TopSpot – Kostengünstige Technik für die Produktion von Microarrays Jens Ducrée IMTEK Institut für Mikrosystemtechnik; Albert-Ludwigs-Universität Freiburg; Georges-Köhler-Allee 103; D-79110 Freiburg; Germany Email: ducree@imtek.uni-freiburg.de HSG-IMIT Institut für Mikro- und Informationstechnik Wilhelm-Schickard-Str. 10, D-78052 Villingen-Schwenningen; Germany Thank you Mr. Chairman, Good afternoon ladies and gentlemen,   The topic of my presentation is”Nanoliter Liquid Handling Devices” Vorlesung Mikrofluidik R. Zengerle, J. Ducrée; WS 1999 / 2000

HSG-IMIT Gemeinnützige Gesellschaft F&E für die Industrie Villingen-Schwenningen (BW) 80 Mitarbeiter 4 Geschäftsbereiche Mikrofluidik Sensorik Mikrotechnologie Informationstechnik At first I want to introduce my organisation. HSG-IMIT is a non-profit organisation located in the South-west of Germany. We are located on the middle between Stuttgart in Germany and Zurich in Switzerland. Both cities are about 1 hour by car away from us. Our bussiness is applied R&D in contract with industry. For doing that we are organized in 4 departments, - micromachining - sensors - microfluidics, - and a department dealing with information technology. HSG-IMIT IMTEK Vorlesung Mikrofluidik R. Zengerle, J. Ducrée; WS 1999 / 2000

IMTEK Albert-Ludwigs-Universität Freiburg Institut für Mikrosystemtechnik Lehrstuhl für Anwendungs-Entwicklung (Prof. Zengerle) Kooperation zwischen HSG-IMIT und IMTEK: Microfluidik-Netzwerk HSG-IMIT IMTEK At first I want to introduce my organisation. HSG-IMIT is a non-profit organisation located in the South-west of Germany. We are located on the middle between Stuttgart in Germany and Zurich in Switzerland. Both cities are about 1 hour by car away from us. Our bussiness is applied R&D in contract with industry. For doing that we are organized in 4 departments, - micromachining - sensors - microfluidics, - and a department dealing with information technology. Vorlesung Mikrofluidik R. Zengerle, J. Ducrée; WS 1999 / 2000

Mikrofluidik-Netzwerk Coordination R. Zengerle, T. Strobelt Microfluidics Internet Service U. Grundmann Microfluidics Monograph J. Ducrée Microarrays B. De Heij Pipettes & Dispenser P. Koltay Microdosage- systems T. Strobelt Micro- valves J. Schaible Fluidic- Disposables J. Ducrée Layout & Construction M. Müller Packaging H. Ashauer Theory of Microfluidics P. Koltay Plastic Technology R. Steger Metrology M. Freygang

Motivation Bisheriger industrieller Partner BioChip Technologies, Freiburg Genescan Europe AG Ziel ... 3.000 Microarrays / 8 Stunden 1.000 verschieden Analyte / Chip 3-fache Wiederholung 9 Mio Spots / 8 h = 100 Spots / s = 10 s pro 1.000-Feature Chip Heutige Microarray-Technologie: On-chip-Synthese Pin-Printing Spotting

Übersicht Einleitung Grundlegende Konzepte der TopSpot-Technologie Die Zweite TopSpot-Generation & Deren Vermarktung Reformatierung von MTPs Schlussfolgerungen The outline of my presentation will be als follows: I will present two new devices for the nanoliter liquid handling. These devices are the NanoJet-Dispenser for the precise dosage of biological liquids in the nanoliter range and the TopSpot printhead, a cheap & simple device for the highly parallel spotting of micro arrays. Concerning the NanoJet dispenser I will discuss drop-on-demand and jet-on-demand procedures. You will see in the results that the Jet-on-demand principle is a much more robust and precise device. Concerning the Top Spot print head, I will give a short introduction of the goals of that development and will present the basic ideas of that printhead. We are working on the commercialisation of that technology and I will present two types of machines which we are developing: - A “Research Machine” which is used for the prototyping of microarrays - A “Production Machine” which is a dedicated machine for producing high numbers of always the same type of Biochip. The basic differences of both machines are costs and output of Biochips / day. Vorlesung Mikrofluidik R. Zengerle, J. Ducrée; WS 1999 / 2000

