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Automated Microfluidic
Cell Separator
Project Group: 16083
Table of Contents

Group Members

Morph/Pugh Chart

Stakeholders

Feasibility

Budget


Project Statement
Experimental Plan and
Designs

Use Scenarios

Future Plans

Engineering Requirements

Risk Management

Phase Two

Function Decomposition

Benchmarking
The P16083 Group
Member
Major
Role
Contact
Jay Dolas
BME
Lead Engineer
[email protected]
Alexandra LaLonde
BME
Microfludics Specialist [email protected]
Vincent Serianni II
BME
Project Manager
[email protected]
Ryan Kinney
EE
Lead Electrical
[email protected]
Chris Molinari
EE
Controls Engineer
[email protected]
Tyler Lisec
ME
Lead Mechanical
[email protected]
Stakeholders
 Customer – Dr. Blanca Lapizco-Encinas
 End Users – Lab Workers, Professors, Researchers, Students
 Sponsors – BME Department at RIT
 Potential Sponsors – Rheonix Inc., Simone Center for Student Innovation
and Entrepreneurship at RIT
 Other Stakeholders – P16083 Group, MSD Team
https://www.rit.edu/research/simonecenter/
http://www.rheonix.com/corporate/careers.php
https://twitter.com/ritbme
Minimum
Estimated Budget
Problem Statement
A cell separator is a device that separates cells in a mixture, based upon preestablished criteria (biomarkers, size, electrical characteristics, etc.). This is necessary
in many cell culture and diagnostic applications where downstream processes occur
after cell culture, such as purification or analysis. Optimally, this device should not
interfere with the viability or characteristics of the cells, while still being cost effective.
Current cell separation devices require some sort of labeling (either fluorescent or
magnetic) which is not only costly but can affect cell behavior and mortality. We
propose an automated microfluidic system that utilizes developing technologies
(dielectrophoretics) to reduce costs drastically while maintaining cell viability.
The goals of this project are to develop a system that not only sorts cells without the
use of labeling, but also fits within a biosafety cabinet, is self-driven, and is automated
(hands-off once the sample is loaded and sequence has started). The expected
result is a functional prototype that fits all of the goals above and is suitable for use in
a teaching laboratory. The design and prototype must conform to intellectual
property and diagnostic laboratory standards so that it may be marketed this as a
definitive step forwards in cell separation technology.
Current State


Flow Cytometry

Fluorescence labeling

Laser to excite and identify the
cells

Additives could alter or damage
cells

Labeling is expensive
http://www.appliedcytometry.com/flow_cytometry.php
Hydrodynamic Cell Separator

Inertial forces to separate cell types

High shear forces can damage
cells

Channel design and
manufacturing is timely and
expensive
http://www.elveflow.com/microfluidic-tutorials/cell-biology-imaging-reviews-andtutorials/microfluidic-for-cell-biology/label-free-microfluidic-cell-separation-andsorting-techniques-a-review/
Desired State

Dielectrophoresis

Uses electric fields to manipulate
the cell location in a stream

No need for additives

No added shear force
http://www.mdpi.com/1422-0067/15/10/18281/htm
Project Goals and Deliverables
Working prototype that can:

Demonstrate in a class setting the use of dielectrophoretics in cell
separation

Act as a partially automated system

User only has to load the sample and set the target specifications

Maintain cell viability during sorting process

Accurately sorts the target cells
Documentation of the prototype that illustrates:

Proper use and care of the device

Target specifications

Voltage amplitude and frequency standards to sort a given cell type
Key Constraints

Device start-up cost below $5000

Lightweight - able to be moved by 1 person

Electrically shielded and insulated

Bio-hazard containment

Footprint (1.5' x 1.5')

120V outlet compatible

Reusable channel

Perform process within one hour
Use Scenarios – Teaching Aid
Use Scenarios – Medical Field
Engineering Requirements
Scale: 1 = Less Important, 3 = Moderately Important, 9 = Very Important
Team Vision for Phase Two


What did your team plan to do during this phase?

Use the problem statement in constructing functionality of the device

From the functional decomposition, the concepts for each subsystem are
determined

Finally, a decision that best fits the customer requirements will be chosen for
the development of subsystems
What did your team actually accomplish during this phase?

