pre-printImmobilization is not only useful for preserving enzyme activity, but also to adhere an enzyme to a surface, such as an electrode, so that the enzyme does not leach into solution during testing. Current immobilization approaches do not readily allow for adjustments to the distance between the enzyme and the electrode or other enzymes. The ability to control the distance of enzymes relative to each other on an electrode can allow for optimal placement and improved current responses. In this report, we investigate the use of comb-branched DNA for enzyme immobilization. A DNA foundation strand was covalently attached to multiwalled carbon nanotubes on a glassy carbon electrode. Comb-branched DNA was then successfully formed using a previously-identified deoxyribozyme to attach DNA strands at specific locations on this foundation strand. By changing the foundation strands, the placement of the DNA strands can be adjusted, allowing for distance changes between the enzyme and the electrode surface. Using standard bioconjugation methods, alcohol dehydrogenase and glucose dehydrogenase were attached to these comb-branched DNA structures, resulting in enzyme immobilization on electrode surfaces. Amperometric analysis revealed both distance and DNA foundation strand length dependence for current response of these enzymes in the presence of their appropriate substrates
