NL9022056A - Synthese van geimmobiliseerd polymeer op zeer grote schaal. - Google Patents
Synthese van geimmobiliseerd polymeer op zeer grote schaal. Download PDFInfo
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- NL9022056A NL9022056A NL9022056A NL9022056A NL9022056A NL 9022056 A NL9022056 A NL 9022056A NL 9022056 A NL9022056 A NL 9022056A NL 9022056 A NL9022056 A NL 9022056A NL 9022056 A NL9022056 A NL 9022056A
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Classifications
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- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
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Description
Synthese van geïirnnnh-i liseerd polymeer op zeer grote schaal
Opmerking ten aanzien van copyright
Een gedeelte van de beschrijving van dit octrooidokument bevat materiaal dat onderhevig is aan copyrightbescherming. De eigenaar van het copyright heeft geen bezwaar tegen facsimile reproduktie door ieder van het octrooidokument of de octrooibeschrijving zoals deze aanwezig is in de Octrooiraad of de registratie ervan, maar reserveert overigens alle copyrightrechten, welke dan ook.
Achtergrond van de uitvinding
De onderhavige uitvinding heeft betrekking op de synthese en het aanbrengen van materialen op bekende locaties. In het bijzonder verschaft een uitvoeringsvorm van de uitvinding een methode en daarmee geassocieerd apparaat voor de bereiding van diverse chemische sequenties bij bekende locaties op een enkelvoudig substraatoppervlak. De uitvinding kan bijvoorbeeld worden toegepast op het gebied van de bereiding van oligomeer, peptide, nucleïnezuur, oligosaccharide, fosfolipide, polymeer, of soortgelijke geneesmiddelbereiding, in het bijzonder voor het scheppen van bronnen van chemische diversiteit voor toepassing bij het screenen op biologische actitivteit.
Het verband tussen structuur en activiteit van moleculen is een fundamenteel punt bij het onderzoek van biologische systemen. Structuur-activiteitsverhoudingen zijn belangrijk bij het begrijpen van bijvoorbeeld de functie van enzymen, de manier waarop cellen met elkaar communiceren, alsmede cellulaire regel- en terugvoersystemen.
Van bepaalde macromoleculen is bekend dat zij in wisselwerking treden en zich aan andere moleculen binden die een zeer specifieke 3~ dimensionale ruimtelijke en electronische verdeling hebben. Elk groot molecuul met een dergelijke specificiteit kan als een receptor worden beschouwd, hetzij het een enzym katalyserende de hydrolyse van een metabolisch tussenprodukt, een cel-oppervlakproteïne dat als membraanmediator fungeert voor het transport van ionen, een glycoproteine dat dient voor het identificeren van een bijzondere cel voor de buren ervan, een IgG-klasse antilichaam dat in het plasma circuleert, een oligonucleotidese-quentie van DNA in de kern, of dergelijke is. De diverse moleculen, die selectief receptoren binden, zijn bekend als liganden.
Vele methoden zijn beschikbaar voor het meten van de bindingsaffi-niteit van bekende receptoren en liganden, maar de informatie die uit dergelijke experimenten verkregen kan worden wordt dikwijls beperkt door het aantal en het type van de liganden die beschikbaar zijn. Nieuwe li-ganden worden soms bij toeval ontdekt of door toepassing van nieuwe technieken voor het ophelderen van de moleculaire structuur, waaronder kris-talligrafische analyse onder toepassing van röntgenstralen en recombinant genetische technieken voor proteïnen.
Kleine peptiden zijn een voorbeeld van het systeem voor het onderzoeken van het verband tussen structuur en functie in de biologie. Een peptide is een sequentie van aminozuren. Wanneer de 20 in de natuur voorkomende aminozuren gecondenseerd worden tot polymere moleculen, vormen zij een grote verscheidenheid van driedimensionale configuraties, elk resulterende uit een bijzondere aminozuursequentie en oplosmiddelcondi-tie. Het aantal mogelijke pentapeptiden van de 20 in de natuur voorkomende aminozuren, is bijvoorbeeld 205 of 3.2 miljoen verschillende peptiden. De waarschijnlijkheid dat moleculen van deze grootte bruikbaar zouden kunnen zijn in onderzoekingen van receptorbinding, wordt ondersteund door epitoop-analysestudies welke aantonen dat sommige antilichamen sequenties herkennen die zo kort zijn als enkele aminozuren met een hoge specificiteit. Verder brengt het gemiddelde molecuulgewicht van aminozuren kleine peptiden in het groottetraject van vele tegenwoordig bruikbare farmaceutische produkten.
Ontdekking van een farmaceutisch geneesmiddel is een type van research dat gebaseerd is op een dergelijk onderzoek van het verband tussen structuur en activiteit. In de meeste gevallen kan huidig farmaceutisch onderzoek worden beschreven als de werkwijze voor het ontdekken van nieuwe liganden met wenselijke patronen van specificiteit voor biologisch belangrijke receptoren.
Een ander voorbeeld is research voor het ontdekken van nieuwe verbindingen voor toepassing in de landbouw, zoals pesticiden en herbiciden.
Soms is de oplossing voor een rationeel proces van het ontwerpen van liganden moeilijk of zonder resultaat. Methoden van de stand der techniek voor het bereiden van grote aantallen verschillende polymeren zijn angstvallig langzaam bij toepassing op een schaal die voldoende is om een effectieve rationele of willekeurige screening mogelijk te maken. Bijvoorbeeld is de "Merrifield"-methode (J. Am. Chem. Soc. (1963) 85.: 2149-2154 welke publikatie hierin voor alle doeleinden door verwijzing wordt opgenomen) toegepast voor de synthese van peptiden op een vaste drager. Volgens de Merrifield-methode wordt een aminozuur covalent gebonden aan een drager die uit een onoplosbaar polymeer is vervaardigd. Een ander aminozuur met een α-beschermde groep, wordt omgezet met covalent gebonden aminozuur voor de vorming van een dipeptide. Na wassen wordt de beschermende groep verwijderd en een derde aminozuur met een a-bescher-mende groep, wordt aan het dipeptide geaddeerd. Dit proces wordt voortgezet totdat een peptide van een gewenste lengte en sequentie is verkregen. Door toepassing van de Merrifield-methode is het economisch niet praktisch om meer dan een handvol van peptidesequenties in een dag te synthetiseren.
Voor het synthetiseren van grotere aantallen polymeersequenties is ook voorgesteld om een reeks reactievaten voor de polymeersynthese te gebruiken. Bijvoorbeeld kan een buisvormig reactorsysteem worden gebruikt voor het synthetiseren van een lineair polymeer op een drager in de vorm van een vaste fase, door geautomatiseerde sequentiële additie van reagentia. Deze methode maakt de synthese nog niet mogelijk van een voldoend groot aantal polymeersequenties voor een effectieve economische screening.
Methoden voor de bereiding van een veelvoud van polymeersequenties zijn ook bekend waarbij een geperforeerde houder een bekende hoeveelheid reactieve deeltjes omvat, welke deeltjes groter zijn dan de geperforeerde openingen van de houder. De houders kunnen selectief worden omgezet met gewenste materialen voor het synthetiseren van gewenste sequenties van produktmoleculen. Zoals met andere methoden welke in deze techniek bekend zijn, kan deze methode praktisch niet worden gebruikt voor de synthese van een voldoende groot aantal polypeptiden voor effectieve screening.
Andere technieken zijn ook beschreven. Deze methoden omvatten de synthese van peptiden op 96 uit kunststof bestaande pennen, die passen in het formaat van standaard microtiterplaten.
Ongelukkigerwijs, hoewel deze technieken enigszins bruikbaar zijn, blijven er aanzienlijke problemen. Bijvoorbeeld zijn deze methoden steeds beperkt ten aanzien van de diversiteit van sequenties die economisch gesynthetiseerd en gescreend kunnen worden.
Uit het bovenstaande blijkt dat een verbeterde methode en een verbeterd apparaat voor de synthese van een grote verscheidenheid van chemische sequenties op bekende locaties gewenst is.
Samenvatting van de uitvinding
Een verbeterde methode en apparaat voor de bereiding van een groot aantal polymeren wordt beschreven.
Volgens een de voorkeur verdienende uitvoeringsvorm worden verbin-dingsmoleculen op een substraat verschaft. Een terminale einde van de verbindingsmoleculen wordt verschaft met een reactieve functionele groep die beschermd is met een door licht verwijderbare beschermende groep. Bij toepassing van lithografische methoden wordt de door licht verwijderbare beschermende groep blootgesteld aan licht en in eerste geselecteerde gebieden uit de verbindingsmoleculen verwijderd. Vervolgens wordt het substraat gewassen of op andere wijze in contact gebracht met een eerste monomeer dat reageert met de blootgestelde functionele groepen op de verbindingsmoleculen. Volgens een de voorkeur verdienende uitvoeringsvorm is het monomeer een aminozuur, bevattende een door licht verwijderbare beschermende groep bij de amino- of carboxyterminus ervan en het verbin-dingsmolecuul eindigt in een amino- of carboxyzuurgroep welke groep een door licht verwijderbare beschermende groep draagt.
Een tweede stel van geselecteerde gebieden wordt daarna blootgesteld aan licht en de door licht verwijderbare beschermende groep op het verbindingsmolecuul/beschermde aminozuur, wordt bij het tweede stel gebieden verwijderd. Vervolgens wordt het substraat in contact gebracht met een tweede monomeer, bevattende een door licht verwijderbare beschermende groep voor omzetting met blootgestelde functionele groepen. Deze werkwijze wordt herhaald om op selectieve wijze monomeren toe te passen totdat polymeren van een gewenste lengte en gewenste chemische sequentie zijn verkregen. Voor licht labiele groepen worden vervolgens eventueel verwijderd en de sequentie wordt daarna eventueel van een eindstandig beschermend middel voorzien. Beschermende groepen in de zijketen, indien aanwezig, worden ook verwijderd.
Door toepassing van de hierin vermelde lithogragische technieken is het mogelijk licht te richten op betrekkelijk kleine en nauwkeurig bekende locaties op het substraat. Derhalve is het mogelijk polymeren te synthetiseren van een bekende chemische sequentie bij bekende locaties op het substraat.
Het resulterende substraat zal een grote verscheidenheid van toepassingen hebben, met inbegrip van bijvoorbeeld het screenen van grote aantallen polymeren op biologische activiteit. Voor het screenen op biologische activiteit wordt het substraat blootgesteld aan een of meer receptoren, zoals gehele cellen van antilichaam, receptoren op blaasjes, lipiden, of elk ander van een groot aantal andere receptoren. De receptoren worden bij voorkeur gemerkt met bijvoorbeeld een fluorescerend merk-middel, radio-actief merkmiddel of een gemerkt antilichaam dat met de receptor reactief is. De plaats van het merkmiddel op het substraat wordt bijvoorbeeld gedetecteerd door foton-detectie of autoradiografische technieken. Als gevolg van kennis van de sequentie van het materiaal bij de locatie waar binding is gedetecteerd, is het mogelijk snel te bepalen welke sequentie zich met de receptor bindt, en derhalve kan de techniek worden gebruikt voor het screenen van grote aantallen peptiden. Andere mogelijke toepassingen van de uitvinding hierin omvatten diagnostische toepassingen waarin diverse antilichamen voor bijzondere receptoren op een substraat worden gebracht, en bijvoorbeeld bloedsera op immuun-defi-ciënties worden gescreend. Nog verdere toepassingen omvatten bijvoorbeeld het selectief "doteren" van organische materialen in semi-geleiderinrich-tingen, en dergelijke.
In verband met een aspect van de uitvinding wordt ook een verbeterd reactorsysteem voor het synthetiseren van polymeren beschreven. Het reac-torsysteem omvat een substraatverhoging die samenwerkt met een substraat rondom een omtrek ervan. De substraatverhoging verschaft een reactorruim-te tussen het substraat en de verhoging door of waarin reactievloeistof-fen worden gepompt of stromen. Een masker wordt geplaatst of gericht op het substraat en verlicht zodat geselecteerde gebieden van de structuur in de reactieruimte worden gedeprotecteerd. Een monomeer wordt gepompt door de reactorruimte of op andere wijze met het substraat in contact gebracht en reageert met de gedeprotecteerde gebieden. Door het selectief deprotecteren van gebieden op het substraat en laten stromen van vooraf bepaalde monomeren door de reactorruimte, kunnen gewenste polymeren op bekende locaties worden gesynthetiseerd.
Een verbeterd detectieapparaat en verbeterde methoden worden ook beschreven. De detectiemethode en het apparaat maken gebruik van een substraat met een grote verscheidenheid van polymeersequenties op bekende locaties op een oppervlak ervan. Het substraat wordt blootgesteld aan een met een fluorescerend middel gemerkte receptor welke zich bindt aan een of meer van de polymeersequenties. Het substraat wordt geplaatst in een microscoop-detectieapparaat voor het identificeren van locaties waar een binding plaatsvindt. Het microscoop-detectieapparaat omvat een monochro-matische of polychromatische lichtbron voor het richten van licht op het substraat, middelen voor het detecteren van gefluoresceerd licht van het substraat, en middelen voor het bepalen van een locatie van het gefluoresceerde licht. De middelen voor het detecteren van licht, dat op het substraat is gefluoresceerd, kan in sommige uitvoeringsvormen een foton-telinrichting omvatten. De middelen voor het bepalen van een locatie van het gefluoresceerde licht kan een x/y-translatietabel voor het substraat omvatten. Translatie van het objectglaasje en van de verzameling van gegevens worden geregistreerd en verwerkt door een op geschikte wijze geprogrammeerde digitale computer.
Een verder begrip van de aard en de voordelen van de uitvinding hierin kunnen worden gerealiseerd door verwijzing naar de resterende gedeelten van de beschrijving en de bijgaande tekeningen.
Korte beschrijving van de figuren
Figuur 1 illustreert het aanbrengen van een masker en het bestralen van een substraat bij een eerste locatie. Het substraat wordt in doorsnede getoond; figuur 2 illustreert het substraat na aanbrengen van een monomeer "AM; figuur 3 illustreert de bestraling van het substraat op een tweede locatie; figuur 4 illustreert het substraat na aanbrengen van monomeer "B”; figuur 5 illustreert het bestralen van het "A" monomeer; figuur 6 illustreert het substraat na een tweede aanbrenging van "B"; figuur 7 illustreert een voltooid substraat; figuren 8A en 8B illustreren alternatieve uitvoeringsvormen van een reactorsysteem voor het vormen van een veelvoud van polymeren op een substraat; figuur 9 illustreert een detectieapparaat voor het localiseren van fluorescerende merkmiddelen op het substraat; figuren 10A-10M illustreren de methode zoals deze toegepast wordt op de produktie van de trimeren van de monomeren "A” en "B"; figuren 11A, 11B en 11C zijn fluorescentiesporen voor standaardflu-orescentiekorrels; figuren 12A en 12B zijn fluorescentiekrommen voor NVOC glaasjes (= slides) welke respectievelijk niet en wel aan licht zijn blootgesteld; figuren 13A en 13B illustreren de vorming van een glaasje (slide) met een schaakbordpatroon van YGGFL en GGFL welke blootgesteld zijn aan gemerkt Herz antilichaam; en de figuren l4A en l4B illustreren het in kaart brengen van zestien sequenties welke op twee verschillende objectglaasjes zijn gesynthetiseerd.
Gedetailleerde beschrijving van de de voorkeur verdienende uitvoeringsvormen
INHOUD
I Verklarende woordenlijst II Algemeen III Polymeersynthese IV Details van een uitvoeringsvorm van een reactorsysteem V Details van een uitvoeringsvorm van een detectie-inrichting van fluorescentie VI Bepaling van de relatieve bindingssterkte van receptoren VII Voorbeelden A. Vervaardiging van een "objectglaasje" (= slide) B. Synthese van acht trimeren van "A” en "B" C. Synthese van een dimeer van een aminopropylgroep en een fluorescerende groep D. Demonstratie van signaalvermogen E. Bepaling van het aantal moleculen per oppervlakte-eenheid F. Verwijdering van NVOC en bevestiging van een fluorescerend merkmiddel
G. Toepassing van een masker bij het verwijderen van NVOC
H. Bevestiging van YGGFL en daaropvolgende blootstelling aan Herz antilichaam en geite-anti-muis I. Monomeer-bij-monomeervorming van YGGFL en daaropvolgende blootstelling aan gemerkt antilichaam
J. Monomeer-bij-monomeersynthese van YGGFL en PGGFL
K. Monomeer-bij-monomeersynthese van YGGFL en YPGGFL
L. Synthese van een baan van 16 verschillende aminozuursequenties en bepaling van de relatieve bindingsactiviteit aan Herz H antilichaam VIII Toelichtende alternatieve uitvoeringsvorm IX Conclusie I. Verklarende woordenlijst
De volgende termen zijn bedoeld om de volgende algemene betekenissen te hebben als zij hierin worden gebruikt.
1. Complementair: Verwijst naar de topologische compatibiliteit of het samenpassen van in interactie tredende oppervlakken van een ligand-molecuul en de receptor ervan. Derhalve kunnen de receptor en het ligand ervan worden beschreven als complementair, en verder zijn de eigenschappen van het contactoppervlak complementair ten opzichte van elkaar.
2. Epitoop: Het gedeelte van een antigenmolecuul dat begrensd wordt door het oppervlak van de interactie met de subklasse van receptoren, bekend als antilichamen.
