Browsing by Subject "Nanotechnology"

Osteoporosis and low bone density affect an estimated 54 million adults of 50 years and over in the United States, resulting in $19 billion in costs for osteoporosis-related bone breaks. Current treatments include the use of antiresorptive and anabolic drugs to decrease the rate of bone resorption and increase the rate of bone formation, respectively. However, these current treatments are unable to completely normalize skeletal integrity. As bone diseases become increasingly prevalent, there is an urgent need to identify novel therapies to improve quality of life and reduce economic burden on the healthcare system.

To identify novel therapeutic targets, we must first begin to understand the cellular complexity of the bone marrow niche and how cellular function is regulated within the bone tissue. Bone-resident cells, such as skeletal progenitors and their descendants, are critically influenced by extrinsic signals derived from the local microenvironment. Previous studies have identified hypoxia as a key microenvironment factor in bone. Thus, the ability to target the hypoxic bone marrow niche presents an attractive and untapped potential for regenerative medicine.

Much of the work investigating the role of hypoxia and HIF signaling have focused on mature osteoblast and chondrocyte populations. In contrast, studies investigating the contribution of HIF signaling on skeletal progenitors and marrow adipocyte populations are scarce. In this dissertation, I investigate the role of hypoxia and HIF signaling in skeletal lineage cells, chiefly skeletal progenitor cells and marrow adipogenic lineage cells. Using cellular, genetic, and pharmacological-based approaches, I characterize the roles of HIF-1α and HIF-2α in both homeostatic and pathological contexts in the aforementioned cell populations.

First, I propose an optimized cell-based system to investigate the function of skeletal progenitors in vitro. Here, I highlight the limitations of current in vitro isolation techniques and introduce a relatively simple method of bone marrow stromal cell purification using hypoxia. Using this system, I assess how skeletal progenitors respond to hypoxic cues and interrogate skeletal progenitor cell differentiation and functional responses in my subsequent research. Next, using genetic and pharmacological approaches, I investigate the role of HIF-2α in bone formation following radiation-injury where I identify HIF-2α as a negative regulator of bone recovery. Additionally, with the assistance of my collaborators, I develop and characterize a bone-targeting nanocarrier to ameliorate radiation-induced bone loss. Lastly, I detail early work I conducted to investigate the role of HIF signaling in marrow adipogenic lineage cells. Here, I establish and characterize animal models to determine how hypoxia and HIF signaling influences adipogenic lineage commitment and expansion in an early and mature marrow adipogenic population.

In summary, this dissertation aims to expand our limited understanding on how the hypoxic bone microenvironment and HIF signaling regulate skeletal lineage cells in vivo, with a special focus on skeletal progenitor and marrow adipogenic populations. In terms of boarder impacts, understanding the signaling networks that regulate bone homeostasis and recovery processes will not only expand our basic understanding of the molecular mechanisms underlying skeletal development, but also provide novel insights for developing therapies to treat bone loss.

Active plasmonic nanostructures with tunable resonances promise to enable smart materials with multiple functionalities, on-chip spectral-based imaging and low-power optoelectronic devices. A variety of tunable materials have been integrated with plasmonic structures, however, the tuning range in the visible regime has been limited and small on/off ratios are typical for dynamically switchable devices. An all optical tuning mechanism is desirable for on-chip optical computing applications. Furthermore, plasmonic structures are traditionally fabricated on rigid substrates, restricting their application in novel environments such as in wearable technology.

In this dissertation, I explore the mechanisms behind dynamic tuning of plasmon resonances, as well as demonstrate all-optical tuning through multiple cycles by incorporating photochromic molecules into plasmonic nanopatch antennas. Exposure to ultraviolet (UV) light switches the molecules into a photoactive state enabling dynamic control with on/off ratios up to 9.2 dB and a tuning figure of merit up to 1.43, defined as the ratio between the spectral shift and the initial line width of the plasmonic resonance. Moreover, the physical mechanisms underlying the large spectral shifts are elucidated by studying over 40 individual nanoantennas with fundamental resonances from 550 to 720 nm revealing good agreement with finite-element simulations.

To fully explore the tuning capabilities, the molecules are incorporated into plasmonic metasurface absorbers based on the same geometry as the single nanoantennas. The increased interaction between film-coupled nanocubes and resonant dipoles in the photochromic molecules gives rise to strong coupling. The coupling strength can be quantified by the Rabi-splitting of the plasmon resonance at ~300 meV, well into the ultrastrong coupling regime.

Additionally, fluorescent emitters are incorporated into the tunable absorber platform to give dynamic control over their emission intensity. I use optical spectroscopy to investigate the capabilities of tunable plasmonic nanocavities coupled to dipolar photochromic molecules. By incorporating emission sources, active control over the peak photoluminescence (PL) wavelength and emission intensity is demonstrated with PL spectroscopy.

Beyond wavelength tuning of the plasmon resonance, design and characterization is performed towards the development of a pyroelectric photodetector that can be implemented on a flexible substrate, giving it the ability to be conformed to new shapes on demand. Photodetection in the NIR with responsivities up to 500 mV/W is demonstrated. A detailed plan is given for the next steps required to fully realize visible to short-wave infrared (SWIR) pyroelectric photodetection with a cost-effective, scalable fabrication process. This, in addition to real-time control over the plasmon resonance, opens new application spaces for photonic devices that integrate plasmonic nanoparticles and actively tunable materials.

There is an urgent need to develop practical and effective systems to detect diseases, such as cancer, infectious diseases and cardiovascular diseases.

Nanotechnology is a new, maturing field that employs specialized techniques to detect and diagnose infectious diseases. To this end, there have been a wealth of techniques that have shown promising results, with fluorescence and surface-enhanced Raman scattering being two important optical modalities that are utilized extensively. The progress in this specialized niche is staggering and many research groups in academia, as well as governmental and corporate organizations, are avidly pursuing leads which have demonstrated optimistic results.

Although much fundamental science is still in the pipeline under the guise of both ex-vivo and in-vivo testing, it is ultimately necessary to develop diagnostic devices that are able to impact the greatest number of people possible, in a given population. Such systems make state-of-the-art technology platforms accessible to a large population pool. The development of such technologies provide opportunities for better screening of at-risk patients, more efficient monitoring of disease treatment and tighter surveillance of recurrence. These technologies are also intrinsically low cost, facilitating the large scale screening for disease prevention.

Fluorescence has long been established as the optical transduction method of choice, because of its wealth of available dyes, simple optical system, and long heritage from pathology. The intrinsic limitations of this technique, however, have given rise to a complementary, and more recent, modality: surface-enhanced Raman scattering (SERS). There has been an explosive interest in this technique for the wealth of information it provides without compromising its narrow spectral width.

A number of novel studies and advances are successively presented throughout this study, which culminate to an advanced SERS-based platform in the last chapter.

The finite element method algorithm is critically evaluated against analytical solutions as a potential tool for the numerical modeling of complex, three-dimensional nanostructured geometries. When compared to both the multipole expansion for plane wave excitation, and the Mie-theory with dipole excitation, this algorithm proves to provide more spatially and spectrally accurate results than its alternative, the finite-difference time domain algorithm.

Extensive studies, both experimental and numerical, on the gold nanostar and Nanowave substrate for determining their potential as SERS substrates, constituted the second part of this thesis. The tuning of the gold nanostar geometry and plasmon band to optimize its SERS properties were demonstrated, and significant 3-D modeling was performed on this exotic shape to correlate its geometry to the solution's exhibited plasmon band peak position and large FWHM. The Nanowave substrate was experimentally revived and its periodic array of E-field hotspots, which was until recently only inferred, was finally demonstrated via complex modeling.

