טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentTenenbaum-Gonikman Elena
SubjectDevelopment and Characterization of Optical Biosensors for
Bacteria Detection
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Ester H. Segal
Full Thesis textFull thesis text - English Version


Abstract

This research aimed towards designing a label-free biosensor for rapid bacteria detection, using Escherichia coli (E. coli) as a model microorganism. The biosensor design is based on a Fabry-Pérot porous silicon (PSi) thin film, used as the optical transducer, functionalized with natural and synthetic recognition elements. Three types of bioreceptors were studied: (i) Fab fragments, which are produced from intact specific antibodies, (ii) synthetic antimicrobial peptides, and (iii) biomimetic supported lipid bilayers. Fab fragments were successfully immobilized onto the PSi surface, while maintaining their immunoactivity and antigenic specificity. However, the resulting biosensors have demonstrated poor bacteria capture in comparison to intact antibodies. An alternative biosensing scheme employs the sequence K-7α12, a synthetic antimicrobial peptide, as the bioreceptor. This compound is a member of a family of oligomers of acylated lysines (OAKs), mimicking the hydrophobicity and charge of natural antimicrobial peptides. The OAK was tethered to the PSi film and changes in the reflectivity spectrum were monitored upon exposure to E. coli suspensions and their lysates. We show that capture of bacterial cell fragments induces predictable changes in the reflectivity spectrum, proportional to E. coli concentration, thereby enabling rapid detection of bacteria at concentrations as low as 103 cells mL-1. The biosensor performance was also studied upon exposure to model Gram- positive and negative bacteria lysates, suggesting preferential capture of E. coli cell fragments in the presented scheme. These OAK-based biosensors offer significant advantages in comparison to conventional antibody-based assays, in terms of their simple and cost-effective production, while providing numerous possible sequence combinations for designing new detection schemes. Our third biosensing approach employed the construction of a supported lipid bilayer (SLB) within the porous nanostructure, mimicking the cellular membrane. This generic system allows the detection of molecules secreted by the bacteria, such as their toxins e.g., Shiga and Cholera toxins. In nature, these toxin’s receptors are anchored to host cells by their lipid moieties, integrated within the membrane. Conventionally, SLBs formation involves the spreading of lipid vesicles on hydrophilic solid supports. Our work presents an alternative facile approach for the construction of tethered SLB within PSi nanostructure, while avoiding liposome preparation. First, a lipid layer was tethered to the pore walls, resulting in a stable monolayer, and a subsequent solvent exchange step induced the self-assembly of the unbound lipids into a robust SLB. Changes in the reflectivity of the PSi-SLB system, in terms of its effective optical thickness, were employed to monitor the partitioning of model amphiphilic molecules and peptides within the SLB. Thus, these self-reporting SLB platforms provide a generic approach for bottom-up construction of complex lipid architectures for performing biological assays at the micro- and nano-scale.