Research Motivations

With the advantages of scattering and absorption enhancement in the long-wavelength range that allows deeper tissue penetration [35], Au NRI is an effective agent for biomedical applications, like photothermal therapy and optical coherence tomography. However, limited by the fabrication process, the size of Au NRI is always between 90~180nm,

which is much larger than those of other Au NPs. In this situation, Au NRIs undergo a slow process of cell internalization [39-41] because there is there’s a longer wrapping time for larger Au NPs. On the other hand, a larger NP has more antibodies bio-conjugated on the surface such that the adsorption probability of Au NRIs onto a cell is increased. With the considerations above, we believe that the study of the photothermal cell inactivation efficiency with internalized and adsorbed Au NRIs is needed.

To distinguish the efficiencies of internalized and adsorbed Au NRIs in photothermal cell inactivation, KI/I2 solution can be applied to etch Au NRIs adsorbed on the cell membrane. Through this process, we can compare the cell inactivation efficiency of internalized Au NRIs with that of adsorbed Au NRIs.

Fig. 1.1 Dispersion relations of SPP and LSP. Due to the momentum mismatch between SPP dispersion and light line, light extraction from SPP energy needs certain mechanisms to match the momentum, such as prism, grating or rough surface structures. In contrast, there is no need to match the momentum for the energy exchange between light and LSP.

Fig. 1.2 Sketch of a homogeneous metallic sphere surrounded by an isotropic dielectric medium in a uniform static electric field.

Fig. 1.3 Relative absorption and scattering spectra of tissue.

X Y

E=E

r a

ε

(ω)

ε

(ω)

Fig. 1.4 (a) TEM image of Au nanospheres with the average size at 99 nm.(b) Extinction spectra of Au nanospheres of 9, 22, 48, and 99 nm in diameter within water [22].

Fig. 1.5 (a) Typical TEM image of Au NRs prepared via the seed-mediated growth method. (b) TEM image of Au NRs with the aspect ratio around 3, the LSP resonance peak around 700 nm. (c) Extinction spectra of gold nanorods with the aspect ratios at 1.7, 2.8, 3.5, 4.0, and 4.4, respectively [29].

Fig. 1.6 (a) Horizontal cut and (b) vertical cross section of the near field distribution and field enhancement of the LSP dipole mode in a ring structure (ring radius: 60 nm, ring thickness: 10 nm, ring height: 22.7 nm), the scale bar shows the field enhancement [36].

(a)

(b)

Fig. 1.7 Variations of LSP resonance wavelengths of the cross-ring dipole and axial dipole modes of an Au NRI as functions of the ring height, H, at various ring thicknesses, d, with the outer radius of the NRI fixed at a = 50 nm. The horizontal dotted line at 965 nm passes the intersection point of the two curves of d = 7.5 nm for the cross-ring and axial dipole modes [36].

Fig. 1.11 Simulation results of Au nanorings with the outer radii at a = 37.5, 62.5, and 87.5 nm in the top, middle, and bottom panels, respectively. The red dashed curve and blue solid curve in the panels represent absorption and scattering cross-section, respectively. The five sets of curve in each panel with their peaks shifting from short to long wavelength correspond to 2.5, 3.33, 5, 6.67, 10 in aspect ratio.

Wavelength (nm) Cross section (10-11 cm 2 )

6000 700 800 900 1000 1100 1200 1300 1400 1500 20

40 60

6000 700 800 900 1000 1100 1200 1300 1400 1500 50

100 150

8000 900 1000 1100 1200 1300 1400 1500 1600 1700 100

Fig. 1.12 Four types of outer material entering into cell: (a) active transport, (b) phagocytosis, (c) pinocytosis, (d) receptor-mediated endocytosis. (pictures are downloaded from

http://commons.wikimedia.org/wiki/File:Endocytosis_types.svg) (a)

(b) (c) (d)

Fig. 1.13 Different stages of the cellular uptake process of _14 nm transferrin-coated Au NPs. (A-D) Schematic depicting the arrival of a NP at the cell membrane, binding of the nanopartcles to surface receptors, membrane wrapping of the NP, and finally internalization into the cell, respectively. (E-F) TEM images capturing each of these steps. Hela cells were used [41].

