GeSiM Gesellschaft für Silizium-Mikrosysteme mbh. Nano-Plotter. Universal Micropipetting System.

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1 GeSiM Gesellschaft für Silizium-Mikrosysteme mbh Nano-Plotter Universal Micropipetting System

2 Nano-Plotter TM Nano-Plotter TM is a product line of modular and flexible automatic pipetting systems for sub-microliter dispensing and arraying applications. The system features piezoelectric pipette tips that accurately dispense liquids in volumes as low as 50 picoliters, following the "drop-on-demand" principle. Multiple pipettors can be positioned in parallel for simultaneous printing (4.5 mm and 9 mm, respectively). This non-contact printing technology allows the immobilization of extremely small spots on a variety of solid surfaces and the dosage into tiny cavities. Ranges of Application DNA and Protein Arrays Cell Pipetting / Tissue Arrays Miniaturized Assays Biosensors Key Benefits No impact on sensitive surfaces through touchless technology Unlimited spot replication from each single sample aspiration Homogeneous spot topology Variation of volume dosage to as low as 0.05 nl without changing the tip Dispensing micro liter quantities through standard disposable plastic tips Array layout can be arbitrary and is independent of pipette head layout Highly effective washing and cleaning system Special humidifying and chilling technology (dew point pipetting) prevents evaporation and conserves nanoliter volumes in applications such as miniaturized assays GeSiM s products are highly amenable to customization. Our expert technicians will adapt hard and software components to integrate our systems into specific robotic or other automated user applications.

3 System Platforms Nano-Plotter NP 2 (NP 2.1 NP 2.1/E) The Nano-Plotter system for high throughput production, and for high density and high precision spot positioning. The system's XYZ robotic stage is available in various sizes. (NP 2.1 bzw. NP 2.1/E). 1 to 16 tips (smallest drop volume 50 picoliters) Positioning speed up to 50 cm/sec Repetition accuracy up ±10 microns Array densities > 2500 cm -2 Extensive line of accessories available The XYZ stage can be configured to support a variety of dispense targets such as slides, membranes, chips and microwell plates. For convenient handling, the dispense targets can be attached to a removable tray that is positioned above the XYZ stage. To increase throughput, additional trays can be used for loading or unloading of dispense targets during the machine s run. Dispense targets can be fixated on the tray. NP 2.1 for max. 55 slides NP 2.1/E for 120 slides Nano-Plotter NP 1.2 The Nano-Plotter TM system for Research and Development. It's cost effective robotic handling system will fit smaller budgets, and yet allows easy and custom specific array spotting. Both pipet tips and fluidics are identical to the larger Nano-Plotter TM 2. 1 to 8 Tips (smallest drop volume 50 picoliters) Positioning speed up to 40 cm/sec Repetition accuracy ± 50 microns Array density up to 1200 cm -2 Fixed slide tray ( < = 40 slides) Extensive line of accessories available The plane surface slide deck allows mounting of (up to 40) standard slides, or customer specific targets, such as custom-fabricated membranes.

4 Piezoelectric Pipetting (Standard-) Micro Pipet Nano-Tip drop volume approx nl Micro Pipet Pico-Tip drop volume approx. 0.1 nl Micro Pipet Pico-Tip A drop volume approx nl Max. dosage frequency: approx Hz The Nano-Plotter TM piezoelectric pipetting tips consist of glass and silicon. They tie into the instrument s fluidic system through metal shafts. Each pipet connects to a dedicated syringe that supplies both sample and washing fluids. Highly effective washing technology prevents cross contamination between pipetting cycles. A thorough rinsing cycle washes the piezoelectric tips inside and out. For standard DNA solutions (such as oligo samples at 0.1 μg/ml), wash cycles of approximately 5 seconds are sufficient. For proteins and peptides, an additional washing station can be purchased, using a dedicated wash fluid, such as a detergent, acid, or base. Washing station with wash wells ONE PIPETTING CYCLE SAMPLE ASPIRATION TESTING / SPOTTING CLEANING Dilutor system to supply with and remove of system liquid Wash Fluid System Fluid Sample VIDEO Sample Aspiration Optimizing dispense parameter and functionality test Spotting Emptying / Washing Additional rinse cycle with specific wash fluid (optional)

