2-1 Description of the remaining chapters
This thesis is composed of two separated but closed related research works on nanodiamonds. A short abstract of each experimental work and instrumentation sections are given below. More details and discussion are presented in chapter 3 and 4.
Studies related to the nitrogen-doped ultrananocrystalline diamond films generated by microwave plasma are put in the appendix attached to this thesis.
2-1-1 Protein Functionalized Nanodiamond Arrays
Nanodiamonds possess remarkable features such as low bio-cytotoxicity, good optical property in fluorescent and Raman spectra, and good photostability for bio-applications. In chapter 3, we devise techniques to position functionalized nanodiamonds on self-assembled monolayer (SAMs) arrays adsorbed on silicon and ITO substrates surface using electron beam lithography techniques. The nanodiamond arrays were functionalized with lysozyme to target a certain bio-molecule or protein specifically. The optical properties of the nanodiamond-protein complex arrays were characterized by a high throughput confocal microscope. The synthesized nanodiamond-lysozyme complex arrays were found to still retain their functionality in
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interacting with E. coli. This work is elaborated in chapter 3.
2-1-2 Plasmon-enhanced photoluminescence from bioconjugated gold
nanoparticle and nanodiamond assembly
In this part of the work, we coupled NDs with gold nanoparticles of different sizes using two complementary DNA sequences. After hybridizing the gold nanoparticles on the NDs, we observed the enhancement of the photoluminescence (PL) signals originating from the nitrogen-vacancy (N-V) center of the ND. The enhancement was attributed to the plasmon field created by the gold nanoparticles. The lineshape of the enhanced PL spectra was also affected by the sizes of the attached nanoparticles due to their different resonant plasma frequencies. The signal enhancement can be used as an indexing tool for bio-sensing applications. This work is elaborated in chapter 4.
2-2 Instrumentation
2-2-1 E-beam lithography
Generally speaking , electron beam lithograph system is constructed from four main parts ; including electron optical column、chamber、handling system、and control unit. The major part is electron optical column, which determines the shape of
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electron. It controls the image resolution and exposing quality. Electron optical column is generally composed of electron gun、blanking、condense lens、stigmator、
objective lens、deflector、and electron detector (figure 2-1). These accessories enable the electron beam ejecting from electron gun to be well-controlled and expose to the right place.
There are three common phenomena occur during the exposure of electron beam system ; including forward scattering、back scattering、and charging effect. Each of them can influence the quality of the e-beam exposed patterns, especially when the pattern size is down to sub-50nm. The effect of forward and back scattering will broaden the pitch size and produce some proximity effect. The charging effect is the results of the electron accumulation phenomenon. These effects will blur the exposed image and produce a poor exposure quality (figure 2-2).
2-2-2 Confocal microscopes
The principle of confocal imaging was patented in 1957 by Marvin Minsky and aims to overcome the limitation of traditional wide filed fluorescence microscopes [2.1]. In wide field fluorescence microscope, the entire specimen is flooded in light from a light source. The whole specimen in the optical path was totally excited at the same time and the fluorescence of the specimen was detected by the microscope’s detector including the unfocused background parts. The principle of cofocal
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microscopes is to use both point-wise illumination and detection. As shown in figure 2-3, the point illumination and a pinhole were in an optically conjugate plane in front of the detector in order to eliminate out-of-focus signals. The name confocal stems from this configuration. As the only signals very close to the focal plane can be detected, the image, the image's optical resolution, particularly in the sample depth direction, is much better than that of wide-field microscopes. However, as much of the light from sample fluorescence is blocked at the pinhole, this increased resolution is at the cost of decreased signal intensity – so long exposures are often required.
As only one point in the sample is illuminated at a time, 2D or 3D imaging requires scanning over a regular raster (i.e. a rectangular pattern of parallel scanning lines) in the specimen. The achievable thickness of the focal plane is defined mostly by the wavelength of the used light divided by the numerical aperture of the objective lens, but also by the optical properties of the specimen. The thin optical sectioning possible makes these types of microscopes particularly good at 3D imaging and surface profiling of samples.
2-2-3 Raman spectrum
Although the inelastic scattering of light was predicted by Adolf Smekal in 1923, it is not until 1928 that it was observed in practice. The Raman effect was named after one of its discoverers, the Indian scientist Sir C. V. Raman who observed the effect by
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means of sunlight (1928, together with K. S. Krishnan and independently by Grigory Landsberg and Leonid Mandelstam). Raman won the Nobel Prize in Physics in 1930 for this discovery accomplished using sunlight, a narrow band photographic filter to create monochromatic light and a "crossed" filter to block this monochromatic light.