Novel gold- and silver- coated magnetic nanoparticles were synthesized after extensive tinkering of the experimental conditions. These plasmonics-active magnetic nanoparticles were small and displayed high stability, were easy to synthesize, exhibited a homogeneous distribution, and were easily functionalizable with Raman dye or thiolated molecules.

Finally, bowtie-shaped cobalt micromagnets were designed, modeled and fabricated to allow the controllable and reproducible concentrating of plasmonics-active magnetic nanoparticles. The external application of an oscillating magnetic field was accompanied by a cycling of the detected SERS signal as the nanoparticles were concentrated and re-dispersed in the laser focal spot. This constituted the first demonstration of magnetic-field modulated SERS; its simplicity of design, fabrication and operation opens doors for its integration into diagnostic devices, such as a digital microfluidic platform, which is another novel concept that is touched upon as the final section of this thesis.

Biological systems have inspired interest in developing artificial molecular self-assembly techniques that imitate nature's ability to harness chemical forces to specifically position atoms within intricate assemblies. Of the biomolecules used to mimic nature's abilities, nucleic acids have gained special attention. Specifically, deoxyribonucleic acid is a stable molecule with a readily accessible code that exhibits predictable and programmable intermolecular interactions. These properties are exploited in the revolutionary structural DNA nanotechnology method known as scaffolded DNA origami. For DNA origami to establish itself as a widely used method for creating self-assembling, complex, functional materials, current limitations need to be overcome and new methods need to be established to move forward with developing structures for diverse applications in many fields. The limitations discussed in this dissertation include 1) pushing the scale of well-formed, fully-addressable origami to two and seven times the size of conventional origami, 2) testing cost-effective staple strand synthesis methods for producing pools of oligos for a specified origami, and 3) engineering mechanical properties using non-natural nucleotides in DNA assemblies. After accomplishing the above, we're able to design complex DNA origami structures that incorporate many of the current developments in the field into a useful material with applicability in wide-ranging fields, namely cell biology and photonics.

Silver nanoparticles (AgNPs) are increasingly being integrated into a wide range of consumer products, such as air filters, washing machines, and textiles, due to their antimicrobial properties [1]. However, despite the beneficial applications of AgNPs into consumer products, it is likely that their use will facilitate the release of AgNPs into wastewater treatment plants, thereby possibly negatively impacting key microorganisms involved in nutrient removal. For this reason, it is important to characterize the effects of AgNPs in natural and engineered systems and to measure the antimicrobial effect of AgNPs on wastewater microorganisms. Polyvinyl alcohol coated AgNPs have already been linked to decreased nitrifying activity [2] and it is important to determine if AgNPs coated with other materials follow similar trends. Furthermore, it is likely that, with repeated exposure to AgNPs microbial communities could evolve and develop resistance to silver. Thus, a long-term effect of silver nanoparticle exposure could be a reduction of the efficacy of such products in a similar fashion to the development of microbial antibiotic resistance [3]. Therefore, it is critical that the impacts of these materials be ascertained in wastewater treatment systems to prevent long-term negative effects.

The objectives of this dissertation were to: 1) characterize the effect of several different AgNPs on the ammonia oxidizing bacterium (AOB) Nitrosomonas europaea and investigate possible mechanisms for toxicity, 2) test the effects of consumer product AgNPs on a wide range of heterotrophic bacteria, 3) evaluate the effects of AgNPs on bench scale wastewater sequencing batch reactors, and lastly 4) assess the impacts on microbial communities that are applied with AgNP spiked wastewater biosolids.

First, Nitrosomonas europaea was was selected because wastewater nitrifying microorganisms carry out the first step in nitrification and are known to be sensitive to a wide range of toxicants [4].The antimicrobial effects of AgNPs on the AOB N. europaea were measured by comparing nitrite production rates in a dose response assay and analyzing cell viability using the LIVE/DEAD® fluorescent staining assay. AgNP toxicity to N. europaea appeared to be largely nanoparticle coating dependent. While PVP coated AgNPs have shown reductions up to 15% in nitrite production at 20 ppm, other AgNPs such as gum arabic (GA) coated showed the same level of inhibition at concentrations of 2 ppm. The first mechanism of inhibition appears to be a post-transcriptional interference of AMO/HAO by either dissolved Ag or ROS, in treatments where membranes are not completely disrupted but nitrite production decreased (2 ppm GA AgNP and 2 ppm PVP AgNP treatments). The disruption of nitrification is dependent on AgNP characteristics, such as zeta potential and coating, which will dictate how fast the AgNP will release Ag+ and ROS production Finally, total membrane loss and release of internal cellular matter occur.

In order to test the effects of AgNP products available to consumers, simple bacterial toxicity tests were carried out on well-studied heterotrophic bacteria. A model gram-positive and gram-negative bacterium (B. subtilis and E. coli, respectively) was selected to assess any differences in sensitivity that may occur with the exposure to AgNPs. A third model gram-negative bacterium (P. aeruginosa) was chosen for its biofilm forming capabilities. In addition to testing pure nanoparticles, three silver supplements meant for ingestion, were randomly chosen to test with these three bacteria. Growth curve assays and LIVE/DEAD staining indicate that the consumer product AgNPs had the most significant inhibition on growth rates, but not membrane integrity. Overall, P. aeruginosa was most negatively affected by all AgNPs with nearly 100% growth inhibition for all 2 ppm AgNP treatments. TEM imaging also confirmed cell wall separation in P. aeruginosa and internal density differences for E. coli. The effects on B. subtilis, a gram-positive bacterium, were not as severe but toxicity was observed for several AgNPs at concentrations greater than 2 ppm. Citrate AgNPs appeared to have the most impact on membrane integrity, while other mechanisms such as internal thiol binding might have been at work for other AgNPs.

The effects of varying concentrations of pure AgNPs on complex microbial wastewater reactors are currently being tested. Eight bench-scale sequencing batch reactors were set up to follow the typical "fill, react, settle, decant, idle" method with an 8 hour hydraulic retention time and constant aeration. Reactors were fed synthetic wastewater and treatment efficiency is measured by monitoring effluent concentrations of COD, NH4+, and NO3-. The reactors were seeded with 500 mL of activated sludge from a local wastewater treatment plant. After reaching steady state, the reactors were spiked with 0.2 ppm gum arabic and citrate coated AgNPs. Treatment efficiency was monitored and results showed significant spikes and ammonia and COD immediately following the first spike, but the microbial community appeared to adapt for future AgNP spikes. Microbial community analysis (terminal restriction fragment length polymorphism) showed confirmed this hypothesis.

Overall, this dissertation asserts that by examining AgNP coating type, Ag+ dissolution rates and Stern layer surface charge, it may be possible to predict which AgNPs may be more detrimental wastewater treatment, but not all AgNPs will have the same effect. The results obtained herein must be expanded to other types of AgNPs and microorganisms of ecological importance.

