SSBII3
Probing diffusion and reactions at biologically-important interfaces using ultramicroelectrodes
Patrick R. Unwin, Julie V. Macpherson, Jie Zhang, Anna L. Barker, Marylou Gonsalves and Nicola J. Gray
Department of Chemistry
University of Warwick
Coventry CV 4 7AL
UK
The transport of ions and molecules through membranes and tissues plays a key role in many biologically-important systems. Ultramicroelectrodes (UMEs), in general, and scanning electrochemical microscopy (SECM), in particular, are proving powerful approaches for quantitatively probing and imaging diffusion processes with high spatial and temporal resolution, in a diversity of systems. This talk will describe several recent advances from our group on the application of SECM to biologically-important interfaces, from model membranes (monolayers at air/water interfaces) to tissue samples.
To understand how monolayers influence the rates of elementary processes such as the transfer of small molecules across interfaces and lateral diffusion along interfaces, a combined SECM-Langmuir trough instrument has been developed [1]. An attribute of the approach is that well-defined molecular interfaces can readily be assembled at the air/water interface. We have developed methods for quantitatively measuring (i) the lateral diffusion of redox-active amphiphiles and (ii) proton hopping rates at acid-base amphiphiles [2], including phospholipids. In the former case we have introduced a three potential-step method, involving "electrochemical bleaching", "recovery" and "analysis". This method, which may be viewed as an electrochemical analogue of fluorescence recovery after photobleaching, provides quantitative information on electron transfer kinetics between a tip-generated species and a surface-confined electroactive amphiphile, as well as the surface diffusion coefficient of the latter. Lateral proton diffusion measurements utilise both steady-state and transient SECM methods [2]. The ultimate goal of this work is to resolve a longstanding question in the biophysical community concerning the mechanism for the movement of protons between source and sink sites in cell membranes [3]. SECM techniques can be extended to quantitatively probe the permeability of tissues, at the micrometre scale, with examples ranging from cartilage [4] to dentine and enamel.
To advance SECM as a mapping technique, it has recently been combined with atomic force microscopy (AFM) [5,6]. Using a hand-made tip capable of functioning as both an electrode and a force sensor, the topography and chemical properties of an interface can be mapped independently. Early examples of the application of this new technique include imaging membrane transport processes and diffusion fields. Prospects for further developments in SECM-AFM instrumentation will be highlighted.
References
1. C. J. Slevin, S. Ryley, D. J. Walton and P. R. Unwin, Langmuir, 1998, 14, 5331.
2. C. J. Slevin and P. R. Unwin, J. Am. Chem. Soc., 2000, 122, 2597.
3. P. Scherrer, Nature, 1995, 374, 222, and refs therein.
4. (a) M. Gonsalves, J. V. Macpherson, D. O'Hare, C. P. Winlove and P. R. Unwin, BBA – Gen. Subj., 2000, 1524, 66;
(b) M. Gonsalves, A. L. Barker, J. V. Macpherson, P. R. Unwin, D. O'Hare and C. P. Winlove, Biophys. J., 2000, 78, 1578.
5. J. V. Macpherson and P. R. Unwin, Anal. Chem., 2000, 72, 276.
6. J. V. Macpherson and P. R. Unwin, Anal. Chem., 2001, 73, 550.
Acknowledgements
We thank the EPSRC, BBSRC, Wellcome Trust, Royal Society, Unilever and Avecia for support.
Probing diffusion and reactions at biologically-important interfaces using ultramicroelectrodes
Patrick R. Unwin, Julie V. Macpherson, Jie Zhang, Anna L. Barker, Marylou Gonsalves and Nicola J. Gray
Department of Chemistry
University of Warwick
Coventry CV 4 7AL
UK
The transport of ions and molecules through membranes and tissues plays a key role in many biologically-important systems. Ultramicroelectrodes (UMEs), in general, and scanning electrochemical microscopy (SECM), in particular, are proving powerful approaches for quantitatively probing and imaging diffusion processes with high spatial and temporal resolution, in a diversity of systems. This talk will describe several recent advances from our group on the application of SECM to biologically-important interfaces, from model membranes (monolayers at air/water interfaces) to tissue samples.
To understand how monolayers influence the rates of elementary processes such as the transfer of small molecules across interfaces and lateral diffusion along interfaces, a combined SECM-Langmuir trough instrument has been developed [1]. An attribute of the approach is that well-defined molecular interfaces can readily be assembled at the air/water interface. We have developed methods for quantitatively measuring (i) the lateral diffusion of redox-active amphiphiles and (ii) proton hopping rates at acid-base amphiphiles [2], including phospholipids. In the former case we have introduced a three potential-step method, involving "electrochemical bleaching", "recovery" and "analysis". This method, which may be viewed as an electrochemical analogue of fluorescence recovery after photobleaching, provides quantitative information on electron transfer kinetics between a tip-generated species and a surface-confined electroactive amphiphile, as well as the surface diffusion coefficient of the latter. Lateral proton diffusion measurements utilise both steady-state and transient SECM methods [2]. The ultimate goal of this work is to resolve a longstanding question in the biophysical community concerning the mechanism for the movement of protons between source and sink sites in cell membranes [3]. SECM techniques can be extended to quantitatively probe the permeability of tissues, at the micrometre scale, with examples ranging from cartilage [4] to dentine and enamel.
To advance SECM as a mapping technique, it has recently been combined with atomic force microscopy (AFM) [5,6]. Using a hand-made tip capable of functioning as both an electrode and a force sensor, the topography and chemical properties of an interface can be mapped independently. Early examples of the application of this new technique include imaging membrane transport processes and diffusion fields. Prospects for further developments in SECM-AFM instrumentation will be highlighted.
References
1. C. J. Slevin, S. Ryley, D. J. Walton and P. R. Unwin, Langmuir, 1998, 14, 5331.
2. C. J. Slevin and P. R. Unwin, J. Am. Chem. Soc., 2000, 122, 2597.
3. P. Scherrer, Nature, 1995, 374, 222, and refs therein.
4. (a) M. Gonsalves, J. V. Macpherson, D. O'Hare, C. P. Winlove and P. R. Unwin, BBA – Gen. Subj., 2000, 1524, 66;
(b) M. Gonsalves, A. L. Barker, J. V. Macpherson, P. R. Unwin, D. O'Hare and C. P. Winlove, Biophys. J., 2000, 78, 1578.
5. J. V. Macpherson and P. R. Unwin, Anal. Chem., 2000, 72, 276.
6. J. V. Macpherson and P. R. Unwin, Anal. Chem., 2001, 73, 550.
Acknowledgements
We thank the EPSRC, BBSRC, Wellcome Trust, Royal Society, Unilever and Avecia for support.
