Use of self‐assembled monolayers for the sequential and independent immobilisation of enzymes

The precise spatial control of the immobilisation of enzymes is necessary for the sequential localization of an enzymatic cascade. In this contribution, the sequential immobilisation of alcohol dehydrogenase (ADH), formaldehyde dehydrogenase (FLDH) and formate dehydrogenase (FoDH) on self-assembled monolayer modified electrodes has been demonstrated. A range of thiols were screened. Optimal enzymatic activity was obtained with thiols bearing -OH, a positively charged heterocyclic aromatic ring and -COOH for the immobilisation of ADH, FLDH and FoDH, respectively. The application of a potential pulse sequence was utilised to separately modify individual electrodes with the appropriate thiol and enzyme to deliver a three enzyme system with catalytically active enzyme. The immobilised enzymes displayed a range of stability (40-70%) on storage for a period of two weeks.