Design Alternatives for Micropatterning of Macromolecules GROUP 3: Sailaja Akella Caroline LaManna Teresa Mak Rupinder Singh Advisor: Emilia Entcheva

Design Project Design a system that facilitates cell deposition and micropatterning to be used in the creation of cellular and polymer based circuits. Customer Criteria Suitable for printing proteins for cell adhesion Fibronectin Collagen IV Collagen I –fluorescence labeled Laminin Biocompatible/Sterile Prints accurate & precise patterns High resolution Cost-effective (<$500) Neural cell patterning1

Design Alternatives Choose Best Design! Each alternative design consists of a Droplet-Depositing-Device (DDD), Substrate-Positioning-Device (SPD), and Solution-Supplying-Device (SSD) that may or may not be the same X-Y Stepper Board with Stationary Print Head Modified Thermal Inkjet Printer Modified Piezoelectric Inkjet Printer Choose Best Design!

Technical Considerations Resolution- 635 dpi Diameter of droplet (2R): 20um Separation of drops (e): 20-25um Fibronectin Solution 20ug/ml - 50ug/ml concentration in H20 Surface Polymer Print on cover slides (150um thick) Hydrophobic Polystyrene, PDMS, Plastic Increase cell patterning PEG, Pluronics, BSA

Assessment of Design Alternatives DDD: For a resolution of 635 dpi, it is necessary to minimize the printed droplet diameter Velocity of Droplet (Ejection Frequency) Surface tension and viscosity of solution Clogging should be prevented Prevent ink spray phenomenon SPD: Movement of substrate should be minimized for high resolution System should allow for uniquely dimensioned substrates to printed on SSD: Could make system robust for different viscosities and surface tensions Different static pressures Software handling for all three should be feasible

Solution-Supplying-Device (SSD) Ink Cartridge Can’t control pressure where the ink leaves cartridge It is difficult to decontaminate printer cartridge Difficult to prevent protein loss in the porous membrane of an ink cartridge (clogging) Height Controlled Reservoir Variation in potential energy will yield different pressures Easily decontaminated Clogging is not an issue Syringe Pump Could be used to change pressure; because, small displacements yield large pressure changes Clogging and Contamination can be easily resolved with a syringe pump

Syringe Pump

X-Y Printing System Automated X-Y stepper board (SPD) Stepper motor Stationary print head (DDD) Piezoelectric Software is available System that uses stationary piezoelectric print head to deposit solution onto motorized X-Y stage1

X-Y Printing System Piezoelectric head (DDD): MicroFab: Printed droplet diameter is 65um (MIT1) Separation of droplets is 10um Maximum Resolution is 339dpi Cost is $500 Can increase resolution by coupling the ejection of the droplets with the movement of the board Piezo drop-on-demand print head1

X-Y Printing System X-Y Movement (SPD) Movement of X-Y stage relative to droplet velocity

X-Y Printing System Model Price Resolution Speed $750 >1350 0.5 μm AB Tech Mini X-Y Linear Motor Positioning Stage 0.5 μm 100+ mm/s Newmark Systems inc. NLS4 Series >1350 0.3 μm 12.5mm/s Rockwell Automation 0.1 μm 4mm/s Arrick Robotics XY-9 $750 .005" per .9 degree step 0.15m/s

X-Y Printing System Advantages Disadvantages Substrate-Dimension-Freedom Software-Ready Viscosities and surface tensions can be varied Disadvantages Costly Low Resolution (339dpi) Time consuming to couple the x-y board with the printer head Slow (5min/coverslip)

Thermal Inkjet Printers Hewlett-Packard Model 550C Print head (DDD) Thermal characteristics Boland et al.2 Printed droplet diameter 2um Separation of droplets is .25mm Maximum Resolution is 100dpi Software Capabilities Printer head element may only change the ejection frequency Droplet size can not be changed Voltage through resistor is constant HP 550C3