5 Micro Pipetting Software NPC8/16* * NPC8 für Nano-Plotter NP 1.2 NPC16 für Nano-Plotter NP 2 Another flexible feature of GeSiM's non-contact dispensing technology is its ability to individually control each single pipette. Using multi-channel pipetting heads, the software allows an operator to program independent and individual spot patterns in applications such as biosensors. One cockpit for all airplanes: All NPCx- versions come with the same user interface and functionalities. Pipetting scripts can easily be transferred between different device platforms. Source plate and dispense target positions can be arranged quickly and simply in the system's unique work plate editor. Individual Application Programming versus Standard Scripts NPC8/16 includes an application development tool similar to a high-level computer language (NPL = Nano-Plotter Language). NPL scripts offer control and feedback from current work plate settings as well as through user interface input screens. The NPCx software functions allow accessing all hardware components, including image processing system of Nano-Plotter TM 2. A A1 B C D D4 Sequentially NPL-Scripts A E B F C G D H 9 mm Simultaneously (four tips) No programming effort is required for standard applications. The NPC software comes with various ready-to-use NPLscripts for frequent pipetting procedures. GeSiM's experienced programmers will support NanoPlotter TM customers in creating NPL-scripts for any special A3 C2 E4 H3 D2 F2 By transfer list B1 D A B C D E 9 mm applications. The standard NPL scripts allow various pipette head configurations and individually check the function of each tip after sample aspiration. Sequential pipetting: For straightforward sample tracking, a defined area of a source plate can be transferred to dispense targets, exactly copying the layout of the source plate. The user may also define replica spots for each sample. Simultaneous pipetting: All tips dispense simultaneously and arrange spots at a distance of 4.5 mm or 9 mm, respectively. Replicas and sample spots of later cycles can be positioned in between at user-defined distances. 9 mm Compiler for NPL scripts Input screen programmed in NPL The transfer list: The user arbitrarily assigns sample wells to target spots by defining a simple transfer list.

6 System Components and Accessories Automatic Plate Exchange* Plate handler with temperature and humidity control for up to 56 micro well plates, including rail connection to the Nano-Plotter TM. Cooling and Humidifying Micro plate holder with refrigeration Exchangeable slide tray, suitable for refrigeration and designed to allow fixation of dispense targets* Ultrasonic humidifier system, fully adjustable humidification of up to 80 % relative humidity. Searching and Recognizing An integrated image processing system locates smallest objects and automatically positions pipette heads. Video microscope, including light* * for Nano-Plotter TM NP 2 only Observation camera, featuring two different magnification levels for manual teaching of XYZ positions.

7 Applications Functionality Test DNA and Protein Arrays** 10 drops = 3 nl 0,1 mg/ml Oligo Piezoelectric technology allows different spot sizes by varying the number of drops per spot without changing the tip itself. 2 mm 100 drops = 30 nl A stroboscope allows the operator to visualize and manually optimize dispense parameters to fit a certain sample consistency. Non-contact arraying technology allows for highly consistent spots showing little or no "donut" effect. The minimum spotting distance depends on the surface structures. Hydrophobic substrates allow grid sizes down to 100 microns. Piezoelectric GeSiM tips are perfect to process all of the most common buffer solutions such as 3x SSC, PBS, Tris, but also glycerol up to a concentration of 40 %. Microarrays also can be generated onto or inside 3-dimensional objects like well bottoms of micro plate wells. The picture shows spots of about 1 nl volume in a 0.4 mm grid. An image processing module of the NPC software allows automatic testing of the functionality of each piezoelectric pipette tip. The Nano-Plotter TM Z-heightsensor identifies their Z-position and automatically adjust spotting height. Microliter Pipetting View through a microscope of a membrane with 2.5 nl spots in a 0.4 mm grid. At a volume range of 0.1 to 50 nl, the Nano-Plotter TM is an excellent platform to tether probes onto membranes. Adapter for standard disposable plastic tips: The Nano-Plotter's TM syringe modules can alternatively be set to dispensing following to the "displacement" principle. Microstructured substrates are well suited to center spots. Combining piezoelectric and passive dispensing technology, nano and microliter volumes can be spotted in applications such as the preparation of MALDI targets, or cryoconservation of cells. ** We are not licensed under any patents owned by Oxford Gene Technology Limited (OGT) or related companies and cannot pass any such license to our customers. A license under OGT s patents may be necessary to manufacture or use oligonucleotide arrays. GeSiM mbh