The antimicrobial properties of silver have been taken advantage of by societies for thousands of years. Its use has come back in favor in the form of silver nanoparticles, which are highly efficacious antimicrobial agents. Silver nanoparticles are incorporated into a myriad of products specifically designed for clinical use, but also for general use by consumers. Silver nanoparticles can be found in textiles such as clothing and stuffed toys, and in home appliances including washing machines and curling irons. A large number of products specifically marketed for use by children are also available to consumers, including pacifiers, sippy cups, and even breast milk storage bags. The hazards and toxicities associated with silver nanoparticles are not well understood, however modes of toxicity have been reported for ionic silver. It is assumed that the main mechanism of toxicity of silver nanoparticles relates to the release of ionic silver, however studies have indicated an additional nano-effect, likely due to nanoparticle size, differential coatings, and means of sustained dosing of ionic silver. However we are sure that these silver nanoparticles will accumulate in the waste stream, likely arriving during different stages of a product’s lifespan. A main sink of these nanoparticles travelling through both natural and engineered environments is wastewater treatment plants. As a society we rely on the biological removal of nutrients, which takes place primarily in the activated sludge of secondary treatment. Studies have already indicated possible, temporary decreases in removal efficiencies as well as changes in microbial communities, including losses of diversity, following exposure to silver nanoparticles. Therefore, it is of paramount importance to examine the effects of both silver nanoparticles and ionic silver on the community and function of wastewater bacteria.

Sequencing batch reactors were operated to mimic wastewater treatment. They were fed synthetic wastewater and after reaching acclimation, were dosed over time with varying concentrations of both ionic and nanosilver. Cell samples were collected periodically to assess the presence and identity of cultivable silver resistant bacteria and to map the microbial community changes taking place under different treatments using Next Generation Sequencing. Isolates were tested for the presence of known silver resistance (sil) genes as were activated sludge samples from a collection of domestic wastewater treatment plants, by designing TaqMan probe assays and performing quantitative PCR. The silver resistant isolates were also used to test the growth implications, as well as sil gene expression changes, following treatment with ionic silver and a variety of silver nanoparticles with various coatings, all at multiple concentrations. This was accomplished by performing multiple batch experiments and then using the TaqMan assays and reverse transcription-quantitative PCR.

Overall, microbial community changes were observed in the sequencing batch reactors, and there were differences noted based on treatment, including ionic silver versus nanosilver and between the two silver nanoparticle coatings. Most notably, the possibility of nitrification in wastewater treatment being particularly susceptible was strongly indicated. Individual wastewater bacteria isolates all contained sil genes, as did the majority of the wastewater treatment plant activated sludge, however the levels of actual sil gene expression were inconsistent. This particular finding supports a current body of work indicating that there are alternate modes of bacterial silver resistance in play that we are just becoming aware of.

Project 1: Chest Phantom Development for Chest X-ray Radiation Protection Surveys

Purpose: Develop an acrylic phantom to accurately represent an average adult’s chest for use in radiographic chest unit radiation protection surveys.

Materials and Methods: 6 sheets of 3.81 cm thick acrylic were cut and assembled to form a 30.5 x 30.5 x 20.3 cm hollow box phantom. The acrylic served as tissue equivalent material and the hollow center simulated lungs in a human patient. Six sheets of 1 mm thick aluminum were cut to line the inner walls of the acrylic phantom to potentially boost scatter radiation. Three phantoms underwent posterior-anterior (PA) and lateral chest protocol radiographic scans: the acrylic phantom (with and without the aluminum lining), a 3 gallon water bottle filled with water, and an adult male anthropomorphic phantom. The phantoms were set up as though they were adult patients and scanned with automatic exposure control. Scatter radiation was measured with ion chamber survey meters at 4 points within the room for each phantom and protocol. The scatter data from the acrylic phantom and water bottle were compared to the anthropomorphic phantom to determine which one more accurately represented an adult patient.

Results: For the PA protocol, the average percent difference in measurements between the acrylic phantom and anthropomorphic phantom was 33.3±28.8% with the aluminum lining and 33.0±21.2% without the lining. The percent difference between the water bottle and anthropomorphic phantom was 66.5±42.0%. For the lateral protocol, the average percent difference in measurements between the acrylic phantom and anthropomorphic phantom was 157.6±5.6% with the aluminum lining and 143.0±17.6% without the lining. The percent difference between the water bottle and anthropomorphic phantom was 78.3±22.8%.

Conclusions: The acrylic phantom provided a more accurate comparison to the anthropomorphic phantom than the water bottle for the PA protocol. For the lateral protocol, neither the acrylic phantom nor water bottle provided an adequate comparison to the anthropomorphic phantom.

Project 2: Internal Beta Dosimetry of an Iodine-131 Labelled Elastin-Like Polypeptide

Purpose: Develop a model and simulation to better understand the dosimetry of an I-131 labeled elastin-like polypeptide (ELP) brachytherapy technique.

Materials and Methods: To develop the model, an average scenario based on mouse trials was explored. A 125 mg tumor was approximated as a sphere, with the I-131 ELP injected into its center. The ELP solidifies into a spherical depot – approximately 1/3 the volume of the tumor – and becomes a permanent brachytherapy source. The injected activity of I-131 was 1.25 mCi. I-131 primarily emits β radiation with an average energy of 182 keV, therefore it was determined that all such emissions were confined within the bounds of the tumor. Gamma emissions associated with I-131 were ignored as they were determined to have enough energy to escape the bounds of the tumor without any interaction. This model was implemented into a simulation using the Monte Carlo program FLUKA. From this simulation, the absorbed dose to the tumor and ELP depot, along with the dose profile, was calculated.

Results: The tumor received an absorbed dose of 72.3 Gy while the ELP received 1.14×10^3 Gy. From the dose profile, it was determined that 99% of the absorbed dose to the tumor was highly localized to a 0.3 mm region surrounding the ELP depot.

Conclusions: The model and simulation provided a better understanding of the dosimetry underlying the novel ELP brachytherapy technique. Results obtained demonstrated that the ELP method delivers doses that are comparable to current conventional brachytherapy techniques.

Project 3: I-131 Beta Detection Using a Scintillating Nanoparticle Detector

Purpose: Determine if a scintillating nanocrystal fiber optic detector (nano-FOD) could detect β emissions from I-131.

Materials and Methods: The nano-FOD’s β response was tested using a source vial containing 101 mCi of I-131 in 2 mL of stabilizing solution. A glass vial containing the I-131 was placed inside a lead pig for shielding. A 1 mm diameter hole was drilled through the tops of the vial and pig to allow insertion of the nano-FOD. Measurements were taken every day over a 17 day period by repeatedly submerging the nano-FOD in the I-131 solution and recording the voltage signal it produced. The activity at the time of measurement was calculated based on the time and date of data acquisition. The net signal and signal-to-noise ratio (SNR) were then calculated and plotted as functions of I-131 concentration.

Results: The nano-FOD produced a measurable response when exposed to the β emissions of I-131. The net signal and SNR both demonstrated a linear correlation with the concentration of I-131.

Conclusions: The nano-FOD was demonstrated to be capable of β detection with a linear correlation to activity. If the signals measured can be calibrated to radiation exposure, then the nano-FOD has promising applications as a novel β detector.

