagpur
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.
41
Microflow Cytometer
Counting number of cells/ particles in a fixed volume of sample
41 SMST, Indian Institute of Technology Kharagpur
42
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
42 SMST, Indian Institute of Technology Kharagpur
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.
43
Schematic representation of the impedance flow cytometer
Analogous Model
43 SMST, Indian Institute of Technology Kharagpur
Fabricated microfluidic device after covering and punching
Microscopic view of the electrode and microchannel
Common electrode Microchannel
Sensing electrode
44 SMST, Indian Institute of Technology Kharagpur
45
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
45 SMST, Indian Institute of Technology Kharagpur
46
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
46 SMST, Indian Institute of Technology Kharagpur
47
•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
47 SMST, Indian Institute of Technology Kharagpur
48
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
48 SMST, Indian Institute of Technology Kharagpur
49
SMST, Indian Institute of Technology Kharagpur
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
SMST, Indian Institute of Technology Kharagpur
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
51
SMST, Indian Institute of Technology Kharagpur
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
52SMST, Indian Institute of Technology Kharagpur
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.
53SMST, Indian Institute of Technology Kharagpur
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.
54SMST, Indian Institute of Technology Kharagpur
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
SMST, Indian Institute of Technology Kharagpur
55
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
THANK YOU