Point-of-need analyte detection using dsDNA-templated fluorescent copper nanoparticles
The detection of various types of pollutants, toxins, and adulterants that are detrimental to the environment and human health is essential for monitoring air, water, soil, and food quality. Technologies that enable the detection and quantification of analytes at the point-of-need can enable decision makers to take timely and effective actions to minimize damage and avert widespread crisis. In recent years, different types of nanomaterials in conjunction with various sensing technologies have led to the creation of novel and highly sensitive analyte detection assays. Of these nanomaterials, fluorescent copper nanoparticles templated by dsDNA have garnered considerable research interest due to their low-cost, wide availability, and relative ease of synthesis. The analyte detection assays utilizing dsDNA-templated copper nanoparticles have the potential to be deployed in point-of-need settings as they do not involve complex procedures and generate results within a short period of time. However, there are a few challenges inherent to copper nanoparticle-based assays that prevent their use outside laboratory settings. The detection of the weak fluorescence emission from the nanoparticles requires the use of highly sensitive and expensive detectors. Further, the fluorescence emission intensity from the nanoparticles varies with time, making the technique sensitive to the time of measurement. Introducing specificity into analyte detection assays using these nanoparticles also remains a challenge. This thesis proposes techniques to address these challenges to facilitate the adaptation of copper nanoparticle-based fluorescence assays for point-of-need applications. Additionally, the thesis proposes three new analyte detection assays. Firstly, a chemical technique to improve the fluorescence properties of dsDNA-templated copper nanoparticles is proposed. When compared to conventional synthesis techniques, the proposed method achieves 11 times higher fluorescence signal intensity from the dsDNA-templated copper nanoparticles and 4 times faster attainment of maximum fluorescence signal. The utility of this enhancement strategy for analyte measurement is demonstrated through a novel assay for melamine detection from milk samples which could achieve a limit of detection of 0.1 ppm melamine. Utilizing the proposed fluorescence enhancement technique, a handheld fluorometer capable of detecting DNA-templated copper nanoparticles is developed. The fluorometer was constructed with low-cost, off-the-shelf components like a UV-LED and a PIN photodiode. The performance of the developed system is demonstrated through the detection of melamine in milk samples via the interference synthesis of copper nanoparticles. The developed fluorometer can easily be adapted to detect different types of analytes at the point-of-need and can be deployed as a screening tool for environmental and food quality testing. The fluorescence of copper nanoparticles decays within a few minutes making the quantitative estimation of analytes sensitive to the time of measurement. Despite the loss of fluorescence, the nanoparticles remain intact as evidenced by microscopy techniques like TEM and AFM. To overcome the issue of time sensitivity of fluorescence measurement, etched fibre Bragg grating (eFBG) sensors are presented as a detection/characterization tool for copper nanoparticles. The in-situ formation of the copper nanoparticles on the DNA template attached to the eFBG sensor causes a significant shift in the reflected Bragg wavelength signal. Moreover, the shift in the Bragg wavelength signal was found to be constant over time after the formation of copper nanoparticles was completed, thus overcoming the issue of time sensitivity. The application of the proposed eFBG sensors for analyte detection utilizing the interference synthesis of copper nanoparticles is also demonstrated. Finally, the use of aptamers to improve the specificity of copper nanoparticle-based assays is proposed. As the growth of the copper nanoparticles is template specific, there are several challenges in developing aptamer-templated copper nanoparticle assays. A method to use a fully hybridized aptamer-duplex as template for copper nanoparticle formation is proposed. A novel fluorescence assay using aptamer-duplex templated copper nanoparticles for the detection of lipopolysaccharides (LPS) is demonstrated. The proposed assay has a wide range of detection from 1-10^5 ng/ml and a limit of detection of 1 ng/ml LPS. The mechanism of the proposed assay was verified with the SYBR Green intercalating dye. This led to the development of an alternative assay for LPS detection using SYBR Green and the aptamer-duplex which was found to have a wide range of detection from 0.1-10^5 ng/ml and a limit of detection of 0.1 ng/ml LPS.