1. Konzept: Reformatierung Oberseite Oberseite: Horizontale Kanäle zur Formatkonversion Unterseite The size of the reservoir on top is comparable to the size of wells on a 384 well microtiterplate. It is importand that the reservoirs can be filled very easily by a conventional pipetting robot. The distance of the nozzles at the the bottom is 500 µm in this case and once again these are the buried channels connecting the reservoir to the nozzles. Vorlesung Mikrofluidik R. Zengerle, J. Ducrée; WS 1999 / 2000

2. Konzept: Auslösen der Düsen And we had also a second idea for actuating the printhead. This idea was to use a pneumatic pressure pulse for actuating the nozzles. For that we cover the print head with a thin plastic film. The film has through holes at the reservoirs for avoiding a low pressure at the reservoirs. Now we have a closed chamber above the nozzles and when we displace the film very fast, we generate a fast pressure pulse in that area above the nozzles. All nozzles are actuated at the same time and every nozzle is ejecting a small droplet. You can see the situation in the following video. Vorlesung Mikrofluidik R. Zengerle, J. Ducrée; WS 1999 / 2000

2. Idee: Auslösen der Düsen And we had also a second idea for actuating the printhead. This idea was to use a pneumatic pressure pulse for actuating the nozzles. For that we cover the print head with a thin plastic film. The film has through holes at the reservoirs for avoiding a low pressure at the reservoirs. Now we have a closed chamber above the nozzles and when we displace the film very fast, we generate a fast pressure pulse in that area above the nozzles. All nozzles are actuated at the same time and every nozzle is ejecting a small droplet. You can see the situation in the following video. Vorlesung Mikrofluidik R. Zengerle, J. Ducrée; WS 1999 / 2000

TopSpot - Qualitätskontrolle 100 % Qualitätskontrolle mit einer nachgeschalteten Bildverarbeitung 500 µm Vorlesung Mikrofluidik R. Zengerle, J. Ducrée; WS 1999 / 2000

Übersicht Einleitung Grundlegende Konzepte der TopSpot-Technologie Die Zweite TopSpot-Generation & Deren Vermarktung Reformatierung von MTPs Schlussfolgerungen The outline of my presentation will be als follows: I will present two new devices for the nanoliter liquid handling. These devices are the NanoJet-Dispenser for the precise dosage of biological liquids in the nanoliter range and the TopSpot printhead, a cheap & simple device for the highly parallel spotting of micro arrays. Concerning the NanoJet dispenser I will discuss drop-on-demand and jet-on-demand procedures. You will see in the results that the Jet-on-demand principle is a much more robust and precise device. Concerning the Top Spot print head, I will give a short introduction of the goals of that development and will present the basic ideas of that printhead. We are working on the commercialisation of that technology and I will present two types of machines which we are developing: - A “Research Machine” which is used for the prototyping of microarrays - A “Production Machine” which is a dedicated machine for producing high numbers of always the same type of Biochip. The basic differences of both machines are costs and output of Biochips / day. Vorlesung Mikrofluidik R. Zengerle, J. Ducrée; WS 1999 / 2000

TopSpot – Anfängliches Design Risiko der Verschleppung durch Benetzung entlang der Pyrex-Kante The outline of my presentation will be als follows: I will present two new devices for the nanoliter liquid handling. These devices are the NanoJet-Dispenser for the precise dosage of biological liquids in the nanoliter range and the TopSpot printhead, a cheap & simple device for the highly parallel spotting of micro arrays. Concerning the NanoJet dispenser I will discuss drop-on-demand and jet-on-demand procedures. You will see in the results that the Jet-on-demand principle is a much more robust and precise device. Concerning the Top Spot print head, I will give a short introduction of the goals of that development and will present the basic ideas of that printhead. We are working on the commercialisation of that technology and I will present two types of machines which we are developing: - A “Research Machine” which is used for the prototyping of microarrays - A “Production Machine” which is a dedicated machine for producing high numbers of always the same type of Biochip. The basic differences of both machines are costs and output of Biochips / day. Vorlesung Mikrofluidik R. Zengerle, J. Ducrée; WS 1999 / 2000

