Receptor-based biological detection techniques often suffer from the problem of non-specific interactions. This is largely due to the presence of weak electrostatic and Van der Waals forces between the receptor and the non-target substances in the analyte that are not easily dissociated in practice. Most existing detection techniques are unable to probe the interaction between the bound entity and the surface and differentiate between specific and non-specific interactions in terms of bond strength or activation energy. The resulting false positive responses lead to various issues, such as misdiagnosis and mistreatment in clinical diagnostics and false alarms in biosecurity. The problem is even more significant with direct direction techniques, such as the resonant frequency shift based detection using quartz crystal microbalance (QCM) or micro-cantilevers, which involve minimal sample processing and washing steps. The work presented in this thesis investigates, through modeling and experiments, the mechanical interactions of a resonator with microparticles attached via biomolecular linkers and analyses the resulting nonlinear acoustic modulation of the resonator from the transduced electrical signal. Physisorbed and specific interactions both in air and liquid medium are studied using thickness shear mode quartz crystal resonators and streptavidin-coated polystyrene microbeads (SCPM) of various sizes. It is found that the modification in the transduced electrical signal measured at the third harmonic (3f), or three times the driving frequency f, is significant in presence of the attached particles and approximately proportional to the number of particles. A detection limit of approximately 2 SCPM of 5.6 µm diameter in air and 6700 SCPM of 0.39 µm diameter in liquid is demonstrated, which corresponds to a mass detection limit of ~200 pg. Most interestingly, the deviation in the magnitude of the 3f signal as a function of the resonator oscillation amplitude is found to hold a distinct relationship with the type of particle-surface interaction. This provides a basis for selectivity in detection over and above the efficacy of the receptor. The function is also found to correlate well with the event of SCPM diffusion on the surface. This detection technique, based on the measurement of deviation in magnitude of the transduced electrical signal measured at a higher odd harmonic of the drive frequency due to the presence of surface-bound particles on a resonator, is termed as the anharmonic detection technique (ADT). A feasibility study with Bacillus subtilis spores in phosphate buffer saline (PBS) is carried out successfully where the modeling and experimental results with SCPM are successfully reproduced. A detection limit of 430 spores is demonstrated, which corresponds to a mass detection limit of ~650 pg. Capability for differentiation of the specifically-captured spores from unwashed physisorbed SCPM of similar dimensions is demonstrated using the shape of the ADT signal. These results indicate that the spore immobilization step may be directly followed by the detection step, which are 9 mins and 2 mins respectively in these experiments. ADT thus potentially enables a rapid, sensitive, reliable and direct detection without the need for any sample processing. Moreover, being an entirely electronic technique, ADT suitably lends itself to multiplexing, large scale fabrication and implementation on a miniaturized low-cost point-of-care detection platform that is of immense need in clinical diagnostics, food and environmental monitoring and biosecurity. Furthermore, fitting the experimental results with modeling estimates enables ADT to determine the force-extension characteristics of the binding biomolecular linker. The force-extension characteristics and the estimated unbinding force for a streptavidin-biotin complex estimated using ADT agrees well with those computed using molecular dynamics (MD) simulation at similar loading rates. Thus ADT contributes a unique force-spectroscopic method, which unlike conventional techniques such as the atomic force microscopy (AFM) provides statistically averaged data for multiple biomolecules in a relatively quicker and simpler experimental format. A method for determination of activation energy of the interaction is also proposed using ADT. This potentially enables a method for rapid and large scale biomolecular screening and studying of interaction networks, which have important applications in drug discovery and individualized therapy.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:557880 |
| Date | January 2011 |
| Creators | Ghosh, Sourav Kumar |
| Contributors | Seshia, Ashwin A. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.repository.cam.ac.uk/handle/1810/243858 |
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