DE60003845T2 - Hybridisierungstest für biomaterial in einem biokanal - Google Patents
Hybridisierungstest für biomaterial in einem biokanal Download PDFInfo
- Publication number
- DE60003845T2 DE60003845T2 DE60003845T DE60003845T DE60003845T2 DE 60003845 T2 DE60003845 T2 DE 60003845T2 DE 60003845 T DE60003845 T DE 60003845T DE 60003845 T DE60003845 T DE 60003845T DE 60003845 T2 DE60003845 T2 DE 60003845T2
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- microfluidic device
- binding
- microchannel
- dna
- test sample
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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Description
- HINTERGRUND DER ERFINDUNG
- Analyseverfahren, die spezifische Bindungspaare wie Antigen/Antikörper; komplementäre DNA/DNA; DNA/RNA; RNA/RNA; Biotin/Avidin enthaltende Paare verwenden, sind im Fachgebiet weithin bekannt. Verfahren zur Herstellung und Verwendung von Mikrofluidikvorrichtungen sind ebenfalls gut bekannt. Der Stand der Technik offenbart verschiedene Verfahren zur DNA-Sequenzierung, die auf der Komplementbindung von DNA beruhen.
- DNA-Sonden-Array-Technologie, die auf das Binden von einstrangiger Ziel-DNA an immobilisierte DNA-Proben zurückgreift, findet weitreichende Anwendung. Vielzählige Forschungs- und Entwicklungsaktivitäten wurden mit unterschiedlichem Technologieschwerpunkt durchgeführt. Beispielsweise konzentrieren sich manche Verfahren auf die Sondenplatzierung mittels mechanischer Mittel. Andere wiederum beschäftigen sich mit In-situ-Sondensynthese, die für die Herstellung großer Arrays vorteilhaft ist. Darüber hinaus setzen wiederum andere Verfahren einen Schwerpunkt auf Gel-Pad-Arrays, die Photopolymerisation und piezoelektrische Flüssigkeitsabgabeverfahren verwenden.
- Eine Herausforderung für alle DNA-Hybridisierungsverfahren stellt das Fehlen der Steuerbarkeit der Stringenz für die jeweilige Sondenstelle dar. Der DNA-Hybridisierungsvorgang erfolgt bei bestimmten Temperatur- und Salinitätsbedingungen und variiert mit den DNA-Sequenzen. Bei DNA-Sonden-Arrays kann die Hybridisierungserkennung unter Bedingungen einheitlicher Stringenz für das gesamte Sonden-Array niemals exakt sein, da die DNA-Sondensequenzen unterschiedlich sind. Das Problem ist bei kurzen Doppel strangabschnitten am offensichtlichsten, wo häufig einzelne Basenübereinstimmungsfehler auftreten. Die Auswirkung der Hybridisierung bei fehlender Übereinstimmung kann durch Verwendung einer sehr großen Anzahl an Sondenstellen minimiert werden. Die Steuerung der Stringenz wurde für jede Sondenstelle durch Steuern der elektrophoretischen Bewegung von Oligonucleotiden bereitgestellt. Um letzteren Ansatz erfolgreich umsetzen zu können, ist eine exakt erzeugte Permeationsschicht erforderlich, um zu verhindern, dass DNA-Moleküle oder Markierungswirkstoffe durch direkte Elektrolyse oder durch das Elektrolyseprodukt beschädigt werden.
- Darüber hinaus ist es aktuellen DNA-Array-Verfahren nicht gelungen, eine wirksame Lösung bereitzustellen, um die Effizienz der Hybridisierung zu maximieren. Für diagnostische Analysen werden häufig DNA-Moleküle in winzigen Mengen verwendet. Die Nachweisgrenze der Analyse ist von der Empfindlichkeit der Detektionsvorrichtung sowie von der Menge an Ziel-Oligo-Sequenzen, die während der Durchführung der Hybridisierung an die Sonden gebunden sind, abhängig. In einer stationären Hybridisierungskammer, bei der kein aktives Mischen erfolgt, ist die Wahrscheinlichkeit, dass ein gegebenes Zielmolekül an seinen Komplementärstrang an der Oberfläche hybridisiert, von der Diffusionsgeschwindigkeit und der Statistik abhängig. Bei niedrigen Zielkonzentrationen kann es bis zu 10 Stunden dauern, bis die Hybridisierung abgeschlossen ist. Um die Zielmoleküle besser nutzen zu können und die Hybridisierung zu fördern, wurden Durchflussverfahren nahegelegt, bei denen Sonden-Arrays senkrecht zur Fluidströmungsrichtung angeordnet sind. Doch selbst bei Durchfluss-Verfahren tritt nur ein Teil der Zielmoleküle mit einer spezifischen DNA-Sondenstelle in Kontakt.