The rapid maturation of the internet of things (IoT) has led to an ever-stronger drive for large-area, flexible, and/or customizable electronics for data collection, display, and communication. The use of printing technology for IoT and thin-film electronics has shown growing promise due to its potential in low-cost and high-throughput manufacturing, as well as the capability to handle a wide array of substrates, materials, and production techniques (e.g., mass-production or customizable). At the heart of many IoT devices are thin-film transistors (TFTs). Carbon nanotubes (CNTs) have been considered promising candidates for printed TFTs due to their extraordinary electronic properties and material attributes, such as mechanical flexibility and low-temperature processability. Despite the significant research progress in printing CNT-TFTs and demonstrating CNT-TFTs in applications, process variability has still remained a major obstacle to the translation of CNT-TFTs out of the lab and into products. In addition, most printing processes reported in the literature involve post-printing thermal treatments, limiting the throughput and efficiency of printing approaches. This dissertation contains scientific discoveries, technical advancements, and innovations that reduce the process variability of CNT-TFTs and lead to the development of print-in-place processes -- a series of rapid, versatile, and streamlined printing approaches for yielding CNT-TFTs without any postprocessing. The key enabling factor of variability reduction is to understand the impact of CNT ink temperature, a commonly overlooked factor, on the resultant ink deposition via aerosol jet printing (AJP). It was discovered that an appropriately lowered ink temperature benefits both long-term (~1 hour) and short-term (~1 minute) stability of AJP, resulting in fully printed TFTs with average mobility of 12.5 cm2/V·s and mobility variation as small as 4%. The streamlining of the print-in-place processes for CNT-TFTs involved identifying low-temperature processable materials and formulating corresponding printable inks from these materials. By printing silver nanowires (AgNW) as the electrical contacts and hexagonal boron nitride (h-BN) as the gate insulator, the maximum processing temperature of the print-in-place process was reduced to 80 °C with no additional thermal treatment required. Notably, the resultant devices (known as 1D-2D TFTs) showed relatively good performance, including an on/off-current ratio up to 3.5×105, channel mobility up to 10.7 cm2/V∙s, small gate hysteresis, and superb mechanical stability under bending. In addition, another print-in-place process was demonstrated by employing a side-gate configuration with an ion gel dielectric and graphene contacts. Compared to the fabrication process for 1D-2D TFTs, this 3-step process was even more streamlined, and the resulting devices exhibited more uniform performance at the cost of the on/off-current ratio. Besides variability reduction and streamlining, the versatility and environmental friendliness of printed CNT-TFTs were also enhanced by studying the use of all-aqueous inks for CNT-TFTs. The study unveiled the printing challenges imposed by aqueous CNT inks compared to the more commonly used inks that depend on harsh solvents, such as toluene. It was discovered that the ionic surfactant in the aqueous inks hinders the CNT adhesion with the substrate, and a multi-step process with interstitial rinsing was proposed to mitigate this issue. Through the combination of aqueous CNT, graphene, and crystalline nanocellulose (CNC) inks, water-only TFTs were printed without the use of any harsh chemical solvents, and most device layers are either recyclable or biodegradable. In addition, although this dissertation is primarily focused on the printing techniques used to make CNT-TFTs, the materials and methods developed in the works were also utilized to demonstrate a surface acoustic wave (SAW) tuning device, an uncommon application for CNT-TFTs. The phase-velocity tunability (∆v/v = 2.5% ) was exceptional and close to the theoretical limit, suggesting the promise of CNT-TFTs for SAW applications and that there exist numerous unexplored areas where CNT-TFTs could potentially be advantageous. Overall, the findings and advancements contributed by this dissertation advance the printing of CNT-TFT by addressing obstacles and demonstrating possibilities. These include the developed methodologies, such as the print-in-place approaches and the multi-step aqueous printing with interstitial rinsing, along with the discoveries that may trigger follow-up studies, such as the correlation between ink temperature and process variability. Combined, these contributions help to reduce the variability, lower the process duration, and enhance the versatility of printed CNT-TFTs, pushing the field toward ubiquitous use in real-world applications.

Solid-state quantum emitters embedded in carefully engineered nanostructures could enable a new generation of quantum information and sensing technologies, including networked processors for quantum computing and precise monitors of temperature and strain at the nanoscale. The primary goal when designing these nanostructures is to utilize the Purcell effect to improve the emission rate, directionality and brightness of quantum emitters, as long decay times, nondirectional emission and weak fluorescence limit their applications. One particularly promising emitter is the silicon vacancy (SiV) in diamond, which offers excellent photostability and minimal spectral diffusion, in addition to coherent emission at its zero-phonon line (ZPL) comprising 80% of its total fluorescence. In this dissertation, up to 121-fold enhancement of the spontaneous emission rate of SiVs coupled to plasmonic nanogap cavities is demonstrated. The vacancy centers are implanted into a monolithic diamond thin film, which is then etched to nanometer-scale thickness, an approach with a clear path towards wafer-scale fabrication. A novel approach to creating film-coupled nanogap metasurfaces was developed to support this research and consists of transferring EBL-fabricated nanoparticles by using a PDMS stamp. Up to seven orders of magnitude of enhancement of nonlinear frequency conversion was also observed in diamond thin films coupled to these metasurfaces. Furthermore, a robust mechanism for actively tuning the nanocavity absorption resonance by integrating sub-10-nm films of the phase-change material vanadium dioxide. This platform opens up opportunities for on-chip quantum networks and nanoscale sensors based on nanocavity-coupled SiVs with the potential for in-situ frequency conversion to outcouple to photonic circuits and reconfigurable properties by incorporating VO2 thin films.

Electrostatic interaction is a strong force that attracts positively and negatively charged molecules to each other. Such an interaction is formed between positively charged polycationic polymers and negatively charged nucleic acids. In this dissertation, the electrostatic attraction between polycationic polymers and nucleic acids is exploited for applications in oral gene delivery and nucleic acid scavenging. An enhanced nanoparticle for oral gene delivery of a human Factor IX (hFIX) plasmid is developed using the polycationic polysaccharide, chitosan (Ch), in combination with protamine sulfate (PS) to treat hemophilia B. For nucleic acid scavenging purposes, the development of an effective nucleic acid scavenging nanofiber platform is described for dampening hyper-inflammation and reducing the formation of biofilms.

Non-viral gene therapy may be an attractive alternative to chronic protein replacement therapy. Orally administered non-viral gene vectors have been investigated for more than one decade with little progress made beyond the initial studies. Oral administration has many benefits over intravenous injection including patient compliance and overall cost; however, effective oral gene delivery systems remain elusive. To date, only chitosan carriers have demonstrated successful oral gene delivery due to chitosan’s stability via the oral route. In this study, we increase the transfection efficiency of the chitosan gene carrier by adding protamine sulfate to the nanoparticle formulation. The addition of protamine sulfate to the chitosan nanoparticles results in up to 42x higher in vitro transfection efficiency than chitosan nanoparticles without protamine sulfate. Therapeutic levels of hFIX protein are detected after oral delivery of Ch/PS/phFIX nanoparticles in 5/12 mice in vivo, ranging from 3 -132 ng/mL, as compared to levels below 4 ng/mL in 1/12 mice given Ch/phFIX nanoparticles. These results indicate the protamine sulfate enhances the transfection efficiency of chitosan and should be considered as an effective ternary component for applications in oral gene delivery.

Dying cells release nucleic acids (NA) and NA-complexes that activate the inflammatory pathways of immune cells. Sustained activation of these pathways contributes to chronic inflammation related to autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, and inflammatory bowel disease. Studies have shown that certain soluble, cationic polymers can scavenge extracellular nucleic acids and inhibit RNA-and DNA-mediated activation of Toll-like receptors (TLRs) and inflammation. In this study, the cationic polymers are incorporated onto insoluble nanofibers, enabling local scavenging of negatively charged pro-inflammatory species such as damage-associated molecular pattern (DAMP) molecules in the extracellular space, reducing cytotoxicity related to unwanted internalization of soluble cationic polymers. In vitro data show that electrospun nanofibers grafted with cationic polymers, termed nucleic acid scavenging nanofibers (NASFs), can scavenge nucleic acid-based agonists of TLR 3 and TLR 9 directly from serum and prevent the production of NF-ĸB, an immune system activating transcription factor while also demonstrating low cytotoxicity. NASFs formed from poly (styrene-alt-maleic anhydride) conjugated with 1.8 kDa branched polyethylenimine (bPEI) resulted in randomly aligned fibers with diameters of 486±9 nm. NASFs effectively eliminate the immune stimulating response of NA based agonists CpG (TLR 9) and poly (I:C) (TLR 3) while not affecting the activation caused by the non-nucleic acid TLR agonist pam3CSK4. Results in a more biologically relevant context of doxorubicin-induced cell death in RAW cells demonstrates that NASFs block ~25-40% of NF-ĸβ response in Ramos-Blue cells treated with RAW extracellular debris, ie DAMPs, following doxorubicin treatment. Together, these data demonstrate that the formation of cationic NASFs by a simple, replicable, modular technique is effective and that such NASFs are capable of modulating localized inflammatory responses.