3· Ligand: Een ligand is een molecuul dat door een bijzondere receptor herkend wordt. Voorbeelden van liganden die door deze uitvinding onderzocht kunnen worden omvatten, maar zijn niet beperkt tot, ago-nisten en antagonisten voor celmembraanreceptoren, toxinen en veno-men, virale epitopen, hormonen (b.v. opiaten, steroxden, enz.), hormoonreceptoren, peptiden, enzymen, enzymsubstraten, cofactoren, geneesmiddelen, lectinen, suikers, oligonucleotiden, nucleïnezuren, oligosacchariden, proteïnen en monoclonale antilichamen.
4. Monomeer: Een lid van het stel van kleine moleculen die zich samen kunnen verbinden onder vorming van een polymeer. Het stel monomeren omvat, maar is niet beperkt tot, bijvoorbeeld het stel van gewone L-aminozuren, het stel van D-aminozuren, het stel van synthetische aminozuren, het stel van nucleotiden en het stel van pentosen en hexosen. Zoals hierin gebruikt wijst monomeer naar elk lid van een basisstel voor de synthese van een polymeer. Bijvoorbeeld vormen dimeren van L-aminozuren een basisstel van 400 monomeren voor de synthese van polypeptiden. Verschillende basisstellen van monomeren kunnen gebruikt worden bij opeenvolgende trappen in de synthese van een polymeer.
5· Peptide: Een polymeer waarin de monomeren a-aminozuren zijn en waarin deze samen verbonden zijn door amidebindingen en die alternatief als een polypeptide worden aangeduid. In de context van deze beschrijving zal worden ingezien dat de aminozuren het L-optische isomeer of het D-optische isomeer kunnen zijn. Peptiden zijn meer dan twee aminozuurmonomeren lang, en dikwijls meer dan 20 amino-zuurmonomeren lang. Standaardafkortingen voor aminozuren worden gebruikt (bijvoorbeeld P voor proline). Deze afkortingen zijn opgenomen in Stryer, Biochemstry, derde druk, 1988, welke publikatie hierin voor alle doeleinden door verwijzing wordt opgenomen.
6. Bestraling: Energie die selectief kan worden toegepast waaronder energie met een golflengte tussen ÏO'1^ en 10* meter, bijvoorbeeld met inbegrip van electronenstralen, gammastralen, röntgenstralen, ultraviolette stralen, zichtbaar licht, infrarode stralen, micro-golfstralen en radiogolven. "Bestraling' verwijst naar de toepassing van stralen op een oppervlak.
7· Receptor: Een molecuul dat een affiniteit heeft voor een bepaald ligand. Receptoren kunnen in de natuur voorkomende of door de mens gesynthetiseerde moleculen zijn. Zij kunnen ook worden toegepast in hun niet gewijzigde toestand of als aggregaten met andere species. Receptoren kunnen covalent of niet covalent aan een bindingslid zijn gebonden, hetzij direkt of via een specifieke bindende stof. Voorbeelden van receptoren die door deze uitvinding kunnen worden toegepast omvatten, maar zijn niet beperkt tot, antilichamen, cel-membraanreceptoren, monoklonale antilichamen en antisera welke reactief zijn met specifieke antigene determinanten (zoals op virussen, cellen of andere materialen), geneesmiddelen, polynucleotiden, nucleïnezuren, peptiden, cofactoren, lectinen, suikers, polysaccha-riden, cellen, cellulaire membranen en organellen. Receptoren worden soms in de techniek aangeduid als anti-liganden. Zoals de term receptoren hierin wordt gebruikt, is er geen verschil in betekenis ervan bedoeld. Een "ligandreceptorpaar" wordt gevormd wanneer twee macromoleculen gecombineerd zijn door moleculaire herkenning onder vorming van een complex.
Andere voorbeelden van de receptoren die door deze uitvinding kunnen worden onderzocht, omvatten maar zijn niet beperkt tot: a) Micro-organisme-recentoren: Bepaling van liganden die zich binden aan receptoren, zoals specifieke transportproteïnen of enzymen welke essentieel zijn voor de overleving van micro-orga-nismen, is bruikbaar in een nieuwe klasse van antibiotica. Van bijzonder waarde zijn antibiotica tegen opportunistische fungi, protozoa en die bacteriën welke resistent zijn tegen de tegenwoordig toegepaste antibiotica.
b) Enzymen: Bijvoorbeeld, de bindingsplaats van enzymen zoals de enzymen welke verantwoordelijk zijn voor het splitsen van neurotransmitters; bepaling van liganden welke zich binden aan bepaalde receptoren voor het moduleren van de werking van de enzymen die de verschillende neurotransmitters splitsen, is bruikbaar bij de ontwikkeling van geneesmiddelen welke gebruikt kunnen worden bij de behandeling van kwalen van de neurotrans-missie.
c) Antilichamen: Bijvoorbeeld, de uitvinding kan nuttig zijn bij het onderzoeken van de ligand-bindingsplaats op het antili-chaammolecuul die zich combineert met de epitoop van een van belang zijnd antigen; bepaling van een sequentie die een anti-geen epitoop imiteert kan leiden tot de ontwikkeling van vac- eins waarvan het immunogen gebaseerd is op een of meer van dergelijke sequenties of kan leiden tot de ontwikkeling van verwante diagnostische middelen of verbindingen welke bruikbaar zijn bij therapeutische behandelingen, zoals voor auto-immuun-ziekten (bijvoorbeeld door het blokkeren van het binden van de "zelf" antilichamen).
d) Nucleïnezuren: Sequenties van nucleïnezuren kunnen worden gesynthetiseerd voor het vormen van DNA of RNA bindingssequen-ties.
e) Katalytische polvnentiden: Polymeren, bij voorkeur polypepti-den, die in staat zijn tot het bevorderen van een chemische reactie waarbij de omzetting van een of meer reagentia aan een of meer produkten is betrokken. Dergelijke polypeptiden omvatten in het algemeen een bindingsplaats die specifiek is voor ten minste een reactiemiddel of reactietussenprodukt en een actieve functionaliteit die proximaal is ten opzichte van de bindingsplaats, welke functionaliteit in staat is tot het chemisch modificeren van het gebonden reactiemiddel. Katalytische polypeptiden worden bijvoorbeeld beschreven in de US aanvrage, Serial Nr. 40*1.920, die voor alle doeleinden hierin door verwijzing wordt opgenomen.
f) Hormoonreceptoren: Bijvoorbeeld, de receptoren voor insuline en groeihormoon. Bepaling van de liganden die zich met een hoge affiniteit aan een receptor binden is bruikbaar bij de ontwikkeling van bijvoorbeeld een oraal vervangingsmiddel van de dagelijkse injecties welke diabetici moeten nemen om de symptomen van diabetes te verlichten, en in het andere geval, een vervanging voor het schaarse humane groeihormoon dat slechts verkregen kan worden uit kadavers of door recombinant DNA technologie. Andere voorbeelden zijn de vasoconstrictieve hormmonrecep-toren; bepaling van die liganden welke zich binden aan een receptor kan leiden tot de ontwikkeling van geneesmiddelen voor het regelen van de bloeedruk.
g) Opiaatrecentoren: Bepaling van liganden die zich binden aan opiaatreceptoren in de hersenen is bruikbaar bij de ontwikkeling van minder-addictieve vervangingen voor morfine en verwante geneesmiddelen.
8. Substraat; Een materiaal met een stijf of half stijf oppervlak. In vele uitvoeringsvormen zal ten minste een oppervlak van het sub- straat nagenoeg vlak zijn, ofschoon in sommige uitvoeringsvormen het gewenst kan zijn om fysisch gescheiden synthesegebieden te hebben voor verschillende polymeren met bijvoorbeeld putjes, verhoogde gebieden, geëtste gleuven, of dergelijke. Volgens andere uitvoeringsvormen kunnen kleine korrels op het oppervlak worden aangebracht die bij voltooiing van de synthese losgelaten kunnen worden. 9· Beschermende groep: Een materiaal dat gebonden is aan een monomeer-eenheid en dat ruimtelijk verwijderd kan zijn bij selectieve blootstelling aan een activator, zoals electromagnetische straling. Voorbeelden van beschermende groepen die hierin gebruikt kunnen worden, omvatten nitroveratryloxycarbonyl, nitrobenzyloxycarbonyl, dimethyldimethoxybenzyloxycarbonyl, 5“broom-7-nitroIndolinyl, o-hydroxy-a-methylcinnamoyl en 2-oxymethyleenanthrachinon. Andere voorbeelden van activatoren omvatten ionenstralen, electrische velden, magnetische velden, electronenstralen, röntgenstralen en dergelijke.
10. Van te voren gedefinieerd gebied; Een van te voren gedefinieerd gebied is een gelocaliseerd gebied op een oppervlak dat is, was, of is bestemd om geactiveerd te worden voor de vorming van een polymeer. Het vooraf bepaalde gebied kan elke geschikte vorm hebben, bijvoorbeeld cirkelvormig, rechthoekig, ellipsvormig, wigvormig, enz. Kortheidshalve worden hierin soms "vooraf bepaalde gebieden" eenvoudig aangeduid als "gebieden".
11. Nagenoeg zuiver: Een polymeer wordt als "nagenoeg zuiver" binnen een vooraf bepaald gebied van een substraat beschouwd wanneer het eigenschappen vertoont welke zich onderscheiden van andere vooraf bepaalde gebieden. Kenmerkend zal de zuiverheid worden gemeten in termen van biologische activiteit of functie als resultaat van een uniforme sequentie. Dergelijke kenmerken worden typisch gemeten door binden met een geselecteerd ligand of receptor.
II. Algemeen
De onderhavige uitvinding verschaft methoden en een apparaat voor de bereiding en toepassing van een substraat met een veelvoud van poly-meersequenties in vooraf bepaalde gebieden. De uitvinding wordt hierin primair beschreven ten aanzien van de bereiding van moleculen welke ami-nozuursequenties bevatten, maar gemakkelijk bij de bereiding van andere polymeren toegepast zouden kunnen worden. Dergelijke polymeren omvatten bijvoorbeeld zowel lineaire als cylische polymeren van nucleïnezuren, polysacchariden, fosfolipiden en peptiden met of α-, β-, of tO-aminozuren, heteropolymeren waarin een bekend geneesmiddel covalent gebonden is aan elk van de bovenstaande, polyurethanen, polyesters, polycarbonaten, polyurea, polyamiden, polyethyleeniminen, polyaryleensulfiden, polysil-oxanen, polyamiden, polyacetaten of andere polymeren die bij een herlezen van deze publikatie duidelijk zullen worden. In een de voorkeur verdienende uitvoeringsvorm wordt de hierin beschreven uitvinding gebruikt bij de synthese van peptiden.
Het bereide substraat kan bijvoorbeeld toegepast worden bij het screenen van een groot aantal polymeren als liganden voor het binden met een receptor, ofschoon het duidelijk zal zijn dat de uitvinding gebruikt zou kunnen worden voor de synthese van een receptor voor het binden met een ligand. Het hierin beschreven substraat heeft een grote verscheidenheid van andere toepassingen. Louter bij wijze van voorbeeld kan de onderhavige uitvinding worden toegepast bij het bepalen van peptide- en nucleïnezuursequenties welke zich aan proteïnen binden, het vinden van sequentie specifieke bindende geneesmiddelen, identificeren van epitopen welke door antilichamen worden herkend, en evaluatie van een groot aantal geneesmiddelen voor klinische en diagnostische toepassingen, alsmede combinaties van de bovenstaande.
De uitvinding verschaft bij voorkeur de toepassing van een substraat "S" met een oppervlak. Verbindingsmoleculen "L" worden eventueel verschaft op een oppervlak van het substraat. Het doel van de verbindingsmoleculen is in sommige uitvoeringsvormen het vergemakkelijken van de receptorherkenning van de gesynthetiseerde polymeren.
Eventueel kunnen de verbindingsmoleculen voor opslagdoeleinden chemisch beschermd zijn. Een voor chemische opslag beschermende groep zoals tert-BOC (tert-butoxycarbonyl) kan in sommige uitvoeringsvormen worden toegepast. Dergelijke chemische beschermende groepen zouden chemisch verwijderd kunnen worden door bijvoorbeeld blootstelling aan een zure oplossing en zouden dienen voor het beschermen van het oppervlak tijdens opslag en verwijderd kunnen worden alvorens de polymeerbereiding plaats vindt.
Op het substraat of een distaai einde van de verbindingsmoleculen wordt een functionele groep met een beschermende groep P0 verschaft. De beschermende groep P0 kan worden verwijderd bij blootstelling aan bestraling, electrische velden, electrische stromen of andere activatoren voor het blootstellen van de functionele groep.
In een de voorkeur verdienende uitvoeringsvorm is de straling ul- traviolet (UV), infrarood (IR) of zichtbaar licht. Zoals hieronder vollediger zal worden beschreven kan de beschermende groep alternatief een electrochemisch-sensitieve groep zijn die verwijderd kan worden bij aanwezigheid van een electrisch veld. In nog verdere alternatieve uitvoeringsvormen kunnen ionenstralen, electronenstralen of dergelijke voor deprotectie worden gebruikt.
In sommige gevallen zijn de blootgestelde gebieden en derhalve het gebied waarop elke afzonderlijke polymeersequentie gesynthetiseerd wordt, kleiner dan ongeveer 1 cm2 of minder dan 1 mm2. In de voorkeur verdienende uitvoeringsvormen is het blootgestelde gebied minder dan ongeveer 10.000 urn2 of meer bij voorkeur minder dan 100 μηι2 en kan in sommige uitvoeringsvormen de bindingsplaats voor zo weinig als een enkel molecuul omvatten. Binnen deze gebieden wordt elk polymeer bij voorkeur gesynthetiseerd in nagenoeg zuivere vorm.
Gelijktijdig met of na blootstelling van een bekend gebied van het substraat aan licht, wordt het oppervlak in contact gebracht met een eerste monomeereenheid Mi die reageert met de functionele groep die blootgesteld is door de deprotectiegroep. Het eerste monomeer omvat een beschermende groep Ρχ. Px kan al of niet hetzelfde zijn als P0.
Dienovereenkomstig kunnen na een eerste cyclus bekende eerste gebieden van het oppervlak de sequentie omvatten:
terwijl resterende gebieden van het oppervlak de sequentie omvatten:
Daarna worden tweede gebieden van het oppervlak (die het eerste gebied kunnen omvatten) aan licht blootgesteld en in contact gebracht met een tweede monomeer M2 (dat al of niet hetzelfde als Mx kan zijn) dat een beschermende groep P2 kan bevatten. P2 kan al of niet hetzelfde zijn als P0 en Pi· Na deze tweede cyclus kunnen verschillende gebieden van het substraat een of meer van de volgende sequenties omvatten:
De bovenstaande werkwijze wordt herhaald totdat het substraat gewenste polymeren van gewenste lengten omvat. Door het regelen van de locaties van het substraat dat blootgesteld is aan licht en de reagentie welke blootgesteld zijn aan het substraat na blootstelling, zal de locatie van elke sequentie bekend zijn.
Daarna worden de beschermende groepen verwijderd uit enkele of alle van het substraat en de sequenties worden eventueel voorzien van een afsluitende eindstandige groep onder toepassing van een beschermende eenheid C. De werkwijze resulteert in een substraat met een oppervlak met een groot aantal polymeren met de volgende algemene formule:
waarin de vierkante haken eventuele groepen aangeven, en Mj...Mx elke sequentie van monomeren aangeeft. Het aantal monomeren zou een groot aantal waarden kunnen dekken, maar in een de voorkeur verdienende uitvoeringsvorm zullen zij liggen in het traject van 2 tot 100.
In sommige uitvoeringsvormen bevatten een veelvoud van plaatsen op de substraatpolymeren een gewone monomeersubsequentie. Het kan bijvoorbeeld gewenst zijn om een sequentie S-M1-M2-M3 bij eerste locaties te synthetiseren en een sequentie S-M^-M2-M3 bij tweede locaties. De werkwijze begint bij bestraling van de eerste locaties, gevolgd door het in contact brengen met M-l-P, hetgeen resulteert in de sequentie S-Mj-P bij de eerste locatie. De tweede locaties zouden dan bestraald worden en in contact worden gebracht met Μ/,-Ρ, resulterende in de sequentie S-Μ/,-Ρ bij de tweede locaties. Daarna zouden zowel de eerste als de tweede locaties bestraald worden en in contact worden gebracht met het dimeer M2-M3, hetgeen resulteert in de sequentie S-M1-M2-M3 bij de eerste locaties en S-Mi,-M2-M3 bij de tweede locaties. Natuurlijk zouden gewone subsequenties van elke lengte gebruikt kunnen worden met inbegrip van die in een traject van 2 of meer monomeren, 2 tot 100 monomeren, 2 tot 20 monomeren, en een meest de voorkeur verdienend traject van 2 tot 3 monomeren.
Volgens andere uitvoeringsvormen wordt een stel maskers gebruikt voor de eerste monomeerlaag en daarna licht met gevarieerde golflengten toegepast voor selectieve deprotectie. Bijvoorbeeld worden in het hierboven besproken proces de eerste gebieden eerst blootgesteld door een masker en omgezet met een eerste monomeer met een eerste beschermende groep Plf die verwijderbaar is bij blootstelling aan een eerste lichtgolflengte (bijvoorbeeld IR). Tweede gebieden worden gemaskeerd en omgezet met een een tweede monomeer met een tweede beschermende groep P2 die verwijderbaar is bij blootstelling aan een tweede golflengte van licht (bijvoorbeeld UV). Daarna worden maskers overbodig bij de synthese omdat het totale substraat alternatief blootgesteld kan worden aan de eerste en aan de tweede golflengte van licht in de deprotectiecyclus.