Thermal Inkjet Printers Can’t change the size of the droplet since pressure in channel or the voltage applied to the resistor is predetermined for the particular ink solution The water-based ink becomes superheated (far above its normal boiling point) and finally reaches the critical temperature for bubble nucleation at around 280 °C. At this point no further heat is applied to the bubble Vapor bubble expands until all of the heat stored in excess to the boiling point is used to convert liquid to vapor. Once the excess heat is removed, the bubble collapses on to the resistor, which is no longer being heated. The bubble expansion and formation sequence occurs in 10us. Then there must be a minimum dwell time of 100-500us to enable the channel to be refilled through capillary action Maximum ejection frequency is 50kHz

Modified Thermal Inkjet Printer Caddy System (SPD) Re-design paper feed mechanism of HP printer To allow 1mm+ thick material to be inserted without rolling Involves re-directing feed and increasing height of rollers Resolution may decrease Printer with modified paper feed system4

Modified Thermal Inkjet Printer Advantages Larger nozzle diameter Mammalian Cells (100um) Fast (50,000 dots/min) Disadvantages Thermal stress on biological materials Poor resolution (100dpi) Need to design a caddy system Can’t control droplet size

Piezoelectric Inkjet Printer Epson® Stylus R200 Print head (DDD) Built-in Caddy System (SPD) Necessary to print on our substrate Resolution of 5760x1440 dpi Maximum Diameter of Droplet (4.5um) Diameter of droplet based on experiments Microencapsulation group achieved a resolution of 254dpi (diameter of droplet 100um) Software Use default Modify voltage supply to actuator Cost $99 Epson Stylus R2005

Modified Piezoelectric Printer The size of the droplets is defined by the voltage applied to the deflection plate, the pulse duration, and the diameter of the orifice6

Modified Piezoelectric Printer Advantages Caddy-ready system High resolution Greater than 1200dpi7 Can vary pressure in channel Might be necessary for solutions of different viscosities and surface tensions Disadvantages Difficult to modify software EPROM based Microprocessor to work in conjunction with different power supply settings

Ink Solution How do we increase the resolution? Current inks have a surface tension of 33.5 dyn/cm (0.0335N/m) and a viscosity of 7.4 cP at 20 ◦C The surface tension of water (73 dyne/cm) would increase the force required to deform droplet Leading to an increase in resolution Viscosity of water (0.89cp) would decrease the time required to wet the surface Leading to a decrease in resolution

Experimental Protocol Hypothesis: By increasing the concentration of proteins in our solution, the viscosity of our ink will increase; hence, we will achieve higher resolution By increasing the surface tension, we will prevent wetting of our substrate A high surface tension means low attraction and a low surface tension means a high degree of attraction Image printed solution of fluorescent collagen (type I) fluorescence microscopy

Best Design Solution Modified Piezoelectric Printer Droplet-Depositing-Device (DDD) High Resolution (5760x1440dpi)  Droplet Size,Frequency Substrate-Positioning-Device (SPD) Built in Caddy System Solution-Supplying-Device (SSD) Syringe Pump Software Easily Modified Versatile and Flexible cell patterning Epson Stylus R2005

Best Design Solution Modified Piezoelectric Printer QFD Analysis Print Head (121.5) Easily Modified Resolution Droplet Size Frequency Low Cost (105) Print Head Movement System

References Sanjana NE, et.al. A fast flexible ink-jet method for patterning dissociated neurons in culture. J Neurosci Meth (2004); 136: 151-163. Boland T, et al. Cell and organ printing 1: protein and cell printers. Anat Rec A Discov Mol Cell Evol Biol. 2003 Jun;272(2):491-6. “HP 550C.” PC Universe (2005). <http://www.pcuniverse.hpsupplieslink.com>. 16 Nov. 2005. “An idiot's guide to making a CD printer from an Epson 640.” VideoHelp.com (2005) <http://www.videohelp.com/forum/viewtopic.php?t=177053> 15 Nov. 2005. “Epson Stylus R200.” Epson America, Inc (2005). <http://www.epson.com>. 16 Nov. 2005. US Patent Office <http://www.uspatent.org> Setti L et al., An amperometric glucose biosensor prototype fabricated by thermal ink jet printing. Biosen & Bioelec 20 (2005) 2019-2026.