1 PicoFill-Systems Outlicensing Electrospray Deposition onto Small Targets Target Applications:  MicroArray Applications: spraying of enzymes, metabolites, co- factors, yeast cells, antibodies: addition of up to 20 wt% of carbohydrates like trehalose to fully preserve enzyme activities after spraying followed by storage. Activities of enzymes, metabolites and cofactors are not reduced by the electric field.  Dotting of Microliter & Nanoliter Fractions onto MALDI targets  Coating Industry: spraying of liquids containing particles, polymer, etching or curing agents, etc. Spraying of viscous materials can be achieved at high temperature. 0.4 millimeter Dot

2 Special Features  The charged droplets are pulled to the grounded substrate on-the-flight, so, no contamination of adjacent targets.  The sprayed amounts of liquid is controlled and flexible by varying the spraying time and/or flow rate. Dot-size is varied by adjusting the spray-distance.  The uniform charged droplets repel each other on-the-flight resulting in a layer of droplets instead of a droplet. This eliminates the donut effect (coffee stain effect).  The droplet spray is always directed toward grounded elements, meaning that if grounded conductive targets are embedded within non-conductive material, the droplets will only fly to these conductive targets like metal wiring or pins.  Capillaries are bendable, low-cost and may be 50 micron in outer diameter, allowing spraying of spots or lines at widths in the order of 10–100 micron.  Parallel spraying was achieved with three capillaries (outer diameter of 150 micron) that were placed 600 micron apart.

3 Spraying Parameters Dot Size Dot size is related to the distance between the capillary-tip and the substrate (spraying distance) Dot size is related to the capillary outer diameter: the smaller the outer diameter, the smaller the cone size Dot size is related to the flow rate: at low flow rates the liquid evaporates rapidly resulting in a solid dot instead of a donut like dot. Typical dot sizes for a capillary-OD of 150 micron and flow rates of 100–400 picoliter/second : 150–400 microns at spraying distances of 250–500 micron, respectively MicroArray Applications Flow rates: picoliters-nanoliters/second Distance between capillary-tip and substrate (spraying distance): 200–500 micron Dot size microarray: 100–400 micron at spraying distances of 200–500 micron Preferred capillary outer diameter: 50–200 micron Maximum speed of x-y-z table depends on actuators: 1.6 inch/sec (4 cm/s) for high speed actuators Dotting of fractions onto MALDI plate targets Flow rates: 1 nanoliter/second – 0.6 microliter/second Dot sizes: 400–1500 micron at spraying distances of 500–2000 micron, respectively

4 Spraying of Arrays of Dots 1-2 kV Spray of droplets MicroArray or slide 1–2 kV Spot A. Piston pushes liquid through syringe while electric field is applied Slide is moved downward and sideways: typical 200 – 1000 micron Second spot is sprayed onto slide B. Slide is moved downward to lower the electric field: spraying stops C. Slide is moved upward and spraying resumes 1-2 kV

5 Parallel Spraying Spots: 0.6 millimeter apart