TopSpot - Überarbeitetes Design Modifiziertes Kanalsystem  Keine Verschleppung Kolben anstelle einer Plastikfolie  vereinfachtes Handling Piezostack anstelle einer pneumatischen Tropfenauslösung  bessere Kontrolle Kühlplatte  geringere Verdunstung Höhere Integration  24- und 96-Kanal Druckköpfe And we had also a second idea for actuating the printhead. This idea was to use a pneumatic pressure pulse for actuating the nozzles. For that we cover the print head with a thin plastic film. The film has through holes at the reservoirs for avoiding a low pressure at the reservoirs. Now we have a closed chamber above the nozzles and when we displace the film very fast, we generate a fast pressure pulse in that area above the nozzles. All nozzles are actuated at the same time and every nozzle is ejecting a small droplet. You can see the situation in the following video. Vorlesung Mikrofluidik R. Zengerle, J. Ducrée; WS 1999 / 2000

TopSpot /24 24 Reservoirs Flüssigkeitsvolumen: 5 µl – 20 µl Abstand der Reservoirs entspricht 384er Mikrotiterplatte

TopSpot /96 96 Reservoirs Flüssigkeitsvolumen: 5 µl Abstand der Reservoirs entspricht 1536er Mikrotiterplatte

Vermarktung TopSpot /E „TopSpot Entry Arrayer“ Weitgehend manueller Betrieb 1 Druckkopf (24 – 96 Medien) TopSpot /M „TopSpot Modular Arrayer“ Halb-automatisiert (Kleinserien) Bis zu 10 Druckköpfe (960 Medien) Integrierte Qualitätskontrolle TopSpot /P „TopSpot Production System“ Vollständig automatisiert (Massenproduktion) 3.000 Microarrays / Tag And we had also a second idea for actuating the printhead. This idea was to use a pneumatic pressure pulse for actuating the nozzles. For that we cover the print head with a thin plastic film. The film has through holes at the reservoirs for avoiding a low pressure at the reservoirs. Now we have a closed chamber above the nozzles and when we displace the film very fast, we generate a fast pressure pulse in that area above the nozzles. All nozzles are actuated at the same time and every nozzle is ejecting a small droplet. You can see the situation in the following video. Vorlesung Mikrofluidik R. Zengerle, J. Ducrée; WS 1999 / 2000

TopSpot Entry Arrayer

TopSpot Entry Arrayer t 1. Vorbereitung des Druckkopfs 2. Einführen des Substrats 3. Auslösen des Druckkopfs t 4. Herausnahme des Substrats

Übersicht Einleitung Grundlegende Konzepte der TopSpot-Technologie Die Zweite TopSpot-Generation & Deren Vermarktung Reformatierung von MTPs Schlussfolgerungen The outline of my presentation will be als follows: I will present two new devices for the nanoliter liquid handling. These devices are the NanoJet-Dispenser for the precise dosage of biological liquids in the nanoliter range and the TopSpot printhead, a cheap & simple device for the highly parallel spotting of micro arrays. Concerning the NanoJet dispenser I will discuss drop-on-demand and jet-on-demand procedures. You will see in the results that the Jet-on-demand principle is a much more robust and precise device. Concerning the Top Spot print head, I will give a short introduction of the goals of that development and will present the basic ideas of that printhead. We are working on the commercialisation of that technology and I will present two types of machines which we are developing: - A “Research Machine” which is used for the prototyping of microarrays - A “Production Machine” which is a dedicated machine for producing high numbers of always the same type of Biochip. The basic differences of both machines are costs and output of Biochips / day. Vorlesung Mikrofluidik R. Zengerle, J. Ducrée; WS 1999 / 2000

Reformatierung von MTPs Häufiges Problem: Wechsel zwischen verschiedenen MTP-Formaten z.B. von 96 (Abstand: 9,0 mm) zu 1536 (Abstand: 2,25 mm) Typische Volumina von 50 nl Mögliche Lösung mit TopSpot: Maßgeschneiderter Düsenabstand Definiertes Volumen aus den Mikrokanälen Pneumatisches Auslösen And we had also a second idea for actuating the printhead. This idea was to use a pneumatic pressure pulse for actuating the nozzles. For that we cover the print head with a thin plastic film. The film has through holes at the reservoirs for avoiding a low pressure at the reservoirs. Now we have a closed chamber above the nozzles and when we displace the film very fast, we generate a fast pressure pulse in that area above the nozzles. All nozzles are actuated at the same time and every nozzle is ejecting a small droplet. You can see the situation in the following video. Vorlesung Mikrofluidik R. Zengerle, J. Ducrée; WS 1999 / 2000

Reformatierung mit TopSpot Testaufbau: TopSpot 24 Druckkopf, 100 µm Düsen Mit 1000 µm Abstand, Wasser als Druckmedium Variable Schaltzeiten

