Browsing by Author "VoDinh, Tuan"

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.

The development of new nucleic acid detection techniques for molecular diagnostics at the point-of-care (POC) and resource-limited settings has attracted great interest. Signal amplification-based nucleic acid detection can be an alternative to enzymatic amplification-based methods (e.g., PCR, i.e., polymerase chain reaction). Compared to enzymatic amplification, some advantages of signal amplification include simple detection workflow without the need of nucleic acid extraction and purification, being more resistant to contamination and inhibitors, etc. However, current signal amplification methods are usually not sufficiently sensitive or too complicated and require skill operators, thus affecting its translation to POC applications.

Advances in nanotechnology and nanomaterials offer new, versatile, and exciting platforms for POC diagnostics. Using nanobiosensors and surface-enhanced Raman scattering (SERS), we have been developing various novel nucleic acid detection methods for in vitro molecular diagnostics. Emphasis was placed on sensitivity and easy-to-use, two of the main requirements of POC molecular diagnostics. Two different and complementary strategies for nucleic acid detection including (1) a chip-based strategy and (2) a nanoparticle-based strategy were investigated. The chip-based strategy involves single-step multiplex detection using SERS-active Nanowave chips. The nanoparticle-based strategy involves sandwich hybridization using magnetic beads, target sequences, and SERS nanorattle nanoparticles.

First, we developed a method for fabrication of large area high enhancement SERS-active Nanowave chip. Using the method, wafer scale of SERS-active Nanowave chip with particularly high enhancement factor of ~108 were achieved. Based on the Nanowave chip platform, we developed molecular sentinel-on-chip assay and inverse molecular sentinel-on-chip assay for nucleic acid detection. To show the assays’ usefulness for molecular diagnostics, genetic biomarkers for respiratory infection and a specific DNA sequence of Dengue virus were used as test models.

Second, method for synthesis of ultrabright SERS nanorattles were developed. The nanorattles are suitable for sandwich hybridization-based nucleic acid detection due to its strong SERS signal and high stability. Using the synthesized nanorattles, we developed a sandwich hybridization assay to detect specific DNA sequence of malaria parasite P. falciparum and genetic biomarker of squamous cell carcinoma (SCC). For the malaria target DNA, detection limit of 3 pM using synthetic DNA was achieved. For the SCC biomarker, we could detect real biological samples by detecting real-time PCR products of RNA extracted from SCC cell lines. Sensitivity of 93% and specificity of 100% were achieved.

Third, we developed an integrated device for automation of the nanorattle sandwich assay. Using the device, we could directly detect malaria RNA in malaria-infected blood lysate. The detection process was quite simple by adding nanorattles pre-functionalized with reporter probes and magnetic beads pre-functionalized with capture probes to the blood lysate followed by several hour incubation and an automated washing by a second-generation lab-in-a-stick device. To our knowledge, this is the first time the SERS-based detection of pathogen nucleic acid in blood lysate without any nucleic acid extraction or enzymatic amplification was reported.

The results showed potential of our nanobiosensors, methods, and systems for nucleic acid-based molecular diagnostics. The nanorattle sandwich assay’s sensitivity and compatibility for automation make them suitable for POC applications. In combination with the lab-in-a-stick device, we can now directly detect malaria nucleic acid in blood lysate. Future works will be to improve the limit of detection so that the technique can be used for detection of bloodborne pathogens and genetic biomarkers at lower clinical-relevant concentrations.

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.

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.

The prevalence of cancer has increasingly become a significant threat to human health and as such, there exists a strong need for developing novel methods for early detection and effective therapy. Nanotheranostics, a combination of diagnostic and therapeutic functions into a single nanoplatform, has great potential to be used for cancer management by allowing detection, real-time tracking, image-guided therapy and therapeutic response monitoring. Gold nanostars (GNS) with tip-enhanced plasmonics have become one of the most promising platforms for cancer nanotheranostics. This work is aimed at addressing the challenges of sensitive cancer detection, metastasis treatment and recurrence prevention by combining state-of-the-art nanotechnology, molecular imaging and immunotherapy. A multifunctional GNS nanoprobe is developed with capabilities ranging from non-invasive, multi-modality cancer detection using positron emission tomography (PET), magnetic resonance imaging (MRI) and X-ray computed tomography (CT), to intraoperative tumor margin delineation with surface enhanced Raman spectroscopy (SERS) and high-resolution nanoprobe tracking with two-photon photoluminescence (TPL), as well as cancer treatment with photoimmunotherapy. The GNS nanoprobe with PET scans is particularly exceptional in detecting brain malignancies as small as 0.5 mm. To the best of our knowledge, the developed GNS nanoprobe for PET imaging provides the most sensitive means of brain tumor detection reported so far. In addition, the GNS nanoprobe exhibits superior performance as photon-to-heat transducer and can be used for specific photothermal therapy (PTT). More importantly, GNS-mediated PTT combined with checkpoint inhibitor immunotherapy has been found to trigger a memorized immunoresponse to treat cancer metastasis and prevent recurrence in mouse model studies. Furthermore, a 6-month in vivo toxicity study including body weight monitoring, blood chemistry test and histopathology examination demonstrate GNS nanoparticles’ biocompatibility. Therefore, the multifunctional GNS nanoprobe exhibits superior cancer detection and treatment capabilities and has great promise for future clinical translation in cancer management.

The advancement in nanotechnology creates a new perspective on future medicine. With more and more understanding on controlling the functional behavior of the nanoplatform, scientists nowadays are aiming to improve the health care system by offering personalized medicine through nanotechnology. Lots of emphasis have been placed on a promising field called theranostics, which integrate imaging and therapeutic functions into one, that not only offers monitoring and imaging of the biological process, but also provides diagnosis and drug delivery simultaneously. Plasmonic gold nanostars, because of its anisotropic geometry and unique plasmonic property, have become one of the most anticipated nanoplatform in the field of nanotheranostics, aiming to achieve superior plasmonic properties for biomedical applications. The work herein will provide an introduction to the related field on plasmonics, nanobiophotonics and nanotheranostics. A facile plasmon-tunable surfactant-free nanostars synthesis method is described followed by an extensive characterization both computationally and experimentally. Its superior plasmon behavior on two-photon photoluminescence imaging and surface-enhanced Raman scattering detection are demonstrated both in cells and in animals. Therapeutic function assessment is carried out both as drug carriers (photodynamic therapy) and as endogenous stimulus responsive agents (photothermal therapy). Finally, the nanostars' cellular uptake mechanism is investigated based on nanostars' endogenous contrast; an enhanced photothermal therapy is achieved using an ultralow irradiance that has ever published. With nanostars being a novel and powerful theranostic agent, the potentials implication lies in the study of their pharmacokinetics, targeted delivery, diagnostic imaging, and toxicity.