De polymeren welke bereid zijn op een substraat volgens de bovenstaande methoden, zullen een verscheidenheid van toepassingen hebben, waaronder bijvoorbeeld het screenen op biologische activiteit. In dergelijke screeningsactiviteiten wordt het substraat, dat de sequenties bevat, blootgesteld aan een niet gemerkte of gemerkte receptor, zoals een antilichaam, receptor op een cel, fosfolipideblaasje, of elk ander van een verscheidenheid van andere receptoren. In een de voorkeur verdienende uitvoeringsvorm worden de polymeren blootgesteld aan een eerste, niet gemerkte, van belang zijnde receptor en daarna blootgesteld aan een gemerkte receptor-specifiek herkenningselement, dat bijvoorbeeld een antilichaam is. Deze werkwijze zal in de detectietrap een signaalversterking verschaffen.
De receptormoleculen kunnen zich binden met een of meer polymeren op het substraat. De aanwezigheid van de gemerkte receptor en derhalve de aanwezigheid van een sequentie, die zich aan de receptor bindt, wordt volgens een de voorkeur verdienende uitvoeringsvorm gedetecteerd door toepassing van autoradiografie, detectie van fluorescentie met een la-dingsgekoppelde inrichting, fluorescentiemicroscopie, of dergelijke. De sequentie van het polymeer bij de locaties waar de receptorbinding gedetecteerd wordt, kan worden toegepast voor het bepalen van alle of van een gedeelte van een sequentie die complementair is ten opzichte van de receptor.
Toepassing van de onderhavige uitvinding wordt primair toegelicht onder verwijzing naar het screenen op biologische activiteit. De uitvinding zal echter vele andere toepassingen vinden. Bijvoorbeeld kan de uitvinding worden gebruikt bij opslag van informatie (bijvoorbeeld op optische schijven), produktie van molecualaire electronische apparaten, produktie van stationaire fasen in scheidingswetenschappen, produktie van kleurstoffen en helderheid verlenende middelen, fotografie en bij het immobiliseren van cellen, maar proteïnen, lectinen, nucleïnezuren, poly-sacchariden en dergelijke, in patronen op een oppervlak via moleculaire herkenning van specifieke polymere sequenties. Door het synthetiseren van dezelfde verbinding in naast gelegen, progressief verschillende concentraties, zal een gradiënt worden verkregen voor het regelen van de chemo- taxis of voor het ontwikkelen van diagnostische dompelstaven die bijvoorbeeld een antilichaam kunnen titreren tegen een stijgende hoeveelheid antigen. Door het synthetiseren van verscheidene katalysatormoleculen in dichte nabijheid van elkaar, kunnen meer efficiënte, uit meerdere trappen bestaande conversies worden uitgevoerd door "coördinaat-immobilisatie". Coördinaat-immobilisatie kan ook worden toegepast voor electronen-over-drachtsystemen, alsmede voor het verschaffen van zowel structurele integriteit als andere gewenste eigenschappen voor materialen, zoals smeren, bevochtiging, enz.
Volgens alternatieve uitvoeringsvormen kunnen de moleculaire bio-distributie- of farmacocinetische eigenschappen worden onderzocht. Bijvoorbeeld teneinde resistentie ten opzichte van intestinale of serumpro-teasen vast te stellen, kunnen polymeren van een eindstandig beschermingsmiddel met een fluorescerend middel worden voorzien en blootgesteld worden aan van belang zijnde biologische vloeistoffen.
III. Polvmeersvnthese
Figuur 1 licht een uitvoeringsvorm toe van de uitvinding, welke hierin is vermeld, waarbij een substraat 2 in doorsnede wordt getoond. In wezen kan elk denkbaar substraat in deze uitvinding worden toegepast. Het substraat kan biologisch, niet-biologisch, organisch, anorganisch, of een combinatie van een van deze zijn, bestaande als deeltjes, strengen, pre-cipitaten, gelen, vellen, buizen, bolletjes, houders, capillairen, kussens, plakjes, films, platen, glaasjes, enz. Het substraat kan elke geschikte vorm hebben, zoals een schijf, vierkant, bolletje, rond voorwerp, enz. Het substraat is bij voorkeur vlak maar kan een grote verscheidenheid van alternatieve oppervlakteconfiguraties bezitten. Bijvoorbeeld kan het substraat verhoogde of verzonken gebieden bevatten waarop de synthese plaats vindt. Het substraat en zijn oppervlak vormen bij voorkeur een stijve drager waarop de hierin beschreven reacties worden uitgevoerd. Het substraat en zijn oppervlak worden ook gekozen voor het verschaffen van geschikte lichtabsorberende eigenschappen. Het substraat kan bijvoorbeeld een gepolymeriseerde Langmuir Blodgett film zijn, gefunctionaliseerd glas, Si, Ge, GaAs, GaP, Si02, SiN*,, gemodificeerd silicium, of elk ander van een groot aantal gelen of polymeren zoals (poly)tetrafluoretheen, (poly)vinylideendifluoride, polystyreen, polycarbonaat, of combinaties ervan. Andere substraatmaterialen zullen gemakkelijk aan de vakman duidelijk zijn bij herlezing van deze publikatie. In een de voorkeur verdienende uitvoeringsvorm is het substraat vlak glas of uit een enkelvoudig kristal bestaand silicium met oppervlaktereliëfvariaties van minder dan 10 X.
Volgens sommige uitvoeringsvormen wordt het oppervlak van het substraat geërtst onder toepassing van algemeen bekende technieken ter verschaffing van gewenste oppervlakte kenmerken. Bijvoorbeeld kunnen voor de vorming van sleuven, v-groeven, mesastructuren, of dergelijke, de synthe-segebieden dichter binnen het brandpunt van opvallend licht worden aangebracht, hetgeen verschaft wordt door reflectieve "spiegel” structuren voor het maximaliseren van lichtinval uit fluorescerende bronnen, of dergelijke.
Oppervlakken op het vaste substraat zullen gebruikelijk, ofschoon niet altijd, samengesteld zijn uit hetzelfde materiaal als het substraat. Derhalve kan het oppervlak samengesteld zijn uit elk van een groot aantal materialen, bijvoorbeeld polymeren, kunststoffen, harsen, polysacchari-den, siliciumoxide of op siliciumoxide gebaseerde materialen, koolstof, metalen, anorganische glazen, membranen, of elk van de hierboven vermelde substraatmaterialen. In sommige uitvoeringsvormen kan het oppervlak de toepassing verschaffen van gekooide bindingsorganen die stevig gebonden zijn aan het oppervlak van het substraat in overeenstemming met de leer van de eveneens hangende verwante aanvrage Serial Nr. 404.920, die hierin door verwijzing is opgenomen. Bij voorkeur zal het oppervlak reactieve groepen bevatten die carboxyl, amino, hydroxyl of dergelijke kunnen zijn. Het meest bij voorkeur zal het oppervlak optisch transparant zijn en zal aan het oppervlak Si-OH functionaliteiten bezitten, zoals die welke op uit siliciumoxide bestaande oppervlakken worden aangetroffen.
Het oppervlak 4 van het substraat is bij voorkeur voorzien van een laag van verbindingsmoleculen 6, ofschoon het duidelijk zal zijn dat de verbindingsmoleculen geen vereiste elementen van de uitvinding zijn. De verbindingsmoleculen zijn bij voorkeur van een voldoende lengte om het mogelijk te maken dat polymeren in een voltooid substraat een vrije interactie kunnen aangaan met moleculen welke aan het substraat zijn blootgesteld. De verbindingsmoleculen moeten 6-50 atomen lang zijn ter verschaffing van een voldoende blootstelling. De verbindingsmoleculen kunnen bijvoorbeeld arylacetyleen, ethyleenglycololigomeren met 2-10 monomeer-eenheden, diaminen, dizuren, aminozuren, of combinaties ervan zijn. Andere verbindingsmoleculen kunnen in het licht van deze publikatie worden toegepast.
Volgens alternatieve uitvoeringsvormen worden verbindingsmoleculen geselecteerd op basis van hun hydrofiele/hydrofobe eigenschappen ter verbetering van de presentatie van gesynthetiseerde polymeren aan bepaalde receptoren. Bijvoorbeeld zullen in het geval van een hydrofiele receptor, hydrofiele verbindingsmoleculen de voorkeur verdienen om de receptor in staat te stellen om het gesynthetiseerde polymeer dichter te benaderen.
Volgens een andere alternatieve uitvoeringsvorm worden verbindingsmoleculen ook verschaft met een op een tussengelegen positie door licht splitsbare groep. De door licht splitsbare groep is bij voorkeur splitsbaar bij een golflengte die verschillend is van de beschermende groep. Dit maakt de verwijdering mogelijk van diverse polymeren na de voltooiing van de synthese door blootstelling aan de verschillende golflengten van licht.
De verbindingsmoleculen kunnen worden gebonden aan het substraat via koolstof-koostofbindingen bij toepassing van bijvoorbeeld (poly)tri-fluorchlooretheenoppervlakken, of bij voorkeur door siloxaanbindingen (bijvoorbeeld bij toepassing van glas- of siliciumoxide-oppervlakken). Siloxaanbindingen met het oppervlak van het substraat kunnen in een uitvoeringsvorm worden gevormd via reacties van verbindingsmoleculen welke trichloorsilylgroepen dragen. De verbindingsmoleculen kunnen eventueel gebonden zijn in een geordende baan, dat wil zeggen als delen van de hoofdgroepen in een gepolymeriseerde Langmuir Blodgett film. In alternatieve uitvoeringsvormen worden de verbindingsmoleculen geadsorbeerd aan het oppervlak van het substraat. '
De hierin toegepaste verbindingsmoleculen en monomeren worden verschaft met een functionele groep waaraan een beschermende groep is gebonden. De beschermende groep is bij voorkeur aan het distale of terminale einde van het verbindingsmolecuul tegenover het substraat. De beschermende groep kan of een negatieve beschermende groep zijn (dat wil zeggen de beschermende groep maakt bij verwijdering de verbindingsmoleculen minder reactief met een monomeer) of een positieve beschermende groep (dat wil zeggen, de beschermende groep maakt bij blootstelling de verbindingsmoleculen meer reactief met een monomeer). In het geval van negatieve beschermende groepen zal een additionele trap van reactivering vereist zijn. In sommige gevallen zal dit door verhitting worden uitgevoerd.
De beschermende groep op de verbindingsmoleculen kan gekozen worden uit een groot aantal positieve, voor licht reactieve groepen, bij voorkeur met inbegrip van nitro-aromatische verbindingen, zoals o-nitroben-zylderivaten of benzylsulfonyl. In een de voorkeur verdienende uitvoeringsvorm wordt 6-nitroveratryloxycarbonyl (NVOC), 2-nitrobenzyloxycarbo- nyl (NBOC) of α,α-dimethyl-dimethoxybenzyloxycarbonyl (DDZ) gebruikt. In een uitvoeringsvorm wordt een nitro-aromatische verbinding, bevattende een benzylwaterstof ortho ten opzichte van de nitrogroep, toegepast, dat wil zeggen een verbinding met de formule:
waarin Rx is alkoxy, alkyl, halogeen, aryl, alkenyl of waterstof; R2 is alkoxy, alkyl, halogeen, aryl, nitro of waterstof; R3 is alkoxy, alkyl, halogeen, nitro, aryl of waterstof, R4 is alkoxy, alkyl, waterstof, aryl, halogeen of nitro; en R5 is alkyl, alkynyl, cyano, alkoxy, waterstof, halogeen, aryl of alkenyl. Andere materialen die gebruikt kunnen worden omvatten o-hydroxy-a-methylcinnamoylderivaten. Door licht verwijderbare beschermende groepen worden bijvoorbeeld beschreven in Patchornik, J. Am. Chem. Soc. (1970) 22: 6333 en Amit et al., J. Org. Chem. (1974) 32: 192, welke beide hierin door verwijzing worden opgenomen.
In een alternatieve uitvoeringsvorm wordt de positieve reactieve groep geactiveerd voor reactie met reagentia in oplossing. Bijvoorbeeld ondergaaat een 5“broom-7-nitro-indolinegroep, wanneer gebonden aan een carbonyl, een reactie bij blootstelling aan licht bij 420 nm.
In een tweede alternatieve uitvoeringsvorm wordt de reactieve groep op het verbindingsmolecuul gekozen uit een grote verscheidenheid van negatieve, door licht reactieve groepen, waaronder een cinnamaatgroep.
De reactieve groep wordt op alternatieve wijze geactiveerd of gede-activeerd door toepassing van electronenstraallithografie, röntgenstraal-lithografie of door elke andere straling. Geschikte reactieve groepen voor electronenstraallithografie omvatten sulfonyl. Andere methoden kunnen worden gebruikt, waaronder bijvoorbeeld blootstelling aan een stroombron. Andere reactieve groepen en methoden voor het activeren kunnen in het licht van deze beschrijving worden toegepast.
Zoals getoond in figuur 1 worden de verbindingsmoleculen bij voorkeur blootgesteld aan bijvoorbeeld licht door een geschikt masker 8 onder toepassing van fotolithografische technieken van het type dat bekend is in de halfgeleiderindustrie en bijvoorbeeld beschreven is in, Sze, VLSI Technology, McGraw-Hill (1983). en Mead et al., Introduction to VLSI
Systems, Addison-Wesley (19δ0), die hierin voor alle doeleinden door verwijzing worden opgenomen. Het licht kan worden gericht op hetzij het oppervlak dat de beschermende groepen bevat of op de achterkant van het substraat, zolang als het substraat transparant is voor de golflengte van licht dat voor de verwijdering van de beschermende groepen nodig is. In de in figuur 1 getoonde uitvoeringsvorm wordt licht gericht op het oppervlak van het substraat dat de beschermende groepen bevat. Figuur 1 illustreert de toepassing van dergelijke maskerende technieken als zij worden toegepast op een positieve reactieve groep teneinde verbindingsmoleculen te activeren en functionele groepen in de gebieden 10a en 10b bloot te stellen.
Het masker 8 is in een uitvoeringsvorm een transparant dragermate-riaal dat selectief bekleed is met een laag troebel materiaal. Delen van het troebele materiaal worden verwijderd, waarbij troebel materiaal in het exacte gewenste patroon op het substraatoppervlak achterblijft. Het masker wordt in dichte nabijheid gebracht met, afgebeeld op, of direct in contact gebracht met het substraatoppervlak zoals in figuur 1 is getoond. "Openingen" in het masker corresponderen met locaties op het substraat waar het gewenst is de door licht verwijderbare beschermende groepen van het substraat te verwijderen. Het brengen in een rechte lijn kan worden uitgevoerd onder toepassing van conventionele technieken voor het brengen in een rechte lijn, waarin lijnmerken (niet weergegeven) gebruikt worden voor het nauwkeurig bedekken van op eenvolgende maskers het eerdere patroon aanbrengende stappen, of meer ingewikkelde technieken kunnen worden toegepast. Bijvoorbeeld kunnen interferometrische technieken, zoals de techniek beschreven in Flanders et al., "A New Interferometric Alignment Technique," App. Phys. Lett. (1977) 31· 426-428, die hierin door verwijzing wordt opgenomen, worden gebruikt.
Ter verhoging van het contrast van licht dat op het substraat wordt aangebracht, is het volgens enkele uitvoeringsvormen gewenst contrast versterkende materialen tussen het masker en het substraat aan te brengen. Deze contrastversterkingslaag kan een molecuul omvatten dat door licht ontleed wordt, zoals chinondiazide of een materiaal dat op tran-siënte wijze gebleekt wordt bij de van belang zijnde golflengte. Transient bleken van materialen zal een grotere penetratie mogelijk maken wanneer licht wordt toegepast waardoor het contrast wordt verhoogd. Con-trastversterking kan ook worden geleverd door middel van een uit beklede vezels bestaande optische bundel.
Haf. Iran van ppn pnmrpn^i'nnple β·1 ρνοτ'Ιτρηη * aan laser', een la- serdiode of dergelijke afkomstig zijn. Indien niet gerichte lichtbronnen worden gebruikt kan het gewenst zijn een uit dikke of uit vele lagen bestaand masker te verschaffen ter verhindering van het spreiden van het licht op het substraat. Verder kan het in sommige uitvoeringsvormen gewenst zijn groepen te gebruiken die gevoelig zijn voor verschillende golflengten voor het regelen van de synthese. Bijvoorbeeld door toepassing van groepen die gevoelig zijn voor verschillende golflengten, is het mogelijk vertakkingsplaatsen bij de synthese van een polymeer te selecteren of bepaalde maskerende trappen te elimineren. Verscheidene reactieve groepen, tezamen met hun corresponderende golflengten voor deprotectie, worden in tabel 1 vermeld.
Hoewel de uitvinding primair hierin wordt toegelicht bij wijze van de toepassing van een masker voor het illumineren van selecte gebieden van het substraat, kunnen ook andere technieken worden toegepast. Bijvoorbeeld kan het substraat getranslateerd worden onder een gemoduleerde laser of diodelichtbron. Dergelijke technieken worden bijvoorbeeld besproken in US-A-4.7i9.6i5 (Feyrer et al.), dat hierin door verwijzing wordt opgenomen. Volgens alternatieve uitvoeringsvormen wordt een laser-galvanometrische aftastinrichting toegepast. In andere uitvoeringsvormen kan de synthese plaats vinden op of in contact met een conventioneel vloeibaar kristal (hierin aangeduid als een "lichtklep") of een uit vezels bestaande optische lichtbron. Door het op geschikte wijze moduleren van vloeibare kristallen, kan licht selectief worden geregeld teneinde het mogelijk te maken dat licht met geselecteerde gebieden van het substraat in contact komt. Ook kan de synthese plaats vinden aan het einde van een reeks optische vezels waarop licht selectief wordt toegepast.
Andere middelen voor het regelen van de locatie van blootstelling aan licht zullen aan de vakman duidelijk zijn.