He found that light of changed frequency passed through the "crossed" filter.
Systematic pioneering theory of the Raman effect was developed by Czechoslovak physicist George Placzek between 1930 and 1934. The mercury arc became the principal light source, first with photographic detection and then with spectrophotometric detection. At the present time, lasers are used as light sources.
The Raman effect is a light scattering phenomenon. While light of frequency V0 (usually from a laser) irradiates a sample, it can be scattered. The frequency of the scattered light can either be at the original frequency which referred to as Rayleigh scattering or at some shifted frequency VS = V0 Vinternal (referred at as Raman scattering). The frequency Vinternal is an internal frequency corresponding to rotational, vibrational, or electronic transitions. In discussing the Raman effect, some commonly used terms need to be defined. As shown in figure 2-4, radiation scattering to the lower frequency side (to the red) of the exciting line is call Stockes scattering. The scattering radiation at the same frequency as the incident radiation is called Rayleigh scattering. While the light scattered at higher frequency than exciting line (to the blue)
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is referred to as the anti-Stokes scattering. Finally, the magnitude of this shift between
the Stokes or the anti-Stokes line and the exciting line is called the Raman shift, ΔV = ∣V0 – Vinternal ∣. The energy diagram for Stokes and anti-Stokes scattering was
shown in figure 2-4.
2-2-4 Luminescence
Luminescence is the emission of light from any substance and occurs from electronically excited states. Luminescence can be of two types: fluorescence and phosphorescence. Phosphorescence is emission of light from triplet-excited states, in which the electron in the excited orbital has the same spin orientation as the ground-state electron. Transitions to the ground state are forbidden and the emission rates are slow (103-100 s-1), so phosphorescence lifetimes are typically milliseconds to seconds. Phosphorescence is usually not seen in fluid solutions at room temperature, but there are many deactivation processes that compete with emission, such as nonradiative decay and quenching processes. Fluorescence is emission light from singlet-excited states, in which the electron in the excited orbital is paired (of opposite sign) to the second electron in the ground-state orbital. Return to the ground state is spin-allowed and occurs rapidly by emission of a photon. Those emission rates of fluorescence typically are 108 s-1, so that a typical fluorescence lifetime is near 10 ns.
Fluorescence spectral data are generally presented as emission spectra. Emission
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spectra vary widely and are dependent upon the chemical structure of the fluorophore and the solvent in which it is dissolved.
A fluorophore is usually excited to some higher vibrational level of either S1 or S2. With a few rare exceptions, molecules in condensed phases rapidly relax to the lowest vibrational level of S1. This process, called internal conversion, is nonradiative and takes place in 10-12 seconds or less. Return to the ground state occurs to a higher excited vibrational ground-state level, which then quickly reaches thermal equilibrium.
An interesting consequence of emission to a higher vibrational ground state is that the emission spectrum is typically a mirror image of the absorption spectrum of the S0S1 transition.
Molecules in the S1 state can also undergo a spin conversion to the first triplet state, T1. Emission from T1 is termed phosphorescence and is generally shifted to longer wavelengths (lower energy) relative to fluorescence. Transition from the T1 to the singlet ground state is forbidden, and as a result, the rate constants for triplet emission are several orders of magnitude smaller than those for fluorescence. As shown in figure 2-5, processes which occur between the absorption and emission of light are usually illustrated by a Jablonski diagram.
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2-3 Reference
[2.1] Paul Daviddovits and M. David Egger, "Scanning Laser Microscope," nature, vol. 223, 831 (1969)
[2.2]ELS-7500EX Electron Beam Lithography System Instrument Manual, ELIONIX Inc.
.
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Figure 2-1: Overlay of electron beam system and the electron optical column of electron beam system [2.2]
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Figure 2-2: Illustration and simulation result of forward and back scattering effect and the charge effect during the electron beam process
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Figure 2-3: Illumination of confocal microscope
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Figure 2-4: Energy diagram for Rayleigh, Stokes, and anti-Stokes scattering
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Figure 2-5: Simplified Jablonski diagram with absorbance, internal conversion, fluorescence, intersystem crossing, and phosphorescence
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