An understandable way to clinically apply the NASF is as a wound bandage. Chronic wounds are a serious clinical problem that is attributed to an extended period of inflammation as well as the presence of biofilms. An NASF bandage can potentially have two benefits in the treatment of chronic wounds by reducing the inflammation and preventing biofilm formation. NASF can prevent biofilm formation by reducing the NA present in the wound bed, therefore removing large components of what the bacteria use to develop their biofilm matrix, the extracellular polymeric substance, without which the biofilm cannot develop. The NASF described above is used to show the effect of the nucleic acid scavenging technology on in vitro and in vivo biofilm formation of P. aeruginosa, S. aureus, and S. epidermidis biofilms. The in vitro studies demonstrated that the NASFs were able to significantly reduce the biofilm formation in all three bacterial strains. In vivo studies of the NASF on mouse wounds infected with biofilm show that the NASF retain their functionality and are able to scavenge DNA, RNA, and protein from the wound bed. The NASF remove DNA that are maintaining the inflammatory state of the open wound and contributing to the extracellular polymeric substance (EPS), such as mtDNA, and also removing proteins that are required for bacteria/biofilm formation and maintenance such as chaperonin, ribosomal proteins, succinyl CoA-ligase, and polymerases. However, the NASF are not successful at decreasing the wound healing time because their repeated application and removal disrupts the wound bed and removes proteins required for wound healing such as fibronectin, vibronectin, keratin, and plasminogen. Further optimization of NASF treatment duration and potential combination treatments should be tested to reduce the unwanted side effects of increased wound healing time.

This study is focused on designing and characterizing protein-based building blocks in order to construct self-assembled nano-structured biomaterials. In detail, this research aims to: (1) investigate a new class of proteins that possess nanospring behaviors at a single-molecule level, and utilize these proteins along with currently characterized elastomeric proteins as building blocks for nano-structured biomaterials; (2) develop a new method to accurately measure intermolecular interactions of self-assembling two or more arbitrary (poly)peptides, and select some of them which have appropriate tensile strength for crosslinking the proteins to construct elastomeric biomaterials; (3) construct well-defined protein building blocks which are composed of elastomeric proteins terminated with self-oligomerizing crosslinkers, and characterize self-assembled structures created by the building blocks to determine whether the elasticity of proteins at single-molecule level can be maintained.

Primary experimental methods of this research are (1) atomic force microscope (AFM) based single-molecule force spectroscopy (SMFS) that allows us to manipulate single molecules and to obtain their mechanical properties such as elasticity, unfolding and refolding properties, and force-induced conformational changes, (2) AFM imaging that permits us to identify topology of single molecules and supramolecular structures, and (3) protein engineering that allows us to genetically connect elastomeric proteins and self-assembling linkers together to construct well-defined protein building blocks.

Nanospring behavior of á-helical repeat proteins: We revealed that á-helical repeat proteins, composed of tightly packed á-helical repeats that form spiral-shaped protein structures, unfold and refold in near equilibrium, while they are stretched and relaxed during AFM based SMFS measurements. In addition to minimal energy dissipation by the equilibrium process, we also found that these proteins can yield high stretch ratios (>10 times) due to their packed initial forms. Therefore, we, for the first time, recognized a new class of polypeptides with nanospring behaviors.

Protein-based force probes for gauging molecular interactions: We developed protein-based force probes for simple, robust and general AFM assays to accurately measure intermolecular forces between self-oligomerization of two or more arbitrary polypeptides that potentially can serve as molecular crosslinkers. For demonstration, we genetically connected the force probe to the Strep-tag II and mixed it with its molecular self-assembling partner, the Strep-Tactin. Clearly characterized force fingerprints by the force probe allowed identification of molecular interactions of the single Strep-tag II and Strep-Tactin complex when the complex is stretched by AFM. We found a single energy barrier exists between Strep-tag II and Strep-Tactin in our given loading rates. Based upon our demonstration, the use of the force probe can be expanded to investigate the strength of interactions within many protein complexes composed of homo- and hetero-dimers, and even higher oligomeric forms. Obtained information can be used to choose potential self-assembling crosslinkers which can connect elastomeric proteins with appropriate strength in higher-order structures.

Self-assembled nano-structured biomaterials with well-defined protein-based building blocks: We constructed well-defined protein building blocks with tailored mechanical properties for self-assembled nano-structured materials. We engineered protein constructs composed of tandem repeats of either a I27-SNase dimer or a I27 domain alone and terminated them with a monomeric streptavidin which is known to form extremely stable tetramers naturally. By using molecular biology and AFM imaging techniques, we found that these protein building blocks transformed into stable tetrameric complexes. By using AFM based SMFS, we measured, to our knowledge for the first time, the mechanical strength of the streptavidin tetramer at a single-molecule level and captured its mechanical anisotropy. Using streptavidin tetramers as crosslinkers offers a unique opportunity to create well-defined protein based self-assembled materials that preserve the molecular properties of their building blocks.

DNA origami has been a paradigm-shifting technique for synthesizing and manipulating matter with nanoscale precision. The simple design principle of using numerous short (<100 nts) oligonucleotides to "fold" a long (>1000 nts) DNA strand achieved both simplicity in design and greatly increased yields in comparison to previous motifs for DNA nanostructure design. Various approaches have been explored that have resulted in DNA nanostructures rapidly growing in mass and complexity while also becoming more accessible for a wide scientific community, such as developing computer-aided design graphical user interfaces, establishing design principles for classes of structures with algorithmic regularity, and refining synthesis strategies and the respective design criteria to exploit them.

These directions are all fundamentally a straight extension of the DNA origami technique and pursuits towards large, functional DNA origami have been amply rewarded. Yet due to the nature of how a primary driving factor of scaling designs upwards has been the exploitation of repeatable motifs, several assumptions underlie conventional strategies for the DNA origami design of complex shapes. This thesis formally classifies a geometry of curved DNA origami nanostructures and discusses how such structures do not align with existing assumptions for DNA nanostructure design. While it is class of structures that has high biotechnological relevance, the tedium of design challenges arising from this departure have limited accessibility and enthusiasm for utilizing them. To achieve greater functional relevance, DNA origami must undoubtedly retread on the establishment of strategies for scaling up mass and shape complexity in DNA nanostructures; this time beyond regular, repeating subunits, and towards supramolecular assemblies with distinct, bespoke geometric features. As such, this thesis entreats an approach towards formalizing local and global properties in DNA origami design that can be quantified and characterized for their effects on DNA nanostructure yield and stability. Thus, a generalized strategy for DNA origami design can be born.

This thesis first consolidates and proposes a hierarchy of properties active in DNA origami design. It then suggests and evaluates two heuristic optimization algorithms to attempt a multi-variable optimization of those properties to achieve rapid generation of oligonucleotide sequences to generate desired DNA origami shapes. This thesis then discusses the existing challenges and potential applications of curved DNA origami nanostructures. Lastly, the application of the aforementioned optimization algorithms are applied to generate examples in this class of nanostructures, and the results are hither reported and discussed.