- Die vorliegende Erfindung überwindet obige technische Problemstellungen, indem sie die DNA-Sondenstellen in Mikrofluidikkanälen nacheinander anordnet, so dass die DNA-Sonde mit ihren Bindungspartnern wirksam in Kontakt kommen kann.
- Die US-A-5.147.607 beschreibt eine Vielfalt von Mikrobestimmungsvorrichtungen, die Mikrokanäle aus Kunststoffmaterialien mit einem Reagens, wie z.B. einem Antikörper oder DNA, aufweisen, das an verschiedenen Stellen auf dem Kanal immobilisiert ist. Es werden Verfahren zum Binden von Antikörpern an die Mikrokanalwand, jedoch keine Verfahren zur Bindung von DNA beschrieben. Das Binden von Sonden an die Mikrokanalwand stellt keinen optimalen Kontakt zwischen Sonde und Testprobe her. Die US-A-5.843.767 beschreibt mikrofabrizierte poröse Durchflussvorrichtungen zur diskreten Detektion von Bindungsreaktionen wie DNA/DNA. Die WO 98/43739 offenbart poröse Strömungskanäle, die in der Kammer immobilisierte Reagenzien aufweisen. Die WO 93/22053 beschreibt Mikrokanäle mit getrennten Bereiche zum Binden von Substanzen, die auf Polymer-Perlen immobilisiert sind.
- KURZBESCHREIBUNG DER ABBILDUNGEN
-
1 zeigt eine schematische Ansicht eines Fluidkanals von oben, der mit porösem Gel und punktierten DNA-Sonden gefüllt ist. -
2 zeigt auf lithographischem Weg gemusterte Gel-Pads innerhalb eines Mikrofluidikkanals. -
3 zeigt Mikrofluidikkanäle mit geformten Kunststoff-Mikrostrukturen zur DNA-Anbindung. -
4 zeigt einen Mikrofluidikkanal, der mit Perlen gepackt ist und bei dem getrennte Perlenbereiche einen spezifischen Bindungswirkstoff wie DNA aufweisen. -
5 veranschaulicht einen einfachen Anfangsfluss, der in zahlreiche Kanäle gelenkt wird. -
6 veranschaulicht eine zirkulierende Mikrfluidikkanalvorrichtung. - ZUSAMMENFASSUNG DER ERFINDUNG
- Die Erfindung umfasst Mikrofluidikvorrichtungen, die einen Abschnitt aus festem Material, wie z.B. einen Chip, mit einem Mikrokanal mit einer Zutrittsöffnung und einer Austrittsöffnung zum Transport von Fluids durch die Kanäle umfassen. Der Mikrokanal besitzt getrennte definierte Bereiche, in denen spezifische Bindungspaarelemente auf einem porösen Polymer immobilisiert sind und Mikrostrukturen in den Mikrokanälen oder gepackten Perlen ausgebildet sind. Diese Strukturen stellen optimalen Kontakt der immobilisierten Bindungspaarelemente mit einem im durch den Mikrokanal strömenden Fluid enthaltenen Bindungspaarelement bereit. Die porösen Polymer-Perlen oder Mikrostruktur müssen für Durchfluss sorgen und dürfen den Kanal nicht blockieren. Der Mikrokanal ist wirksam mit einem Detektor und einer ein Fluid antreibenden Komponente verbunden, um Flüssigkeiten in den Kanal zu transportieren, und kann zudem an den Zutritts- und Austrittsöffnungen Elektroden aufweisen. Des Weiteren ist ein Rezirkulationsarm zum Rezirkulieren einer Testprobe durch die Bereiche des Mikrokanals vorgesehen.