Het substraat kan worden bestraald, hetzij in contact of niet in contact met een oplossing (niet weergegeven) en wordt bij voorkeur bestraald in contact met een oplossing. De oplossing bevat reagentia voor het verhinderen dat volgens enkele uitvoeringsvormen bijprodukten, gevormd door bestraling, interfereren met de synthese van het polymeer. Dergelijke bijprodukten kunnen bijvoorbeeld omvatten kooldioxide, nitro-socarbonylverbindingen, styreenderivaten, indoolderivaten en produkten van hun fotochemische reacties. De oplossing kan ook reagentia bevatten welke gebruikt worden om te passen bij de brekingsindex van het substraat. Reagentia, welke aan de oplossing worden toegevoegd, kunnen verder bijvoorbeeld omvatten zure of basische buffers, thiolen, gesubstitueerde hydrazinen en hydroxylaminen, reducerende middelen (bijvoorbeeld NADH) of reagentia waarvan bekend is dat ze met een bepaalde functionele groep reageren (bijvoorbeeld arylnitroso + glyoxylzuur -* arylformhydroxa-maat + C02).
Of gelijktijdig met of na de bestralingstrap worden de verbindings-moleculen gewassen op of andere wijze in contact gebracht met een eerste monomeer, toegelicht door "A" in de gebieden 12a en 12b in figuur 2. Het eerste monomeer reageert met de geactiveerde functionele groepen van de verbindingsmoleculen die blootgesteld zijn aan licht. Het eerste monomeer, dat bij voorkeur een aminozuur is, wordt ook geleverd met een licht beschermende groep. De licht beschermende groep op het monomeer kan dezelfde alsof verschillend van de beschermende groep zijn die in de verbindingsmoleculen wordt toegepast, en kan worden gekozen uit elk van de hierboven beschreven beschermende groepen. In een uitvoeringsvorm worden de beschermende groepen voor het A monomeer gekozen uit de groep NBOC en NVOC.
Zoals getoond, in figuur 3» wordt de werkwijze van het bestralen daarna herhaald onder toepassing van een masker dat opnieuw zodanig wordt geplaatst dat de verbindende beschermende groepen worden verwijderd en functionele groepen worden blootgesteld in gebieden 14a en 14b, welke toegelicht zijn als zijnde gebieden die beschermd waren in de voorafgaande maskeringstrap. Als een alternatief voor het opnieuw positioneren van het eerste masker, zeil in vele andere uitvoeringsvormen een tweede masker worden gebruikt. In andere alternatieve uitvoeringsvormen kunnen enkele trappen worden verschaft voor het belichten van een gemeenschappelijk gebied in opeenvolgende trappen. Zoals getoond in figuur 3 kan het ge wenst zijn een scheiding te verschaffen tussen bestraalde gebieden. Bijvoorbeeld kan een scheiding van ongeveer 1-5 μΐη geschikt zijn om toleranties in het in rechte lijn aanbrengen op te vangen.
Zoals getoond in figuur 4 wordt het substraat vervolgens blootgesteld aan een tweede beschermd monomeer "B", waardoor B gebieden l6a en 16b worden gevormd. Daarna wordt het substraat opnieuw gemaskeerd ter verwijdering van de beschermende groepen en blootstelling aan reactieve groepen op A gebied 12a en B gebied 16b. Het substraat wordt weer blootgesteld aan monomeer B, hetgeen resulteert in de vorming van de structuur die in figuur 6 is getoond. De dimeren B-A en B-B zijn op het substraat gevormd.
Een daaropvolgende reeks van maskerende en contact leverende trappen, soortgelijk aan die welke hierboven beschreven zijn met A (niet weergegeven) verschaft de in figuur 7 getoonde structuur. De werkwijze verschaft alle mogelijke dimeren van B en A, dat wil zeggen B-A, A-B, A-A en B-B.
Het substraat, het gebied van de synthese, en het gebied voor de synthese van elk afzonderlijk polymeer zou elke grootte of vorm kunnen hebben. Bijvoorbeeld kunnen vierkanten, ellipsoïden, rechthoeken, driehoeken, cirkelvormige of delen ervan, tezamen met onregelmatige geometrische vormen, worden gebruikt. Duplicaatsynthesegebieden kunnen ook aangebracht worden op een enkelvoudig substraat voor doeleinden van overbodigheid.
In een uitvoeringsvorm zullen de gebieden 12 en 16 op het substraat een specifiek oppervlak hebben gelegen tussen ongeveer 1 cm2 en 1CT10 cm2. In sommige uitvoeringsvormen hebben de gebieden 12 en 16 specifieke oppervlakken van minder dan ongeveer 10-1 cm2, 10-2 cm2, 10"3 cm2, 10"* cm2, 10“5 cm2, 10"6 cm2, 10-7 cm2, 10"8 cm2 of 10"10 cm2. In een de voorkeur verdienende uitvoeringsvorm zijn de gebieden 12 en 16 gelegen tussen ongeveer 10 x 10 pm en 500 x 500 pm.
In sommige uitvoeringsvormen ondersteunt een enkelvoudig substraat meer dan ongeveer tien verschillende monomeersequenties en bij voorkeur meer dan ongeveer 100 verschillende monomeersequenties, ofschoon in sommige uitvoeringsvormen meer dan ongeveer 10'3, 10*, 105, 106, 107, of 108 verschillende sequenties op een substraat worden verschaft. Binnen een gebied van het substraat waarin een monomeersequentie wordt gesynthetiseerd, verdient het natuurlijk de voorkeur dat de monomeersequentie nagenoeg zuiver is. In sommige uitvoeringsvormen bevatten de gebieden van het substraat polymeersequenties die ten minste ongeveer 1%, 5%», 10%, 15%, 20%t 25%, 30%, 35%, *iO%, *5%, 50%, 60%, 10%, Βθ%, 9O%, 95%, 96%, 97%, 96% of 99% zuiver zijn.
Volgens sommige uitvoeringsvormen worden verscheidene sequentieer-inrichtingen met opzet geleverd binnen een enkelvoudig gebied teneinde een aanvankelijke screening te verschaffen op biologische activiteit, waarna materialen binnen gebieden welke significante binding vertonen, verder worden geëvalueerd.
IV. Bijzonderheden van een uitvoeringsvorm van een reactorsvsteem
Figuur 8A licht schematisch een de voorkeur verdienende uitvoeringsvorm van een reactorsysteem 100 toe voor het synthetiseren van polymeren op het substraat, bij toepassing van een aspect van de uitvinding. Het reactorsysteem omvat een lichaam 102 met een holte 104 op een oppervlak ervan. In de voorkeur verdienende uitvoeringsvorm is de holte 104 tussen ongeveer 50 en 1.000 μΐη diep waarbij een diepte van ongeveer 500 μΐη de voorkeur verdient.
De bodem van de holte is bij voorkeur voorzien van een baan gleuven 106 die zich zowel in het vlak van de figuur als parallel aan het vlak van de figuur uitstrekken. De gleuven zijn bij voorkeur ongeveer 50 tot 200 pm diep en op een afstand van 2 tot 3 mm van elkaar aangebracht. Het doel van de gleuven is het doen verkrijgen van een turbulente stroom voor een betere menging. Het bodemoppervlak van de holte is bij voorkeur licht absorberend teneinde terugkaatsing van invallend licht te voorkomen.
Een substraat 112 wordt aangebracht boven de holte 104. Het substraat wordt langs het bodemoppervlak 114 ervan voorzien van een door licht verwijderbare beschermende groep, zoals NV0C met of zonder een tussenkomend verbindingsmolecuul. Het substraat is bij voorkeur transparant voor een breed spectrum van licht, maar in sommige uitvoeringsvormen is transparantie slechts bij een golflengte waarbij de beschermende groep verwijderd kan worden (zoals UV in het geval van NVOC). Het substraat is in sommige uitvoeringsvormen een conventioneel objectglaasje van een microscoop of een bedekkingsslip. Het substraat is bij voorkeur zo dun mogelijk terwijl het nog een adequate fysische ondersteuning verschaft. Bij voorkeur is het substraat minder dan ongeveer 1 mm dik, meer bij voorkeur minder dan 0,5 mm dik, meer bij voorkeur minder dan 0,1 mm dik, en het meest bij voorkeur minder dan 0,05 mm dik. Volgens alternatieve de voorkeur verdienende uitvoeringsvormen is het substraat kwarts of silicium.
Het substraat en het lichaam dienen voor het afdichten van de holte met uitzondering van een inlaatopening 108 en een uitlaatopening 110. Het lichaam en het substraat kunnen in sommige gevallen in elkaar grijpen voor het af dichten met een of meer afdichtingsringen. Volgens een de voorkeur verdienende uitvoeringsvorm wordt het lichaam voorzien van twee concentrische afdichtingsringen en de tussengelegen ruimte wordt onder vacuum gehouden teneinde een nauwkeurig passen van het substraat aan de afdichtingsringen te verzekeren.
Vloeistof wordt door de inlaatopening in de holte gepompt door toepassing van een pomp 116, die bijvoorbeeld van een model nr. B-120-S kan zijn, vervaardigd door Eldex Laboratories. Geselecteerde vloeistoffen worden door de pomp in de holte gecirculeerd, door de holte, en uit de uitlaatopening voor recirculatie of afvoer. De reactor kan worden onderworpen aan ultrasone straling en/of worden verhit om in sommige gevallen bij te dragen tot de beweging.
Boven het substraat 112 wordt een lens 120 aangebracht die bijvoorbeeld een 2" 100 mm focale lengte gesmolten siliciumoxidelens kan zijn. Ter wille van een compact systeem kan een reflectieve spiegel 122 worden verschaft voor het richten van licht uit een lichtbron 124 op het substraat. De lichtbron 124 kan bijvoorbeeld een Xe (Hg) lichtbron zijn, vervaardigd door Oriel en van het model nr. 66024. Een tweede lens 126 kan zijn aangebracht met het doel van het projecteren van een maskeer-beeld op het substraat in combinatie met lens 112. Deze vorm van lithografie wordt hierin als projectie-druk aangeduid. Zoals uit deze beschrijving duidelijk zal zijn kan proximiteit-druk en dergelijke ook volgens sommige uitvoeringsvormen worden toegepast.
Licht uit de lichtbron kan slechts geselecteerde locaties op het substraat bereiken als gevolg van het masker 128. Het masker 128 kan bijvoorbeeld een objectglaasje zijn met chroom daarop geëtst. Het masker 128 wordt in een uitvoeringsvorm verschaft met een rooster van transparante locaties en troebele locaties. Dergelijke maskers kunnen bijvoorbeeld worden vervaardigd door Photo Sciences, Ine. Licht gaat vrij door de transparante gebieden van het masker, maar wordt gereflecteerd door of geabsorbeerd door andere gebieden. Derhalve worden slechts geselecteerde gebieden van het substraat aan licht blootgesteld.
Zoals hierboven is besproken kunnen lichtkleppen (LCD's) gebruikt worden als een alternatief voor conventionele maskers voor het selectief blootstellen van gebieden van het substraat. Uit vezels bestaande optische voorplaten, zoals die welke verkrijgbaar zijn van Schott Glass, Inc, kunnen ook worden gebruikt met het doel van contrastverhoging van het masker of als het enige middel voor het beperken van het gebied waarop licht wordt toegepast. Dergelijke voorplaten worden direct boven of op het substraat in de reactor, getoond in figuur 8A, aangebracht. In nog verdere uitvoeringsvormen kunnen vliegooglenzen, tapstoelopende, uit vezels bestaande optische voorplaten, of dergelijke, voor contrastverho-ging worden toegepast.
Ter verschaffing van illuminatie van de gebieden kleiner dan een golflengte van licht, kunnen meer uitgewerkte technieken worden toegepast. Bijvoorbeeld wordt volgens een de voorkeur verdienende uitvoeringsvorm licht op het substraat gericht door toepassing van moleculaire mi-crokristallen op de punt van bijvoorbeeld micropipetten. Dergelijke inrichtingen worden beschreven in Lieberman et al., "A Light Source Smaller Than the Optical Wavelength”, Science (1990) 247: 59“6l, welke publikatie hierin voor alle doeleinden door verwijzing wordt opgenomen.
Bij het in bedrijf zijn wordt het substraat op de holte geplaatst en daarmee afdichtend verbonden. Alle bewerkingen in het proces van het bereiden van het substraat worden uitgevoerd in een kamer die primair of geheel verlicht is door licht met een golflengte buiten het lichttraject waarbij de beschermende groep wordt verwijderd. Bijvoorbeeld moet in het geval van NVOC de kamer worden verlicht met een conventionele donkere kamerverlichting die weinig of geen UV licht levert. Alle bewerkingen worden bij voorkeur bij kamertemperatuur uitgevoerd.
Een eerste deprotecterende vloeistof (zonder een monomeer) wordt door de holte gecirculeerd. De oplossing is bij voorkeur 5 inmol zwavelzuur in dioxaanoplossing welke dient om blootgestelde aminogroepen in geprotoneerde toestand te houden en hm reactiviteit met bijprodukten van de fotolyse te verminderen. Absorptieve materialen, zoals N,N-diethylami-no 2,4-dinitrobenzeen, kan bijvoorbeeld in de deprotectievloeistof worden opgenomen welke dient voor het absorberen van licht en het verhinderen van reflectie en ongewenste fotolyse.
Het objectglaasje wordt daarna geplaatst in de lichtbaan uit een masker, zodanig dat eerst locaties op het substraat worden verlicht en daarna gedeprotecteerd. In de voorkeur verdienende uitvoeringsvormen wordt het substraat verlicht tussen ongeveer 1 en 15 minuten waarbij een de voorkeur verdienende verlichtingsperiode ongeveer 10 minuten bij 10-20 mW/cm2 is met licht van 365 nm. De objectglaasjes worden geneutraliseerd (dat wil zeggen gebracht op een pH van ongeveer 7) na fotolyse met bijvoorbeeld een oplossing van di-isopropylethylamine (DIEA) in dichloor-methaan gedurende een periode van ongeveer 5 minuten.
Het eerste monomeer wordt vervolgens op de eerste locaties op het substraat gebracht. Na bestraling wordt het ob jectglaasje verwijderd, behandeld in massa, en vervolgens opnieuw in de stroomcel geïnstalleerd. Een vloeistof, bevattend het eerste monomeer, bij voorkeur ook beschermd door een beschermende groep, wordt op alternatieve wijze gecirculeerd door de holte door middel van toepassen van pomp 116. Indien bijvoorbeeld het gewenst is het aminozuur Y te binden aan het substraat bij de eerste locaties, wordt het aminozuur Y (dragende een beschermende groep aan het α-stikstofatoom ervan), tezamen met reagentia die gebruikt worden om het monomeer reactief te maken, en/of een drager, uit een opslaghouder 118 door de pomp, door de holte en terug naar de inlaat van de pomp gecirculeerd.
De drageroplossing van monomeer is in een de voorkeur verdienende uitvoeringsvorm gevormd door het mengen van een eerste oplossing (hierin aangeduid als oplossing "A") en een tweede oplossing (hierin aangeduid als oplossing "B"). Tabel 2 levert een toelichting van een mengsel dat voor oplossing A kan worden gebruikt.
De samenstelling van oplossing B wordt in tabel 3 toegelicht. Oplossingen A en B worden gemengd en men laat ze gedurende ongeveer 8 minuten bij kamertemperatuur reageren, vervolgens worden ze verdund met 2 ml DMF, en 500 μΐ worden aangebracht op het oppervlak van het objectglaasje of de oplossing wordt gecirculeerd door het reactorsysteem en men laat deze gedurende ongeveer 2 uur bij kamertemperatuur reageren. Het objectglaasje wordt vervolgens gewassen met DMF, dichloormethaan en ethanol.
A Λ Λ Λ Λ Λ
Als de oplossing, bevattende het te binden monomeer, door de holte wordt gecirculeerd, zal het aminozuur of ander monomeer reageren bij de carboxyterminus ervan met aminogroepen op de gebieden van het substraat welke gedeprotecteerd zijn. Hoewel de uitvinding toegelicht wordt bij wijze van circulatie van het monomeer door de holte, zou de uitvinding natuurlijk ook kunnen worden toegepast door het verwijderen van het ob-jectglaasje uit de reactor en dit onder te dompelen in een geschikte monomeeroplossing.
Na toevoeging van het eerste monomeer wordt de oplossing, bevattende het eerste aminozuur, vervolgens uit het systeem gespoeld. Na circulatie van een voldoende hoeveelheid van het DMF/dichloormethaan, zodanig dat verwijdering van het aminozuur verzekerd kan worden (bijvoorbeeld ongeveer 5 x het volume van de holte en de drager leidingen) wordt het masker of het substraat weer in positie gebracht, of een nieuw masker wordt toegepast zodanig dat tweede gebieden op het substraat aan licht zullen worden blootgesteld en het licht 124 voor een tweede blootstelling wordt toegepast. Dit zal tweede gebieden op het substraat deprotecteren en de werkwijze wordt herhaald totdat de gewenste polymeersequenties gesynthetiseerd zijn.
Het totale gederivatiseerde substraat wordt vervolgens blootgesteld aan een van belang zijnde receptor, bij voorkeur gemerkt met bijvoorbeeld een fluorescerend merkmiddel, door circulatie van een oplossing of suspensie van de receptor door de holte of door het in contact brengen van het oppervlak van het objectglaasje in massa. De receptor zal zich bij voorkeur binden aan bepaalde gebieden van het substraat welke complementaire sequenties bevatten.
Antilichamen worden kenmerkend gesuspendeerd in wat algemeen wordt aangeduid als "supercocktail", die bijvoorbeeld een oplossing kan zijn van ongeveer 1% BSA (runderserumalbumine), 0,5# Tween in PBS (met fosfaat gebufferde zoutoplossing) buffer. De antilichamen worden in de supercock-tailbuffer verdund tot een uiteindelijke concentratie van bijvoorbeeld 0,1 tot 4 ug/ml.
