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 concentra on 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 ( 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 = 푃표푖푠푠표푛 푠 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. The gasket seal, chamber enclosure lid, and additional latch allow the micro-bioreactor to create the ideal conditions needed for various cell cultivation laboratory applications.
Customer Needs Addressed:
Requirement 7: Capable of incubation/hibernation periods from one hour to two weeks (Heat Loss Rate Analysis).
Requirement 9: Can culture microbes in fully- enclosed compartments or vessels that are easily interchangeable and that allow for media exchange (Gasket Strain Seal & Material Analysis). DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Gasket Strain Seal & Material Analysis OTS Silicone Rubber Gasket: 1. Good thermal stability and low chemical reactivity. 2. Excellent resistance to ozone, sunlight, and oxygen. 3. Consistent properties at high and low temperatures. 4. Repels water, resists moisture, and forms watertight seals. 5. Good electrical insulation and excellent gas permeability Elastomer (Mooney-Rivilin 2) parameters were utilized to simulate silicone rubber behavior with appropriate Young's modulus and Poisson's ratio. Strain applied from chamber enclosure distributed pressure should be sufficient to establish a seal. DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Heat Loss Rate Analysis
ΔQ = net heat (energy) transfer Δt = time taken (2 weeks in seconds) This heat loss does not account for ΔT = difference in temperature between the cold and hot sides the insulation on the outside of the Δx = thickness of the material conducting heat (¼'' stainless steel) chamber k = thermal conductivity (316 stainless steel material property) A = surface area of the surface emitting heat DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Chamber Enclosure Cost Table: Expense Cost Details OTS $135.70 Rubber grommets, gas canisters, and air regulator.
Raw Material $1391.48 316 Stainless Steel Sheet metal (welded) and Polyurethane insulation (adhesive variety). Manufacturing $43.82 Sheet metal welding and chamber holes.
Assembly $90.00 3 hr approximate assembly time for skilled labor at 30$/hr.
Total $1661.00 -
Technology Readiness Level (TRL): Chamber enclosure concept has been extensively demonstrated through various analysis therefore falls somewhere within technology readiness Level 3. DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Chamber Control Subsystem
Subsystem Purpose:
The Chamber Control Sub-system is the temperature control for the Chamber Enclosure. This includes 40 Phase Change Materials (PCM), PolyUrethane Foam Insulation Sheets, and a PCM holder that all are necessary to keep the chamber at a stable temperature.
Customer Needs Addressed: Requirement 18: Capable of incubation/hibernation periods from one hour to two weeks. Requirement 20: Can culture at a temperature range from 4C to 70C. DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Phase Changing Material Properties
Phase change materials (PCM) are substances that absorb and release thermal energy during the process of melting and freezing. Pure Temp® provides a wide range of Phase Change Materials available that can heat or cool from temperatures between -40°C and 150 °C.
1) Heat energy storage capacities that average 200J/g. 2) Consistent, repeatable performance over thousands of melting and solidifying cycles. 3) Readily biodegradable, 100% renewable and nontoxic 4) Stores 5 to 14 times more heat per unit volume than materials such as water and masonry. 5) High-density energy storage allows to store heat within a 2 °C range. DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Energy Required to Change Chamber Temperature from Ambient to 70ºC
∆푇 =푇 −푇 = 70℃ − 20℃ = 50℃ = 323.15 퐾 In the above equation, the ambient temperature is taken as room temperature, as defined by the Oxford English Dictionary (20ºC).
푘푔 푘퐽 푄 =푚푐 ∆푇 = 휌푉푐 ∆푇 = 1.225 0.04657 푚 0.718 323.15 퐾 = 13.236 푘퐽 = 13,236 퐽 푚 푘푔∗퐾
The phase changing wax has a heat energy storage capacity of 200 J/g. Therefore, the amount of phase changing wax required to achieve the required energy for temperature change is:
13236 퐽 푎푚표푢푛푡 표푓 푝ℎ푎푠푒 푐ℎ푎푛푔푖푛푔 푤푎푥 푟푒푞푢푖푟푒푑 = = 66.18 푔 = 0.146 푙푏 퐽 200 푔 푉 = 퐶ℎ푎푚푏푒푟 푉표푙푢푚푒 (푚 ) 휌 = 퐷푒푛푠푖푡푦 표푓 푎푖푟 (푘푔/푚 ) 푚 = 푀푎푠푠 표푓 푎푖푟 푖푛 푐ℎ푎푚푏푒푟 (푘푔)
푐 = 푆푝푒푐푖푓푖푐 ℎ푒푎푡 푐푎푝푎푐푖푡푦 표푓 푎푖푟 푎푡 푐표푛푠푡푎푛푡 푣푎푙푢푚푒 (푘퐽/푘푔 ∗ 퐾)
푇 = 퐴푚푏푖푒푛푡 푡푒푚푝푒푟푎푡푢푟푒 (℃)
푇 = 퐷푒푠푖푟푒푑 푓푖푛푎푙 푡푒푚푝푒푟푎푡푢푟푒 (℃) 푄 = 퐸푛푒푟푔푦 푟푒푞푢푖푟푒푑 푓표푟 푡푒푚푝푒푟푎푡푢푟푒 푐ℎ푎푛푔푒 (퐽) DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Heat Loss Rate with Insulation
ℎ = 2.55 푊/푚 *K 푘 = 13.5 푊/푚*K 푘 = 0.025 푊/푚*K A= 0.0612 푚 퐿 = 0.00635 푚 퐿 = 0.00635 푚
푅 = 27.3 K/W ∆
( . . ) 27.3 K/W
1.65 J/s (W) DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING
Chamber Control Costs
Expense Cost Details OTS $200.00 Phase Changing Wax (40 types) Raw Materials $2.70 Sheet Metal for Wax Cup Manufacturing $9.50 Welding Wax cup to Chamber, Welding $19/hour for 30 minutes Assembly $0.00 No assembly required Total $212.20 -
Technology Readiness Level (TRL): Chamber Control concept falls somewhere within technology readiness Level 3. DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING
Optical Density and Fluorescent Intensity DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Optical Density & Fluorescent Intensity
Subsystem Purpose: The OTS optical density and fluorescent intensity subsystem plays and integral role in the function of the Micro Bio Reactor. This system parametrizes the turbidity, optical density, and fluorescent intensity of a bacterial or cellular culture.