Carbon nanotubes (CNTs) have recently emerged as promising candidates for electron field emission (FE) cathodes in integrated FE devices. These nanostructured carbon materials possess exceptional properties and their synthesis can be thoroughly controlled. Their integration into advanced electronic devices, including not only FE cathodes, but sensors, energy storage devices, and circuit components, has seen rapid growth in recent years. The results of the studies presented here demonstrate that the CNT field emitter is an excellent candidate for next generation vacuum microelectronics and related electron emission devices in several advanced applications.

The work presented in this study addresses determining factors that currently confine the performance and application of CNT-FE devices. Characterization studies and improvements to the FE properties of CNTs, along with Micro-Electro-Mechanical Systems (MEMS) design and fabrication, were utilized in achieving these goals. Important performance limiting parameters, including emitter lifetime and failure from poor substrate adhesion, are examined. The compatibility and integration of CNT emitters with the governing MEMS substrate (i.e., polycrystalline silicon), and its impact on these performance limiting parameters, are reported. CNT growth mechanisms and kinetics were investigated and compared to silicon (100) to improve the design of CNT emitter integrated MEMS based electronic devices, specifically in vacuum microelectronic device (VMD) applications.

Improved growth allowed for design and development of novel cold-cathode FE devices utilizing CNT field emitters. A chemical ionization (CI) source based on a CNT-FE electron source was developed and evaluated in a commercial desktop mass spectrometer for explosives trace detection. This work demonstrated the first reported use of a CNT-based ion source capable of collecting CI mass spectra. The CNT-FE source demonstrated low power requirements, pulsing capabilities, and average lifetimes of over 320 hours when operated in constant emission mode under elevated pressures, without sacrificing performance. Additionally, a novel packaged ion source for miniature mass spectrometer applications using CNT emitters, a MEMS based Nier-type geometry, and a Low Temperature Cofired Ceramic (LTCC) 3D scaffold with integrated ion optics were developed and characterized. While previous research has shown other devices capable of collecting ion currents on chip, this LTCC packaged MEMS micro-ion source demonstrated improvements in energy and angular dispersion as well as the ability to direct the ions out of the packaged source and towards a mass analyzer. Simulations and experimental design, fabrication, and characterization were used to make these improvements.

Finally, novel CNT-FE devices were developed to investigate their potential to perform as active circuit elements in VMD circuits. Difficulty integrating devices at micron-scales has hindered the use of vacuum electronic devices in integrated circuits, despite the unique advantages they offer in select applications. Using a combination of particle trajectory simulation and experimental characterization, device performance in an integrated platform was investigated. Solutions to the difficulties in operating multiple devices in close proximity and enhancing electron transmission (i.e., reducing grid loss) are explored in detail. A systematic and iterative process was used to develop isolation structures that reduced crosstalk between neighboring devices from 15% on average, to nearly zero. Innovative geometries and a new operational mode reduced grid loss by nearly threefold, thereby improving transmission of the emitted cathode current to the anode from 25% in initial designs to 70% on average. These performance enhancements are important enablers for larger scale integration and for the realization of complex vacuum microelectronic circuits.

The largest obstacle in nanoscale microscopy is the diffraction limit. Although several means of achieving sub-diffraction resolution exist, they all have shortcomings such as cost, complexity, and processing time, which make them impractical for widespread use. Additionally, these technologies struggle to find a balance between a high resolution and a large field of view. In this introduction of dissertation, we provide an overview of various microsphere based super resolution techniques that address the shortcomings of existing platforms and consistently achieve sub-diffraction resolutions. Initially, the theoretical basis of photonic nanojets, which make microsphere based super resolution imaging possible, are discussed. In the following sections, different type of acoustofluidic scanning techniques and intelligent nanoscope are explored. The introduction concludes with an emphasis on the limitless potential of this technology, and the wide range of possible biomedical applications.First, we have documented the development of an acoutofluidic scanning nanoscope that can achieve both high resolution and large field of view at the same time, which alleviates a long-existing shortcoming of conventional microscopes. The acoutofluidic scanning nanoscope developed here can serve as either an add-on component to expand the capability of a conventional microscope, or could be paired with low-cost imaging platforms to develop a stand-alone microscope for portable imaging. The acoutofluidic scanning nanoscope achieves high-resolution imaging without the need for conventional high-cost and bulky objectives with high numerical apertures. The field of view of the acoutofluidic scanning nanoscope is much larger than that from a conventional high numerical aperture objective lens, and it is able to achieve the same resolving power. The acoutofluidic scanning nanoscope automatically focuses and maintains a constant working distance during the scanning process thanks to the interaction of the microparticles with the liquid domain. The resolving power of the acoutofluidic scanning nanoscope can easily be adjusted by using microparticles of different sizes and refractive indices. Additionally, it may be possible to further improve the performance of this platform by exploring additional microparticle sizes and materials, in combination with various objectives. Altogether, we believe this acoutofluidic scanning nanoscope has potential to be integrated into a wide range of applications from portable nano-detection to biomedicine and microfluidics. Next, we developed a dual-camera acoustofluidic nanoscope with a seamless image merging algorithm (alpha blending process). This design allows us to precisely image both the sample and the microspheres simultaneously and accurately track the particle path and location. Therefore, the number of images required to capture the entire field of view (200 × 200 μm) by using our acoustofluidic scanning nanoscope is reduced by 55-fold compared with previous designs. Moreover, the image quality is also greatly improved by applying an alpha blending imaging technique, which is critical for accurately depicting and identifying nanoscale objects or processes. This dual-camera acoustofluidic nanoscope paves the way for enhanced nanoimaging with high resolution and a large field of view. Next, we developed an acoustofluidic scanning nanoscope via fluorescence amplification technique. Nanoscale fluorescence signal amplification is a significant feature for many biomedical and cell biology research area. Different types of fluorescence amplification techniques were studied; however, those technologies still need a complex process and rely on an elaborate optical system. To conquer these limitations, we developed an acoustofluidic scanning nanoscope via fluorescence amplification with hard PDMS membrane technique. The microsphere magnification by photonic nanojets effect with the hard PDMS could deliver certain focal distance to maximize the amplification. Moreover, a bidirectional acoustofluidic scanning device with an image processing also developed to perform 2D scanning of large field of view area. In the image processing procedure, we applied a correction of lens distortion to provide a restored distortion image. This fluorescence amplification via acoustofluidic nanoscope allow us to afford a nanoscale fluorescence imaging. Next, we developed an intelligent nanoscope that combines machine learning and microsphere array-based imaging to: (1) surpass the diffraction limit of the microscope objective with microsphere imaging to provide high-resolution images; (2) provide large field-of-view imaging without the sacrifice of resolution by utilizing a microsphere array; and (3) rapidly classify nanomaterials using a deep convolution neural network. The intelligent nanoscope delivers more than 46 magnified images from a single image frame so that we collected more than 1,000 images within 2 seconds. Moreover, the intelligent nanoscope achieves a 95% nanomaterial classification accuracy using 1,000 images of training sets, which is 45% more accurate than without the microsphere array. The intelligent nanoscope also achieves a 92% bacteria classification accuracy using 50,000 images of training sets, which is 35% more accurate than without the microsphere array. This platform accomplished rapid, accurate detection and classification of nanomaterials with miniscule size differences. The capabilities of this device wield the potential to further detect and classify smaller biological nanomaterial, such as viruses or extracellular vesicles. Lastly, this chapter serves a conclusion. Here, I discuss current issues regarding the acoustofluidic scanning nanoscope across review the current limitations of the technology and give suggestions for different direction of microsphere imaging. Moreover, I provide my perspective on the future development of acoustofluidic scanning nanoscope and potential new applications. I discuss how the technologies developed in this dissertation can be improved and applied to new applications in nanoimaging.

