Team Vision for System-Level Design Phase
- What did your team plan to do during this phase?
We plan to use our finalized problem statement to construct an in depth functional decomposition of our device, which will drive the thought process behind establishing the concepts to construct each subsystem. By the end of this phase, we will have decided on which concepts to pursue to develop a system level plan for our device that will fit most, if not all, the customer requirements. We will then start the development of these subsystems further.
- What did your team actually accomplish during this phase?
In this phase we established the functions that the automated cell separator will accomplish. Potential solutions to each of these functions were then generated. Of the list of possible solutions for each function, one specific solution was chosen to construct a device concept. These concepts were compared against each other and to other already existing devices. This phase resulted in the development and validation of a general concept of our system design.
Morphological Chart and Concept Selection
Concept GenerationUsing the morphological chart above, four potential concepts were developed to be compared to each other and the benchmark instrument.
Concepts 1 and 2 are strongly considered models for this project. Concept 3 is a high budget project. And concept 4 has a minimal budget.
- See live document for morphological chart and concept selection here.
Concept Comparison and Selection
- See live document here.
This chart compares each concept to the datum device used in benchmarking, the autoMACS made by Miltenyi Biotec.
- See live document here.
Feasibility: Prototyping, Analysis, Simulation
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.
Yeast Growth Curve
E.coli Growth Curve
- 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
Load Cells into Device: Put in Syringe
- Using standard syringe aspiration techniques, cells in fluid will be added to the syringe. This involves 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 to set the resistance both the resistors for the micro-controller and of the Rf resistor value of the op-amp, which will set decide between the different signal types, frequencies, and the gain of the input voltage. (See below, Modulate Voltages and Modulate Frequencies).
Initiate Process: Hardware
- Using the power supply for the lower voltage devices, a simple toggle button with an electro-mechanical relay, will be used for initiating the start of the test. This button will allow for fluid to pass through the channel. Once toggled on, a timer will begin counting for the users’ sake. This will work simultaneously work with the higher voltages and frequencies where the voltages and frequencies for each cell must be set beforehand.
Drive Flow: Syringe Pump
- In order to induce flow into the channel, the device will use syringe pumps. 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.
- Once a channel design has been finalized the flow rate calculations will be made in order to design a syringe pump that will be able to accurately induce the correct flow rate. This will need to be characterized once the prototype has been build.
Monitor Flow: Flow Meter
- We plan on using a flow meter in order to measure and verify the flow rate going into the channel. This is necessary in order to ensure the flow through the channel stays laminar, a Reynolds Number less that 1. This flow meter will be implemented in series with the buffer flow inlet in order to not adversely effect the cells, which will join the flow further downstream.
- Flow rate monitoring is needed because the fluid properties can change with the use of different cells and buffer. Since the Reynolds number is inversely related to the viscosity, the flow field will change with different fluid types.
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 insulator-based dielectrophoresis which can require hundreds of volts.
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
Modulate Voltages: Operational AmplifierRun small scale testing with an OP Amp and potentiometer to make sure assumptions of inverting amplifier equations are valid and that the potentiometer will act as expected and that the frequency will not be affected.
To test if the frequency will be affected small scale testing will be done on a similar Op Amp using function generator to create a signal and then will be analyzed using an oscilloscope.
The Op amp will need to have rails that are high enough to allow the Op amp to amplify the voltages to the upper voltages that are needed. So to check for an available transformer the Ideal Transformer equation will be used to make sure there is a transformer on the market that can achieve the upper rails that are needed.
The ratio of the transformer that will be needed is 1:8.3. There are commercially available ones that are rated for these voltages.
Modulate Frequency: Micro-controller
- Using a micro-controller, a waveform generator will be replicated to obtain a either a sine, triangle, pulse, or saw signal with a range of frequencies from 1-50kHz. The frequency, pulse width, and amplitude (gain) will be controlled using potentiometers, as well as displayed using an LED.
Monitor Signals: Current MonitoringTo monitor the frequency 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 save components from being damaged if there is a current spike and will be easily replaced if such an event occurs.
Display Signals: Digital
- An LED display will be attached to the circuit and used like a multi-meter would be used, connected the positive wire to the node where the voltage will be read, and the negative wire to ground.
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
Cost to Run Machine (excluding consumables)
Proposed Device Design
Proposed Channel Design
Proposed Electrical Design
Risk AssessmentThe risk assessment chart can be found here.
Research1. Yafouz, Bashar, Nahrizul Adib Kadri, and Fatimah Ibrahim. "Microarray dot electrodes utilizing dielectrophoresis for cell characterization." Sensors 13.7 (2013): 9029-9046.
- Theory behind use of planar electrodes in
- Possible replacement for embedded electrodes that would be disposed of with the channel.
- Reduce the risk of damaging the electrodes during molding of the micro-channel.
2. Gascoyne, Peter “Isolation of Rare Cells from Cell Mixtures by Dielectrophoresis” Electrophoresis. 2009 Apr; 30(8): 1388–1398. doi: 10.1002/elps.200800373
What was done:
- Separated three different cultured tumor cell types from blood without labeling and compromising tumor cell viability.
- Recovered more than 90% of tumor cells in small samples
- Device channel: 0.6 mm high × 25 mm wide × 300 mm long: device floor lined with interdigitated 50 micron wide gold-on-copper electrodes spaced 50 microns apart on a Kapton substrate. 3000 electrode elements ran widthwise across the chamber with alternate elements being connected to two bus lines
Useful to our project:
- We are doing a separation where viability needs to be considered and we want to construct a device with embedded electrodes.
3. Tai, Chien-Hsuan “Automatic microfluidic platform for cell separation and nucleus” Biomed Microdevices (2007) 9:533–543 DOI 10.1007/s10544-007-9061-7 What was done:
- New biochip capable of cell separation and nucleus
collection utilizing dielectrophoresis (DEP) forces in a
microfluidic system comprising of micropumps and
microvalves, operating in an automatic format.
- DEP forces operated at a low voltage (15 Vpp) and at a specific frequency (16 MHz) can be used to separate cells in a continuous flow, which can be subsequently collected.
Useful to our project:
- Important to benchmark and consider during our design phase
Design Review Materials
- See presentation here.
Plans for next phaseSee below for each team members plan for the next phase:
- As an individual on the team, what are you doing to help your team achieve this vision? (Use the individual 3-week plan template for this).
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