Physics And Chemistry Basis Of Biotechnology - De Cuyper - tiera.ru

Britta Lindholm-Sethson
reservoir lipids were self-assembled to the gold electrode surface and then the bilayer
was formed in situ with a solvent/dilution technique. The length of the hydrophilic
spacer precisely determines the size of the reservoir. For a number of lipids the
capacitance and thickness of these bilayer membranes are in good agreement with
literature values. A potassium sensor with an analytical range over physiologically
relevant concentrations was obtained with valinomycin incorporated in this bilayer.
In the other paper the channel-forming ionophore was exploited as an essential part
of the biosensor [43]. Gramicidin spans only half a cellmembrane, and when a dimer is
formed with a gramicidin in the opposite part of the membrane a channel is opened that
passively transports monovalent cations. In this work two types of reservoir lipids were
employed, one of them a synthetic archaebacterial membrane-spanning lipid possessing
antibody fragments (Fab') and one half-membrane spanning lipid. Both of them were
attached to the gold surface via a hydrophilic spacer terminated with a disulfide group.
Gramicidin was inserted in the lower membrane and immobilised with a similar spacer
as the anchor lipids. Mobile gramicidin linked to antibodies with biotin/streptavidin
technology was incorporated in the upper leaflet. When a targeted analyte is added to
the surrounding electrolyte it crosslinks the Fabs on the membrane spanning lipid and
the gramicidin. Thus the gramicidin in the upper leaflet is immobilised and prevented
from forming a dimer with its partner in the lower leaflet. This is registered as a
decreasing conductivity in the admittance spectrum. The receptor in this sensor can be
varied and the authors claim that the technology has been applied successfully in the
detection of bacteria, virus particles, DNA, antibodies and electrolytes.
4.2. POLYMER CUSHIONED BILAYER LIPID MEMBRANES
The benefits of using a polymer cushioned lipid bilayer as a support for artificial cell
membranes were clearly pointed out by Sackmann [ 15], who in earlier work evaluated
frictional coefficients between the lipids in a Langmuir-Blodgett transferred film and a
supporting polyacrylamide film [110]. The bilayer was not continuously closed but the
structural, dynamic and thermodynamic properties were preserved after deposition.
The first contribution to this class of supported membranes came several years
earlier and involved mixed monolayers composed of phosphatidylcholine and
cholesterol that were transferred to a polyacrylamide hydrogel by Langmuir-Blodgett
techniques [111]. Only a fraction of the attempts to produce low conductance bilayers
was successful, but when they were transient ion currents were observed upon addition
of valinomycin or phloretin close to the membrane.
In a similar but improved configuration spincoated polyanhydride was used as a
support for Langmuir-Blodgett transfer of phospholipids to form supported bilayers
[ 112]. In both studies the stability problems with fragile black lipid membranes were
recognised, but also the need for an ionic reservoir on both sides of the artificial
membrane.
In a novel approach a copolymer consisting of a hydrophilic main chain,
hydrophobic lipidic parts and finally a disulfide group was self-assembled on a gold
surface with gold/sulphur bonds. Thus a polymer supported monolayer of a lipid-like
phase was formed and after fusion with phospholipid vesicles a bilayer was obtained
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