Advanced Technology and Manufacturing Institute (ATAMI)

Since their discovery in the 1990s, the great potential of carbon nanotubes (CNTs) has made them a focus of many research endeavors, including their application as components of biosensors. The inherent chemical "inertness" of CNTs makes their application to biosensing a challenge. It is necessary to "decorate" their surfaces to endow them with molecular recognition capability along with a signal transduction function. Many research challenges still remain, most importantly in the area of immobilization of bioreceptors, in a controlled orientation, onto the sidewalls of CNTs with sufficient stability and without compromising either the activity of the biomolecule or the electrical properties of the nanotubes. On the other hand, it is also necessary to assess potential environmental toxicity effects in advance of widespread application of CNTs in nanomedicine and clinical devices. Effective functionalization of CNTs could render them very hydrophilic and ensure their stability in suspension and in physiological environments, though it also might alter their potential toxicity. The work described in this dissertation focused on the development of novel techniques for functionalization of carbon nanotubes to enable their use as components of biosensors for use in physiological conditions. The goal was to develop cost effective detection techniques with novel mechanisms of detection. Also presented is a new method of functionalization of CNTs to render them highly hydrophilic and stable in aqueous suspension. CNTs modified using this approach were studied to ascertain their toxicity using a zebrafish model. For biosensor development, carbon nanotubes were functionalized with 4-carboxybenzene diazonium tetrafluoroborate salt, which covalently grafted a carboxyphenyl moiety on the CNT sidewall without excessively compromising the electronic integrity of the nanotube. The carboxyphenyl moieties were then used to chemically attach biorecognition probes to the nanotubes in a controlled orientation, so as to preserve the activity of the probes. Initially, potentiometric biosensors were constructed by covalently coupling the carboxyphenyl moieties on the CNTs via an amine-modified anti-thrombin oligonucleotide (aptamer) or antibody. We explored the performance of the biosensor under physiological conditions in an effort to ensure the sensors would be suitable for biological analyses. The biosensors showed high sensitivity with a limit of detection for thrombin in the picomolar range, and exhibited reusability and reproducibility. A second approach focused mainly on antibodies as bioreceptors on CNT transducers. Because of the fragile nature of antibodies, it was necessary to apply "gentle" immobilization methods that would not compromise the activity of the antibody. This study, therefore, exploited the sulfhydryl group of the intrinsic disulfide groups in the hinge region of the antibody as a point of attachment to the functionalized CNT using 3-(2-pyridyldithio)propionyl hydrazide (PDPH) as a crosslinker. Because the number of disulfide bonds is quite small, a self-assembled monolayer of antibodies on CNTs was envisiaged. This approach was intended to ensure the correct orientation of the antibodies and to preserve their activity. To demonstrate this principle, C-reactive protein was used as the model target using anti-C-reactive protein antibody as biorecognition molecule. This biosensor demonstrated high sensitivity over physiologically relevant concentrations of CRP with limits of detection in the picomolar range. We also demonstrated in these studies the dependence of the sensitivity of potentiometric biosensors on the pH of the electrolyte buffer and proposed a novel mechanism for protein detection by the biosensor. Lastly, we demonstrated effective functionalization of CNTs to render them highly hydrophilic - which led to enhanced suspension stability in physiological solutions. The prepared functionalized nanotubes were well characterized to evaluate their physical morphology and elemental composition. We also evaluated the functionalized CNTs for possible toxicological effects using the zebrafish model. The results showed that none of the CNTs studied caused significant adverse developmental effects. These results support the potential safe use of CNTs as components of indwelling medical devices such as tissue growth scaffolds, monitoring devices, and drug delivery.