Fig. 1.14 Different stages of the cellular uptake process of _50 nm transferrin-coated Au NP. (A-D) Schematic depicting the arrival of a NP at the cell membrane, binding of the nanopartcles to surface receptors, membrane wrapping of the NP, and finally internalization into the cell, respectively. (E-F) TEM images capturing the endocytosis of nanoparticles into Hela cells [41].

Chapter 2

Materials and Methods

2.1 Fabrication of Bio-conjugated Au Nanorings

Figures 2.1(a)-2.1(g) show the fabrication procedures of bio-conjugated Au NRI solution. In the first step, a Si nano-imprint mold is used to impress a polymer substrate for forming a nanopillar array on it.

The diameter and height of the nanopillars are 180 and 100 nm, respectively. The schematic demonstration of this step and the resultant tilted SEM image of the substrate are shown in Figs. 2.2(a) and 2.3(a), respectively. Then, O2 plasma is applied in a reactive ion etching (RIE) process to adjust the diameter and height of the nanopillars, as shown in Figs. 2.1(b) and 2.2(b) for the schematic demonstration and the resultant SEM image, respectively. In one of the implementations leading to the results to be discussed in the following, the nanopillar diameter and height are reduced to ~70 and ~80 nm, respectively. Next, an Au film of

~23 nm in thickness is deposited onto the polymer substrate to serve as the source of secondary sputtering, as depicted in Fig. 2.1(c). The secondary sputtering is implemented through a process of CHF3 RIE under the conditions of 30 SCCM in gas flow rate, 1.3 Pa in pressure, 80 W in RF power, and 565 s in RIE duration. In this process, the Au atoms on the tops of the nanopillars are removed. Meanwhile, the Au atoms on

the substrate surface in the gap regions between nanopillars are sputtered onto the sidewalls of the nanopillars to form a ring shape, as depicted in Fig. 2.1(d). The tilted SEM image after this step is shown in Fig. 2.2(c).

Then, another step of O2 RIE is applied to remove the polymer inside the Au ring structure, i.e., the original pillar body. In this stage, the background substrate level is also lowered to form new pillars with the Au ring structures at the tops, as depicted in Fig. 2.1(e). The plan-view and tilted SEM images of the Au NRIs on substrate after this step are shown in Figs. 2.2(d) and 2.2(e), respectively. To enhance the robustness of the Au ring structure, the sample is thermally annealed at 170-180 ^oC for 10 min. The purposes of the thermal annealing process are to avoid the breakage of Au NRI after liftoff and to enhance the overall CRD LSP resonance strength. We use the ratio of the overall LSP resonance peak level over the background level in the extinction spectrum (within the measurement range of 400-1300 nm) of an Au NRI solution at 4 as the criterion of the CRD LSP resonance strength. It is noted that RIE is a semi-physical and semi-chemical reaction process, depending on the chosen etching chemicals. With O2 as the etching gas, RIE is essentially a chemical process and can easily etch polymers. In our operation with CHF₃, RIE mainly causes a physical process of bombarding the Au atoms to result in the effect of secondary sputtering.

Next, the bio-conjugation process is applied to the Au NRIs when

they are still attached to the substrate. In this process, first, the substrate with Au NRIs is immersed in a biolinker solution for 4 hours to form the carboxyl groups on the surface of Au NRIs. The biolinker solution is prepared by mixing a Nanothinks acid16 (5mM in ethanol, Sigma-Aldrich) solution of 30 µL with 15 mL de-ionized water. Then, the sample is rinsed in de-ionized water for several times to remove the residual biolinker. Next, the sample is immersed in a mixed solution of 100 µL (100 mM) 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), 25 µL (100 mM) N-hydroxysulfosuccinimide (sulfo-NHS), and 15 mL phosphate buffer saline (PBS) solution for 20 min to activate the biolinker. Then, the monoclonal anti-EGFR antibody (20 µL , 0.5 mg/mL, Anti-EGFR (26-125) mAb, Abnova) is added to the mixed solution for antibody connection. After the interaction for 22 hours, the sample is rinsed again to remove the residual antibody and other chemicals. The bio-conjugation process is schematically depicted in Fig.

