Automated Pipetting Device for a 2-D Membrane Electrophoresis. Parsaoran Hutapea Department of Mechanical Engineering Temple University

Transcription

1 Automated Pipetting Device for a 2-D Membrane Electrophoresis Parsaoran Hutapea Department of Mechanical Engineering Temple University

2 Outline Background Impact & Need Challenges Design Process Milestones Future Goals

3 Background The science is to study protein interactions to understand function and regulation of proteins within cells. 2-D Polyacrylamide Gel Electrophoresis (PAGE) system (O Farrell 1975) Uses µg of sample and takes 1 to 2 days to run With an aqueous buffer, hydrophobic proteins such as membrane proteins cannot be separated 2-D gel has to be transferred onto a PVDF membrane 2-D Membrane Electrophoresis (Chang & Yonan 2008) US Patent 7,326,326, 2008 Uses 5 µg (~ 5 µl) of sample and takes 40 minutes With water-miscible organic solvents, both hydrophilic and hydrophobic protein complexes can be separated Directly on PVDF membrane rather than in 2-D gel

4 Background 2-D PAGE system (O Farrell 1975) 2-D Membrane Electrophoresis (Chang & Yonan, 2008) US Patent 7,326,326, 2008

5 Impact & Need Impact Alternative to the current 2-D PAGE system. Biology and Proteomics: separation of hydrophobic proteins and protein-protein interactions to understand function and regulation of proteins within cells (identification of biomarkers, drug design, etc.) Huge market potential

6 Impact & Need Need Due to strong interactions between proteins and the blotting membrane, a very critical aspect is protein sample placement on the porous PVDF membrane ( nm pores) Automated device will eliminate the manual dispensing process (high failure rates) The engineering is to develop an automated pipetting device to deliver protein sample on the blotting membrane as required by the 2-D Membrane Electrophoresis. Polyvinylidene Fluoride (PVDF) nm pores High porosity (~0.7) with less than 100 nm pore spacing 500 nl PVDF membrane

7 Challenges Avoid or delay sample diffusion on the membrane Reduce contact area (varying d) to stack the protein h < 0.7 mm 500 nl PVDF nm pores < 100 nm pore spacing d ~ 0.7 mm Can we reduce contact area??

8 500 nl h < 0.7 mm PVDF nm pores ~100 nm pore spacing d ~ 0.7 mm

9 Challenges Touch-and-Lift (TL) Method (NTI) STEP 1 STEP 2 STEP 3 DISPENSE 70% of 5 µl LOWER PIPET, TOUCH, RELEASE THE REST LIFT PIPET HOW FAST? PVDF Membrane

10 Challenges Push-and-Lift (PL) Method (NTI) PLUNGER HOW FAST? STEP 1 STEP 2 STEP 3 LOWER PIPET LIFT PIPET HOW FAST? DISPENSE ALL OF 5 µl PVDF Membrane

11 Challenges Nano Bubble Method (Temple, proposed to NTI and NSF IDBR) 0.5 µl Can we create nano bubbles to reduce contact area??

12 500 nl nanoporous membrane Can we create nano bubbles to reduce contact area?

13 Challenges Nano Droplet Method (proposed to NTI and NSF IDBR) h < 0.7 mm PVDF Instead of 5 µl, can we instead reduce this to nl droplet?? nm pores < 100 nm pore spacing d ~ 0.7 mm

14 Challenges PERIOD MILESTONES PROGRESS ONGOING Device functional requirements?? MARCH - JUNE 08 Module operation, computer simulations, and assessment of the device manufacturability?? A computer solid model series of part drawings and bills of materials?? JULY - SEPT 08 Construct physical device prototypes??

