Two adjacent electrode surfaces were modified in a sequential manner with self-assembled thiol layers from the same solution using conditions (aqueous buffer at neutral pH) suitable for applications with proteins. A faradaic response was obtained from the redox protein, cytochrome c, 10 independently immobilised at each surface.Biofuel cells rely on the use of biocatalysts, predominantly as either isolated enzymes, 1 or in the form of mitochondrial 2 or microbial 3 fuel cells to generate power from a fuel source. Both types of biofuel cell have been extensively investigated for 15 potential applications ranging from clinical uses to the remediation of waste materials. [1][2][3][4] One of the challenges in the development of more efficient biofuel cells is to perform multistep oxidation of a fuel such as glucose to enhance the power output of the cell. 5 Enzymatic biofuel cells in particular face a 20 significant limitation in this regard due to the specificity of enzymes, limiting their use to single substrates. Enzyme cascades can alleviate this limitation by enabling the sequential and more complete oxidation of the fuel. 6, 7 Such sequential reactions mimic cascade reactions in cells where the product of one 25 enzyme serves as the substrate for an adjacent enzyme with the rate of reaction controlled by the concentrations of substrates and co-factors and the activity of the enzymes involved. A prototype biofuel cell using this approach was described for the conversion of methanol to carbon dioxide by employing three NAD + 30 dependent enzymes: alcohol (ADH), aldehyde (AldDH) and formate (FDH) dehydrogenases resulting in a cell with an overall power output of 0.68 mW cm -2 . 6 A similar type of cell with the addition of a quaternary ammonium bromide modified Nafion membrane provided a higher power output of 1.55 mW cm -2Recently a biofuel cell prepared using a hydrogel containing three immobilised NAD + dependent enzymes: ADH, AldDH, and FDH had a current density of 26 mA cm -2 when utilising ethanol as a fuel. 7 On using formaldehyde and formate as intermediate fuels (two and one enzyme systems, respectively) lower current 40 densities of 16 and 6 mA cm -2 were observed. While substantial increases in output (>4 fold for the aADH, aAldDH, FDH system 7 ) can be enabled through the use of multiple enzymes, methods of precisely locating the enzymes at the desired location and sequence have not been described. Such an approach has the 45 potential to enable more efficient oxidation of the fuel.A wide range of methods of immobilizing enzymes has been described. 9, 10 One of the most widely used methods of immobilising enzymes on electrodes entails the use of selfassembled-monolayers (SAMs). interactions with redox enzymes such as cytochrome c oxidase. These residues can also be utilised to promote electron transfer at SAM modified electrodes and enable rapid rates of electron transfer when the haem prosthetic group is correctly oriented at a short distance from the surface. 11-13, 16 65 Electrochemical method...

Dedicated to our friend and colleague, Prof. Wolfgang Schuhmann on the occasion of his 65 th birthdayThe precise spatial control of the immobilisation of enzymes is necessary for the sequential localisation 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 and 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. Scanning of the applied potential was utilised to separately modify individual electrodes with the appropriate thiol and enzyme to deliver a threeenzyme system with catalytically active enzyme. Immobilised ADH, FLDH and FoDH retained 46, 32 and 76 % of initial activity, respectively, on storage in aqueous solution at 4 °C for a period of two weeks.