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?