15 Design Process Electric Actuator CAD (BRAINSTORMING) CFD ASSEMBLY (CAM) Blotting Membrane Air Slide Air Solenoids Air Linear Stage Control Switch Air Pressure Regulator 2-D MEMBRANE ELECTROPHORESIS DROPLET MECHANICS

16 Design Process Computer Aided Design (CAD) Electric Actuator Blotting Membrane Air Slide Air Solenoids Air Linear Stage Control Switch Air Pressure Regulator

17 Design Process CAD Touch-and-Lift (TL) Method

18 Design Process CAD Push-and-Lift (PL) Method

19 Design Process Computational Fluid Dynamics (CFD) L B θ D = L B L + B Optimum à L = B à D = 0 Assumption: We use water (Newtonian), protein sample is a non-newtonian fluid. We use glass instead of the porous substrate

20 Design Process CFD The rate of plunger motion We = ρu 2 r /σ ρ = density r = tip radius u = velocity σ = interfacial tension Propagation of fluid-air interface from ms at We = 8, u = 0.2 m/s Propagation of fluid-air interface from ms at We = 16, u = 0.4 m/s Small We is preferable!

21 Design Process CFD The rate of lifting Transient image of pressure contours with superimposed velocity vectors of a free standing drop shortly after the removal of the catheter tip Drop Deformation Parameter D Deformation D Rate of lifting (m/s) Drop deformation parameter D plotted as a function of the rate of lifting. The most ideal dispensing situation occurs at a velocity of approximately m/s

22 Design Process TL Assembly (CAM) Firgelli L12 Actuator Norgren air slide: M/46125/B Gilson, Pipetman P2 (0.2-2 µl) F Reed Switch Blotting Membrane Plate Mitutoyo micrometer stages Mitutoyo granite base Control System: Air pressure gages/regulators Switches Pneumatic solenoids Potentiometer Power supply

23 Design Process PL Assembly (CAM) Trombetta Q613-A1V12 Al mount to reduce vibration

24 Design Process Droplet Mechanics 500 fps highspeed camera (Few samples from ten-thousands of pictures taken) Touch-and-Lift Method Push-and-Lift Method Step 1 Step 2 Step 3 Step 1 Step 2 Step 3

25 Design Process 2-D Membrane Electrophoresis TL Method Blue = ok Green = no PL Method

26 TL Method

27 Milestones (6 months) PERIOD MILESTONES PROGRESS ONGOING Device functional requirements Designed and achieved CAD models to perform the proposed TL & PL methods MARCH - JUNE 08 Module operation, computer simulations, and assessment of the device manufacturability CAD/CAM and CFD analyses to analyze device operation and manufacturability (Very) extensive numerical work is in progress to take into account non- Newtonian fluid and porous membrane A computer solid model series of part drawings and bills of materials Developed CAD/CAM models and defined bills of materials More device optimization and tests JULY - SEPT 08 Construct physical device prototypes Developed and tested two Proof-of-Concept prototypes must be performed New ideas are currently in progress, nano bubble and nano droplet methods

28 Future goals Continue device optimization and tests Develop a PoC prototype for the nano bubble method Develop a PoC prototype for the nano droplet method using smart material actuators Develop an electrophoretic unit to complement the pipetting device

29 Future goals 0.5 µl Can we create nano bubbles to reduce contact area??

30 Future goals Develop prototype for the nano droplet method using smart material actuators (proposed to NSF IDBR) Based on our current work on smart actuators for aircraft (Hutapea et al, Aircraft Eng. & Aerospace Technology 2008; Hutapea et al, Aeronautical Journal 2008) and a smart bone transport device (Luo & Hutapea, J. Medical Devices 2008) TERFENOL-D ROD BAR 2 SLIDES ON THE WALL PIPET SLIDES ON BAR 4 ELECTRIC SOLENOID 2 4 METAL SPRING NITINOL SPRING PLUNGER NITINOL SPRING BAR 1 FIXED ON THE WALL 3 1 I = CURRENT (POWER SUPPLY)

31 Future goals Develop an electrophoretic unit (NSF IDBR) 3D CAD/CAM MODEL 3D CAM RAPID PROTOTYPE INSPECTION (REVERSE ENG.) MANUFACTURING 3-AXIS CNC

32 QUESTIONS??