Towards pheromone binding protein based sensors for detection of Disparlure. (2021)
Type of ContentTheses / Dissertations
Thesis DisciplineElectrical Engineering
Degree NameDoctor of Philosophy
PublisherUniversity of Canterbury
The development of sensing methodologies and apparatus is fundamental in today’s world. From controlling airborne pollution levels to the detection of new infectious diseases, sensors have become indispensable in our lives. Biosensor are also being highly requested for the control and pursuit of invasive alien species (IAS). This is of particular importance to isolated ecosystems, which might possess more delicate biomes whose balance can be easily upset. In the case of New Zealand, past incursions of IAS have left irreparable damage in the country’s ecosystem, leading to stricter biocontrol rules being implemented at the border. Lymantria dispar is one such IAS capable of causing great damage to the NZ flora as a voracious defoliating agent. Because of this, significant investment into the investigation of new biosecurity methodologies has been made, with the work presented in this thesis representing the particular case of L. dispar.
This thesis reports from an engineering perspective on the development of functionalized electrodes for electrochemical sensing of volatile organic compounds (VOCs) using a biomimetic approach. The project was developed with the goal of investigating the possibility of using L. dispar proteins for the development of a sensor capable of detecting female L. dispar inside and in the vicinity of shipping containers arriving from overseas.
The main strategy was to mimic the in vivo system that the male moth uses to detect the presence of females by "smelling" the female produced pheromone, Disparlure. For this purpose, pheromone binding proteins (PBPs) from the L. dispar moth were produced and used to create functionalized gold interdigitated electrodes (IDEs) for the performance of electrochemical impedance measurements.
The genes pbp1 and pbp2 were obtained from Genbank, and sequences treated and converted for expression E. coli expression system. A new purification, denaturation and renaturation scheme was developed to improve the expression of hydrophobic proteins PBP1 and PBP2. Expressed proteins were characterized using circular dichroism spectroscopy. Binding and competition assays were performed on PBP1 to evaluate its affinity for both enantiomeric forms of Disparlure. The binding assays yielded Kds in the same order of magnitude as the literature, while competitive assays demonstrated the unsuitability of N-Phenylnaphthalen-1-amine (1-NPN) as a competitor against hydrophobic ligands.
A platform was microfabricated for the functionalization of the expressed proteins. A 150 nm gold film was deposited on a glass slide and lithography was used for etching of an IDE pattern. Flame annealing was used for the relaxation of the gold grains and generation of more  gold facets. These allowed for the adhesion of a tightly packed self-assembled monolayer of the previously conjugated CS2 and PBP1 complex. Annealing was evaluated using atomic force microscopy (AFM) and X-ray diffraction (XRD) and the functionalized IDEs were characterized by AFM, Fourier transform infrared (FTIR) spectroscopy and contact angle goniometry measurements.
Electrochemical impedance spectroscopy (EIS) was used to evaluate the stability of the impedance signal from gold IDEs before and after functionalization using potassium hexacyanoferrate (HCF) and phosphate-buffered saline as probes. The reliability of Faradaic and non-Faradaic EIS as sensing methodologies for the detection of Disparlure was also evaluated. The HCF probe demonstrated to be more unstable when used in conjunction with gold electrodes, confirming existing literature reports regarding the effects of this probe when in contact with gold. Faradaic EIS did also present higher sensitivity to the presence of Disparlure at the sensor surface.
The work in this thesis demonstrates how changes in the impedance at the surface of a PBP functionalized electrode can be used as a sensing methodology for the detection of a VOC. The data obtained adds to the body of literature on biofunctionalized transducers, and presents the first exploratory research on the use of soluble L. dispar proteins, specifically functionalized for the selective detection of a unique VOC.
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