- DNA/DNA-, DNA/RNA- und RNA/RNA-Komplementärbindungspaare sind bevorzugt. Der Mikrokanal ist wirksam mit der mit einem Fluorophor markierten Ziel-DNA, einer Anregungsquelle und einem Detektor verbunden, um von den Bindungspaaren emittierte Fluoreszenzsignale zu detektieren. Es ist ein Ziel der Erfindung, ein Verfahren zur Sequenzierung von DNA oder RNA bereitzustellen, indem der oben definierte Chip mit DNA- oder RNA-Sonden versehen wird, die in den getrennten definierten Bereichen immobilisiert sind, um Fluoreszenz-markierte Ziel-DNA zu binden.
- Ein weiteres Ziel der vorliegenden Erfindung ist es, ein Mittel zum Bestimmen genetischer Defekte bereitzustellen. Die Erfindung sieht zudem ein Mittel zum Identifizieren von Pathogenen durch DNA-Analyse vor.
- Die Mikrokanäle können eine Vielzahl von Konfigurationen, Rückführarmen, Ventilen und Öffnungen zum Steuern der Fluidströmung aufweisen. Es können Einzel- oder Mehrfachkanäle vorliegen. Die Erfindung ermöglicht wirksamen Kontakt zwischen immobilisierten Bindungssubstanzen und Bindungspartnern, die im durch den Kanal strömenden Fluid vorhanden sind. Die Erfindung sieht eine verbesserte Steuerung der Hybridisierungsstringenz durch Strömungsmodulation, verkürzte Analysezeiten durch eine Erhöhung der Hybridisierungsgeschwindigkeit mittels Strömungs-induzierter Bewegung und durch ein nahes Zueinanderführen des Ziels und der Sonde innerhalb des Mikrofluidikkanals, sowie erhöhte Hybridisierungseffizienz vot, wodurch die Empfindlichkeit verbessert wird. Zusätzlich kommt es zu keiner Beeinträchtigung durch Hydrolyse.
- DETAILLIERTE BESCHREIBUNG DER ERFINDUNG
- Die Mikrofluidikkanäle der vorliegenden Erfindung sind Kanäle mit im Allgemeinen weniger als 200 μm an Kunststoff, die im Guß- oder Prägeverfahren hergestellt wurden. Die Kanäle müssen so dimensioniert sein, dass sie das Pumpen des Mikrofluidiksystems unterstützen. Die Mikrofluidikkanäle können beliebig geformt sein, z.B. linear, schlangenförmig, bogenförmig und dergleichen. Der Querschnitt des Kanals kann quadratisch, rechteckig, halbrund etc. sein. Es können mehrere, miteinander verbundene Mikrokanäle mit Ventilen bereitgestellt sein, um eine Rezirkulation zu ermöglichen.
- Der aus festem Material bestehende Abschnitt können Chips aus Glas, Keramik, Silizium oder Kunststoff sein. Die Chips sind vorzugsweise aus Kunststoffen wie Epoxidharz, Polyacrylharz, Polyesterharz, Polystyrol, Polycarbonat, Polyvinylchlorid und dergleichen hergestellt. Spezifische Bindungspaare sind DNA/DNA- oder DNA/RNA-Bindungspaare.
- Fluid antreibende Komponenten wie Druckgas, Vakuum, elektrisches Feld, Magnetfeld und Zentrifugalkraftvorrichtungen sind wirksam mit dem Mikrokanal verbunden, um Fluid durch den Mikrokanal zu bewegen. Darüber hinaus können geladene Testproben verändert werden, indem das elektrische Feld in Gegenrichtung zur oder in Richtung der Strömung oder senkrecht zur Strömung moduliert wird. Dadurch kann die Strömungsgeschwindigkeit des Fluids im Mikrokanal verändert werden, um das Binden von Bindungspaaren, z.B. die Hybridisierung von DNA/DNA- oder DNA/RNA-Paaren, voranzutreiben. Mit dem Mikrokanal ist zudem wirksam ein Detektor, wie z.B. ein optischer, elektrischer oder elektrochemischer Detektor, verbunden.
