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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