2.1(f). After this process, the tilted SEM image of the on-substrate Au NRIs is shown in Fig. 2.2(f). From the SEM images of several samples at this stage, it is estimated that ~5 % of Au NRIs can be lost during the bio-conjugation process. To transfer the Au NRIs into water solution, the substrate is placed in a glass bottle with a proper amount of PBS.

Sonication (NeyTech 104H with a power of 340 W for 5 min) is applied

until >95 % of the NRIs, which still remain on the substrate after the bio-conjugation process, are transferred into water, as depicted in Fig.

2.1(g). Figure 2.3 shows a tilted SEM image of the substrate after Au NRI liftoff with sonication. The only Au NRI remained on the substrate is indicated by the arrow. To avoid NRI aggregation in solution, PEG-Thiol (mPEG-Thiol-5000, Laysan Bio Inc., Arab, AL) is added to the NRI solution. The concentration of PEG-Thiol in the NRI solution is 1×10^-4 M.

Fig. 2.4(a) shows the SEM image of the fabricated Au NRIs when the dried-up Au NRIs are placed on a Si substrate. Figure 2.4(b) shows the photograph of Au NRI solution in a container. Its concentration is estimated to be 10¹¹ per mL. The Au NRI concentration in the solution is estimated by evaluating the number of the on-substrate Au NRIs and by assuming that 90 % of the Au NRIs are transferred into the solution [35].

To confirm the surface modification procedures, the zeta potential levels under different surface modification conditions of Au NRIs are measured. First, we measure the zeta potential of pure NRIs and obtain the value is -34.51 mV. It shows there are negative charges left on the surface of NRIs during the fabrication process. After the biolinker is applied to the Au NRIs, the zeta potential is -34.55 mV. The zeta potential is basically the same because the carboxyl groups in the biolinker are also possess negative charge. Then, after antibody is applied to the Au NRIs besides the biolinker, the zeta potential becomes -23.51 mV, which is a

sign for we’ve successfully bio-conjugated the biolinker to the antibody.

The zeta potential magnitude is reduced because the carboxyl groups of part of the biolinker are linked to the amine groups of the antibody such that the negative surface charge of the Au NRIs is decreased. Next, when the biolinker, antibody, and PEG-Thiol are applied to the Au NRIs, the zeta potential becomes -21.08 mV. If only PEG-Thiol is applied, the zeta potential becomes -17.55 mV. The above two data show that PEG-Thiol will not replace biolinker-antibody. Although the antibody can be directly linked to an Au NP without using a biolinker [57], we use the biolinker in this work for connecting the antibody and the Au NRIs to assure the strong bonding between them. Our zeta potential measurements confirm that the function of the biolinker works.

Consider the cell uptake efficiency is better for small NPs, we attempt to fabricate NRIs with CRD LSP resonance wavelength near 1064 nm, which is the output wavelength of our CW laser, as small as possible to promote the amount of NRIs taken up by SAS cells. In chapter 1.3, we’ve talked about the characteristics of Au NRI. In order to fabricate smaller NRIs and make the resonance wavelength remain near 1064 nm at the same time, we have to reduce the diameter, which leads to blue shift, the height and the thickness, both which leads to red shift. In order to reduce the diameter, we have to increase the first CHF3-O2 RIE process time to ~220 s and control the diameter in the range from 65 nm

to 70 nm. Then, we attempt to decrease the thickness by reducing the thickness of deposited Au film to 21~23 nm. Because the thickness cannot increase further after all Au atoms are sputtered onto sidewalls of polymer nanorods, reducing the thickness of the deposited Au film can effectively reduce the thickness of NRIs. Finally, we deal with height by increasing the second CHF3 RIE process time. With the second CHF3 RIE process, the Au atoms attached to the sidewall of a polymer nanorod can be removed from the top to make the fabricated Au NRI shorter (smaller h), as schematically shown in Fig. 2.5. Because the stability of RIE process is not perfectly good, SEM is used to check if the wanted dimensions of diameter, thickness, and height are achieved. As a result, NRIs with diameter near 105 nm, thickness near 18 nm, and height near 75 nm perform LSP resonance near 1065 nm. Figure 2.6 shows six examples of NRIs with similar geometry diameters. From (a) to (f) the diameter, thickness, height (represented by D, d, h) are (a): (102 nm,18 nm,73 nm), (108 nm,20 nm,80 nm), (100 nm,16 nm,75 nm), (109 nm,19 nm,80 nm), (100 nm,20 nm,70 nm), (108 nm,20 nm,70 nm). All of them have similar CRD LSP resonance wavelength near 1065 nm as in Fig.

