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P16083 – Automated Microfluidic Cell Separator
Subsystems Design Review
Table of Contents
Risk Assessment
Subsystem Definitions
System Overview
Power
Controls
Fluid Flow
EM Field Generation
Monitoring/Visualization
Safety
Risk Assessment
ID Category Risk Item Effect Cause
Likelihood
Severity
Importance Action to Minimize Risk Owner
What type of risk is this? Describe the risk briefly
What is the effect on any or all of the project deliverables if the
cause actually happens?
What are the possible cause(s) of this risk? L S L*S
What action(s) will you take (and by when) to prevent, reduce the impact of, and/or transfer the risk of this occurring?
Who is responsible for following through on
mitigation?
1 Resource Time ConstraintsThe project will fall behind schedule and may not be
finished
This project has multiple subsystems that rely on the previous system to work so
if one falls behind everything is behind
2 3 6
Scheduling will be done with buffers so that if something goes over the expected amount of time it will not greatly impact
the work flow
Vincent Serianni
2 Resource Outsourced Work Project will be behind schedule Outsourced work may take extensive time
2 3 6Proper planning and scheduling will be used to reduce urgency of outsourced
workVincent Serianni
3 Resource Expense of Components Project will be over budget
Some Components will be quite expensive and difficult
to fit within the limited budget
2 3 6Purchase orders will be looked over to see
if equivalent components can be purchased for less
Jay Dolas
4 Safety Electric Shock Injury may occurBuilding and testing of the Power Supply is inherently
hazardous2 3 6
National Electric Code will be referenced during the design stages, and proper safety measures will be taken during
testing
Ryan Kinney/ Christopher Molinari
5 Safety Lab Work Injury may occurWorking in a lab around chemicals is inherently
hazardous1 2 2
Sufficient safety training will be completed by all involved Alexandra LaLonde
6 Safety LeaksPotentially biohazard fluids are
sprayed on workerHigh Pressure and error with a assembly may cause leak 2 1 2
Pressure release system will be added and start up check will be performed Tyler Lisec
7 Technical Power Supply Failure If the power supply fails then the project will be over budget
Errors in design or assembly 2 3 6Proper design review and assembly is
done correctly and checked before start up
Ryan Kinney
8 Technical Size/Weight Requirement: Size and Weight Requirements will no be met
With current parts it may be difficult to meet
requirements3 2 6 Orders will be double checked to see if
lighter components can be purchasedJay Dolas
9 Technical Fabrication ErrorsReproducibility can be difficult
in a sensitive microfluidic system
May cause air bubbles to form which will hinder the
separation process2 2 4
Designs of channels will be tested to make sure they can prevent this Jay Dolas
10Environment
alPotential Air Born
ChemicalsPotentially hazardous
chemicals may be ingested
Heat may cause of burning of plastics which will cause airborne chemicals to form
1 1 1Proper PPE will be used throughout
experimentation, proper heat management will be put into place
Tyler Lisec
1=lowest, 2=moderate, 3=highest
Subsystems Definitions
SubsystemsSubcompone
nt Power Controls Safety Fluid FlowEM Field
GenerationMonitoring/
Visualization
1 Transformer Start Button E-stopSyringe Pump Electrodes Voltage Displays
2Aux Power Supplies Potentiometers Relays Microchannel Current Display
3Signal
GeneratorTemperature
Sensor Receptacles Frequency Displays
4 Op-amp Fuses Run Indicator
5 Stepper Motor Insulation Error Indicator
6 Device Geometry Microscopy
System Flow Chart
System Flow Chart (Controls)
System Flow Chart (Fluid Flow)
System Flow Chart (Power)
Systems Level Design
Rev 1
Features:• Syringe Pump w/o
Stepper Motor• Microchannel• Basic Control Panel• Ventilation• Receptacle Locations
Sub-Systems Level Design
Rev 2
Updated Features:• Syringe Pump• Microchannel w/
Fixture• Updated Control
Panel• Ventilation• Receptacle Locations• Aesthetics
Power Transformer, Auxiliary Power Supplies
Main Power
Voltage and Frequency
120V
Inverting Op-Amp
𝑉 𝑜𝑢𝑡=−𝑅𝐹
𝑅𝑖𝑛
𝑉 𝑖𝑛
ControlsStart Button, Potentiometers, Signal Generator, Op-amp, Stepper
Motor
Monitoring, Fans,and Op Amps
Op AmpFeasibilityTesting
• Achieved variable voltage
• Maintained input Hz
• Can be used to reach specific voltages
Electronic TimerTesting
• Counts down from any time necessary
• Highly accurate
• Programmable
Variable Frequency Drive
• Arduino Uno failed to output variable frequencies
Reasons for incompletion:
• Lacked Proper Components:
• Filter
• Amplifier
• Leads
• Larger Breadboard
• Arduino Due (has analog outputs)
• Planning to retest with additional components
Fluid FlowSyringe Pump, Microchannel, Receptacles
Syringe Pump
Microchannel
Microchannel Designs
All length dimensions in µm
Microchannel Designs
All length dimensions in µm
Run Time Calculations
Using:
(Reynold’s Number Formula)
(Conservation of mass with constant density)
We find that:
Run time is:
Variables:V = velocityµ = 1.002x10-3 (kg/m-s)– Dynamic ViscosityRe =1 – Reynold’s NumberB = 2x10-3 (m) – Base of the microchannelH = 40x10-6 (m) – Height of the microchannelρ = 998.2 (kg/m3) – Density ID = 0.017 (m) – Inner DiameterQ = 10 (mL) – Volume of fluid
Sensitivity Analysis
Reducing the Run Time
Based on the sensitivity analysis: Increasing the channel base
Increasing the channel height
Decreasing the volume of fluid run through the device
Run multiple channels in parallel
EM Field GenerationElectrodes
Feasibility- Cell Type Ideal cells
• Yeast and E.coli
Escherichia coli Saccharomyces cerevisiae
Cell Size (µm) Length: 2.38±0.32Width: 0.96±0.21
Diameter:6.23±0.77
Medium LB medium YMB medium Incubation Temperature
37 oC 30 oC
Would we need to purchase?