The functions of the device were defined

Potential solutions to each of these functions were generated

A device concept was chosen from the function morphological chart

A comparison of concepts using the Pugh Chart

Secured funding
Functional Decomposition
Functions-Requirements Mapping
Benchmarking –
Cell Separation
Benchmarking –
Electrical Components
Morph Chart
Morph Chart
Morph Chart
Morph Chart
Concept Generation
Selection Criteria
Pugh Chart
System Architecture
Feasibility –
Acquire, Culture, Prepare Cells



Acquire Cells: Borrow Cells

Cells will be acquired from the Microscale BioSeparations Laboratory run by our customer,
Dr. Blanca Lapizco-Encinas

We will use E. coli and S. cerevisiae for benchmarking separations
Culture Cells: Standard Culture

E. coli will be cultured in liquid LB media

S. cerevisiae will be cultured in liquid YM media
Prepare Cells for Separation: Re-suspend in Buffer

Cells will be used that are no older than 12 hrs and their optical density (OD) will be
determined using a spectophotometer. The measured OD will be compared to a
predetermined cell growth curve for the cell sample of interest. Cell culture should be near
the transition from log phase to stationary phase

For E.coli an optimal OD is around 0.6. For yeast an optimal OD is around 1.2

From the OD and equation relating that to the cell concentration, the cell concentration in
the culture will be calculated
Feasibility –
Prepare Cells Continued

Using the equation (Concentration_1)
(Volume_1) = (Concentration_2)
(Volume_2) the volume needed of
buffer solution to resuspend the cell
culture in will be solved for

Vfinal = (Cinitial x Vinitial) / Cfinal

The final volume desired of each cell
type will be determined by
experimentation

Once this value is calculated a
centrifuge is used to spin down the
culture to get a nice pellet of cells
(8000 rpm for 10 minutes)

The supernatant is discarded and
Vfinal is added which is dependent
on the desired concentration
Feasibility –
Load Cells and Initiation


Load Cells into Device: Put in Syringe

Using standard syringe aspiration techniques, cells in fluid will be added to
the syringe

Fully compressing the syringe's plunger, followed by placing the needle into
cell filled fluid

By extending the plunger, the fluid and cells will fill the barrel of the syringe
Set Voltages and Frequencies: Hardware


Potentiometers will be used and adjusted to set the resistance of the microcontroller and of the Rf resistor value of the op-amp
Initiate Process: Hardware

A toggle button, with an electro-mechanical relay, will be used for initiating
the start of the test

Once toggled (ON), a timer will begin counting for the users’ sake
Feasibility –
Drive Flow: Syringe Pump

These pumps will use a stepper
motor to control the rotation of a
screw that will push/pull a plunger
block

The plunge block will convert the
rotational speed of the screw to
linear speed of the syringe plunger
in order to control the flow from the
syringe to the channel

This device will use two pumps, one
to control the flow of buffer in order
to prime the channel, drive the
cells, and clear the channel of cells
and the second to control the flow
of suspended cells into the flow of
buffer
Feasibility –
Drive Flow: Syringe Pump
Feasibility –
Monitor and Separate Cells

Monitor Flow: None

Separate Cells: eDEP

Dielectrophoresis (DEP) can selectively trap or repel targeted particles

Metal electrode arrays can be used to implement DEP

This requires as little as 10 Vpp to polarize the array compared to insulatorbased dielectrophoresis which can require hundreds of volts.
Feasibility –
Separate Cells Continued

The dielectrophoretic force for a spherical
particle is given by:

Key constraints using planar electrodes: An
electric field gradient is only effectively
established in sample volumes less than ~30
microns away from the bottom of the device
References: http://onlinelibrary.wiley.com/doi/10.1002/elps.201200242/abstract;jsessionid=F2A6707B83E7EE5CEE779F3
9BC66341A.f04t04
http://www.rsc.org/suppdata/lc/c1/c1lc20307j/c1lc20307j.pdf
http://www.sciencedirect.com/science/article/pii/0005272896000242
Feasibility –
Modulate Voltages: Op-Amp

Small scale testing will be ran with an OP Amp and
potentiometer to confirm the op amps validity and the
potentiometer will increase the voltages
Small scale testing will be done on a similar Op Amp using a
function generator to create a signal
 One major concern of Op Amps is that the rails need to have
a large enough limit to allow for amplification to the voltages
 An Ideal Transformer equation will be used to determine
values needed by the transformer to meet the voltage of the
rails for the Op Amp


The ratio of the transformer that will be needed is 1:8.3
Feasibility –
Frequency and Displays



Modulate Frequency: Micro-controller

A waveform generator will be replicated to obtain a either a sine, triangle,
pulse, or saw signal

Will have a range of frequencies from 1-50kHz

Reference: http://www.instructables.com/id/Arduino-Waveform-Generator/
Monitor Signals: Current Monitoring

A digital current meter will be used to give a visual representation to the user
about of how much current is flowing through the electrodes

The rest of the device will use fuses in areas that are at risk of drawing high
current, this will prevent or minimize any damage
Display Signals: Digital

An LED display will be attached to the circuit and used like a multi-meter,
displaying any values where connected
Feasibility –
Access and Analyze


Access Separated Cells: 15mL Tube

Each channel output will be connected to tubing which terminates inside of a sterile 15mL
centrifuge tube

After separation is completed, the tubes can be removed and capped
Analyze Cells: Hemocytometer
Experimental Plan
Device Design
Channel Design
*All units are in microns
Electrical Design
Risk Management
Risk Management
Risk Management
Subsystem Design Plans
Questions and Comments