-
1 veranschaulicht einen schlangenförmigen Mikrodluidikkanal1 , der mit porösem Gel2 mit diskreten, getrennten Bereichen3 gefüllt ist, an denen wiederum ein Element eines spezifischen Bindungspaares wie DNA anhaftet. Die Probe strömt an Stelle4 in den Mikrofluidikkanal und tritt an Stelle5 wieder aus dem Kanal aus. Bei diesem Ansatz wird er Kanal mit porösem Gelmaterial wie Agarose oder Polyacraylamid gefüllt. Die Poren des Gels werden groß genug ausgebildet, indem verdünnte Gelierungslösungen verwendet werden, um eine wesentliche Fluidströmung durch das Gel zu ermöglichen. Elemente der spezifischen Bindungspaare werden auf die Gele gespottet, so dass die Sonden chemisch gebunden werden. -
2 veranschaulicht einen Mikrofluidikkanal10 , der innerhalb des Kanals gemusterte Gel-Pads aufweist. Die Gel-Pads werden durch Photopolymerisation von Acrylamid mittels lithographischer Verfahren hergestellt. -
3 veranschaulicht einen Mikrofluidikkanal15 , bei dem hohe Oberflächenmikrostrukturen in den Kanal geformt sind.3a zeigt eine Reihe von Säulen16 in einem bestimmten Bereich und3b eine bestimmten Bereich mit Kuppeln, die in den Kanal15 geformt sind. Diese Mikrostrukturen sind chemisch modifiziert, und spezifische Bindungssubstanzen sind an diese gebunden. -
4 veranschaulicht einen Mikrofluidikkanal20 , der abwechselnd mit Abschnitten aus zusatzfreien Perlen21 und Perlen22 mit einer spezifischen Bindungssubstanz wie DNA gepackt ist. -
5 veranschaulicht einen Mikrodluidikkanal25 , der sich in eine Vielzahl von Mikrofluidikkanälen26a,b,c etc. verzweigt, von denen jeder einen einen bestimmten mit einer wie oben beschriebenen Bindungssubstanz27 versehenen Bereich aufweist. Diese Ausführungsform ermöglicht, dass eine Probe parallel untersucht und auf ihre Reaktivität bezüglich derselben oder unterschiedlicher spezifischer Bindungssubstanzen getestet werden kann. -
6 veranschaulicht einen Chip30 mit einem rezirkulierenden Mikrofluidikkanal34 . Der Mikrofluidikkanal besitzt diskrete Bereiche mit spezifischen Bindungssubstanzen32 , wie oben beschrieben wurde, sowie einen Rezirkulationsarm33 und ein Ventil34 zum Ablassen nach der Rezirkulation. Die Testprobe wird an der Stelle des Bindungspartners vorbei rezirkuliert. Somit kann die spezifische Bindungssubstanz an verdünnten Proben bzw. langsam reagierenden Proben vorbeiströmen. - Mikrohergestellte Kunststoff-Kapillarelektrophoresevorrichtungen wurden auf dem Gebiet eingesetzt. Thermoplastisch geformte Polymethylmethacrylat-Kapillarelektrophorese-Vorrichtungen wurden von R.M. McCormick et al. in „Microchannel electrophoretic separations of DNA in injection-molded plastic substrates" (Anal. Chem. 69, 2626 (1997)) beschrieben. Eckstrom et al. untersuchten elastomere Polymere wie PDMS („PCT-Anmeldung WO 91/16966" (1991)). Aktuellere Veröffentlichungen, z.B. von C.S. Effenhauser et al. in „Integrated Capillary Electrophoresis on Flexible Silicone Microdevices" (Anal. Chem, 69, 3451 (1997)), beschäftigen sich mit der elektrophoretischen Trennung von DNA-Leitern in PDMS-Vorrichtungen. Mastrangelo et al. beschreiben die Herstellung von Mikro-Kapillarelektrophoresevorrichtungen auf Basis von Parylen-Polycarbonat-Substraten mittels eines Oberflächen-Mikromaterialberarbeitungsverfahrens („An Inexpensive Plastic Technology for Microfabricated Capillary Electrophoresis Chip", vorgestellt auf der Micro-TAS '98 in Banff 1998). Verfahren zur Herstellung von Mikrokanälen sind somit verfügbar. Die Erfindung schließt das Anbringen von Bindungssubstanzen im Mikrokanal durch poröses Polymer, Perlen oder Strukturen, um den Bindungsvorgang effizienter zu unterstützen.
