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Localized Surface Plasmon Resonance: Single Particle to Ensemble Binding Measurements



Localized Surface Plasmon Resonance: Single Particle to Ensemble Binding Measurements.


Jamie Nicholas Kulp

Doctor of Philosophy in Chemistry

University of California, Berkeley

Professor Marcin Majda, Chair

Plasmonic nanoparticles are an emerging technology, which demonstrates immense utility in the field of sensitive, label-free, molecular detection assays. Plasmonic nanoparticles exhibit a physical phenomenon known as Localized Surface Plasmon Resonance or LSPR. LSPR is a phenomenon based on the collective oscillations of free, surface electrons in certain metals such as silver and gold. The surface electrons couple to incident light at a specific frequency corresponding to the resonance frequency of these surface electron's oscillations. These oscillations scatter the incident light at the resonant frequency with a very high efficiency. The frequency of these oscillations and the scattered light is not only dependent on physical characteristics of the particles, such as their material composition, geometric shape and size, but also on the refractive index of both the particle and the surrounding medium in which the particles are located. Changes in the refractive index at the interface between the particle's surface and the surrounding medium, is the physical basis of analytical methods utilizing plasmonic nanoparticles. The sensitivity of the LSPR frequency to the surrounding medium's refractive index allows for the ability to observe changes in the refractive index at the particles surface due to molecular binding events. The monitoring of changes in the refractive index is accomplished by analyzing the peak LSPR frequency as a function of the refractive index at the surface interface of the particle and the surrounding medium. As molecular binding events occur at the surface interface between the particle and the surrounding medium, an increase in the local refractive index occurs. This increase in the local refractive index is accompanied by a red shift in the maximum LSPR wavelength of the scattered light. By monitoring changes in the maximum wavelength of scattered light, a calibration curve can be constructed, which relates the changes in the LSPR frequency to changes in the local refractive index at the particles surface. Changes in refractive index are ultimately related to a change in the mass of absorbed particles at the particle's surface due to molecular binding.

There are various instrumental methods that have been shown to be capable of detecting these refractive index changes at the plasmonic particles surface. These methods include solution-based ensemble techniques, as well as single particle scattering measurements. Solution-based ensemble measurements commonly utilize UV-Visible spectrophotometers, but can also include the use of the backscattering interferometer. Single particle methods are predominately performed using the dark field scattering microscopy. The dark field scattering microscope is ideal for single particle measurements on plasmonic nanoparticles due to the particles remarkably high scattering coefficients relative to their geometrical size. The dark field scattering microscope also has relatively low background noise, which is particularly important for experiments on the single particle level where the signal-to-noise ratio must be maximized. Single particle dark field scattering methods involve the coupling of a dark field scattering microscope with a spectrometer in order to collect and analyze the scattered light of individual nanoparticles. The limitations and benefits of each of these experimental designs are discussed further in the body of this dissertation. Briefly, solution-based assays offer the benefit of being performed in colloidal suspensions of nanoparticles without the need to utilize specialized surface chemistry as a means to immobilize the particles to the underlying substrate. Solution-based measurements are also compatible, in most situations, with instrumentation that is commonly found in laboratories. As a result of the compatibility of solution-based measurements with common laboratory instruments, such as the UV-visible spectrometer, measurements do not generally require dedicated, specialized instrumentation. This of course is not universally true as will be seen with experiments are performed using the backscattering interferometer. However, many examples of solution-based assays utilizing more common laboratory instrumentation are now available in the literature. Despite these advantages, solution-based colloidal measurements are ensemble measurements and due to the statistical nature of these measurements, individual, unique features that may occur on the single particle level are obscured due to the averaging of many particles contribution to the collected signal. To overcome this statistical limitation, single particle assays have been developed. Most notably among these single particle experiments is the use of dark field scattering microscopy, coupled with spectroscopic measurements of the scattered light to examine the optical properties of individual nanoparticles. While this platform has shown that it is possible to interrogate individual particles, as well molecular binding events occurring at the surface of the particles, there are a few technical drawbacks to this method as well. The first of these technical drawbacks is the need to immobilize the particle onto the underlying substrate. This is to prevent random Brownian motion of the particles when performing experiments in aqueous environments. When observing the particles in a vacuum or air environment this need is not as rigorous as the electrostatic interactions between the particle and the underlying substrate are sufficient to immobilize the particle during measurements. In order to explore binding events occurring in aqueous systems, which include receptor-ligand binding, DNA-DNA base pairing and other solution-based molecular recognition events, it is necessary to develop a method of immobilizing the particle to the surface of the underlying substrate utilizing surface chemistry techniques. In this dissertation it is shown that the use of the supported lipid bilayer is a very effective and versatile means of accomplishing this immobilization of the particle to the underlying substrate used in dark field microscopy. This can be accomplished by incorporating biotin-functionalized lipids into the composition of a supported lipid bilayer formed on the particles, as well as the supported bilayer formed on the underlying substrate. The formation of a supported lipid bilayer containing a fraction of biotin-functionalized lipids on the underlying substrate, as well as the nanoparticle, it is possible to tether the particles to the substrate surface through streptavidin-biotin molecular interactions. This proves to be a robust and simple method of immobilizing particles to a substrate capable of supporting the formation of a supported lipid bilayer. Silica oxide or glass is a material, which allows for the spontaneous formation of a supported lipid bilayer from single unilaminar vesicles. Presented here is the synthesis of cubic geometry nanoparticles that are surrounded with a thin silica shell of nanometer thickness. This thin silica shell allows for the spontaneous formation of a supported lipid bilayer on individual nanoparticles. This feature facilitates the immobilization of the particles for studies using the dark field scattering microscope. Nanoparticle-based analytical platforms, regardless of being implemented on the single particle level or using colloidal, solution-based ensemble measurements, offer a very robust and sensitive, label-free analytical technique for quantify molecular binding events occurring at the particle's surface.

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