Genetic modification of dendritic cells with plasmid DNA is plagued with low transfection efficiencies because DNA taken up by non-dividing dendritic cells rarely reaches the nucleus. But this difficulty can be overcome by the use of messenger RNA (mRNA), which exerts its biological function in the cytoplasm and obviates the need to enter the nucleus. Since pioneering work of Boczkwoski et al, the ex-vivo application of mRNA-transfected dendritic cells as a vaccine has been evaluated in numerous phase I trials worldwide and is still currently being actively optimized in clinical trials.

However, a major disadvantage of using mRNA-transfected DCs as a vaccine is that it requires patients to undergo at least one 4-hour leukapheresis procedure, followed by separation of the peripheral blood mononuclear cells (PBMCs), from which monocytes are isolated and cultured for a week in a defined medium with cytokines. The resulting DCs are matured after being loaded with mRNA and frozen for storage. Aliquots are subsequently thawed prior to administration to patients. This process of harvesting, culturing and loading DCs is more time- and resource-intensive than Provenge, the first FDA approved cell based tumor vaccine in 2011.Recent evidence has confirmed a lack of broad translation of Provenge due to complexity and cost of treatment. This predicates a similar fate for mRNA-transfected dendritic cell vaccine going forward.

This thesis presents alternative delivery strategies for mRNA mediated tumor vaccination. Through the application of synthetic and natural biomaterials, this thesis demonstrates two viable approaches that reduce or eliminate the need for extensive manipulation and cell culture.

The first approach is the direct in vivo delivery of mRNA encapsulated in nanoparticles for tumor vaccination. A selected number of synthetic gene carriers that have been shown to be effective for other applications are formulated with mRNA into nanoparticles and evaluated for their ability to transfect primary DCs. The best performing formulation is observed to transfect primary murine and human dendritic cells with an efficiency of 60% and 50% (based on %GFP+ cells) respectively. The in vivo transfection efficiency and expression kinetics of this formulation is subsequently evaluated and compared with naked mRNA via various routes of delivery. Following this, a proof-of-concept study is presented for a non-invasive method of mRNA tumor vaccination using intranasally administered mRNA encapsulated in nanoparticles. Results show that intranasally administered mRNA induces tumor immunity only if it is encapsulated in nanoparticles. And anti-tumor immunity is observed in mice intranasally immunized under both prophylactic as well as therapeutic models.

The second approach evaluates whole blood cells as alternative cell based mRNA carriers. A method is developed to encapsulate intact and functional mRNA in murine whole blood cells. Whole blood cells loaded with mRNA not only include erythrocytes but also T cells (CD3+), monocytes (CD11b), antigen presenting cells (MHC class II) as well as plasmacytoid DCs (CD45R-B220). Mice immunized with mRNA-loaded whole blood cells (intravenously) develop both humoral and cellular antigen-specific immune responses, and demonstrate delayed tumor onset and progression in a melanoma therapeutic immunization model (using tyrosinase related protein -2, TRP-2, as an antigen). Importantly, the therapeutic efficacy of mRNA-loaded whole blood cell vaccine formulation is found to be comparable to mRNA-transfected dendritic cell vaccine.

In conclusion, this thesis presents new methods to the delivery of mRNA tumor vaccines that reduce or eliminates the need for extensive cell manipulation and culture. Results presented in this thesis reveal viable research directions towards the development and optimization of mRNA delivery technologies that will address the problem of broad translation of mRNA tumor vaccines in the clinics.

Plasmonic nanoplatforms have fundamentally changed the landscape of biomedical sciences, particularly in the fields of early disease detection and treatment. Metallic nanoparticles with unique geometries and compositions such as gold nanostars (GNS) and nanorattles (NR) have allowed for the development of highly sensitive and effective platforms for detecting early disease biomarkers such as RNA without the need for laboratory-based sample amplification tools such as polymerase chain reaction (PCR). Furthermore, these plasmonics-active particles have also enabled novel optical methods for deep tissue tumor detection without the associated energy concerns and technical complexity of traditional imaging methods such as X-Ray computed tomography (CT) or magnetic resonance imaging (MRI). Finally, these particles can also be used for their effective photon to heat conversion capabilities for highly specific treatment of cancer tissue. The body of work described here is a culmination of several applications of plasmonic nanoparticles ranging from biomarker disease detection to deep tumor localization and photothermal treatment.

Recent advances in the of plasmonic nanoplatforms utilizing gold nanoparticles have resulted in many applications for point-of-care (POC) diagnostics. Upon laser excitation, the surface plasmons on the gold nanoparticles strongly oscillate, generating a strong electromagnetic field (EF) in the vicinity of the nanoparticle surface. This EF field enhancement, often referred to as the plasmonic effect, can be utilized to greatly increase the Raman scattering signal of molecules near the particle’s surface. This phenomenon called Surface-Enhanced Raman Scattering (SERS) can then be utilized for highly specific diagnostic and therapeutic applications. Our group has developed numerous biosensors that take advantage of this unique plasmonic property for use in non-invasive and non-amplifying biomarker detection. Due to its strong SERS signal, the ultrabright SERS nanorattles were developed as a unique sandwich hybridization biosensor for nucleic acid detection. We have demonstrated their successful use in detecting unamplified RNA genetic biomarkers of squamous cell carcinoma (SCC) for Head and Neck Cancers (HNCs) in a joint project with our clinical collaborator, Dr. Walter Lee, MD.

Nanoparticle platforms have also allowed for the development of novel optical and spectroscopic detection of deeply seated tumors. The unique spectroscopic fingerprint of SERS spectra on Raman-labelled GNS can be paired with optical techniques that separate the excitation laser source from the detector, which allows for deep tissue interrogation. approach This Surface-Enhanced Spatially Offset Raman Spectroscopy (SESORS) modality has allowed for the detection of GNS in tissue model systems such as through the centimeter-thick bone of a monkey skull. This spatial offset detection mechanism was further developed into a more general system known as Optical Recognition of Constructs using Hyperspectral Imaging and Detection (ORCHID). This system takes advantage of the two-dimensional charge-coupled detection (CCD) system itself as a means of physical separation between the source and detector, and by binning pixels of specific radial distances, a novel and digital-based spatial offset system can be utilized for probing deep tissue layers.

Finally, nanoparticles are utilized for the improved and highly targeted treatment of cancer tissue by taking advantage of the enhanced permeation and retention (EPR) effect in tumors. The photothermal heat treatment with GNS allows for highly specific targeted treatment of tumor, thereby minimizing off-target healthy tissue heating. We have demonstrated this in a brain tumor in a mouse model in a collaborative project with our clinical collaborator Dr. Peter Fecci, MD. We have also developed several simulation models utilizing Monte Carlo Photon propagation as well as analytical thermal diffusion models to demonstrate this effect in tissue containing GNS accumulated in a tumor volume. These simulations were then complemented with experimental studies showing the extent of heat using MRI heat imaging and direct contact thermocouples.

