Inkjet printing enzymes for biosensor and biotechnology applications

Inkjet printing is a non-contact printing method which could be used to manufacture enzyme based sensors and devices. Because it is a precise metered dosing technique, expensive material is only deposited where required and different materials can be deposited in sequence with no loss of register. However there are significant fluid shears that occur during inkjet printing and these may damage complex biological molecules such as proteins and enzymes. Preliminary work at the University of Manchester has demonstrated that it is possible to deposit the enzyme glucose oxidase, suitably protected through a formulated packaging solution of sugars and polyalcohols, onto carbon electrodes by inkjet printing. These can then be used as demontrator glucose sensors. However, this work has also shown that under some printing conditions the enzyme is damaged, as identified by a quantitative fluorescence assay. Although glucose oxidase is a very important enzyme that is used in commercial biosensors, it is a relatively robust enzyme. The objective of this proposal is to extend our understanding of the response of enzymes to inkjet printing using example enzymes that are known to be less robust and more difficult to package and stabilize. In discussion with our industrial partner, Applied Enzyme Technology, we have selected lactate dehydrogenase as a suitable enzyme with potential biosensor applications. This will be compared with lactate oxidase that has been selected because of its known fragility. Alcohol oxidase will also be studied, this is currently one of AET's most commercially important enzymes used in sensor applications and is an obvious target for an 'inkjet ready' product. We have also selected laccase, a powerful enzyme with applications in biofuel cells as another candidate material. Alcohol oxidase will also be studied as an enzyme for biofuel cell applications. It is possible to control the stress and strain rate that is characteristic of droplet generation in the inkjet printer by varying the electrical pulse that drives the piezoelectric actuator in a printer and this will be used to vary the stress state that the enzymes are exposed to. We will explore how the pulse amplitude and shape influences enzyme activity after printing. This can be achieved using electrochemical methods or through fluorescence assays. Protein structure will be studied using light scattering and circular dichroism to study whether this has been affected by printing. Working closely with AET the student will develop suitable packaging formulations of polyalcohols and sugars to minimise enzyme damage and investigate how the packaging influences damage mechanisms. We will investigate the interaction of the protein with the electrode, of particular interest is to determine if the enzyme penetrates the porous electrode, spreads across its surface, or whether 'coffee staining' results in inhomogenous distribution of the protein as the inkjet deposited drop dries. Standard electrode structures will be provided by AET and their protocols will be used for the test programme. Enzyme electrochemistry will be characterised in Manchester and at AET. Alternative electrode materials such as carbon nanotubes and metallic nanowires will be explored as potential all-printed electrode structures.