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Date of Award

Fall 2011

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Chemical Engineering

Supervisor

Carlos Filipe

Co-Supervisor

Robert Pelton

Language

English

Committee Member

Yingfu Li, Todd Hoare

Abstract

This thesis describes a new type of biosensor, which reports the presence of a target by generating a smell that can be easily detected by the human nose. This approach is radically different from, but complementary to, colorimetric based reporting and it paves the way for the development of multi-sensory biosensors that can be used in a variety of fields, such as diagnostic device, food processing and environmental monitoring

Biosensors typically consist of two parts: a bio-recognition element and a signal transducer. The biorecognition element is the component that can specifically interact with its cognate target, while the transducer produces a signal that can be easily identified. The key element of the smell generating biosensor is the enzyme tryptophanase (TPase), which was used as the signal transducer. This enzyme uses either L-tryptophan or S-methyl-L-cysteine as substrates, to produce either indole or methyl mercaptan as final products- both molecules are easily detectable by the human nose. Proof-of-concept for this biosensor was achieved by performing an enzyme-linked immunosorbent assay (ELISA) on magnetic beads with detection of IgG from rabbit serum (the target) in a sample and reporting the presence of the target through the generation of a smell (either indole or methyl mercaptan, depending on the substrate used).

The potential use of TPase for biosensing was further expanded by creating a bienzyme system that allows specifically detecting of adenosine-5’-triphosphate (ATP) and reporting its presence by generating a smell. This bienzyme system is based on the fact that TPase activity is greatly affected by the concentration of pyridoxal phosphate (PLP)- which acts as a cofactor that modulates enzyme activity. The enzyme pyridoxal kinase PKase catalyzes the phosphorylation of pyridoxal to PLP in the presence of ATP. The more ATP presents, the more PLP is produced per unit time. If this occurs in the presence of TPase, larger concentrations of ATP in samples will result in higher amounts and faster rates of PLP formation, leading to increased activity of TPase, hence faster generation of either indole or methyl mercaptan is achieved. This bienzyme was used for the detection of DNA molecules with a specific sequence as well as for the detection of microbial cells through smell generation.

Most widely used biosensors require immobilization of the biologically active elements on a stable surface. Paper, being a cheap and easy accessible substrate, was used for fabrication of the olfactory-based biosensor. Poly(N-isopropylacrylamide-co-vinylacetic acid) (PNIPAM-VAA) microgels with functional groups present on their surface were modified by biotinylation and loaded with streptavidin/avidin (to be prepared as a platform for further biomolecule immobilization). The microgels were then used as a supporter for the bienzyme system on filter paper to construct a paper-based smell-generating biosensor, which opens the way for the creation of printable smell-reporting printable bio-inks.

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