Multienzyme Inkjet Printed 2D Arrays.

The use of printing to produce 2D arrays is well established, and should be relatively facile to adapt for the purpose of printing biomaterials; however, very few studies have been published using enzyme solutions as inks. Among the printing technologies, inkjet printing is highly suitable for printing biomaterials and specifically enzymes, as it offers many advantages. Formulation of the inkjet inks is relatively simple and can be adjusted to a variety of biomaterials, while providing nonharmful environment to the enzymes. Here we demonstrate the applicability of inkjet printing for patterning multiple enzymes in a predefined array in a very straightforward, noncontact method. Specifically, various arrays of the enzymes glucose oxidase (GOx), invertase (INV) and horseradish peroxidase (HP) were printed on aminated glass surfaces, followed by immobilization using glutardialdehyde after printing. Scanning electrochemical microscopy (SECM) was used for imaging the printed patterns and to ascertain the enzyme activity. The successful formation of 2D arrays consisting of enzymes was explored as a means of developing the first surface confined enzyme based logic gates. Principally, XOR and AND gates, each consisting of two enzymes as the Boolean operators, were assembled, and their operation was studied by SECM.

Biomolecular AND logic gate based on immobilized enzymes with precise spatial separation controlled by scanning electrochemical microscopy.

A surface-localized enzymatic AND gate based on scanning electrochemical microscopy was designed and studied. The gate is composed of an insulating glass surface modified with the enzyme glucose oxidase (GOx) and another surface opposing it made of a microelectrode. The latter was modified with a second enzyme, invertase (INV). The distance separating the modified microelectrode and surface controlled the output of the AND gate produced upon the biocatalytic reaction of the confined enzymes. Specifically, as the GOx-modified glass substrate entered the diffusion layer of the microelectrode, it catalyzed the regeneration of an electron-transfer mediator, ferroceniummethanol, generated electrochemically at the tip by oxidizing glucose, also generated at the tip, by catalytic cleaving of sucrose by INV. To enhance the activity of the GOx, mutarotase was added to convert α- to ß-glucose to be further consumed by GOx. Hence, an increase of the current at the microelectrode was obtained by approaching the glass surface only in the presence of all the components. This is the first micrometer-sized biomolecular logic gate, of which we are aware, that is surface-confined and shows the promise held by the localization of biomolecular information-processing species.