Wellenlänge [nm]
8. Experimenteller Teil
8.2. Versuchs- und Messbedingungen
8.2.4. Reflektometrische Inteferenzspektroskopie (RIfS)
Über einen Y-Lichtleiter (Quarz, 1mm Durchmesser, Laser Components) wurde Weißlicht von einer Wolfram-Halogenlampe (20 W) zu einem Transducer-Chip geleitet. Das reflektierte Licht wurde über denselben Lichtleiter zu einem Diodenzeilenspektrometer (Spekol 1100, Analytik Jena) gesendet und dort spektral aufgezeichnet.
Die Auswertung erfolgte über einen PC. Der Transducer-Chip befindet sich in einer Flusszelle und wird mittels Glycerin mit dem Lichtleiter in Kontakt gebracht.
Flusszelle (RIfS)
Sie besteht aus einem Transducer-Chip (1212 mm², d ~1 mm, Glas beschichtet mit 10 nm Ta2O5 und 330 nm SiO2, Schott, siehe Abbildung 8.7), einer Silikonmaske mit elliptischer Öffnung 57 mm, 0.5 mm dick und einem Quarzfenster (Hellma) mit 2 Kanülen, durch die mittels Schläuchen und einer Schlauchpumpe (Ismatec, 0.5 ml/min) Lösung gepumpt werden kann.
Abbildung 8.7: Transducer-Chip.
Bestrahlungsbedingungen (RIfS)
Die Bestrahlungen wurden mit der zu Beginn des Kapitels beschriebenen UV-Lampe (Interfe-renzfilter 365 nm, I = 6.098 10-8 Es-1) über einen Lichtleiter durchgeführt. Das Ende des zweiten Lichtleiters kann zur Belichtung der Substrate von oben in die Flusszelle eingeführt werden.
Ein Kantenfilter (420 nm) vor dem Diodenzeilenspektrometer blockiert das UV-Licht, damit die Messung nicht gestört wird.
Oberflächenmodifizierung der RIfS-Transducerchips
1) Reinigung
- Transducer-Chips etwa 1 min in 6 M NaOH einlegen - mit Millipore Wasser spülen
- Transducer-Chips 15 min in Piranha-Lösung (60% H2SO4 und 40% H2O2, frisch herstellen!) geben und ins Ultraschallbad stellen
- mit Millipore Wasser und Ethanol p.a. spülen und mit Stickstoff trockenblasen 2) Silanisierung mit GOPTS (wasserfrei)
- Transducer-Chips werden paarweise verarbeitet
- auf einen der gereinigten Transducer-Chips 10 µl GOPTS geben und mit einem zweiten Chip abdecken (d.h. die zu funktionalisierenden Seiten der Transducer-Chips aufeinander legen)
- in einer abgedeckten Petrischale 1 h einwirken lassen
- mit trockenem Aceton p.a. (H2O-Gehalt 0.1%) spülen und sofort mit Stick-stoff trockenblasen, schnell weiterverarbeiten
3) Funktionalisierung mit Dicarboxy-PEG
- 4 mg Dicarboxy-PEG in 1 ml Dichlormethan (trocken) lösen - Transducer-Chips werden einzeln verarbeitet
- 30 µl dieser Lösung auf dem Transducer-Chip gleichmäßig verteilen
- Transducer-Chip in eine trockene Petrischale geben und über Nacht im Ofen bei ~ 70 °C, nicht abgedeckt, stehen lassen
- mit Millipore Wasser und Ethanol p.a. spülen und mit Stickstoff trockenblasen 4) Aktivierung der Carboxy-Funktion über Aktivester
- Transducer-Chips werden paarweise verarbeitet
- Aktivierlösung: 7.5 mg NHS in 38 µl trockenem DMF lösen und 12 µl DIC zugeben → Lösung erst kurz vor Gebrauch herstellen !!!
- 10 µl dieser Lösung auf den funktionalisierten Transducer-Chip geben und ei-nen zweiten Transducer-Chip mit der funktionalisierten Seite darauf legen, 4 h in einer DMF-Dampfkammer liegen lassen
- Transducer-Chips mit trockenem DMF und trockenem Aceton spülen und mit Stickstoff trockenblasen
- schnell weiterarbeiten, Aktivester ist hydrolyseempfindlich 5) Anbindung der Probe über den Aminlinker
- Transducer-Chips werden paarweise verarbeitet
- ~ 1 - 2 mg Bt-NPPOC-NH2 in 50 µl trockenem DMF lösen
- 10 µl dieser Lösung auf den aktivierten Transducer-Chip im Dunklen bei Rot-licht geben und einen zweiten Transducer-Chip mit der aktivierten Seite darauf legen, über Nacht in einer abgedeckten DMF-Dampfkammer im Dunklen lie-gen lassen
- Transducer-Chips mit DMF p.a. und Aceton p.a. im Dunklen bei Rotlicht spü-len und mit Stickstoff trockenblasen
- Aufbewahrung der modifizierten Transducer-Chips in Aufbewahrungsbox im Exsikkator über Trockenmittel (Silica Orange), eingepackt in Alufolie
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