Figuur 8b illustreert een alternatieve de voorkeur verdienende uitvoeringsvorm van de in figuur 8A getoonde reactor. Volgens deze uitvoeringsvorm wordt het masker 128 direct in contact gebracht met het substraat. Bij voorkeur wordt het geëtste gedeelte van het masker met de voorkant naar beneden aangebracht teneinde de effecten van lichtdispersie te verminderen. Volgens deze uitvoeringsvorm zijn de beeldvormende lenzen 120 en 126 niet noodzakelijk omdat het masker in dichte nabijheid van het substraat wordt gebracht.
Voor doeleinden van het verhogen van de signaal-tot-geluid verhouding van de techniek, verschaffen sommige uitvoeringsvormen van de uitvinding het blootstellen van het substraat aan een eerste gemerkte of niet gemerkte receptor, gevolgd door blootstelling van een gemerkte tweede receptor (bijvoorbeeld een antilichaam) die zich op meerdere plaatsen aan de eerste receptor bindt. Indien bijvoorbeeld de eerste receptor een antilichaam is verkregen uit een eerste species van een dier, is de tweede receptor een antilichaam verkregen uit een tweede species, gericht op epitopen welke geassocieerd zijn met de eerste species. In het geval van een antilichaam van een muis, kan bijvoorbeeld met een fluorescerend middel gemerkt geite-antilichaam of antiserum, dat antimuis is, gebruikt worden voor het binden aan veelvoudige plaatsen op het antilichaam van de muis, waardoor verscheidene malen de fluorescentie wordt verschaft in vergelijking met de binding van een enkelvoudig muizen-antilichaam op elke bindingsplaats. Deze werkwijze kan weer worden herhaald met additionele antilichamen (bijvoorbeeld geit-muis-geit, enz.) voor verdere signaal vers terking.
In de voorkeur verdienende uitvoeringsvormen wordt een geordende sequentie van maskers toegepast. In sommige uitvoeringsvormen is het mogelijk om zo weinig als een enkelvoudig masker toe te passen voor het synthetiseren van alle mogelijke polymeren van een gegeven stel monome-ren.
Indien het bijvoorbeeld gewenst is om alle zestien dinucleotiden uit vier basen te synthetiseren, wordt een 1 cm2 synthesegebied qua concept verdeeld in 16 vierkanten, elk met een breedte van 0,25 cm. De vier monomeereenheden worden aangeduid door A, B, C en D. De eerste reacties worden uitgevoerd in vier verticale kolommen, elk met een breedte van 0,25 cm. Het eerste masker stelt de meest linkse kolom van de vierkanten bloot, waarbij A gekoppeld wordt. Het tweede masker stelt de volgende kolom bloot, waarbij B gekoppeld wordt; gevolgd door een derde masker voor kolom C; en een uiteindelijk masker dat de meest rechtse kolom voor D blootstelt. Het eerste, tweede, derde en vierde masker kan een enkelvoudig masker zijn dat op verschillende locaties wordt getranslateerd.
De werkwijze wordt herhaald in horizontale richting voor de tweede eenheid van het dimeer. Deze keer maken de maskers een blootstelling van horizontale rijen mogelijk, weer met een breedte van 0,25 cm. A, B, C en D worden sequentieel gekoppeld onder toepassing van maskers welke horizontale vierde gedeelten van het reactieoppervlak blootstellen. Het resulterende substraat bevat alle 16 nucleotiden van vier basen.
De acht maskers welke toegepast zijn voor het synthetiseren van het dinucleotide, zijn gerelateerd aan elkaar door translatie of rotatie. In feite kan een masker in alle acht trappen worden gebruikt indien het op geschikte wijze geroteerd en getranslateerd wordt. Bijvoorbeeld zou in het bovenstaande voorbeeld een masker met een enkelvoudig transparant gebied sequentieel gebruikt kunnen worden voor het blootstellen van elk van de verticale kolommen, getranslateerd 90°, en vervolgens sequentieel gebruikt om blootstelling van de horizontale rijen mogelijk te maken.
De tabellen 4 en 5 verschaffen een eenvoudig computerprogramma in "Quick Basic" resp. voor het uitvoeren van een maskeringsprogramma en de output van een monster, voor de synthese van een polymeerketen van drie monomeren ("residuen") met drie 'verschillende monomeren in het eerste niveau, vier verschillende monomeren in het tweede niveau en vijf verschillende monomeren in het derde niveau, welke zich in een gestreept patroon bevinden. De output van het programma is het aantal cellen, het aantal "strepen" (lichte gebieden) op elk masker, en de hoeveelheid translatie die voor elke blootstelling van het masker vereist is.
Tabel 4
MaskerL_strategiiBTOgramma
DEFINT A-Z
DIM b(20), w(20), 1(500) F$ = "LPT1:" OPEN f$ voor OUTPUT AS #1 jmax = 3 'Aantal residuen b(1) = 3: b(2) = 4: b(3) = 5 'aantal bouwblokken voor res 1, 2, 3 g = 1: lmax(l) = 1 FOR j = 1 TO jmax: g= g * b(j): DAARNA j w(0) = 0: w(l) = g / B(l) DRUK #1, "MASKER2.BAS", GEGEVENS, TIJDEN: Druk #1, DRUK #1, TOEPASSING VAN "aantal residuen = ##"; jmax FOR j = 1 NAAR jmax DRUK #1, TOEPASSING VAN " Residu ## ## bouwblokken"; j; b(j) DAARNA j DRUK #1, " DRUK #1, TOEPASSING VAN "aantal cellen = ####"; g: DRUK #1, FOR j = 2 NAAR jmax lmax(j) = lmax(j - 1) * b(j - 1) w(j) = w(j - 1) / b(j) DAARNA j FOR j = 1 NAAR jmax DRUK #1, TOEPASSING VAN "masker voor residu ##"; j: DRUK #1, DRUK 31, TOEPASSING VAN "aantal strepen = ###"; lmax(j) DRUK #1, TOEPASSING VAN "breedte van elke streep"; w(j) FOR 1 = 1 NAAR lmax(j) a = 1 + (1 - 1) * w(j - 1) ae = a + w(j) - 1 DRUK #1, TOEPASSING VAN "streep ## begint bij locatie ### en eindigt bij ###; 1; a; ae DAARNA 1 DRUK #1, DRUK #1, TOEPASSING VAN "voor elk van ## boublokken, translateer masker door ## cel(len)"; b(j); w(J), DRUK #1,: DRUK #1,: DRUK #1, DAARNA j
Copyright 1990, Affymx N.V.
Tabel 5
Masker strategie output
Aantal residuen = 3
Residu 1 3 bouwblokken
Residu 2 4 bouwblokken
Residu 3 5 bouwblokken
Aantal cellen = 60
Masker voor residu 1
Aantal strepen = 1
Breedte van elke streep = 20
Streep 1 begint bij locatie 1 en eindigt bij 20
Voor elk van 3 bouwblokken, translateer masker door 20 cel(len)
Masker voor residu 2
Aantal strepen = 3 Breedte van elke streep = 5
Streep 1 begint bij locatie 1 en eindigt bij 5
Streep 2 begint bij locatie 21 en eindigt bij 25
Streep 3 begint bij locatie 4l en eindigt bij 45
Voor elk van 4 bouwblokken, translateer masker door 5 cel(len)
Masker voor residu 3
Aantal strepen = 12 Breedte van elke streep = 1
Streep 1 begint bij locatie 1 en eindigt bij 1
Streep 2 begint bij locatie 6 en eindigt bij 6
Streep 3 begint bij locatie 11 en eindigt bij 11
Streep 4 begint bij locatie 16 en eindigt bij 16
Streep 5 begint bij locatie 21 en eindigt bij 21
Streep 6 begint bij locatie 26 en eindigt bij 26
Streep 7 begint bij locatie 31 en eindigt bij 31
Streep 8 begint bij locatie 36 en eindigt bij 36
Streep 9 begint bij locatie 4l en eindigt bij 4l
Streep 10 begint bij locatie 46 en eindigt bij 46
Streep 11 begint bij locatie 51 en eindigt bij 51
Streep 12 begint bij locatie 56 en eindigt bij 56
Voor elk van 5 bouwblokken, translateer masker door 1 cel
Copyright 1990, Affymax N.V. _ V. Details van een uitvoeringsvorm van een fluorescerende detectie- inrichting.
Fig. 9 licht een fluorescerende detectieinrichting toe voor het detecteren van fluorescerend gemerkte receptoren op een substraat. Een substraat 112 wordt aangebracht op een x/y translatie tabel 202. In een de voorkeur verdienende uitvoeringsvorm is het x/y translatietabel een model nr. PM500-A1, vervaardigd door Newport Corporation. De x/y translatietabel wordt verbonden met en geregeld door een geschikt geprogrammeerde digitale computer 204 die bijvoorbeeld een geschikt geprogrammeerde IBM PC/AT of AT verenigbare computer kan zijn. Natuurlijk zouden andere computersystemen, hardware voor speciale doeleinden, of dergelijke gemakkelijk gesubstitueerd kunnen worden voor de AT computer die hierin voor toelichtende doeleinden is gebruikt. Computersoftware voor de functies van translatie en verzameling van gegevens, zoals hierin beschreven, kan worden verschaft op basis van in de handel verkrijgbare software, waaronder bijvoorbeeld "Lab windows", gelicentieerd door National Instruments, hetgeen hierin voor alle doeleinden door verrijzing is opgenomen.
Het substraat en de x/y translatietabel worden onder een microscoop 206 gebracht die een of meer objectieven 208 omvat. Licht (ongeveer 488 nm) van een laser 210, die in sommige uitvoeringsvormen een model nr. 2020-05 argon ionlaser is, vervaardigd door Spectraphysics, wordt gericht op het substraat door toepassing van een dichroitische spiegel 207, die licht doorlaat groter dan ongeveer 520 nm maar licht van 488 nm terugkaatst. De dichroitische spiegel 207 kan bijvoorbeeld een model nr. FT510 zijn, dat door Carl Zeiss is vervaardigd. Licht dat uit de spiegel is teruggekaatst komt vervolgens in de microscoop 206 die bijvoorbeeld een model nr. Axioscop 20 kan zijn, vervaardigd door Carl Zeiss. Door een fluorescentiemiddel gemerkte materialen op het substraat zullen >488 nm licht fluoresceren, en het gefluoresceerde licht zal door de microscoop worden verzameld en door de spiegel worden geleid. Het gefluoresceerde licht van het substraat wordt vervolgens gericht door een golflengtefliter 209 en daarna door een opening in plaat 211. De golflengte filter 209 kan bijvoorbeeld een model nr. OG530 zijn, vervaardigd door Melles Griot en openingsplaat 211 kan bijvoorbeeld een model nr. 477352/477380 zijn, vervaardigd door Carl Zeiss.
Het gefluoresceerde licht komt dan in een lichtvermenigvuldigings-buis 212, die in sommige uitvoeringsvormen een model nr. R943~02 is, vervaardigd door Hamamatsu, waarbij het signaal versterkt wordt in een voorversterker 212 en fotonen door de fotontelinrichting 216 worden ge- teld. Het aantal fotonen wordt geregistreerd als een functie van de locatie in de computer 204. Pre-Amp 214 kan bijvoorbeeld een model nr. SR400 zijn, vervaardigd door Stanford Research Systems en de fotontelinrichting 216 kan een model nr. SR400 zijn, vervaardigd door Stanford Research Systems. Het substraat wordt vervolgens bewogen naar een daaropvolgende locatie en het proces wordt herhaald. In de voorkeur verdienende uitvoeringsvormen worden de gegevens elke 1 tot 100 μΐη verkregen waarbij een datacollectie diameter van ongeveer 0,8 tot 10 pm de voorkeur verdient. In uitvoeringsvormen met een voldoende hoge fluorescentie wordt een CCD detector met belichting in een breed veld toegepast.
Door het tellen van het aantal fotonen ontwikkeld in een bepaald gebied in respons op de laser, is het mogelijk te bepalen waar de fluorescerend gemerkte moleculen op het substraat zijn gelocaliseerd. Voor een objectglaasje dat bijvoorbeeld een matrix van polypeptiden bevat, gesynthetiseerd op het oppervlak ervan, is het derhalve mogelijk te bepalen welk van de polypeptiden complementair voor een fluorescerend gemerkte receptor is.
Volgens de voorkeur verdienende uitvoeringsvormen wordt de intensiteit en de duur van het op het substraat toegepaste licht geregeld door het variëren van het laservermogen en de aftasttrapsnelheid voor verbeterde signaal-tot-geluid verhouding door het maximaliseren van de fluorescentie emissie en het minimaliseren van achtergrondgeluid.
Hoewel het detectieapparaat hierin primair is toegelicht met betrekking tot de detectie van gemerkte receptoren, zal de uitvinding in andere gebieden kunnen worden toegepast. Bijvoorbeeld zou het hierin beschreven detectieapparaat gebruikt kunnen worden op het gebied van katalyse, aftasten van DNA of proteïnegel, en dergelijke.
VI. Bepaling van de relatieve bindingssterkte van receptoren.
De signaal-tot-geluid verhouding van de onderhavige uitvinding is voldoende hoog dat niet alleen de aanwezigheid of afwezigheid van een receptor op een ligand kan worden gedetecteerd, maar ook de relatieve bindingsaffiniteit van receptoren aan een groot aantal sequenties kan worden bepaald.
In de praktijk is gebleken dat een receptor zich in een baan aan verscheidene peptidesequenties zal binden, maar zich aan sommige sequenties veel sterker zal binden dan aan andere. Een sterke bindingsaffiniteit wordt getoond door een sterk fluorescerend of radiografisch signaal aangezien vele receptormoleculen zich binden in een gebied van een sterk gebonden ligand. Daarentegen zal een zwakke bindingsaffiniteit aangetoond worden door een zwak fluorescerend of radiografisch signaal als gevolg van het betrekkelijk kleine aantal receptormoleculen die zich in een bepaald gebied van een substraat binden welk substraat een ligand heeft met een zwakke bindingsaffiniteit voor de receptor. Dientengevolgde wordt het mogelijk om een relatieve bindingsaffiniteit (of affiniteit in het geval van univalente interacties) van een ligand te bepalen door de intensiteit van een fluorescerend of radiografisch signaal in een gebied dat het ligand bevat.
Semikwantitatieve gegevens van affiniteiten zouden ook verkregen kunnen worden door het variëren van de wasomstandigheden en de concentraties van de receptor. Dit zou uitgevoerd kunnen worden door vergelijking met bijvoorbeeld bekende ligandreceptorparen.
VII. Voorbeelden.
De volgende voorbeelden worden verschaft voor het toelichten van de effectiviteit van de onderhavige uitvinding. Alle bewerkingen werden uitgevoerd bij ongeveer omgevingstemperaturen en -drukken tenzij het tegengestelde is aangegeven.
A. Vervaardiging van het obiecterlaasie.
Alvorens het binden van reactieve groepen verdient het de voorkeur het substraat schoon te maken, dat in een de voorkeur verdienende uitvoeringsvorm een glassubstraat is, zoals een microscoop objectglaasje of een bedekkingsslip. Dienovereenkomstig wordt volgens een uitvoeringsvorm het objectglaasje geweekt in een alkalisch bad, bijvoorbeeld bestaande uit 1 liter 95%'s ethanol met 120 ml water en 120 gram natriumhydroxide welke behandeling 12 uur duurt. Vervolgens worden de objectglaasjes onder leidingwater gewassen en men laat ze aan de lucht drogen, en nog eenmaal gespoeld met een oplossing van 95%'s ethanol.
Vervolgens worden de objectglaasjes geamineerd met bijvoorbeeld aminopropyltriethoxysilaan met het doel om aminogroepen op verbindingsmo-leculen aan het glas te hechten, ofschoon elk omega gefunctionaliseerd silaan ook voor dit doel gebruikt zou kunnen worden. In een uitvoeringsvorm wordt 0,1% aminopropyltriethoxysilaan gebruikt, ofschoon oplossingen met concentraties van 10~7% tot 10% gebruikt kunnen worden, waarbij ongeveer 10"3# tot 2/ de voorkeur wordt gegeven. Een 0,1%1s mengsel wordt bereid door toevoegen aan 100 ml van een 95%'s ethanol/5#'s watermengsel, 100 microliter (μΐ) aminopropyltriethoxysilaan. Het mengsel wordt bij omgevingstemperatuur gedurende ongeveer 5 minuten geroerd op een roterende schudinrichting. 500 μΐ van dit mengsel wordt vervolgens op het oppervlak van een kant van elk schoongemaakt objectglaasje gebracht. Na 4 mi nuten wordt de oplossing van deze objectglaasjes afgeschonken en driemaal gespoeld door bijvoorbeeld onder te dompelen in 100#’s ethanol.
Nadat de platen droog zijn worden zij gedurende ongeveer 20 minuten in een zich op 110-120°C bevindende vacuümoven gebracht en vervolgens laat men ze gedurende ongeveer 12 uur in een argonomgeving bij kamertemperatuur harden. De objectglaasjes worden vervolgend gedompeld in DMF (dimethylformamide) oplossing, gevolgd door een grondige wasbehandeling met dichloormethaan.
Het geamineerde oppervlak van het objectglaasje wordt vervolgens blootgesteld aan ongeveer 500 μΐ van bijvoorbeeld een 30 millimolair (mM) oplossing van NVOC-GABA (gamma-aminoboterzuur) NHS (N-hydroxysuccinimide) in DMF voor het binden van een NVOC-GABA aan elk van de aminogroepen.
