DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING SEMI-AUTONOMOUS MICROBIOREACTOR EML4501- GROUP TWO CARLOS BELLO, EDUNN DALAL, JUAN MEJIA, BRANDON GODEFROY, NATALIA GONZALEZ, GABRIEL GUTIERREZ, JESSICA ROZEN DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Outline Product Overview Subsystem Details Liquid Handling Liquid Control Well Tray Movement Chamber Control Chamber Enclosure Optical Density and Fluorescent Intensity System Interface Framework Questions DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Product Overview The semi-autonomous bioreactor we have designed has several stages: Set-up: Person places cells, stressors, and cleaning fluid into the beakers, and a well tray into the machine. The machine fills the tray with cells and varying amounts of stressor. A person closes the well tray chamber. Experimental stage: The well tray is consistently shaken in linear, orbital and double orbital patterns and enclosed in a chamber which can be set at a temperature between 4℃ - 70 ℃. A person opens the lid to the well tray chamber and an ODFI sensor observes the effect of the stressor on the cells. Cell proliferation stage: The living cells in each well are identified and moved to the turbidostats. Once new cells have grown, they will be distributed back into the well plate for another round of experimentation with a higher concentraon of stressor. DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Liquid Handling Subsystem DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Liquid Handling Subsystem Subsystem Purpose: The liquid handling system is responsible for collecting and dispensing fluids between the beakers and well trays. It consists of 4 parts: Peristaltic pump Flexible Tube Tube Tip Dispenser Mount How it works: The peristaltic pump has 1 flexible tube running through it. One end of the tube is suspended over a waste bin and the other end is attached to the tube tip in the dispenser mount. As the pump rotates, its rollers push fluid through the tube. The pump both collects and dispenses fluid, depending on which direction the motor rotates. The flow rate depends on how fast the motor rotates and the amount of fluid dispensed depends on how much the motor rotates. Submerge in Collect Navigate to desired Dispense Submerge in Flush the desired fluid fluid dispensing location fluid cleaning fluid line DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Liquid Handling Subsystem Customer needs: The customer needs relevant exclusively to this system are: Requirement #33: Achieve a dispense rate of 300 휇L/s and a minimum of 225 휇L/s for well plates. Requirement #34: Deposit a minimum and maximum volume of fluid in the range of 5-20,000 Requirement #35: Dispose and neutralize waste Cost Table: Expense Cost Details Technology Readiness Level (TRL): OTS $65.00 Peristaltic pump OTS $1.60 Flexible tube (5 ft) (PVC Plastic) Although the peristaltic pump has met requirements for proper operational use on Modified OTS $0.48 Tube tip (Polypropylene Plastic) its own (TRL 9), the combination of all 4 Raw material $0.53 3D-printed dispenser mount (ABS) parts in this subsystem have not been tested in a laboratory environment for this Manufacturing $6.00 Cost to 3D print dispenser mount specific application. However, the system Assembly $6.50 1/2 hr approximate assembly time for unskilled labor at 13$/hr has been analytically verified. Thus, this subsystem receives a TRL of 3. Total $74.11 DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING OTS Peristaltic Pump Flow Rate Specs 1.8 degrees per step, 24 Vdc Bipolar Stepping Peristaltic Pump, 4 rollers, self-priming 3/16” ID ultra-chemical resistant flexible tubing DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Peristaltic Pump Flow Rate vs. Motor Speed Flow Rate vs. Motor Speed 1200 Flow rate meets the customer 1000 requirement of 300 L/s 800 Flow rate varies linearly with L/s) 휇 motor speed 600 165, 333.33 Variable flow rate is ideal for 400 this application Flow rate ( rate Flow 200 Volume dispensed is variable and is directly related to how 0 much the motor turns 0 100 200 300 400 500 600 Motor Speed (rpm) DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Liquid Control Subsystem DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Subassembly Description Subsystem Purpose: The liquid control subsystem is responsible for moving the liquid handling and ODFI subsystems between the beakers, chamber and turbidostats. The subsystem moves along three axes (XYZ) using a belt-pulley system in a Cartesian configuration. The dimensions of the Liquid Handling subsystem allow for precise movement to every possible receptacle. Customer Needs Addressed: Requirement 25: Capable of automated liquid handling that permits new fluid addition and subtraction from each well/tube. Requirement 28: Can measure OD and FI in all individual wells of any accommodated well plate readings to provide culture condition data. DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Size Analysis • A 2mm pitch diameter in the belts and pulleys allows the Liquid Control system to move in 2mm increments. • The smallest possible well diameter, in a 384 well plate, is 3.5mm. • Since 3.5mm > 2mm, the Liquid Control subsystem can reach each individual well for liquid collecting/dispensing (25) and ODFI readings (28). DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Liquid Control Subsystem Cost Table: Expense Cost Details OTS $847.31 Pulleys (8, $67.68), Belts (4, $189.12), Axles (4, $17.97), Limit Switches (7, $36.54), Motors (4, $56.00), carriage & guide rail (6, $480.00) Raw Material $190.70 Bar stock for brackets (Motors and Belt, 2ft, $2.00), Sheetmetal for hinges (holding X to Y, 2ft2, $4.70), and stock material for aluminum extrusions (23ft of 2x2, $184.00) Manufacturing $60.00 Machining and drilling aluminum extrusions, forming brackets from bar stock, skilled labor $30/hour for 2 hours Assembly $75.00 2.5 hours approximate assembly time for skilled labor at 30$/hour Total $1173.01 - Technology Readiness Level (TRL): Liquid Control concept falls somewhere within technology readiness Level 3. DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Well Tray Movement Subsystem DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Subassembly Description Subsystem Purpose: The main purpose of movement in a microbioreactor is to increase the availability of nutrients and improve the transfer of oxygen to the cell cultures. The design that our team has created involves the use of two linear actuators pushing against the sides of a platform where well trays of different sizes reside. The actuations cause vibrations in the plate at speeds of up to 500 rpm with a 10 mm diameter orbit. Customer Needs Addressed: Requirement 22: Accommodates each of the following culture plates sizes: 6, 24, 48, 96, deep 96, 384. Requirement 31: Can achieve linear, orbital, and double orbital shaking patterns. DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Well Tray Shaking Patterns Linear, Orbital, and Double Orbital Linear motion is achieved from the actuation of one motor by itself. Orbital motion is achieved from the actuation of the x and y motors at the same rate and same time. Double orbital motion is achieved when one motor is actuating at double the rate as the other. DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Natural Frequency of Base Plate Analysis 퐸ℎ 28 × 10 푝푠푖 0.06 푖푛 퐷 = = = 543.63 푙푏 푖푛 12 1− 휇 12 1 − 0.270 푊 4.804 푙푏 푙푏 푠 휌 = = = 0.0002 푖푛 푔푎푏 386 9.842 푖푛 6.299 푖푛 푖푛 푓 = 푛푎푡푢푟푎푙 푓푟푒푞푢푒푛푐푦 푖푛 퐻푧 푠 퐷 = 푝푙푎푡푒 푏푒푛푑푖푛푔 푠푡푖푓푓푛푒푠푠 푓푎푐푡표푟 푙푏 푖푛 / / 휌 = 푚푎푠푠 푝푒푟 푢푛푖푡 푎푟푒푎 (푙푏 푠/푖푛) 휋 퐷 1 1 휋 543.63 푙푏 푖푛 1 1 푓 = + = + 2 휌 푎 푏 2 푙푏 푠 9.842 푖푛 6.299 푖푛 푎 = 푝푙푎푡푒 푙푒푛푔푡ℎ 푖푛 = 250 푚푚 = 9.842 푖푛 0.0002 푖푛 푏 = 푝푙푎푡푒 푤푖푑푡ℎ 푖푛 = 150 푚푚 = 6.299 푖푛 = 91.82 퐻푧 퐸 = 푀표푑푢푙푢푠 표푓 퐸푙푎푠푡푖푐푖푡푦 = 28 푀푝푠푖 Maximum operating frequency of the well plate shaker is 40 Hz. ℎ= 푝푙푎푡푒 푡ℎ푖푐푘푛푒푠푠 푖푛 = 0.06 푖푛 휇 = 푃표푖푠푠표푛푠 푟푎푡푖표 = 0.270 Vibration resonance occurs when equipment or a product is exposed to an external forced vibration occurring at one or more of its natural 푊 = 푝푙푎푡푒 푤푒푖푔ℎ푡 푙푏푠 = 4.804 푙푏푠 frequencies. When forced vibration levels are low, they are considered 푔 = 푔푟푎푣푖푡푎푡푖표푛푎푙 푐표푛푠푡푎푛푡 = 32.17 푓푡/푠 = 386 푖푛/푠 non-damaging. DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Natural Frequency of Base Plate Analysis (cont’d) The shaker can be harmful to the system if: Resonance occurs The max input displacement is significantly high Since the maximum input displacement is minute (10 mm) and the operating frequency is much smaller than the natural frequency of the system, the system is safe. Ways to protect from resonance: Do not operate at the natural frequency Increase the stiffness of the equipment (this increases the natural frequency) Reduce the mass of the equipment (this increases the natural frequency) Install a damper Install a tuned dynamic absorber DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Well Tray Movement Costs Expense Cost Details OTS $328.01 Servomotor Precision Flexible Shaft Coupling x 2, Position- Control DC Motor x 2, and Compression Springs found on McMaster-Carr Raw Materials $12.06 Aluminum sheet metal, Aluminum rectangular bar stock, and Aluminum round bar stock material price list found on the Design and Manufacturing Lab of the University of Florida website. Manufacturing $53.08 Precise mill, lathe, and sheet metal cutting skill required. Assembly $30.00 1 hour approximate assembly time for skilled labor at $30/hr Total $423.15 - Technology Readiness Level (TRL): Well tray movement concept falls somewhere within technology readiness Level 3. DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Chamber Enclosure Subsystem DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Chamber Enclosure Subsystem Purpose: The chamber enclosure creates an isolated and controlled cell cultivation environment.