No No
Shape Rod SphereProperties Gram-negative
prokaryotic cellEukaryotic fungalcell
Widely studied using dielectrophoresis
Yes Yes
Cell Strain
Possible Risks:May not be able to see E. coli with microscope
Feasibility- Cell Type Alternative Cells
Source: https://en.wikipedia.org/wiki/Streptococcus
Source: http://www.wikiwand.com/de/Bacillus_subtilis
Bacillus Subtilis
Bacillus Cereus Listeria Monocytogenes*
Saccharomyces Pastorianus
Cell Size (µm) Length: 4.86±0.41Width: 1.94±0.19
Length: 4.94±0.47Width: 1.32±0.13
Length: 3.00Width: 1.00
Diameter: 10.00
Medium TSB medium TSB medium Brain Heart Infusion Broth
YM medium
Incubation Temperature
30 oC 30 oC 37 oC 30 oC
Would we need to purchase?
No No Yes (~$300) Yes (~$300)
Shape Rod Rod Rod SphereProperties Gram-positive
prokaryotic cellGram-positiveprokaryotic cell
Gram-positiveprokaryotic cell
Eukaryotic fungalcell
Studied using dielectrophoresis
Moderately Moderately Moderately Moderately
Cell Strain
Cells not to use:• Lactobacillus• Streptococcus• *Listeria Monocytogenes (BSL
2)
Streptococcus
Bacillus Subtilis
Feasibility- Cell Type under electric field • Gram negative cell:
Outer membrane 7–8 nm thick, with a thin layer 1–3 nm thick of peptidoglycan
• Gram positive cell: a thick peptidoglycan multilayer 20– 80 nm thick, a cytoplasmic membrane, and no outer membrane
• Eukaryotic fungi cells: outer layer of manoproteins, an inner layer of β-1,3-glucan (fibrous polysaccharide), and 70-100 nm thick layer of chitin (polysaccharide of acetylglucosamine)
Peptidoglycan layer 90% of dry weight
Peptidoglycan layer 10% of dry weight
No Peptidoglycan layer
Gram Positive
Gram Negative
Yeast
Source: Assessment of cell viability after manipulation with insulator-based dielectrophoresis
Dielectrophoretic Force
Where
is particle radius
is the permittivity of suspending medium
E is the electric field
K is the Clausius-Mossotti (CM) factor
CM Factor
For simple spherical particles, CM factor is
Where p and m denote the particle and the medium, respectively and is the complex electric permittivity
Where is the conductivity and is the frequency
http://www.rsc.org/suppdata/lc/c1/c1lc20307j/c1lc20307j.pdf
Cell Characteristics
Relative Permittivity Conductivity (S/m) Size (µm)
ShapeCytoplas
m
Cell Membra
neCell Wall
Cytoplasm
Cell Membra
neCell Wall
Cytoplasm
Cell Membra
neCell Wall
E. coli*Double Shell
Spheroid 61 10.8 60 0.19 5.00E-08 0.68a=2.74,
b=c=0.63a=2.75,
b=c=0.64
a=2.79, b=c=0.
68S.
