Current progress on the synthesis and characterization of a biosensing cascade on a patterned gold-silicon substrate

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Wolff, Erich Paul

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The specificity and functionality of enzymes make them ideal for use in a range of devices from biosensors to catalytic reactors. These applications require the immobilization of enzymes onto substrates to preserve activity and reusability of these devices. In order to expand the functionality of these devices, it is increasingly more important to find ways to generate biomimetic devices with multiple enzymes in a controlled manner. These multiple enzymes-based device work together through enzymatic cascades to perform more advanced functions than possible with a single enzyme. Herein, I document my research on generating a substrate composed of Au and Si that will be used to immobilize two enzymes, Choline Oxidase (ChOx) and Horseradish peroxidase (HRP), to detect choline in solution, as a test for controlled enzyme immobilization on biosensing devices. Au and Si substrates were functionalized with alkyl thiol and alkyl trichlorosilane self-assembled monolayers for enzyme immobilization. ChOx will be immobilized on the Si portion of the substrate with electrostatic attachment, and HRP immobilized on the Au portion with (N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride) /(N-hydroxysuccinimide) coupling. Experiments in this study were performed in the manner of least to most complex mediums. Therefore, the enzymes were first studied in solution, then studied on one element substrates composed of either Au or Si, and finally studied on a substrate patterned with Au and Si domains. The properties, including native folding and activity (HRP V [subscript max] = 265.2 ± 20.1 U/mg, ChOx V [subscript max] = 2.4 ± 0.1 U/mg), of both enzymes were first collected in their native solutions; so that they could be compared to the enzymes once immobilized on their corresponding substrates with ultraviolet – visible (UV-Vis) and circular-dichroism spectroscopy. The successful immobilization, surface concentration (1.97 ± 0.27 pmol/cm²), and activity of HRP on uniform Au substrate were determined with infrared spectroscopy, ellipsometry, and UV-Vis. Future experiments in this project will focus on the properties of ChOx on Si, and both enzymes on a patterned Au/Si substrate. Long term, the goal is to expand the complexity of these multi-enzyme biodevices by adding more domains for attachment of enzymes with other metal domains and metal nanoparticles.



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