2.7.

2.2 SAS Cell Culture

SAS cells (see Fig. 2.8) are donated by Yih-Chih Hsu’s laboratory of

Chung Yuan Christian University. It is a poorly differentiated human squamous cell carcinoma line, cultured in DMEM/F-12 medium containing 10% FBS, 400 ng/ml hydrocortisone, 1% sodium bicarbonate and 1% antibiotic in culture flasks in humidified 5% CO, at 37°C [59-61].

The cell inactivation experiment is conducted with 4×10⁵ SAS cells seeded on a 24-well cell culture plate for 12 hours. Then, both bio-conjugated/non-bio-conjugated NRIs are applied for the durations of 8, 10, 12, 16, 20, and 24 hours, respectively. Then, the cell inactivation experiment is conducted. After SAS cells are trypsinized for ICP-MS analysis, we count the number of cell on the cell culture plate for analyzing the number of NRI per cell.

2.3 Inductively Coupled Plasma Mass Spectrometer (ICP-MS) Analysis

In our experiment, we need to measure the amounts of internalized, adsorbed, and non-interacted Au NRIs. For this purpose, we use inductively coupled plasma mass spectrometry (ICP-MS) for Au quantization. The ICP-MS measurement is performed at the Instrumentation Center at National Tsing Hua University. Inductively coupled plasma mass spectrometry (ICP-MS, see Fig. 2.9) is a type of mass spectrometry, which is designed to measure metals and several

non-metals at a concentration as low as one part per 10¹². This kind of precision is achieved by ionizing the sample with inductively coupled plasma and then using a mass spectrometer to separate and quantify those ions. Because the content of Au contained in a sample is in the order of 10^-9 µg/ml, highly-sensitive ICP-MS is needed to measure the Au content.

Before measuring the concentration, we have to dissolve Au NRIs with aqua regia for uniform distribution of the solution.

Fig. 2.1 Fabrication procedures of bio-conjugated Au NRI solution. (a):

Nano-imprint step to prepare the polymer substrate with nanorods; (b):

First O2 RIE step to modify the geometry of the polymer nanorods; (c):

Au deposition step. (d): CHF3 RIE step for secondary sputtering of Au to form the ring shape. (e): Second O₂ RIE step to remove the polymer nanorods and lower the substrate surface. (f): Bio-conjugation step. (g):

Sonication step to transfer Au NRIs into water solution.

Fig. 2.2 SEM images on the substrate showing the results after various fabrication steps. (a): After the nano-imprint step. (b): After the first O₂ RIE step. (c): After the Au deposition and CHF₃ RIE steps; (d) and (e): After the second O2 RIE step (plan-view and tilted-view, respectively). (f): After the bio-conjugation process.

Fig. 2.3 Tilted SEM image of the substrate after Au NRI liftoff with sonication. The Au NRI remained on the substrate is indicated by the arrow.

Fig. 2.4 (a): SEM image of the fabricated Au NRIs taken when the dried-up Au NRIs are placed on a Si substrate. (b): Photograph of an Au NRI solution in a container.

Fig. 2.5: Schematic drawing to show the reduction of ring height in the second CHF3 RIE process.

Fig. 2.6 (a)-(f) are tilted SEM images of NRI arrays on polymer substrate (samples I-IV, respectively). The followings are three geometry parameters (diameter, D, thickness, d, and height, h) of each sample.