- Die Beispiele sind lediglich zur Veranschaulichung der vorliegenden Erfindung gedacht und schränken den Schutzumfang der Erfindung nicht ein.
Claims (22)
- Mikrofluidikvorrichtung, umfassend einen Abschnitt aus festem Material, Folgendes umfassend (i) einen Mikrokanal (
1 ,10 ,15 ,20 ,25 ) mit einer Zutrittssöffnung (4 ) und einer Austrittsöffnung (5 ) für den Transport zumindest einer Testprobe, wobei der Mikrokanal mehrere räumlich getrennte Regionen (3 ) umfasst, auf denen eine Bindungssubstanz, die fähig ist, ein spezifisches Bindungspaar mit einem Bindungspartner zu bilden, auf einem porösen Polymer, auf Perlen oder auf Mikrostrukturen immobilisiert ist, die im Mikrokanal ausgebildet sind; (ii) eine Fluid antreibende Komponente, die fähig ist, die Strömungsgeschwindigkeit der Testprobe einzustellen; (iii) einen Detektor, der die Bindung eines Bindungspartners an die Bindungssubstanz detektiert; (iv) einen Rezirkulationsarm (33 ) und ein Ventil (34 ), um die Testprobe über räumlich getrennte Regionen zirkulieren zu lassen. - Mikrofluidikvorrichtung nach Anspruch 1, worin der Mikrokanal in Schlangenlinien verläuft.
- Mikrofluidikvorrichtung nach einem der vorangegangenen Ansprüche, worin sich der Mikrokanal in mehrere Mikrofluidikkanäle (
26a ,26b ,26c ) verzweigt, die jeweils eine getrennte Region einer Bindungssubstanz (27 ) umfassen. - Mikrofluidikvorrichtung nach einem der vorangegangenen Ansprüche, worin die Vorrichtung aus einem Material hergestellt ist, das aus der aus Silizium, Siliziumdioxid, Glas, Kunststoff und Keramik bestehenden Gruppe ausgewählt ist.
- Mikrofluidikvorrichtung nach einem der vorangegangenen Ansprüche, worin die räumlich getrennten Regionen poröse Polymere umfassen.
- Mikrofluidikvorrichtung nach einem der Ansprüche 1 bis 4, worin die räumlich getrennten Regionen Hydrogelabschnitte (
2 ) umfassen. - Mikrofluidikvorrichtung nach Anspruch 6, worin der Hydrogelabschnitt auf lithographischem Weg gemustert (
11 ) ist. - Mikrofluidikvorrichtung nach einem der vorangegangenen Ansprüche, worin die räumlich getrennten Bereiche im Mikrokanal Perlen umfassen.
- Mikrofluidikvorrichtung nach einem der vorangegangenen Ansprüche, worin die räumlich getrennten Bereiche in den Mikrokanal eingearbeitete Mikrostrukturen umfassen.
- Mikrofluidikvorrichtung nach Anspruch 9, worin die Mikrostrukturen eine Abfolge von in den Mikrokanal geformten Säulen (
16 ) umfassen. - Mikrofluidikvorrichtung nach Anspruch 9, worin die Mikrostrukturen in den Mikrokanal geformte Kuppeln (
17 ) umfassen. - Mikrofluidikvorrichtung nach einem der vorangegangenen Ansprüche, worin die Bindungssubstanzen Nucleinsäuren sind.
- Mikrofluidikvorrichtung nach Anspruch 12, worin die Nucleinsäure eine DNA ist.
- Mikrofluidikvorrichtung nach Anspruch 12, worin die Nucleinsäure eine RNA ist.
- Mikrofluidikvorrichtung nach einem der Ansprüche 1 bis 11, worin die Bindungssubstanzen Polypeptide sind.
- Mikrofluidikvorrichtung nach Anspruch 15, worin die Polypeptide Antigene sind.
- Mikrofluidikvorrichtung nach Anspruch 15, worin die Polypeptide Antikörper sind.