Het oppervlak wordt bijvoorbeeld gewassen met DMF, dichloormethaan en ethanol.
Elk niet omgezet aminopropylsilaan op het oppervlak - dat is diami-nogroepen die niet gebonden zijn aan NVOC-GABA - worden nu beschermd met acetylgroepen (ter verhindering van verdere reactie) door blootstelling aan een 1:3 mengsel van azijnzuuranhydride in pyridine gedurende 1 uur. Andere materialen die deze beschermende functie van deze rest kunnen uitvoeren, omvatten trifluorazijnzuuranhydride, formicaazijnzuuranhydri-de, of andere reactieve acrylerende middelen. Tenslotte worden de objectglaasjes weer gewassen met DMF, dichloormethaan en ethanol.
B. Synthese van acht trimeren van "A" en "B".
Figuur 10 illustreert een mogelijke synthese van de acht trimeren van het stel van twee monomeren; gly, phe (respectievelijk weergegeven door "A" en "B"). Een objectglaasje, dragende silaangroepen die eindigen in 6-nitroveratryloxycarboxamide (NV0C-NH) residuen, wordt als substraat bereid. Actieve esters (pentafluorfenyl, oBt, enz.) van gly en phe, beschermt bij de aminogroep met NV0C, worden als reactiemiddelen bereid. Indien bescherming van zijketengroepen vereist is voor het stel monomeren, hoewel dit niet van toepassing is voor dit voorbeeld, moeten deze niet fotoreactief zijn bij de golflengte van het licht dat gebruikt wordt voor het beschermen van de primaire keten.
Voor een stel monomeren van grootte n, zijn η x 1 cycli vereist voor het synthetiseren van alle mogelijke sequenties met een lengte 1. Een cyclus bestaat uit: 1. Bestralen door een geschikt masker voor het blootstellen van de aminogroepen bij de plaatsen waar het volgende residu moet worden toegevoegd, onder toepassing van geschikte wasbehandelingen ter verwijdering van de bijprodukten van de deprotectie.
2. Toevoegen van een enkelvoudig geactiveerd en beschermd (met dezelfde door fotochemie verwijderbare groep) monomeer, dat slechts zal reageren met de plaatsen welke in trap 1 worden aangesproken, onder toepassing van geschikte wasbehandelingen voor het verwijderen van de overmaat reactiemiddel van het oppervlak.
De bovenstaande cyclus wordt herhaald voor elk lid van het stel monomeren totdat elke locatie op het oppervlak met een residu in een uitvoeringsvorm is verlengd. In andere uitvoeringsvormen worden verscheidene residuen sequentieel geaddeerd op een locatie alvorens naar de volgende locatie wordt gegaan. De duur van de cycli zal in het algemeen worden beperkt door de koppelingssnelheid, welke nu zo kort is als 20 minuten in geautomatiseerde peptidesynthetiseerinrichtingen. Deze trap wordt eventueel gevolgd door additie van een beschermende groep voor het stabiliseren van de baan voor later onderzoek. Voor sommige typen polymeren (bijvoorbeeld peptiden) kan een uiteindelijke deprotectie van het totale oppervlak (verwijdering van fotoprotectieve zijketengroepen) vereist zijn.
Meer in het bijzonder, zoals getoond in figuur 10A, is het glas 20 voorzien van gebieden 22, 24, 26, 28, 30» 32, 3^ en 36. De gebieden 30, 32, 34 en 36 zijn gemaskeerd, zoals getoond in figuur 10B, en het glas wordt bestraald en blootgesteld aan een reactiemiddel dat "A" bevat (bijvoorbeeld gly), waarbij de resulterende structuur in figuur IOC is getoond. Daarna worden de gebieden 22, 24, 26 en 28 gemaskeerd, het glas wordt bestraald (zoals getoond in figuur 10D) en blootgesteld aan een reactiemiddel, bevattende "B" (bijvoorbeeld phe), waarbij de resulterende structuur in figuur 10E is getoond. De werkwijze verloopt verder door het maskeren en blootstellen van de getoonde secties totdat de structuur, getoond in figuur 10M, is verkregen. Het glas wordt bestraald en de terminale groepen worden eventueel door acetylering van een bescherming voorzien. Zoals getoond worden alle mogelijke trimeren van gly/phe verkregen.
In dit voorbeeld is er geen verwijdering van een beschermende groep van de zijketen noodzakelijk. Indien het gewenst is kan deprotectie van de zijketen worden uitgevoerd door behandeling met ethaandithiol en tri-fluoraz i j nzuur.
In het algemeen wordt het aantal trappen, nodig voor het verkrijgen van een bijzondere polymeerketen, gedefinieerd door:
Q Cl 90 A R
(1) waarin: η = het aantal monomeren in het basisstel van de monomeren, en 1 = het aantal monomeereenheden in een polymeerketen.
Daarentegen zou het gesynthetiseerde aantal sequenties met lengte 1 zijn:
(2)
Natuurlijk wordt een grotere diversiteit verkregen door gebruik te maken van maskerstrategieën die ook de synthese van polymeren omvatten welke een lengte hebben van minder dan 1.
Indien in het extreme geval alle polymeren met een lengte minder dan of gelijk aan 1 worden gesynthetiseerd, zal het aantal gesynthetiseerde polymeren zijn:
(3)
Het maximale aantal lithografische stappen welke nodig zijn, zal in het algemeen n zijn voor elke "laag" monomeren, d.w.z. het totale aantal maskers (en derhalve het aantal lithografische stappen), die nodig zijn, zal n x 1 zijn, De grootte van de transparante maskergebieden zal variëren in overeenstemming met het gebied van het substraat dat voor synthese beschikbaar is en met het aantal te vormen sequenties. In het algemeen zal de grootte van de synthesegebieden zijn: grootte van synthesegebieden = (A)/(S) waarin: A is het totale gebied dat voor de synthese beschikbaar is; en S is het aantal in het gebied gewenste sequenties.
Het zal aan de vakman duidelijk zijn dat de bovenstaande methode gemakkelijk gebruikt kan worden voor het simultaan produceren van duizenden of miljoenen oligomeren op een substraat onder toepassing van de hierin beschreven fotolithografische technieken. Dientengevolge resulteert de methode in het vermogen om grote aantallen van bijvoorbeeld di, tri, tetra, penta, hexa, hepta, octapeptiden, dodecapeptiden of grotere polypeptiden (of overeenkomstige polynucleotiden) in de praktijk te testen.
Het bovenstaande voorbeeld heeft de werkwijze bij wijze van een met de hand uit te voeren voorbeeld toegelicht. Het zal natuurlijk duidelijk zijn dat geautomatiseerde of semi-geautomatiseerde methoden gebruikt zouden kunnen worden. Het substraat wordt dan aangebracht in een stroom-cel voor geautomatiseerde toevoeging en verwijdering van reactiemiddelen, voor het minimaliseren van het volume van de benodigde reagentia en voor een meer zorgvuldige regeling van reactieomstandigheden. Opeenvolgende maskers kunnen met de hand of automatisch worden aangebracht.
C. Synthese van een dimeer van een aminopropylgroep en een fluorescerende groep.
Bij het synthetiseren van het dimeer van een aminopropylgroep en een fluorescerende groep werd een gefictionaliseerd durapore membraan als substraat toegepast. Het durapore membraan was een polyvinylideendi-fluoride met aminopropylgroepen. De aminopropylgroepen werden beschermd met de DDZ-groep door omzetting van het carbonylchloride met de amino-groepen, een reactie die algemeen aan de vakman bekend is. Het deze groepen dragende oppervlak werd gebracht in een oplossing van THF en in contact gebracht met een masker dat een schaakbordpatroon van 1 mm troebele en transparante gebieden droeg. Het masker werd blootgesteld aan ultraviolet licht met een golflengte in benedenwaartse richting tot tenminste ongeveer 280 nm gedurende ongeveer 5 minuten bij omgevingstemperatuur, ofschoon een ruim traject van blootstellingstijden en temperaturen in diverse uitvoeringsvormen van de uitvinding geschikt zijn. Bijvoorbeeld kan in een uitvoeringsvorm een blootstellingstijd van tussen ongeveer 1 en 5000 seconden gebruikt worden bij procestemperaturen van tussen -70 en +50°C.
In een de voorkeur verdienende uitvoeringsvorm worden blootstellingstijden van tussen ongeveer 1 en 500 seconden bij ongeveer omgevings-druk toegepast. In sommige de voorkeur verdienende uitvoeringsvormen wordt een druk boven omgevingsdruk toegepast ter verhindering van verdamping.
Het oppervlak van het membraan werd vervolgens gedurende ongeveer 1 uur gewassen met een fluorescerend merkmiddel dat een actieve ester omvatte die gebonden was aan een chelaat van een lanthanide. De wastijden variëren over een ruim traject van waarden van ongeveer enkele minuten tot enkele uren. Deze materialen fluorescerend in het rode en het groene zichtbare gebied. Nadat de reactie met de actieve ester in de fluorofoor volledig was, konden de locaties waarin de fluorofoor gebonden was, worden gevisualiseerd door ze bloot te stellen aan ultraviolet licht en door de rode en de groene fluorescentie waar te nemen. Waargenomen werd dat de gederivatiseerde gebieden van het substraat nauw correspondeerden met het oorspronkelijke patroon van het masker.
D. Demonstratie van signaalvermogen.
Het signaaldetectievermogen werd aangetoond door toepassing van een standaard fluorescerende uitrusting van laag vermogen, vervaardigd door
Flow Cytometry Standarda en van model nr. 824. Deze uitrusting omvat korrels met een diameter van 5»8 v>m, die elk geïmpregneerd zijn met een bekend aantal fluoresceïne moleculen.
Een van de korrels werd in het belichtingsgebied op de aftasttrap aangebracht, zoals getoond in figuur 9» in. een veld van laser die aanvankelijk was af geschermd. Na in het belichtingsveld te zijn geplaatst werd het fotondetectieapparaat aangezet. De laserstraal werd niet geblokkeerd en werd in wisselwerking gebracht met het korrelvormige deeltje, welke deeltje vervolgens gefluoresceerd werd. Fluorescentiecurves van korrels, geïmpregneerd met 7000 en 29.000 fluoresceïnemoleculen, worden respectievelijk in de figuren 11A en 11B getoond. Op elke curve worden ook sporen van korrels zonder fluoresceïnemoleculen weergegeven. Deze experimenten werden uitgevoerd bij een 488 nm excitatie met een laservermogen van 100 pW. Het licht werd gefocusseerd door een 0,75 NA objectief met vermogen 40.
De fluorescentie intensiteit in alle gevallen begon bij een hoge waarde en nam vervolgens exponentieel af. De daling in intensiteit is te wijten aan het bleken van de fluoresceïnemoleculen door licht. De sporen van de korrels zonder fluoresceïnemoleculen worden gebruikt voor het aftrekken van de achtergrond. Het verschil in de aanvankelijke exponentiële afname tussen gemerkte en niet gemerkte korrels wordt geïntegreerd om het totale aantal fotontellingen te leveren, en dit getal wordt gerelateerd met het aantal moleculen per korrel. Derhalve is het mogelijk het aantal fotonen per fluoresceïnemolecuul af te leiden welk aantal gedetecteerd kan worden. Voor de krommen, weergegeven in figuur 11, geeft deze berekening aan dat de straling van ongeveer 40 tot 50 fotonen per fluoresceïnemolecuul wordt gedetecteerd.
E. Bepaling van het aantal moleculen oer oppervlakte-eenheid.
Geaminopropyleerde microscoop objectglaasjes, bereid volgens de hierboven besproken methoden, werden gebruikt voor het vaststellen van de dichtheid van de merkmiddelen van de glaasjes. De vrije aminotermini van de objectglaasjes werden omgezet met FITC (fluoresceïne isothiocyanaat) welke verbinding een covalente binding met de aminogroep vormt. Het ob-jectglaasje wordt vervolgens afgetast voor het tellen van het aantal fluorescerende fotonen, gevormd in een gebied, dat bij toepassing van de geschatte 40-50 fotonen per fluorescerend molecuul, de berekening mogelijk maakt van het aantal moleculen dat zich op het oppervlak per eenheid van oppervlak bevindt.
Een objectglaasje met aminopropylsilaan op het oppervlak ervan werd ondergedompeld in een 1 mM oplossing van FITC in DMF gedurende 1 uur bij ongeveer omgevingstemperatuur. Na de reactie werd het objectglaasje tweemaal gewassen met DMF en vervolgens gewassen met ethanol, water en dan weer met ethanol. Vervolgens werd het gedroogd en in het donker bewaard totdat het gereed was om onderzocht te worden.
Door het gebruik van curven, soortgelijk aan die welke in figuur 11 zijn getoond, en door het integreren van de fluorescentiemiddeltellingen onder het exponentieel af nemende signaal, werd het aantal vrije amino-groepen op het oppervlak na derivatisering bepaald. Bepaald werd dat objectglaasjes met dichtheden aan merkmiddel van 1 fluoresceïne molecuul per 103xl03 tot -2x2 nm reproduceerbaar zouden kunnen worden vervaardigd als de concentratie aan aminopropyltriethoxysilaan varieerde van 10~5% tot 10-1#.
F. Verwildering van NVOC en binding van een fluorescerend merkmiddel.
NVOC-GABA groepen werden gebonden zoals hierboven is beschreven.
Het totale oppervlak van een objectglaasje werd aan licht blootgesteld teneinde een vrije aminogroep aan het einde van het gamma-aminoboterzuur bloot te stellen. Dit objectglaasje, en een duplicaat dat niet was blootgesteld, werden vervolgens blootgesteld aan fluoresceïne isothiocyanaat (FITC).
Fig. 12A toont het objectglaasje dat niet aan licht was blootgesteld, maar dat aan FITC was blootgesteld. De eenheden van de x as zijn tijd en de eenheden van de y as zijn het aantal tellingen. Het spoor bevat een bepaalde hoeveelheid achtergrond fluorescentie. Het duplicaat objectglaasje werd blootgesteld aan een belichting met een breede band van 350 nm gedurende ongeveer 1 minuut (12 mW/cm2, ongeveer 150 nm belichting), gewassen en omgezet met FITC. De fluorescentiecurven voor dit objectglaasje zijn in figuur 12B getoond. Een grote toename in het niveau van de fluorescentie wordt waargenomen, hetgeen aangeeft dat fotoly-se een aantal aminogroepen op het oppervlak van de objectglaasjes voor hechting van een fluorescentie merkmiddel heeft blootgesteld.
G. Toepassing van een masker bii de verwijding van NVOC.
Het volgende experiment werd uitgevoerd met een 0,1% geaminopropy-leerd objectglaasje. Licht van een Hg-Xe booglamp werd door een met laser afgezette chroom-op-glasmasker in direct contact met het substraat als een beeld op het substraat gebracht.
Dit objectglaasje werd gedurende ongeveer 5 minuten belicht onder toepassing van 12 mW van 350 nm breedbandlicht en vervolgens omgezet met de 1 mM FITC oplossing. Vervolgens werd het gebracht op de aftasttrap van laserdetectie en een grafiek werd uitgezet als een tweedimensionale weergave van positie-kleur-gecodeerd voor de fluorescentie intensiteit. Het experiment werd door diverse maskers een aantal keren herhaald. De fluo-rescentiepatronen voor een 100x100 μΐη masker, een 50 μπι masker, een 20 jim masker en een 10 μΐη masker geven aan dat het maskerpatroon verschilt in benedenwaartse richting tot tenminste ongeveer 10 μΐη vierkanten onder toepassing van deze lithografische techniek.
H Hechting van YGGFL en daaropvolgende blootstelling aan Herz anti-Ήrhaam en geite-muis antilichaam-
Teneinde vast te stellen dat receptoren voor een bijzondere polypeptide sequentie zich binden aan een aan een oppervlak gebonden peptide en gedetecteerd kunnen worden, werd Leu encefaline aan het oppervlak gekoppeld en door een antilichaam herkend. Een objectglaasje werd gederi-vatiseerd met 0,1% aminopropyl-triethoxysilaan en beschermd met NVOC. Een schaakbordmasker van 500 μΐη werd gebruikt om het objectglaasje bloot te stellen in een stroomcel onder toepassing van contactdrukken aan de achterkant. De Leu encefalinesequentie (H2N-tyrosine, glycine, glycine, fenylalanine, leucine-C02H, overigens hierin aangeduid als YGGFL) werd via het carboxy uiteinde ervan gebonden aan de blootgestelde aminogroepen op het oppervlak van het objectglaasje. Het peptide werd toegevoegd in DMF oplossing met B0P/H0BT/DIEA koppelingsreagentia en gedurende 2 uur bij kamertemperatuur door de stroomcel gerecirculeerd.
Een eerste antilichaam, bekend als Herz antilichaam, werd op het oppervlak van het objectglaasje aangebracht gedurende 45 minuten bij 2 μg/ml in een supercocktail (bevattende ook in dit geval 1% BSA en 1% ovalbumine). Een tweede antilichaam, geite anti-muis fluoresceïne conju-gaat, werd vervolgens toegevoegd bij 2 μg/ml in de supercocktail buffer, en men liet gedurende 2 uur incuberen.
De resultaten van dit experiment werden uitgezet als fluorescentie intensiteit als functie van de positie. Dit beeld werd genomen bij 10 μΐη trappen en toonde dat niet alleen deprotectie uitgevoerd kan worden in een goed gedefinieerd patroon, maar ook dat (1) de methode een succesvol koppelen van peptiden aan het oppervlak van het substraat verschafte, (2) het oppervlak van een gebonden peptide beschikbaar was voor het binden met een antilichaam, en (3) het vermogen van het detectieapparaat voldoende was om de binding van een receptor te detecteren.