TopSpot for Reformatting (II) Testaufbau: TopSpot 24 Druckkopf, 200 µm Düsen Mit 1000 µm Abstand, Wasser als Druckmedium Variable Schaltzeiten

Übersicht Einleitung Grundlegende Konzepte der TopSpot-Technologie Die Zweite TopSpot-Generation & Deren Vermarktung Reformatierung von MTPs Schlussfolgerungen The outline of my presentation will be als follows: I will present two new devices for the nanoliter liquid handling. These devices are the NanoJet-Dispenser for the precise dosage of biological liquids in the nanoliter range and the TopSpot printhead, a cheap & simple device for the highly parallel spotting of micro arrays. Concerning the NanoJet dispenser I will discuss drop-on-demand and jet-on-demand procedures. You will see in the results that the Jet-on-demand principle is a much more robust and precise device. Concerning the Top Spot print head, I will give a short introduction of the goals of that development and will present the basic ideas of that printhead. We are working on the commercialisation of that technology and I will present two types of machines which we are developing: - A “Research Machine” which is used for the prototyping of microarrays - A “Production Machine” which is a dedicated machine for producing high numbers of always the same type of Biochip. The basic differences of both machines are costs and output of Biochips / day. Vorlesung Mikrofluidik R. Zengerle, J. Ducrée; WS 1999 / 2000

in den Life Science Industrien Schlussfolgerungen “Microarrays”, “Mikrofluidik” und “Nanoliter Liquid Handling” gehören zweifellos zu den Schlüsseltechnologien in den Life Science Industrien TopSpot Technologie Kostengünstige und robuste Technologie zum Spotten von Microarrays Hochparallel (24 or 96 nozzles) auf der Eingangs- und Ausgangsseite Hohe Geschwindigkeit (bis zu 10 Microarrays / s) Verschiedene Konfigurationen: TopSpot /E, /M, /P Eignung zum Reformatieren von MTPs?  erste sehr vielversprechende Tests Now I come to the conclusions: First of all “Microfluidics” and “nanoliter Handling is a key technology in pharmaceutical industry and modern biology” I presented two new principles and devices: The NanoJet device for the high precision dosage of liquids in the nanoliter range. The new sysdtems saves money especially when you are dosing expensive reagents, it is a low cost device and it will be commercialized by Eppendorf. The product name will be Nanozyme. I presented also the TopSpot principle and the TopSpot printhead. It is a cheap and simple method for spotting low cost microarrays. We have 24 nozzles in parallel today and expect to have 96 nozzles in the near future. It is a high speed method, that means we can print up to 10 arrays / s and we are working on two different configurations - a “Research configuration” and - a “production configuration” Concerning the TopSpot principle we are also looking for distribution partners in the USA. Vorlesung Mikrofluidik R. Zengerle, J. Ducrée; WS 1999 / 2000

Danksagung Weitere Informationen: „Microfluidics“-Monography Finanzielle Unterstützung bei der Entwicklung des TopSpot-Dosierverfahrens “Zukunftsoffensive Baden-Württemberg, Förderung der Biotechnologie” BioChip Technologies GmbH, Freiburg Weitere Informationen: „Microfluidics“-Monography by J. Ducrée and R. Zengerle (Springer-Verlag, Frühjahr 2001) Last but not least I want to mention the “Pipetting & Dispensing” Team at HSG-IMIT. They are: - Nicolaus Hey - Stefan Bekesi - Jens Ducree - Michael Freygang - Holger Gruhler - Martin Müller - Michael Willmann Financial support for the development of the NanoJet-principle was given by: - Eppendorf - and the BMBF Financial support for the development of the TopSpot -principle was given by: - Zukunftsinitiative Baden Württemberg - Genescan Europe AG - BioChip Technologies In Kürze: Microfluidics Worldwide Information Service (www.mikrofluidik.de) Vorlesung Mikrofluidik R. Zengerle, J. Ducrée; WS 1999 / 2000

Microfluidik-Team Gerhard Kattinger Jörg Kohnle Peter Koltay Jürgen Merz Gino Raffa Jochen Schaible Michael Sesterhenn Reinhard Steger Manfred Stehr Matthias Storz Bas de Heij Herbert Straatmann Günther Waibel Michael Willmann Gerhard Birkle Roland Zengerle Heidi Ashauer Robert Cernosa Jens Ducrée Ulrike Grundmann