Recent advances in nanotechnology have led to the application of nanoparticles in a wide variety of fields. In the field of nanomedicine, there is great emphasis on combining diagnostic and therapeutic modalities into a single nanoparticle construct (theranostics). In particular, anisotropic nanoparticles have shown great potential for surface-enhanced Raman scattering (SERS) detection due to their unique optical properties. Gold nanostars are a type of anisotropic nanoparticle with one of the highest SERS enhancement factors in a non-aggregated state. By utilizing the distinct characteristics of gold nanostars, new plasmonic materials for diagnostics, therapy, and sensing can be synthesized. The work described herein is divided into two main themes. The first half presents a novel, theranostic nanoplatform that can be used for both SERS detection and photodynamic therapy (PDT). The second half involves the rational design of silver-coated gold nanostars for increasing SERS signal intensity and improving reproducibility and quantification in SERS measurements.

The theranostic nanoplatforms consist of Raman-labeled gold nanostars coated with a silica shell. Photosensitizer molecules for PDT can be loaded into the silica matrix, while retaining the SERS signal of the gold nanostar core. SERS detection and PDT are performed at different wavelengths, so there is no interference between the diagnostic and therapeutic modalities. Singlet oxygen generation (a measure of PDT effectiveness) was demonstrated from the drug-loaded nanocomposites. In vitro testing with breast cancer cells showed that the nanoplatform could be successfully used for PDT. When further conjugating the nanoplatform with a cell-penetrating peptide (CPP), efficacy of both SERS detection and PDT is enhanced.

The rational design of plasmonic nanoparticles for SERS sensing involved the synthesis of silver-coated gold nanostars. Investigation of the silver coating process revealed that preservation of the gold nanostar tips was necessary to achieve the increased SERS intensity. At the optimal amount of silver coating, the SERS intensity is increased by over an order of magnitude. It was determined that a majority of the increased SERS signal can be attributed to reducing the inner filter effect, as the silver coating process moves the extinction of the particles far away from the laser excitation line. To improve reproducibility and quantitative SERS detection, an internal standard was incorporated into the particles. By embedding a small-molecule dye between the gold and silver surfaces, SERS signal was obtained both from the internal dye and external analyte on the particle surface. By normalizing the external analyte signal to the internal reference signal, reproducibility and quantitative analysis are improved in a variety of experimental conditions.

Although various proof-of-concept studies have demonstrated the eventual potential of a multifunctional SERS-active metallic nanostructures for biological applications such as single cell analysis/measurement and drug delivery, the actual development and testing of such a system in vitro has remained challenging. One key point at which many potentially useful biomethods encounter difficulty lies in the translation of early proof-of-concept experiments in a clean, aqueous solution to complex, crowded, biologically-active environments such as the interior of living cells. The research hypotheses for this work state that multifunctional nanoconstructs can be fabricated and used effectively in conjunction with surface-enhanced Raman scattering (SERS) spectroscopy and other photonics-based methods to make intracellular measurements in and deliver treatment to single cells. The results of experimental work address the specific research aims, to 1) establish temporal and spatial parameters of nanoprobe uptake and modulation, 2) demonstrate targeting of functionalized nanoparticles to the cytoplasm and nucleus of single cells, 3) deliver to and activate drug treatment in cells using a multifunctional nanosystem, and 4) make intracellular measurements in normal and disease cells using external nanoprobes,

Raman spectroscopy and two-dimensional Raman imaging were used to identify and locate labeled silver nanoparticles in single cells using SERS detection. To study the efficiency of cellular uptake, silver nanoparticles were functionalized with three differently charged SERS/Raman labels and co-incubated with J774 mouse macrophage cell cultures for internalization via normal cellular processes. The surface charge on the nanoparticles was observed to modulate uptake efficiency, demonstrating a dual function of the surface modifications as tracking labels and as modulators of cell uptake.

To demonstrate delivery of functionalized nanoparticles to specific locations within the cell, silver nanoparticles were co-functionalized with the HIV-1 TAT (49-57) peptide for cell-penetrating and nuclear-targeting ability and p-mercaptobenzoic acid (pMBA) molecules as a surface-enhanced Raman scattering (SERS) label for tracking and imaging. Two-dimensional SERS mapping was used to track the spatial and temporal progress of nanoparticle uptake in PC-3 human prostate cells and to characterize localization at various time points, demonstrating the potential for an intracellularly-targeted multiplexed nanosystem. Silver nanoparticles co-functionalized with the TAT peptide showed greatly enhanced cellular uptake and nuclear localization as compared with the control nanoparticles lacking the targeting moiety.

The efficacy of targeted nanoparticles as a drug delivery vehicle was demonstrated with development and testing of an anti-cancer treatment in which novel scintillating nanoparticles functionalized with HIV-1 TAT (49-57) for cell-penetrating and nuclear-targeting ability were loaded with tethered psoralen molecules as cargo. The experiments were designed to investigate a nanodrug system consisting of psoralen tethered to a nuclear targeting peptide anchored to UVA-emitting, X-ray luminescent yttrium oxide nanoparticles. Absorption of the emitted UVA photons by nanoparticle-tethered psoralen has the potential to cross-link adenine and thymine residues in DNA located in the nucleus. Such cross-linking by free psoralen following activation with UVA light has previously been shown to cause apoptosis in vitro and an immunogenic response in vivo. Experimental results using the PC-3 human prostate cancer cell line demonstrate that X-ray excitation of these psoralen-functionalized Y2O3 nanoscintillators yields concentration-dependent reductions in cell number density when compared to control cultures containing psoralen-free Y2O3 nanoscintillators.

The development and demonstration of a small molecule-sensitive SERS-active fiber-optic nanoprobe suitable for intracellular bioanalysis was demonstrated using pH measurements in single living human cells. The proof-of-concept for the SERS-based fiber-optic nanoprobes was illustrated by measurements of intracellular pH in MCF-7 human breast cancer, HMEC-15/hTERT immortalized normal human mammary epithelial, and PC-3 human prostate cancer cells. Clinical relevance was demonstrated by pH measurements in patient biopsy cell samples. The results indicated that that fiber-optic nanoprobe insertion and interrogation provide a sensitive and selective means to monitor biologically relevant small molecules at the single cell level.

The development of practical and sensitive techniques for screening early biomarkers such as nucleic acid targets related to medical diseases and cancers is critical for early diagnosis, prevention and effective interventions. Recent advances in molecular profiling technology have made significant progress in the discovery of various biomarkers that could serve as important predictors of cancer risk and progression. Fast and precise measurement of biomarkers will help identify molecular signatures critical for the evaluation of cancer risk and early detection. Recently, there has been great interest in the design and fabrication of plasmonics-active biosensing platforms for a wide variety of applications ranging from biomedical diagnostics, food safety, environmental monitoring, to homeland defense. In particular, DNA-functionalized metal nanoparticles (e.g. gold and silver) have been utilized in the development of novel plasmonics-based analytical techniques for the detection of nucleic acid targets. In this study, two novel label-free approaches named "molecular sentinel (MS) nanoprobes", and "plasmonic coupling interference (PCI) nanoprobes" have been developed for multiplex detection of disease biomarkers using surface-enhanced Raman scattering (SERS). The MS approach has been further extended into a unique "molecular sentinel-on-chip" (MSC) technology based on a SERS-active nanowire array substrate, leading to the development of a unique diagnostic tool having multiplexing and high-throughput screening capabilities. Finally, a novel nanoparticle-based colorimetric assay has been developed and implemented for the detection of microRNAs (miRNAs). Direct detection of miRNAs in RNA samples from breast cancer cell lines has been demonstrated. Furthermore, the PCI technique has successfully detected miRNA biomarkers in biopsies of gastrointestinal cancer patients and the results are consistent with established techniques such qRT-PCR. The results of this study demonstrate that these plasmonics-based nanoprobes have great potential as useful point-of-care diagnostic tools for medical applications.