I. Monomeer-bii-monomeer vorming van YGGFL en daaropvolgende blootstelling aan gemerkt antilichaam.
Monomeer-bij-monomeer-synthese van YGGFL en GGFL in alternerende vierkanten werd uitgevoerd op een objectglaasje in een schaakbordpatroon en het resulterende objectglaasje werd blootgesteld aan het Herz antili-chaam. Dit experiment wordt toegelicht in de figuren 13A en 13B.
In figuur 13A wordt een objectglaasje getoond dat gederivatiseerd is met de aminopropylgroep, in dit geval beschermd met tert-BOC (tert-bu-toxycarbonyl). Het objectglaasje werd behandeld met TFA ter verwijdering van de tert-BOC beschermende groep. E-aminocaproïnezuur, dat bij deze aminogroep met tert-BOC was beschermd, werd vervolgens op de aminopropyl-groepen gekoppeld. Het aminocaprolnezuur dient als een ruimte verschaffend middel tussen de aminogroep en het te synthetiseren peptide. Het aminoeinde van het ruimte leverende middel werd gedeprotecteerd en aan NVOC-leucine gekoppeld. Het totale objectglaasje werd vervolgens belicht onder toepassing van 12 mW van 325 nm breedband belichting. Het objectglaasje werd vervolgens gekoppeld met NVOC-fenylalanine en gewassen. Het totale objectglaasje werd weer belicht, vervolgens gekoppeld aan NVOC-glycine en gewassen. Het objectglaasje werd weer belicht en gekoppeld aan NVOC-glycine voor de vorming van de sequentie die in het laatste gedeelte van figuur 13A is getoond.
Zoals getoond in figuur 13B werden vervolgens alternerende gebieden van het objectglaasje belicht onder toepassing van een projectiedruk bij gebruik van een 500 x 500 μΐη schaakborsmasker; op deze wijze werd de aminogroep van glycine slechts in de verlichte gebieden blootgesteld. Wanneer de daaropvolgende koppelingsreactietrap uitgevoerd werd, werd NV0C-tyrosine toegevoegd, en dit koppelde zich slechts op die plaatsen die belicht waren. Het totale objectglaasje werd vervolgens belicht ter verwijdering van alle NVOC-groepen, waarbij een schaakbord van YGGFL in de belichte oppervlakken en in de andere oppervlakken, GGFL, achterbleef. Het Herz antilichaam (dat het YGGFL herkent, maar niet GGFL) werd vervolgens toegevoegd, gevolgd door geite anti-muis fluoresceïneconjugaat.
De resulterende fluorescentie aftasting vertoonde donkere gebieden, bevattende het tetrapeptide GGFL, dat niet herkend wordt door het Herz antilichaam (en derhalve is er geen binding van het geite anti-muis antilichaam met het fluoresceïneconjugaat) en rode gebieden waarin YGGFL aanwezig was. Het YGGFL pentapeptide wordt herkend door het Herz antilichaam en derhalve is er antilichaam in de belichte gebieden voor het fluorescine-geconjugeerde geite anti-muis antilichaam om te herkennen.
Soortgelijke patronen voor een 50 urn masker, toegepast in direct contact ("proximiteit/druk") met het substraat verschafte een patroon dat duidelijker was en de hoeken van het schaakbordpatroon waren "touching” als resultaat van het feit dat het masker in direct contact was gebracht met het substraat (hetgeen de stijging in resolutie onder toepassing van deze techniek reflecteert).
J Monomeer-bi.i-monomeer synthese van YGGFL en PGGFL
Een synthese onder toepassing van een 50 pm schaakbordmasker, soortgelijk aan dat getoond in figuur 13, werd uitgevoerd. Echter werd P toegevoegd aan de GGFL plaatsen op het substraat via een additionele koppelingstrap. P werd geaddeerd door het blootstellen van beschermd GGFL aan licht door een masker, en daaropvolgende blootstelling aan P op de hierboven vermelde wijze. De helft van de gebieden op het substraat bevatten derhalve YGGFL en de resulterende helft bevatte PGGFL.
De fluorescentieuitzetting voor dit experiment toonde dat de gebieden weer gemakkelijk onderscheiden kunnen worden tussen die waarin het binding wel en niet heeft plaatsgevonden. Dit experiment demonstreerde dat antilichamen in staat zijn tot het herkennen van een specifieke sequentie en dat het herkennen niet van de lengte afhankelijk is.
K Monomeer-bii-monomeer synthese van YGGFL en YPGGFL
Teneinde verder de operabiliteit van de uitvinding aan te tonen werd een 50 um schaakbordpatroon van alternerend YGGFL en YPGGFL gesynthetiseerd op een substraat onder toepassing van technieken die gelijk waren aan die welke hierboven zijn vermeld. De resulterende fluorescen-tieafzetting toonde dat het antilichaam duidelijk in staat was tot het herkennen van de YGGFL sequentie en zich niet significant op de YPGGFL gebieden bond.
L Synthese van een baan van zestien verschillende aminozuur- seauenties en bepaling van de relatieve bindings af fini tei t aan Herz antilichaam
Onder toepassing van technieken welke soortgelijk zijn aan die welke hierboven zijn vermeld, werd een baan van zestien verschillende aminozuursequenties (viermaal gerepliceerd) gesynthetiseerd op elk van twee glassubstraten. De sequenties werden gesynthetiseerd door het binden van de sequentie NVOC-GFL over het gehele oppervlak van de glaasjes. Bij toepassing van een reeks maskers werden twee lagen aminozuren vervolgens selectief op het substraat aangebracht. Elk gebied had dimensies van 0,25 cm x 0,0625 cm. Het eerste objectglaasje bevatte aminozuursequenties, bevattende slechts L aminozuren terwijl hét tweede objectglaasje geselecteerde D aminozuren bevatte. De figuren l4A en 14b illustreren een kaart van de diverse gebieden op respectievelijk het eerste en het tweede glaasje. De in figuren 14A en l4B getoonde patronen werden viermaal op elk objectglaasje gedupliceerd. De glaasjes werden vervolgens blootgesteld aan het Herz antilichaam en aan met fluorescine gemerkt geite anit-muis antilichaam.
Een fluorescentieuitzetting van het eerste objectglaasje, dat slechts L aminozuren bevatte, vertoonde rode gebieden (aangevende een sterke binding, dat wil zeggen 149.000 tellingen of meer) en zwarte gebieden (aangevende weinig of geen binding van het Herz antilichaam, dat wil zeggen 20.000 bindingen of minder). De sequentie YGGFL werd duidelijk het sterkste herkend. De sequenties YAGFL en YSGFL vertoonden ook een sterke herkenning van het antilichaam. In tegenstelling daarmee vertoonden de meeste van de resterende sequenties weinig of geen binding. De vier duplicaatporties van het objectglaasje waren buitengewoon consistent ten aanzien van de daarin getoonde hoeveelheid binding.
Een fluorescentieafzetting van het D aminozuurglaasje gaf aan dat de sterkste binding vertoond werd door de YGGFL sequentie. Een significante binding werd ook gedetecteerd voor YaGFL, YsGFL en YpGFL. De resterende sequentie toonde minder binding met het antilichaam. Er werd een lage bindingsefficiency van de sequentie YGGFL waargenomen.
Tabel 6 vat de diverse onderzochte sequenties samen in de volgorde van relatieve fluorescentie, welke informatie verschaft ten aanzien van de relatieve bindingsaffiniteit.
VIII. Toelichtende alternatieve uitvoeringsvorm
Volgens een alternatieve uitvoeringsvorm van de uitvinding verschaffen de methoden voor het binden aan het oppervlak een gekooid bin-dingslid, dat in zijn gekooide vorm een relatief lage affiniteit heeft voor andere potentieel bindende species, zoals receptoren en specifieke bindingsstoffen. Dergelijke technieken worden vollediger beschreven in de verwante hangende aanvrage Serial nr. 404.920, ingediend 8 september, 1989. en hierin voor alle doeleinden door verwijzing opgenomen.
Volgens deze alternatieve uitvoeringsvorm verschaft de uitvinding methoden voor het vormen van voorgedefinieerde gebieden op een oppervlak van een vaste drager, waarbij de voorgedefinieerde gebieden in staat zijn tot het immobiliseren van receptoren. De methoden maken gebruik van gekooide bindingsleden welke gebonden zijn aan het oppervlak om de selectieve activering van de voorgedefinieerde gebieden mogelijk te maken. De gekooide bindingsleden worden in vrijheid gesteld om te werken als bindingsleden welke uiteindelijk in staat zijn tot het binden van receptoren bij selectieve activering van de voorgedefinieerde gebieden. De geactiveerde bindingsleden worden vervolgens gebruikt voor het immobiliseren van specifieke moleculen, zoals receptoren op het voorgedefinieerde gebied van het oppervlak. De bovenstaande procedure wordt herhaald op dezelfde of verschillende plaatsen op het oppervlak ter verschaffing van een oppervlak dat bereid is met een veelvoud van gebieden op het oppervlak, bevattende bijvoorbeeld dezelfde of verschillende receptoren. Wanneer op deze wijze geïmmobiliseerde receptoren een verschillende affiniteit voor een of meer liganden hebben, kan een screening en een bepaling van de liganden worden uitgevoerd in de gebieden van het oppervlak dat de receptoren bevat.
De alternatieve uitvoeringsvorm kan gebruik maken van nieuwe gekooide bindingsleden welke aan het substraat zijn gebonden. Gekooide (niet geactvieerde) leden hebben een betrekkelijk lage affiniteit voor receptoren van stoffen die zich specifiek binden aan niet gekooide bindingsleden in vergelijking met de corresponderende affiniteiten van geactiveerde bindingsleden. Derhalve worden de bindingsleden beschermd tegen reactie totdat een geschikte energiebron toegepast wordt op de gebieden van het oppervlak welke men wenst te activeren. Bij toepassing van een geschikte energiebron worden de gekooide groepen labiel waardoor het geactiveerde lid wordt aangeboden. Licht zal een een typische energiebron zijn.
Wanneer eenmaal de bindingsleden op het oppervlak geactiveerd zijn, kunnen zij aan een receptor worden gebonden. De gekozen receptor kan een monoklonaal antilichaam, een nucleïnezuursequentie, een geneesmiddelre-ceptor, enz., zijn. De receptor zal gebruikelijk, ofschoon niet altijd, worden bereid om daaraan, direct of indirect, het binden aan een bin-dingslid mogelijk te maken. Een specifiek bindende stof met een sterke bindende affiniteit voor het bindingslid en een sterke affiniteit voor de receptor of een conjugaat van de receptor kan bijvoorbeeld desgewenst worden gebruikt als een brug tussen de bindingsleden en de receptoren. De methode maakt gebruik van een receptor die zodanig is bereid dat de receptor zijn activiteit ten opzichte van een bijzonder ligand behoudt.
Het gekooide bindingslid, gebonden aan het vaste substraat, is bij voorkeur een door licht activeerbaar biotinecomplex, dat wil zeggen een biotinemolecuul dat chemisch gemodificeerd is met door licht activeerbare beschermende groepen, zodat het een significant verlaagde bindingsactivi-teit voor avidine of avidineanalogen heeft dan dit bij natuurlijk biotine het geval is. In een de voorkeur verdienende uitvoeringsvorm zullen de beschermende groepen, gelocaliseerd in een vooraf bepaald gebied van het oppervlak, verwijderd worden bij toepassing bij een geschikte stral^ngs-bron voor het leveren van bindingsleden, welke biotine of een functioneel analoge verbinding zijn met praktisch dezelfde affiniteit voor avidine of avidineanalogen zoals dit met biotine het geval is.
Claims (46)
1. Werkwijze voor het bereiden van sequenties op een substraat, omvattende de trappen van: a) blootstellen van een eerste gebied van het substraat aan een activator ter verwijdering van een beschermende groep; b) blootstellen van ten minste het eerste gebied aan een eerste monomeer; c) blootstellen van een tweede gebied aan een activator ter verwijdering van een beschermende groep; en d) blootstellen van ten minste het tweede gebied aan een tweede monomeer.
2. Werkwijze volgens conclusie 1, waarin de trappen van het blootstellen aan een activator gebruik maken van een activator, gekozen uit de groep, bestaande uit ionenstralen, electronenstralen, gammastralen, röntgenstralen, ultraviolette straling, licht, infrarode straling, microgolven, electrische stromen, radiogolven, en combinaties ervan.
3· Werkwijze volgens conclusie 1, waarin de beschermende groepen lichtgevoelige beschermende groepen zijn.
4. Werkwijze volgens conclusie 1, waarin de trappen van het blootstellen aan een activator trappen zijn van het toepassen van licht op geselecteerde gebieden van het substraat.
5. Werkwijze volgens conclusie 1, waarin het eerste en het tweede monomeer aminozuren zijn.
6. Werkwijze volgens conclusie 1, verder omvattende een trap van het screenen van sequenties op het substraat ten aanzien van affiniteit met een receptor, welke screeningstrap verder de trap omvat van het blootstellen van het substraat aan de receptor en het onderzoeken ten aanzien van de aanwezigheid van de receptor in het eerste en in het tweede gebied.
7. Werkwijze volgens conclusie 6, waarin de receptor een antili-chaam is..
8. Werkwijze volgens conclusie 1, waarin het substraat gekozen wordt uit de groep, bestaande uit gepolymeriseerde Langmuir Blodgett film, gefunctionaliseerd glas, germanium, silicium, polymeren, (poly)te-trafluoretheen, polystyreen, galliumarsenide, en combinaties ervan.
9· Werkwijze volgens conclusie 1, waarin de beschermende groep gekozen is uit de groep bestaande uit ortho-nitrobenzylderivaten, 6-ni-troveratryloxycarbonyl, 2-nitrobenzyloxycarbonyl, cinnamoylderivaten, en mengsels ervan.
10. Werkwijze volgens conclusie 1, waarin het eerste en het tweede gebied elk een totaalgebied van minder dan 1 cm2 hebben.
11. Werkwijze volgens conclusie 1, waarin het eerste en het tweede gebied elk een totaal gebied hebben van tussen ongeveer 1 μΐη2 en 10.000 pm2.
12. Werkwijze volgens conclusie 4, waarin het licht monochromatisch coherent licht is.
13· Werkwijze volgens conclusie 1, waarin de trappen van het blootstellen aan een activator uitgevoerd worden met een oplossing die met het substraat in contact is.
14. Werkwijze volgens conclusie 13, waarin de oplossing verder het eerste of het tweede monomeer omvat.
15. Werkwijze volgens conclusie 6, waarin de receptor verder een merkmiddel omvat, gekozen uit de groep bestaande uit radioactieve merk-middelen en fluorescerende merkmiddelen en waarin de trap van het onderzoeken ten aanzien van de aanwezigheid van de receptor een trap van het detecteren van het merkmiddel is.
16. Werkwijze volgens conclusie 11, waarin de trappen van het blootstellen aan een activator verder de trappen omvat van: a) aanbrengen van een masker naast het substraat welk masker nagenoeg transparante gebieden en nagenoeg opake gebieden heeft bij een golflengte van licht; b) illumineren van het masker met een lichtbron, welke lichtbron ten minste de golflengte van licht produceert.
17. Werkwijze volgens conclusie 1, waarin de trappen herhaald worden teneinde 103 of meer verschillende sequenties op het substraat te synthetiseren.
18. Werkwijze volgens conclusie 1, waarin de trappen herhaald worden teneinde 106 of meer verschillende sequenties op het substraat te synthetiseren.
19. Werkwijze voor het synthetiseren van een groot aantal chemische sequenties, welke chemische sequenties ten minste een eerste en een tweede monomeer omvatten, omvattende de trappen van: a) bij een eerste gebied op een substraat met ten minste een eerste en een tweede gebied, welk eerste en tweede gebied een substraat beschermende groep omvatten, activeren van het eerste gebied ter verwijdering van de substraat beschermende groep in het eerste gebied; b) blootstellen van het eerste monomeer aan het substraat, welk eerste monomeer verder omvat een eerste monomeer beschermende groep, welk eerste monomeer gebonden is aan het eerste gebied; c) activeren van het tweede gebied ter verwijdering van de substraat beschermende groep in het tweede gebied, d) blootstellen van het tweede monomeer aan het substraat, welk tweede monomeer verder omvat een tweede monomeer beschermende groep, welk tweede monomeer zich aan het tweede gebied bindt; e) activeren van het eerste gebied ter verwijdering van de eerste monomeer beschermende groep; f) blootstellen van een derde monomeer aan het substraat, welk derde monomeer zich bindt aan het eerste gebied teneinde een eerste sequentie te vormen; g) activeren van het tweede gebied ter verwijdering van de tweede monomeer beschermende groep; en h) blootstellen van een vierde monomeer aan het substraat, welk vierde monomeer zich bindt aan het tweede gebied teneinde een tweede sequentie te vormen, welke tweede sequentie verschillend is van de eerste sequentie.
20. Werkwijze voor het synthetiseren van een groot aantal chemische sequenties, welke chemische sequenties ten minste een eerste en een tweede monomeer omvatten, omvattende de trappen van: a) deactiveren op een substraat met ten minste een eerste en een tweede gebied het eerste gebied ter verschaffing van een eerste beschermende groep in het eerste gebied; b) blootstellen van het eerste monomeer aan het substraat, welk eerste monomeer zich bindt aan het tweede gebied; c) verwijderen van de beschermende groep in het eerste gebied; d) deactiveren van het tweede gebied ter verschaffing van een tweede beschermende groep in het tweede gebied; e) blootstelling van het tweede monomeer aan het substraat, welk tweede monomeer zich bindt aan het eerste gebied; f) verwijderen van de beschermende groep in het tweede gebied; g) deactiveren van het eerste gebied ter verschaffing van een beschermende groep in het eerste gebied; h) blootstellen van een derde monomeer aan het substraat, welk derde monomeer zich bindt aan het tweede gebied voor de vorming van een eerste sequentie; i) verwijderen van de beschermende groep in het eerste gebied; en j) blootstellen van een vierde monomeer aan het substraat, welk vierde monomeer zich bindt aan het eerste· gebied voor de vorming van een tweede sequentie, welke tweede sequentie verschillend is van de eerste sequentie.