cerevisiae**Single Shell
Spheroid 50.6 198.5 0.55 1.00E-04 0.03 r=2.7 r=2.95
* http://www.rsc.org/suppdata/lc/c1/c1lc20307j/c1lc20307j.pdf** http://www.sciencedirect.com/science/article/pii/0005272896000242
Yeast CM Factor
Utilizes a single shell spherical model
In model, cell membrane and cell wall are lumped into the membrane parameter
Utilizes same equation for CM factor as above, but complex permittivity is calculated as
http://www.rsc.org/suppdata/lc/c1/c1lc20307j/c1lc20307j.pdf
Yeast CM Factor
Need overall membrane conductivity (combination of membrane and wall conductivities)
Model of resistances across cell wall and membrane yields the equation
Where
m is trans-membrane, w1 is trans-wall, and w2 is hemi-wall
P is the wall circumference
Yeast CM Factor
E. coli CM Factor
Uses a complicated double shell spheroid model
CM factor calculated as
http://www.rsc.org/suppdata/lc/c1/c1lc20307j/c1lc20307j.pdf
E. coli CM Factor
Electrode BenchmarkingPlanar metal electrodes (2D)• Electrodes on bottom of channel• Alternating sets of grounded and energized
electrodes-- creates nonuniform electric field
• Metals normally used:• Gold• Platinum• Indium tin oxide (ITO)
Key Benefits• Fabrication techniques are very developed
and readily used– can be fabricated in a few hours
• Requires very low voltages to produce DEP force
Source: E.D. Pratt et al. / Chemical Engineering Science 66 (2011) 1508–15221512
• Angled electrodes- most often used for binary separation, creates localized particle pathlines due to the particles’ negative DEP mobilities- net force parallel to the electrodes
Electrode BenchmarkingPlanar metal electrodes (2D) continued… Constraints/disadvantages:• Sample volume must be < 30 µm away
from the bottom for particles to feel DEP force
• Can cause sample electrolysis • Most likely to happen at low
frequencies, high medium conductivity, and high voltage
• Can coat electrodes with inert polymers to resolve this (will increase price)
• Could use a copper-like material, which makes electrodes reusable and inexpensive, but requires more voltage to produce needed DEP force
Alternatives:• Electrodes on top and bottom of
channel• As channel height increases > 50
µm, particles in middle may not experience DEP force
Source: E.D. Pratt et al. / Chemical Engineering Science 66 (2011) 1508–15221512
Electrode Benchmarking3D metal electrodes• Electroplated gold structures
embedded in the walls of a microchannel
• Electrodes span the entire height of the channel walls (30–40 µm)
• Enables the dynamic positioning of a stream of particles anywhere along the width of the channel (lateral flow DEP)
• The use of two different frequencies independently polarizes electrodes embedded on each side of the channel
• Complicated fabrication• Expensive due to metal
electroplating– more material needed
Source: Martinez-Duarte et. al. Microfabrication technologies in dielectrophoreis applications (2012)Constraints/disadvantages:
Key Benefits:
Electrode Benchmarking3D Doped silicon• Made of three parts: a thick, heavily
doped silicon layer containing the microfluidic channel and electrodes sandwiched between two glass layers bound to the silicon layer
Key Benefits• The device does not suffer from sample
leakage since it is fully enclosed • Very beneficial if this device is used for
high-throughput systems• Implementing high-flow rates and
pressure in the channel can cause leakage if using other materials or fabrication techniques
Constraints/disadvantages • The fabrication process is fairly complex
and expensive
Source: Martinez-Duarte et. al. Microfabrication technologies in dielectrophoreis applications (2012)
Electrode BenchmarkingCarbon electrodes • Combines the advantages of metal-
based and insulator-based DEPKey benefits• Possibility of sample electrolysis is
minimized • Carbon has a much wider
electrochemical stability window than metals like gold and platinum
• Fabrication of 3D carbon electrodes is simple and low cost
• Requires low voltages to polarize electrodes
Constraints/disadvantages • Voltage is lost due to narrow leads so
higher voltages are required than for metal electrode DEP • Still relatively low (tens of volts)
Source: Martinez-Duarte et. al. A novel approach to dielectrophoresis using carbon electrodes
Electrode Designs
All length dimensions in µm
Monitoring/Visualization
Voltage Displays, Current Displays, Frequency Displays, Run Indicator, Error Indicator, Microscopy
Microscope Benchmarking
Celestron Handheld
Digital Microscope
Pro
Bodelin Technologies ProScope EDU 5MP Digital
Microscope
Avangard Optics AN-
E500 eScope 500x USB 2.0 Mega Pixel
Digital Microscope
Carson eFlex Handheld
Digital Microscope
Bodelin Technologies ProScope HR2 QC Lab
KitCost $83.95 $119.00 $49.95 $45.99 $999.99
Objective 20-200x 10-300x 500x 75x and 500x
Lenses: 0-10x, 30N, 100x, 200x (50x and 400x are available)
Interface USB USB USB USB USBMP 5-12MP 5MP 2MP - 2MP
Resolution -2592x1944 (image),
1280x960 (video)1600x1200
(video) -1280x1024 (image), 1600x1200 (video)
Size 4.3" x 1.4"(D) 4.3" x 1.4"(D) 4.72" x 1.30"(D) 3.4" x 1.1"(D) 6.0" x 1.8"Focal
Length 15.8mm - - - Depends on lens
Focus Type Manual Manual, 10mm-300mm Manual Manual Manual/DigitalIlluminatio
n None 8 LED 8 LED 4 LED None
SafetyE-Stop, Relays, Temperature Sensor, Fuses, Insulation, Device
Geometry
Safety – Failure of Components
• Arduino Signal Generator – System/Op Amps shut off
• Transformer – Lose voltage for Amplification, Op Amps limits to 0V
• Op Amps – Stops all separation functionality of the device, Current limiter fails causing a spike in current
• Fans – System will heat up, may damage cells
Gantt Chart
See Bill of Materials
Questions/Comments?