8 Applications Loading of Samples into Microfluidic Systems Dispensing sub-microliter volumes bridges the gap between conventional substance libraries (96 and 384 well format) and microfluidic flowthrough cells (Lab-on-a-Chip). Microfluidic system for vitality measurements on cardiovascular and neurological cells A microinjector chip developed by GeSiM, interacting with the Nano- Plotter TM, allows the injection of nanoliter volumes into the flowthrough system. Cells immobilized inside the cell chip can be subjected to different drug components successively. Cell chips can be designed to feature microelectrodes or fluorescent signal detectors. Electro-physiological activity of cardiovascular cells A joint project of GeSiM mbh and MPI for Polymer Physics in Mainz, Germany Pipetting Head Inlet Injected Fluid Outlet Microinjector Chip Cell Chip Gesellschaft für Silizium-Mikrosysteme mbh Rossendorfer Technologiezentrum Bautzner Landstraße Großerkmannsdorf Germany Phone +49 (0) Fax +49 (0) info@gesim.de Subject to changes

9 Specialties GeSiM Gesellschaft für Silizium-Mikrosysteme mbh Process Validation Module As an accessory, GeSiM offers a process validation module (µcp-pvm) consisting only of the Z-drive. This allows method development and optimization of printing, but also kinetic and other tests on high-end inverted microscopes. Control Software A simple to use through comprehensive software that controls every device and the entire printing process accompanies the shipment. It runs under all Windows versions and pretty well also on netbooks. After one has determined the respective positions, one can: pick up a stamp with the print head and eject it again microcontact printing: the head moves fast almost to the surface, then very slowly until full contact µcp-pvm, microsope-adapted process validation module with only Z-drive and slide holder An extra button exists for imprinting (NIL). It starts a complete predefined printing program with heating of the substrate, printing, cooling of the substrate, lifting of print head. Examples of Use PDMS stamp in the light microscope (left) and µcp of fluorescein- and rhodamine-labeled fibronectin, side by side on glass (right). Bar: 50 µm. Multicellular spheroids of MCF-7 breast cancer cells distributed in printed nanowells. Bar: 300 µm. Reference: Prof. M. Deutsch, Jerome Schottenstein Center, Bar-Ilan University, Israel. Gesellschaft für Silizium-Mikrosysteme mbh Rossendorfer Technologiezentrum Bautzner Landstrasse Grosserkmannsdorf, Germany Phone +49 (0) Fax +49 (0) info@gesim.de Front page: scanning EM picture of 175 µm deep hexagonal wells that were manufactured by imprinting in UV resist. Si master from GeSiM, NIL done at the Bar-Ilan University (reference see left). Subject to change without notice µ-cp 3.0 Robotics for Microcontact Printing and NIL