21. Werkwijze voor het synthetiseren van ten minste een eerste polymeersequentie en een tweede polymeersequentie op een substraat, welke eerste polymeersequentie een verschillende monomeersequentie van de tweede polymeersequentie heeft, omvattende de trappen van: a) inlassen van een eerste masker tussen het substraat en een energiebron, welk masker eerste en tweede gebieden heeft, welke eerste gebieden de passage van energie uit de bron mogelijk maken, welke tweede gebieden energie uit deze bron blokkeren; b) richten van energie uit deze bron op het substraat, welke energie een beschermende groep uit de eerste delen van het eerste polymeer onder deze eerste gebieden van het eerste masker verwijdert; c) blootstellen van een tweede portie van het eerste polymeer aan het substraat voor het leveren van een eerste polymeersequentie; d) inlassen van een tweede masker tussen het substraat en de energiebron, welk tweede masker eerste gebieden en tweede gebieden heeft; e) richten van energie uit deze bron op het substraat, welke energie de beschermende groep onder deze eerste gebieden van het tweede masker uit de eerste gebieden van het tweede polymeer verwijdert; en f) blootstellen van een tweede gedeelte van het tweede polymeer aan het substraat, welk tweede gedeelte van het tweede polymeer zich bindt aan het eerste gedeelte van het tweede polymeer voor het leveren van een tweede polymeersequentie.
22. Werkwijze voor het screenen van een groot aantal aminozuurse-quenties voor het binden met een receptor, omvattende de trappen van: a) op een glasplaat met ten minste een eerste oppervlak, welk eerste oppervlak ten minste een licht beschermend materiaal omvat, gekozen uit de groep bestaande uit nitroveratryloxycarbonyl en nitrobenzyloxycar-bonyl, omzetten van ten minste een eerste oppervlak met ter-butoxycarbo-nyl voor opslag, welke glasplaat praktisch transparant is voor ten minste ultraviolet licht; b) blootstellen van ten minste een eerste oppervlak aan TFA ter verwijdering van het tert-butoxycarbonyl; c) plaatsen van de glasplaat op een reactor, welke reactor een reactorruimte omvat waarvan ten minste een eerste oppervlak blootgesteld is aan de reactorruimte; d) aanbrengen van een masker op een eerste positie op de glasplaat. welk masker eerste locaties en tweede locaties omvat, welke eerste locaties praktisch transparant zijn voor ten minste utraviolet licht en welke tweede locaties praktisch opaak zijn voor ten minste ultraviolet licht, welke tweede locaties een licht blokkerend materiaal op een eerste oppervlak van het masker omvatten, welk eerste oppervlak van het masker in contact is gebracht met de glasplaat; e) vullen van de reactorruimte met een reactieoplossing; f) illumineren van het masker met ten minste ultraviolet licht, welk ultraviolet licht het licht beschermende materiaal van ten minste een eerste oppervlak van de glasplaat onder de eerste locaties van het masker verwijdert; g) blootstellen van het eerste oppervlak aan een eerste aminozuur, welk eerste aminozuur zich bindt aan de gebieden van ten minste een eerste oppervlak waarvan het licht beschermende materiaal was verwijderd, welk eerste aminozuur de licht beschermende groep bij een terminus ervan omvat; h) plaatsen van een' masker in contact met de glasplaat op een tweede positie; i) illumineren van het masker met ten minste ultraviolet licht, welk ultraviolet licht het licht beschermende materiaal uit ten minste een eerste oppervlak van de glasplaat onder de eerste locaties van het masker verwijdert; j) blootstellen van ten minste een eerste oppervlak aan een tweede aminozuur, welk tweede aminozuur zich bindt aan gebieden van ten minste een eerste oppervlak waarvan het licht beschermende materiaal was verwijderd, welk tweede aminozuur de licht beschermende groep bij een terminus ervan omvat; k) plaatsen van een masker in contact met de glasplaat op een derde positie; l) illumineren van het masker met ten minste ultraviolet licht, welk ultraviolet licht het licht beschermende materiaal uit ten minste een eerste oppervlak van de glasplaat onder de eerste locaties van het masker verwijdert; m) blootstellen van ten minste een eerste oppervlak aan een derde aminozuur, welk derde aminozuur zich bindt aan gebieden van ten minste een eerste oppervlak waarvan het licht beschermende materiaal was verwijderd; n) plaatsen van een masker in contact met de glasplaat op een vierde positie; o) illumineren van het masker met ten minste ultraviolet licht, welk ultraviolet licht het licht beschermende materiaal van ten minste een eerste oppervlak van de glasplaat onder de eerste locaties van het masker verwijdert; p) blootstellen van ten minste een eerste oppervlak aan een vierde aminozuur, welk vierde aminozuur zich bindt aan de gebieden van ten minste een eerste oppervlak waarvan het licht beschermende materiaal was verwijderd, welk eerste oppervlak ten minste omvat eerste, tweede, derde en vierde aminozuursequenties; q) blootstellen van ten minste een eerste oppervlak aan een van belang zijnd antilichaam, welk van belang zijnd antilichaam zich sterker bindt aan ten minste een van de eerste, tweede, derde of vierde aminozuursequenties ; r) blootstellen van ten minste een eerste oppervlak aan een receptor, welke receptor het van belang zijnde antilichaam herkent en zich aan veelvoudige locaties ervan bindt, welke receptor fluoresceïne omvat; s) blootstellen van ten minste een eerste oppervlak aan licht, welk eerste oppervlak fluoresceert in ten minste een gebied waar de sterker gebonden aminozuursequentie zich bevindt; en t) detecteren en registreren van de intensiteit van gefluoresceerd licht als een functie van de locatie over ten minste een eerste oppervlak.
23. Werkwijze voor het identificeren van ten minste een peptidese-quentie voor het binden met een receptor, omvattende de trappen van: a) op een substraat met een veelvoud van polypeptiden, die elk een door licht verwijderbare beschermende groep bevatten, bestralen van de eerste geselecteerde polypeptiden ter verwijdering van de beschermende groep; b) het in contact brengen van de polypeptiden met een eerste aminozuur voor het vormen van een eerste sequentie, tweede polypeptiden op het substraat omvattende een tweede sequentie; en c) identificeren welke van de eerste of tweede sequentie zich met de receptor verbindt.
24. Apparaat voor de bereiding van een veelvoud van polymeren, omvattende: a) een substraat met een oppervlak, welk oppervlak een reactief gedeelte omvat, welk reactief gedeelte geactiveerd wordt bij blootstelling aan een energiebron teneinde met een monomeer te reageren; en b) middelen voor het selectief beschermen en blootstellen van ge deelten van het oppervlak tegen de energiebron.
25. Apparaat volgens conclusie 24, waarin het reactieve gedeelte verder een beschermende groep omvat, welke beschermende groep de vorm heeft van:
waarin Rx is alkoxym alkyl, halogeen, aryl, alkenyl, of waterstof; R2 is alkoxy, alkyl, halogeen, aryl, nitro of waterstof; R3 is alkoxy, alkyl, halogeen, nitro, aryl of waterstof; R4 is alkoxy, alkyl, waterstof, aryl, halogeen of nitro; en R5 is alkyl, alkynyl, cyano, alkoxy, waterstof, halogeen, aryl of alkenyl.
26. Apparaat volgens conclusie 24, waarin het reactieve gedeelte verder verbindingsmoleculen omvat.
27. Apparaat volgens conclusie 26, waarin de verbindingsmoleculen geselecteerd worden uit de groep bestaande uit ethyleenglycololigomeren, diaminen, dizuren, aminozuren en combinaties ervan.
28. Apparaat volgens conclusie 24, waarin het middel voor het selectief beschermen verder een masker omvat.
29. Apparaat volgens conclusie 24, waarin het middel voor het selectief beschermen verder een lichtbuis omvat.
30. Apparaat volgens conclusie 24, waarin de energiebron een lichtbron is.
31. Apparaat volgens conclusie 24, waarin het reactieve gedeelte verder een samenstelling omvat, gekozen uit de groep bestaande uit nitro-veratryloxycarbonyl, nitrobenzyloxycarbonyl, dimethyldimethoxybenzyl-oxycarbonyl, 5~broom-7-nitroindolinyl, hydroxy-2-methylcinnamoyl en 2-oxymethyleenanthrachinon.
32. Apparaat voor de bereiding van een substraat met een veelvoud van aminozuursequenties daarop, welk apparaat omvat: a) een substraat met een oppervlak; b) een beschermende groep op het oppervlak, welk beschermende groep verwijderbaar is bij blootstelling aan een energiebron, welke energiebron gekozen is uit de groep bestaande uit licht, electronenstralen en rönt- genstralen; c) middelen voor het richten van de energiebron op geselecteerde locaties op het oppervlak; en d) middelen voor het blootstellen van aminozuren aan het oppervlak voor het binden aan het oppervlak.
33· Apparaat voor het screenen van polymeren, omvattende een substraat met een oppervlak, welk oppervlak ten minste twee vooraf bepaalde gebieden omvat, welke vooraf bepaalde gebieden verschillende monomeerse-quenties daarop bevatten, welke vooraf bepaalde gebieden elk een gebied bezetten van minder dan ongeveer 0,1 cm2.
34. Apparaat volgens conclusie 33. waarin het oppervlak minder dan ongeveer 0,01 cm2 is.
35. Apparaat volgens conclusie 33. waarin het oppervlak minder dan 10.000 pm2 is.
36. Apparaat volgens conclusie 33. waarin het oppervlak minder dan ongeveer 100 pm2 is.
37· Apparaat volgens conclusie 33. 34, 35 of 36, waarin de mono-meersequenties nagenoeg zuiver in de vooraf gedefinieerde gebieden aanwezig zijn.
38. Substraat voor het screenen op biologische activiteit, welk substraat 103 of meer verschillende liganden op een oppervlak daarvan in vooraf bepaalde gebieden omvat.
39· Substraat volgens conclusie 38, waarin het substraat 104 of meer verschillende liganden in vooraf bepaalde gebieden omvat.
40. Substraat volgens conclusie 38, waarin het substraat 105 of meer verschillende liganden in vooraf bepaalde gebieden omvat.
41. Substraat volgens conclusie 38, waarin het substraat 106 of meer verschillende liganden in vooraf bepaalde gebieden omvat.
42. Substraat volgens conclusie 38, 39, 40 of 4l, waarin de liganden peptiden zijn.
43. Substraat volgens conclusie 33, waarin de liganden praktisch zuiver in de vooraf gedefinieerde gebieden zijn.
44. Apparaat voor het screenen op biologische activiteit, omvattende: a) een substraat omvattende een veelvoud van polymeersequenties, welke polymeersequenties gebonden zijn aan een oppervlak van het substraat op bekende locaties op het substraat, waarbij elke sequentie een oppervlak bezet van minder dan ongeveer 0,1 cm2; b) middel voor het blootstellen van het substraat aan een receptor, welke receptor gemerkt is met een fluorescerend merkmiddel, welke receptor zich bindt aan ten minste een van de sequenties; en c) middel voor het detecteren van een locatie van het fluorescerende merkmiddel op het substraat.
45· Apparaat voor het vormen van een veelvoud van polymeersequen-ties, omvattende: a) een substraat, welk substraat ten minste een eerste oppervlak en een tweede oppervlak heeft, welk tweede oppervlak een door licht verwijderbaar beschermend materiaal omvat, welk substraat praktisch transparant is voor ten minste licht van een eerste golflengte; b) een reactorlichaam, welk reactorlichaam een stijgend oppervlak heeft met een holte voor een reactievloeistof daarin, welk tweede oppervlak gehandhaafd wordt in een afgedichte verhouding ten aanzien van het stijgende oppervlak; en c) een lichtbron voor het vormen van licht van ten minste een eerste golflengte en gericht op een oppervlak van het substraat.
46. Apparaat voor het detecteren van fluorescerende gemerkte gebieden op een substraat, omvattende: a) een lichtbron voor het richten van licht op een oppervlak van het substraat; b) een middel voor het detecteren van licht dat uit het oppervlak als respons op de lichtbron is gefluoresceerd; c) middel voor het brengen van het substraat van een eerste positie in een tweede positie; en d) middel voor het opslaan van gefluoresceerde lichtintensiteit als een functie van de locatie op het substraat, welk middel voor het opslaan verbonden is met een middel voor het overbrengen en het middel voor het detecteren. Uittreksel Werkwijze en inrichting voor de bereiding van gewenste sequenties op een substraat op bekende locaties. Bekende locaties (10) van een substraat (2) worden bestraald door middel van een masker (8) teneinde een materiaal (4) voor binding te activeren. Vervolgens wordt het substraat blootgesteld aan een eerste materiaal (12) om daaraan te binden. Tweede locaties (14) worden vervolgens bestraald door een masker en blootgesteld aan een tweede materiaal (16). Een groot aantal sequenties kan worden gevormd door selectieve bestraling van het substraat, gevolgd door toepassing van geselecteerde materialen. Een reactorsysteem en een fluorescent iedetectiesys teem worden ook geopenbaard.
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- 1990-06-07 DK DK90909187T patent/DK0476014T3/da active
- 1990-06-07 HU HU9004730A patent/HU223462B1/hu not_active IP Right Cessation
- 1990-06-07 CA CA 2391491 patent/CA2391491A1/en not_active Abandoned
- 1990-06-07 EP EP19980203518 patent/EP0902034A3/en not_active Withdrawn
- 1990-06-07 KR KR1019910701791A patent/KR970001577B1/ko not_active IP Right Cessation
- 1990-06-07 EP EP90909187A patent/EP0476014B1/en not_active Expired - Lifetime
- 1990-06-07 KR KR1019960705897A patent/KR970001578B1/ko not_active IP Right Cessation
- 1990-06-07 BR BR9007425A patent/BR9007425A/pt not_active IP Right Cessation
- 1990-06-07 EP EP19990202455 patent/EP0953835A1/en not_active Withdrawn
- 1990-06-07 NL NL9022056A patent/NL191992C/nl not_active IP Right Cessation
- 1990-06-07 DE DE1990632888 patent/DE69032888T2/de not_active Revoked
- 1990-06-07 DK DK94200059T patent/DK0619321T3/da active
- 1990-06-07 CA CA 2054706 patent/CA2054706C/en not_active Expired - Lifetime
- 1990-06-29 TW TW79105390A patent/TW434254B/zh not_active IP Right Cessation
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1991
- 1991-12-04 FI FI915723A patent/FI109130B/fi active
- 1991-12-06 NO NO914826A patent/NO301233B1/no not_active IP Right Cessation
- 1991-12-06 GB GB9125996A patent/GB2248840B/en not_active Expired - Lifetime
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1992
- 1992-03-12 US US07/850,356 patent/US5405783A/en not_active Expired - Lifetime
- 1992-09-30 US US07/954,646 patent/US5445934A/en not_active Expired - Lifetime
- 1992-09-30 US US07/954,519 patent/US5510270A/en not_active Expired - Lifetime
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1994
- 1994-11-04 AU AU77655/94A patent/AU672723B2/en not_active Expired
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1995
- 1995-01-26 SG SG13595A patent/SG13595G/en unknown
- 1995-04-27 HK HK64195A patent/HK64195A/xx not_active IP Right Cessation
- 1995-04-27 HK HK61395A patent/HK61395A/xx not_active IP Right Cessation
- 1995-06-01 US US08/456,598 patent/US6225625B1/en not_active Expired - Lifetime
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1996
- 1996-12-04 JP JP8324451A patent/JPH1121293A/ja not_active Withdrawn
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1997
- 1997-12-09 US US08/999,188 patent/US6491871B1/en not_active Expired - Fee Related
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1998
- 1998-08-04 US US09/129,470 patent/US6329143B1/en not_active Expired - Fee Related
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1999
- 1999-02-25 JP JP11049217A patent/JPH11315095A/ja not_active Withdrawn
- 1999-04-15 US US09/292,455 patent/US6261776B1/en not_active Expired - Fee Related
- 1999-11-17 US US09/442,028 patent/US6291183B1/en not_active Expired - Fee Related
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2000
- 2000-10-16 US US09/690,191 patent/US6403957B1/en not_active Expired - Fee Related
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2001
- 2001-12-06 US US10/004,501 patent/US6630308B2/en not_active Expired - Fee Related
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2002
- 2002-03-15 US US10/098,203 patent/US6646243B2/en not_active Expired - Fee Related
- 2002-05-15 US US10/150,290 patent/US6660234B2/en not_active Expired - Fee Related
- 2002-05-20 US US10/152,440 patent/US6747143B2/en not_active Expired - Fee Related
- 2002-09-30 US US10/259,391 patent/US20030082831A1/en not_active Abandoned
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2003
- 2003-04-16 JP JP2003112204A patent/JP2004002386A/ja not_active Withdrawn
- 2003-05-02 US US10/428,628 patent/US20030235853A1/en not_active Abandoned
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2004
- 2004-11-23 US US10/997,439 patent/US20050148027A1/en not_active Abandoned
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2005
- 2005-01-21 US US11/040,520 patent/US20050214828A1/en not_active Abandoned
- 2005-01-26 JP JP2005018992A patent/JP2005112867A/ja not_active Withdrawn
- 2005-08-25 US US11/212,551 patent/US20060210452A1/en not_active Abandoned
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