(a)D=102 nm, d=18 nm, h=73 nm (b) D=108 nm , d=20 nm, h=80 nm (c)D=100 nm, d=16 nm, h=75 nm (d) D=109 nm, d=19 nm, h=80 nm (e) D=100 nm, d=20 nm, h=70 nm (f) D=108 nm, d=20 nm, h=70 nm, all of them are similar in these dimensions.

Fig. 2.7 Normalized extinction spectrums of I-IV sample are illustrated in this figure. With similar diameters, thicknesses, and heights, the CRD LSP resonance wavelength of each sample are pretty close.

Fig. 2.8 SAS cells in cell culture medium.

Fig. 2.9 Inductively Coupled Plasma Mass Spectrometer (ICP-MS).

Chapter 3

Cell Inactivation by Localized Surface Plasmon-induced Photothermal Effect 3.1 Experimental Procedures

The insert of Fig. 3.1 shows the SEM image of the used Au NRIs before liftoff. Figure 3.1 shows the normalized extinction spectrum of the Au NRIs in PBS with PBS as the base line of transmission measurement.

The average outer diameter, thickness, and height of the Au NRIs are 102, 18, and 66 nm, respectively. Two Au NRI solution samples are prepared, including the one with antibody (NRI-AB) and another without antibody (NRI-control). The Au NRI concentrations of the NRI-AB and NRI-control solutions are (1.51 0.12)×10¹⁰ and (1.74 0.18)×10¹⁰ cm^-3, respectively. The zeta potentials of the NRI-AB and NRI-control are -21.1 and -17.6 mV, respectively, indicating the effective antibody linkage.

In Fig. 3.1, one can see that the major LSP resonance induced extinction peak is located at 1058 nm, as indicated by the vertical dashed line. The arrow next to the dashed line marks the wavelength of the excitation laser for cancer cell inactivation at 1065 nm. The extinction ratio of the peak with respect to the minimum level around 500 nm is as large as 5.42, indicating the strong cross-ring LSP resonance around 1058 nm.

Figure 3.2 shows the viability of the used cancer cell (SAS oral cancer cell) when it is incubated with Au NRI-AB and NRI-control for different incubation times (8, 12, and 24 hrs). The statistics are obtained based on the cell incubation of 24-well culture plate. From Fig. 3.2, one can see that either Au NRI-AB or NRI-control is essentially non-toxic to the used cancer cells. In the study, we will use KI/I2 solution for etching the absorbed Au NRIs on cell membrane (not internalized yet) without significantly damage the cells. Figure 3.3 shows the normalized extinction spectra of Au NRI-AB solution before and after adding a KI/I2

solution (0.268 mM for KI and 0.088 mM for I₂) for different durations.

The extinction spectrum of the Au NRI solution in Fig. 3.1 is repeated here and labeled by “Before etching”. The extinction spectra after adding the KI/I₂ solution in Fig. 3.3 are obtained by using the same KI/I₂ solution as the measurement base line. Here, one can see that after adding KI/I2 to the Au NRI solution, the major and minor spectral peaks red- and blue-shifts, respectively, due to the increase of the refractive index after KI/I₂ is added. After the addition of KI/I₂, both the major and minor peaks diminish with time as the Au NRIs are dissolved. Also, the major and minor spectral peaks continue red- and blue-shifting, respectively, due to the continuing increase of solution refractive index caused by the increase of Au ion concentration after the Au NRIs are etched. The extinction spectra after KI/I2 is added are measured every 3 min. One can see that

after 60 min etching, the major Au NRI features disappear. The optimized etching time of KI/I2 also relies on the cell viability in KI/I2 solution.

Figure 3.4 shows such data at different etching durations. With 60 min in etching duration, 96.6 % cells can still survive. Therefore, we choose 60 min as the optimized KI/I2 etching duration for almost completely etching the Au NRIs outside cells and maintain reasonably high cell viability.

Figures 3.5(a)-3.5(c) schematically show the different laser illumination and cancer inactivation conditions in this study. As demonstrated in Fig. 3.5(a), after the designed incubation time upon the application of Au NRIs to cancer cell wells, the cancer cell is illuminated by the laser from the bottom. This condition is referred to as

“Pre-washout”, under which the Au NRIs can be internalized into the

“Pre-washout”, under which the Au NRIs can be internalized into the