10 Basics Technical Data The Making of Micro- and Nanostructures The manufacturing of very small structures, e. g. to determine their effect on the growth and migration of cells, is becoming more and more important. Easy and reliable production methods, however, are hard to find. PDMS Master Curing Stamp sizes: 10 mm x 10 mm (Si master chip size: 15 mm x 15 mm), 20 mm x 20 mm; scalable to smaller and likely larger areas Inking and drying station for up to four stamps Substrate holder: mechanical fixation with clamps for up to four slides or petri dishes, 6-well microtiter plates, films, or silicon chips; customization possible Microarray spotting, which is possible with the GeSiM Nano- Plotter (see extra brochure), generates structures of at least 50 µm. For higher resolution down to approx. 50 nanometers, microcontact printing (µcp) is established as the method of choice. On the surface of a structured master, a block of PDMS (polydimethylsiloxane) is molded; the resulting stamp is inked with substrate and pressed onto the surface, thereby transferring sample only from the protruding areas, much like in letterpress printing. GeSiM has developed the new method of "dry inking", in which only the exposed stamp areas take up sample (or glue). Please ask for our dry-inking slides with specially functionalized surface. Not only chemicals, but also biomolecules, nanoparticles, beads, or cells can be printed, which is why the technique is increasingly getting attention in the life sciences. Printed patterns of fibronectin, laminin, gelatin or growth factors can serve as adhesive support to grow cells only on protein-covered areas. This renders possible studies on the influence of defined structures on growth, division, and differentiation of e. g. stem cells. In the research lab, µcp is usually done by hand. But only experts with years of hands-on experience are able to achieve usable results. The semiautomatic microstamping robot GeSiM µ-cp 3.0 was developed to make all steps of micro-contact printing reproducible at a reasonable price (a fully automatic machine with XYZ robotics based on the GeSiM Nano-Plotter will soon be available): Inking Drying of the stamp in a stream of air or N 2 Pneumatically controlled stamping with a precisely guided Z-drive (pat. pend.) The substrate is precisely adjusted in two spatial directions and by rotation (XY table) PDMS PDMS Substrate Microcontact printing: linear PDMS is cast onto a silicon master (top) and cured. The molded stamp (yellow) is impregnated with an "ink" (green) and pressed onto the substrate (bottom). If no inking is used and the stamp is pressed into a polyer that was softened by heating (NIL), 3D structures are obtained. Compressed air Inking, drying Stamp holder Stamp frame Ink Optional: vacuum fixation or heating/cooling (from room temperature to 145 C, heating/cooling in / 60 s) Manually adjustable substrate table; positioning accuracy 5 µm for repeated printing on the same substrate, aided by a built-in microscope Easy to use Windows software with preconfigurable automatic NIL procedure Dimensions: approx. 63 cm x 53 cm x 37 cm (WxLxH) Weight: approx. 35 kg Periphery: 115 or 230 V AC, oil-free compressed air or nitro- 6 gen (max. 10 Pa = 10 bar), vacuum if needed, Windows computer with RS-232- or USB interface Stamping PDMS stamps are molded on a silicon master in the included casting station (pictures below). Teflon-coated silicon masters (structures sizes from less than 100 nm) are available from GeSiM. Inking: by stamp immersion in fluid, time-controlled. Alternative: dry inking (GeSiM specialty!). 5 Stamp drying for seconds: in N 2 flow at 1 bis 8 10 Pa (1 8 bar) Stamping unit of the GeSiM µ-cp 3.0 with the print head above the heatable substrate holder. The vernier drives for substrate adjustment are visible. A built-in video microscope makes it possible to align stamp and substrate and thus to print on defined regions only or to printe multiple layers exactly on top of each other or side by side. This way it is possible to structure e. g. matrix proteins so that nerve cells grow right on microelectrodes. 3D Functionalization Threedimensional procedures such as nanoimprint lithography (NIL), where polymers are molded by the PDMS stamp at higher temperarutes and subequently UV-hardened, are also feasible. Rapid heating and cooling of the substrate and built-in UV illumination guarantee a quick NIL process in which structures can also be tiled together via step & repeat. Slides pre-coated with photoresist such as SU-8 are available from GeSiM. NIL is currently very hot in microelectronics and cell biology. Stamping principle (pat. pend.). The yellow PDMS diaphragm is bulged out by air pressure to contact the surface for printing, resulting in a very even pattern. Inking station 4 stamps in stamp holder PDMS Print head Substrate holder Alignment tool Schematic drawing of µ-cp 3.0 with the different work stations. The system base contains electronics, microscope, and connections/controls for vacuum and compressed air. Stamping ( seconds): Fine positioning including rotation on XY table Controlled, even expansion of PDMS stamp against the substrate via air pressure (patent application: DPA no ) Lowering of the print head in micrometer steps under microscope controld (LED backlight in stamp holder) Contact pressure and duration variable Option: substrate holder with predefined stop positions for fast and exact movement in large steps Option: UV curing for NIL by external UV lamp with optical fiber or UV LED array in the print head PDMS casting: insertion of GeSiM silicon master and Teflon spacer (left) and injection of PDMS into the casting station (center). Right: scanning EM picture of a molded PDMS stamp (bar: 2 µm). Four stamps above the inking/drying station during "wet inking". The air jet nozzles for the subsequent drying step are visible. Two options for the print head with receptacle for the stamp (in the front). Left, single print head. Right, double print head with an additional array of four UV LEDs for the crosslinking for NIL.