- Mikrofluidikvorrichtung nach einem der vorangegangenen Ansprüche, worin die Fluid antreibende Komponente ein Druckgas, Vakuum, ein elektrisches Feld, ein Magnetfeld oder Zentrifugalkraft umfasst.
- Mikrofluidikvorrichtung nach einem der vorangegangenen Ansprüche, worin der Detektor ein optischer, elektrischer oder elektrochemischer Detektor ist.
- Verfahren zum Detektieren eines spezifischen Bindungselements in einer Testprobe, wobei das Verfahren Folgendes umfasst: (a) das Hindurchschicken der Testprobe durch die Mikrofluidikvorrichtung nach einem der vorangegangenen Ansprüche, um ein Bindungspaar zu bilden; (ii) das Detektieren des Bindungspaares.
- Verfahren nach Anspruch 20, worin die Testprobe vor dem Detektieren rezirkuliert wird.
- Verfahren nach Anspruch 20 oder 21, worin die Strömungsgeschwindigkeit der Testprobe mittels der Fluid antreibenden Komponente eingestellt wird, um die Bindung der Bindungspaare zu fördern.
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-
1999
- 1999-11-12 US US09/438,600 patent/US6361958B1/en not_active Expired - Lifetime
-
2000
- 2000-11-09 CA CA002389549A patent/CA2389549A1/en not_active Abandoned
- 2000-11-09 PT PT00993037T patent/PT1233830E/pt unknown
- 2000-11-09 ES ES00993037T patent/ES2202224T3/es not_active Expired - Lifetime
- 2000-11-09 DE DE60017809T patent/DE60017809T2/de not_active Expired - Lifetime
- 2000-11-09 DE DE60003845T patent/DE60003845T2/de not_active Expired - Fee Related
- 2000-11-09 JP JP2001536293A patent/JP2003514221A/ja active Pending
- 2000-11-09 EP EP04027973A patent/EP1520619A3/de not_active Withdrawn
- 2000-11-09 EP EP03010981A patent/EP1350568B1/de not_active Expired - Lifetime
- 2000-11-09 AU AU29238/01A patent/AU773289B2/en not_active Ceased
- 2000-11-09 EP EP00993037A patent/EP1233830B1/de not_active Expired - Lifetime
- 2000-11-09 DK DK00993037T patent/DK1233830T3/da active
- 2000-11-09 AT AT03010981T patent/ATE287765T1/de not_active IP Right Cessation
- 2000-11-09 AT AT00993037T patent/ATE244603T1/de not_active IP Right Cessation
- 2000-11-09 WO PCT/US2000/042047 patent/WO2001034302A2/en active IP Right Grant
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2001
- 2001-12-19 US US10/028,277 patent/US6960467B2/en not_active Expired - Lifetime
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2004
- 2004-08-02 AU AU2004203548A patent/AU2004203548A1/en not_active Abandoned
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US20020094584A1 (en) | 2002-07-18 |
ES2202224T3 (es) | 2004-04-01 |
AU773289B2 (en) | 2004-05-20 |
DK1233830T3 (da) | 2003-09-22 |
AU2923801A (en) | 2001-06-06 |
EP1233830A2 (de) | 2002-08-28 |
WO2001034302A9 (en) | 2002-08-15 |
ATE287765T1 (de) | 2005-02-15 |
EP1350568B1 (de) | 2005-01-26 |
EP1520619A2 (de) | 2005-04-06 |
JP2003514221A (ja) | 2003-04-15 |
DE60003845D1 (de) | 2003-08-14 |
CA2389549A1 (en) | 2001-05-17 |
EP1350568A1 (de) | 2003-10-08 |
EP1233830B1 (de) | 2003-07-09 |
AU2004203548A1 (en) | 2004-08-26 |
EP1520619A3 (de) | 2005-09-21 |
ATE244603T1 (de) | 2003-07-15 |
US6960467B2 (en) | 2005-11-01 |
DE60017809T2 (de) | 2006-01-05 |
WO2001034302A3 (en) | 2002-01-10 |
WO2001034302A2 (en) | 2001-05-17 |
US6361958B1 (en) | 2002-03-26 |
PT1233830E (pt) | 2003-11-28 |
DE60017809D1 (de) | 2005-03-03 |
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