Examination of bilayer lipid membranes for 'pin-hole' character

BLM prepared on electrode substrates by supporting or tethering were tested for 'pin-hole' character, comparing data from cyclic voltammetry (CV), surface plasmon resonance (SPR) and rotating disc electrodes (RDE). 1-hexadecylamine tethered BLMs on SAM modified gold electrodes were compared with BLMs assembled on modified polyHEMA or sol-gel layers. BLM formation followed by SPR showed that the initial phase of the assembly was complete in 5-20 minutes and produced layers of thickness >5 nm, compared with the expected final BLM thickness of approximately 3 nm. The CVs of the K(3)[Fe(CN)(6)] couple were significantly suppressed irrespective of the method of BLM assembly, without major differences emerging for the different methods. However, data from the RDE distinguished the 'pin-hole' character of the different preparations. The data were consistent with incomplete initial (<1 h, SPR estimated BLM thickness >5 nm) vesicle fusion leaving 'pin-holes' of approximately 2 microm (HDA-11-mercaptoundecanoic acid (MUA) tethered BLM) to approximately 3 microm (tetraethylorthosilicate sol-gel supported BLM) followed by a slow maturation (>15 h; impedance spectroscopy estimated thickness approximately 3 nm) and lateral spreading and fusion, resulting in loss of 'pin-hole' character (<1 microm). The BLM could be used in conjunction with potentiometric measurement to observe the incorporation of nystatin into the BLM and the rate of incorporation adjusted according to original permeability of the BLM. The 'pin-hole-free' BLM construction with lowest permeability (TEOS supported, 4 x 10(-10) cm s(-1) compared with HDA-MUA, 3 x 10(-9) cm s(-1)) gave a potentiometric signal independent of bulk ion-concentration across 5 decades change in concentration. Formed on an ion-selective electrode, nystatin incorporation could be followed as a change in potential, over >2 h, whereas the TEOS supported BLM with permeability 1 x 10(-9) cm s(-1) shows nystatin incorporation within 1 h. In this instance, addition of ConA reduced the potential to the same value as prior to nystatin incorporation, consistent with nystatin channel closure.