Etude de différentes méthodes de biofonctionnalisation pour la ...

III. Etudes du film Lan gmuir-Blodgett enzymatique
ATR-FTIR data and AFM results have been discussed. lt should be stressed here that the
information obtained from ATR experiments cannot be comparable to that obtained from AFM
experiments if the substrates used for the different experiments are not the same. Most authors
recognize that the organization of the film on the substrate differs dramatically according to nature of
the substrates. lt means that the need to use the same substrate or at least the same chemical
composition becomes evident. lt is why silanized germanium and silanized silica have been used in
this study. Meanwhile, it will be necessary to firstly confirm that the so-obtained surfaces will be quite
equivalent.
2. Materials and methods
2.1. Materials
Butyrylcholinesterase (BuChE) (EC 3.1.1.8., 13 Units per gram of solid, pseudo-cholinesterase,
from horse serum) purchased from Sigma Chemical Co. was used without any further purification.
Octadecylamine (ODA) dissolved in chloroform at a concentration of i mg mr1 used to form
monolayers on the subphase was bought from Aldrich Chemical Co.. Chloroform used for spreading of
ODA monolayers was an analytical grade reagent (+ 99% purity) from Merck.
Water used as a subphase was purified by using an Elga system including a reverse osmosis,
deionization, filtration and ultra-violet irradiation systems to give water resistance as high as 18.2 Mc.
Its pH was about 5.5 and its surface tension was 71.8 mN m at 25°C in air.
Oxidized silicon wafers provided by LAAS (Toulouse, France) with double identically polished faces
with a 1000 A SiO2 layer were used as our solid substrates for LB film transfer; they were cleaned
with acetone in an ultrasonic bath for 5 min and a sulphochromic acid solution in order to get rid of the
organic and inorganic contamination on the substrate surfaces and then dried under a nitrogen flow
finally. Then, these hydrophilic and clean substrates were treated with 2 mM OTS (noctadecyltrichlorosilane,
98%, Merck) in CCL for 30 min at 4°C and then heated for 2 hours in oven at
100°C 7,8 to make their surfaces hydrophobic by the "SAMs" (SeIf-Assembled-Monolayers) method. At
last the substrates were rinsed with Cd4 and were then ready to use.
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