Metal nanostructure-enhanced fluorescence and its applications in bioassays and bioimaging
thesisposted on 28.03.2022, 02:22 by Wei Deng
Over the last two decades, fluorescence based detection has become one of the leading sensing technologies in biomedical, biological and related sciences. Its sensitivity makes it possible to detect a single biomolecule through labeling with a suitable fluorophore. Two principal fluorophore properties, brightness and photostability, are fundamentally important to achieve a high level of sensitivity and in many conventional fluorophores these often fall short of the requirements. Among the methods used to improve the sensitivity of fluorescence detection, the metal-enhanced fluorescence (MEF) technique has been recently actively developed. The MEF phenomenon occurs when an excited fluorophore is located in close proximity to metals, and it is particularly pronounced near noble metal nanostructures. Electrons in such metal nanostructures exhibit strong resonances often located in the visible part of the spectrum (also known as surface plasmon resonance). They can interact with proximal fluorophores modifying their optical properties and producing increased quantum yield (fluorescence efficiency) and improved photostability. It has been experimentally demonstrated that the MEF technique can increase fluorescence intensity up to several hundreds times. The work through my PhD project mainly focus on siver nanostructures and its potential in fluorescence-based applications requiring very high sensitivity because their strong surface plasmon resonance in the visible matches the absorption and emission bands of most fluorophores. I began with synthesis for Ag nanostructure-coated silica beads in the solution-based platform as a new MEF substrate. My first study employed these nanostructures to enhance the fluorescence readout on individual silica beads. These Ag nanostructure were deposited on micrometer size silica beads. The fluorescence enhancement was investigated using a model AlexaFluor 430 IgG immunoassay and AlexaFluor 430 labeling. Approximately 8.5-fold and 10.1-fold higher fluorescence intensities at 430 nm excitation were, respectively, observed from silvered 400 nm and 5 μm silica beads deposited on glass as compared to the control sample. This achievement allowed us to demonstrate for the first time MEF immunoassays on silica beads by using high-throughput flow cytometry. Furthermore, we discovered that these Ag nanostructure-coated silica beads are able to modify the luminescence decay lifetime of lanthanide fluorophores (BHHCT-Eu3+) for time-gated luminescence bioimaging applications. The fluorescence enhancement factor achieved about 11 times, while the simultaneously measured fluorescence lifetime was reduced twofold. The fluorophore stability was also improved by a factor of three. We applied such bead substrates to time-gated fluorescence imaging of Giardia lamblia cells stained by BHHCT-Eu3+ with improvement in brightness by a factor of two. This will open up a broad range of opportunities for ultrasensitive and low background fluorescence detection using lanthanide fluorophores. [...] Based on these experimental results, we also presented the theoretical analysis for the high fluorescence enhancement factor. This phenomenon observed on the nanostructured silver surfaces is a result of two effects: an increase of a local electromagnetic field near silver nanostructures, leading to increased excitation rate of fluorophores and an increase of the radiative decay rate Γ of fluorophores close to silver nanostructures, reflected both in the fluorescence lifetime and quantum yield. The local electromagnetic field enhancement produces a higher excitation rate but it does not change the lifetime of the fluorophore; this effect is referred as excitation enhancement (Eex). The second effect referred as emission enhancement (Eem), increases the quantum yield and reduces the lifetime of the fluorophore. The detailed mechanism was systematically analysed in the following chapters. In summary, this work mainly covered the fabrication of nanoscaled silver with different geometries and the MEF effect induced by them in biological applications such as bioassays, immunoassays and bioimaging.