Both Optical density and Fluorescent intensity serve as proxies for culture concentration and/or proliferance.
The characterization and manipulation of living systems is critical to their study. Thus, through continuous culture sensing, a closed-loop control of culture parameters can be developed. DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Optical Density (OD) Light is emitted from the top optical train and focused through a collimating aspheric lens into a well or conical tube. The light travels through the culture (getting absorbed and scattered) and through a standard transparent flat-bottom microplate.The light enters the bottom optical train through an aspheric lens into a CCD-based visible-range detector capable of full-spectrum and monochromatic detection. An intensity delta is then calculated, and a measurement of opacity derived. Fluorescent intensity (FI) FI measurements in a vertical arrangement are particularly hard to perform. The culture in a well is exited through 3 narrow-solid-angle LEDs which emit light beyond the acceptance angle of the collimating lens. The LED exitation wavelength can be tailored to the culture measured. The fluorescent light emitted by the culture travels through the aspheric lens into the CCD detector. λ = 470, 525, and 610 nm DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Optical Density & Fluorescent Intensity DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Optical Density & Fluorescent Intensity
Customer Need: # 30 Light Intensity refers to the light needed for photosensitive cultures, that is, cell cultures which need light to grow.
(1) (2) The power used by the concept was calculated through equation (1) and the distance from the light emitting source to the culture is 1 cm. Intensity can be calculated from equation (2) as 25.06 kW/cm^2. This is above the specified intensity of 1 kW/cm^2. Cost Table: Expense Cost Details TRL:
OTS $1491.60 Cost of assembly for the Open-Source Microplate Reader ODFI This Open Source Microplate
Raw Material $7.40 Sheet pieces and a 3D printed mount reader concept has been tested by 3rd party users and Manufacturing $15.70 Laser/ Waterjet parts and 3D printed parts developers. The systems seem Assembly $70.00 2 hr approximate assembly time for skilled labor at 30$/hr to fall somewhere between 4/5.
Total $1584.70 - DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING System Interface Subsystem DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING System Interface
Subsystem Purpose: The system interface is the means by which the user can control any necessary parameters and view necessary information about the processes being performed. Our bioreactor uses an iPad as the system interface. The iPad will be used to run the application that enables control of all necessary experimental parameters.
Customer Needs Addressed: -Requirement 6: Includes a fail-safe system, and a software/phone alert that informs the user of culture condition failure.
-Requirement 7: Includes an emergency shut-off that can be actuated by the user or nearby personnel that safely stops all functions.
-Requirement 9: Has a visual indicator that is easily seen by the user and nearby personnel that shows: system on/off, the mode of function and duration, and if an error is present.
-Requirement 13: Programmable: control parameters can be changed, or more complex control can be added.
-Requirement 14: Have an intuitive user interface.
-Requirement 19: Can perform culture condition closed loop control. DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING
System Interface Design System On/Off System Alert Buttons on bottom right allow (Emergency shut-off) Center user to control experimental stages. Buttons on bottom left allow user to set up Fill Receptacle: experiment. • Beaker or turbidostat • How much per well/tube Receptacle: • Reminder to open lid and • Well tray – Type prepare the phase change wax • Conical tube – Size Start Experiment: Beakers: • Alert to close lid & insert phase • Contents of beakers change wax • Which are used • Starting temperature • Cleaning • Time duration
Shaking Pattern: Data Collection: • Pause experiment • Linear/Orbital/ • Run ODFI sensor Double Orbital • Prep setup before proceeding • Speed Fill Turbidostat: Gas control: • Recent ODFI data • Chemical makeup of • Wells to corresponding chamber turbidostats • Amount of each gas • 96 squares with % of living cells & 16 turbidostat options DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING System Interface
Cost Table:
Expense Cost Details (OTS) iPad Air $429.99 Can be purchased at Best Buy or through Apple (OTS) iPad Stand $13.99 Can be purchased through Amazon Assembly $0.00 No assembly required No manual manufacturing required, coding costs for software Manufacturing $0.00 would need to be determined by an expert in that field Total $443.98 -
Technology Readiness Level (TRL): This subsystem would likely earn a score of 5 on the TRL scale as the iPad is a device that has already gone through reliable testing by Apple Inc. before being brought to market. However, the software run on the iPad which will be designed specifically for the purposes of the bioreactor has not been tested. Therefore, the subsystem has yet to be proven reliable for the specific purpose of this device. DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING Framework
List of Parts: Expense Cost Details • Aluminum extrusions OTS $2050.00 Beakers, Beaker Tray, • Shelves Turbidostats • Turbidostats • Beaker Tray Raw Material $116.50 Aluminum extrusions • Beakers ($85.00), sheetmetal for shelves (6.3ft2, $31.50) Assembly $30.00 Skilled labor, $30/hour for 1 hour Welding, $19/hour for 5 Manufacturing $95.00 hours - Total $2291.50 DEPARTMENT OF MECHANICAL AND AEROSPACE ENGINEERING