Date of Award

8-2007

Level of Access

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Biological Engineering

Advisor

Michael Mason

Second Committee Member

Douglas W. Bousfield

Third Committee Member

Nancy Kravit

Abstract

Research interest on bio-detection of biological warfare agents (BWA) and infectious diseases (ID) has tremendously increased over the pas few years because of the threats; these agents pose in the society. There are two current bio-detection methods - field and laboratory methods. The major advantages of the field methods over the laboratory methods are; they are relatively inexpensive, portable, and give rapid results. However, there are drawbacks associated with the field methods such as; low sensitivity (false negatives) and lack of specificity (false positives). This projects aims to address some of these drawbacks by utilizing the combination of biochemical and chemical amplification techniques on a paper-based substrate. Paper-based biochemical and chemical amplification (BCA) technique is a model technique for bio-detection in the field. It utilizes both biochemical amplification from enzymatic reactions and chemical amplifications from photolithography chemistry, which drastically improves specificity and sensitivity over existing methods. Amongst the various enzymes, lipases were chosen because of their commercial availability and their robustness in acid generation - a key parameter in biochemical amplification. Thermomyces lanuginose lipase (TLL) and Candida antartica lipase (CALA) were used as candidate lipases in the phase I of this project. The turnover efficiency, kcat/Km for CALA and TLL were KT'S and lOOOS respectively. In solution, CALA has proved to a better enzyme with faster turnover rates as indicated from their turnover efficiencies. Spectroscopic determination of the pH change as a function of time for both CALA and TLL showed similar trend as with the direct pH measurements from the pH meter. Understanding the spectroscopic measurements of the lipolytic reactions in solution forms the basis of using the technique to characterize the lipolytic reaction on a paper (surface) where the pH meter measurements are not feasible. Conjugation of CALA and TLL with a heterobifunctional cross-linker (NHS-PEO4) was carried out and the conjugation was monitored by taking UV-Vis spectra of the reaction mix before and after the purification step. For CALA, the conjugate was further tested for activity by assaying with the substrate for both the unpurified and purified conjugate. Result showed a decline in activity for the conjugated CALA when compared to the native (unbound) CALA. A rapid decrease in activity was also observed after the conjugated CALA has been purified with a desalting column. This implies that, during the purification stage, the conjugated CALA is further diluted and this can account for the further loss in activity.

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