Biomedical Engineering Reference
In-Depth Information
10.2.2.3
Protein Immobilization
Proteins were immobilized on the gold surfaces of the quartz crystals using two
different methodologies:
• direct covalent immobilization
• Covalent immobilization—Self assembled monolayer (SAM)
Before immobilization of proteins, the gold surface was rinsed with absolute etha-
nol and double distilled H
2
O in case of new crystals. If quartz crystals were to be
reused, they were cleaned by dipping the crystal in Piranha solution (1:3 = 30 %
H
2
O
2
: H
2
SO
4
) for few minutes to remove any organic residues from the surface.
10.2.2.4
Direct covalent immobilisation
For direct covalent immobilization, we modified the OBP by adding a cysteine
residue at the N-terminal end of the protein—allowing a stable bond to be formed
between the thiol group of cysteine and the gold surface. The immobilisation was
carried out by spreading 5 μl of proteins (2.3 mg ml
−1
) on the gold surface. The
protein was added about every 2 hours for at least four times. After that, the surface
was gently rinsed with sterile double distilled H
2
O and was left to dry. The same
procedure was applied for the gold electrode on the other side of the QCM.
10.2.2.5
Covalent Immobilization
OBPs were immobilised on the gold surface through covalent immobilization via
a Self assembled monolayer (SAM). Thioctic acid (TA) ((R)-5-(1,2-dithiolan-3-yl)
pentanoic acid). The sulphur atoms of the TA forms a strong bond with the gold,
while the other end of the molecule is free to bind to the proteins. TA (100 mM in
absolute ethanol) was dropped on the gold surface of the crystal, repeatedly ev-
ery 20 min, for at least 2 hours. The same procedure was used for the gold elec-
trode on other side of the quartz crystal. The SAM procedure was carried out in
a controlled environment under nitrogen. The quartz crystal was then rinsed with
an excess of absolute ethanol and was left to dry at air. In order to activate the
carboxylic acid groups of the SAM, 20 μl of a solution consisting of a mixture
of ethyl(dimethylaminopropyl)carbodiimide (30 mM) and N-hydroxysuccinimide
(60 mM) was placed on the gold surface for 2 hours. The solution was then rinsed
with distilled water and was dried at air. These procedures for the SAM activation
were applied to both sides of the gold surfaces for all the quartz crystals. The im-
mobilization of proteins on the activated SAM layer was carried out by pipetting the
OBP solution onto the gold surface and leaving it for 1 hour before gently rinsing
with distilled water and drying in air. The amount of protein deposited on to the gold
surface corresponded to about 10 µg of OBP.
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