Browsing by Author "Vo-Dinh, Tuan"

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.

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.

Fluorescence-based methodologies have been used extensively for biosensing and to analyze molecular dynamics and interactions. An emerging, promising diagnostic tool are fluorescence-based microarrays due to their high throughput, small sample volume and multiplexing capabilities. However, their low fluorescence output has limited their implementation for in vitro diagnostics applications in a point-of-care (POC) setting. Here, by integration of a sandwich immunoassay microarray within a plasmonic nanogap metasurface, we demonstrate strongly enhanced fluorescence enabling readout by a fluorescence microarray even at low sensitivities. We have named this plasmonic architecture the plasmonically enhanced D4 (PED4) assay. The immunoassay consists of inkjet-printed capture and fluorescently labeled detection antibodies on a polymer brush which is grown on a gold film. Colloidally synthesized silver nanocubes (SNCs) are placed on top of the brush through a polyelectrolyte layer and interacts with the underlying gold film creating a nanogap plasmonic structure supporting local electromagnetic field enhancements of ~100-fold. By varying the thickness of the brush between 5 and 20 nm, a 151-fold increase in fluorescence and a 14-fold improvement in the limit-of-detection (LOD) is observed for the cardiac biomarker B-type natriuretic peptide (BNP) compared to the unenhanced assay, paving the way for a new generation of point-of-care clinical diagnostics.

To move the PED4 towards a single step point of care test (POCT), SNCs are conjugated with a secondary antibody that attaches specifically to the detection antibody. This allows SNCs to deposit on the surface without the need of a charged polyelectrolyte layer. In addition, multiplexing capabilities are demonstrated in this plasmonic platform where NT-proBNP, Galectin-3, and NGAL are simultaneously detected and fluorescently enhanced. Microfluidics integration and use of a low-cost detector is also demonstrated.

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In this thesis, we present an overview of the development and application of surface-enhanced Raman scattering (SERS) and plasmonic nanoplatforms developed in our laboratory for sensing applications. Plasmonic nanomaterials, such as gold nanostars (AuNS) a hallmark particle pioneered by the Vo-Dinh group, increase inherently weak Raman signals from molecules providing an intense and unique SERS spectrum allowing for targets to be sensitively detected and easily identified. For this work, we investigated biosensing in plants and murine models using plasmonic nanoplatforms. In the first part of the work, we developed a wide variety of plasmonics-active substrates and nanoparticle-based sensing systems including inverse molecular sentinels (iMS) utilizing two platforms silver-coated gold nanostars (AuNS@Ag) and nanorods (AuNR@Ag). The AuNS@Ag were decorated onto fiber-optrodes for chemical fiber sensing and monitoring of genomic biomarkers in plants for renewable bioenergy research. Rapid chemical sensing of illegal food additives proves to be a challenge at the site of exposure thus requiring the need for in situ fiber detection. Also, the fiber sensing approach is necessary for facilitating field analyses of microRNAs since the gold standard methods can only be performed in laboratory settings on the timescale of days. The fiber-optrodes were capable of functioning as chemical and biological sensors for analytes such as illegal food additives and plant microRNAs. The work is also aimed at microRNA sensing but directly inside of plant tissue with spatial and temporal resolutions that PCR cannot achieve. Thus, the silver-coated nanorod (AuNR@Ag) was developed with the purpose of infiltrating plant cells. We designed the plasmonic nanorod to have a dimension smaller than the plant cell wall exclusion limit to permit cellular uptake, while improving SERS properties through a silver coating on the particle. We confirmed particle uptake in plant cells using a multi-modal approach consisting of confocal microscopy, transmission electron microscopy, and x-ray fluorescence microscopy. Dye coated AuNR@Ag served as a strong contrast agent for two-photon imaging, photoacoustic imaging, and Raman mapping during in vivo experiments in Tobacco leaves. The AuNR@Ag was further functionalized with iMS technology and was applied for sensing for microRNA targets in leaf tissue. To our knowledge, this is the first demonstration of intracellular SERS sensing in vivo of leaf tissue treated with the AuNR@Ag nanoprobes. In another chapter of the work, we used standoff shifted-excitation difference spectroscopy (SERDS) for remote detection of biomarkers in plants under ambient light conditions. Monitoring plant molecular targets in field conditions remain an elusive task for standard optical methods such as fluorescence and Raman spectroscopy. However, recent developments in nanoprobe technology and remote optical techniques have ushered in a novel mechanism for highly specific molecular monitoring of living organisms at different stages of growth and other phenotypic cues. We have successfully demonstrated nanoprobe detection in a live plant leaf at a minimal distance of 2 meters. This work brings the remote monitoring of plant genetic biomarkers closer to in vivo tracking and analysis without the need of a dark laboratory as required by traditional optical sensing. Applications of plasmonic AuNS for bioimaging of tumors in combination with SERDS is also presented. During surgery accurately removing the entire tumor without harming surrounding healthy tissue is critical; however, due to the lack of intraoperative imaging techniques, surgeons rely on visual and physical inspection to identify tumors. To address this problem, we established the first use of SERDS for in vivo tumor detection in a murine model under ambient light conditions, mimicking an intraoperative environment.  

Recent advances in nanotechnology have led to the application of nanoparticles in a wide variety of fields. 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 sensing, diagnostics, and therapy can be synthesized. The work described herein is divided into two main themes. The first half demonstrates the development and application of a novel label-free inverse molecular sentinel (iMS) nanoprobe for detection of microRNA biomarkers related to cancer progression as well as those related to gene expression in plants. This work also describes the initial proof-of-concept for a SERS-based electrowetting-on-dielectric (EWD) digital microfluidic platform as a diagnostic platform requiring samples of nanoliter volume. The second half demonstrates the utility of plasmonic nanoparticles for SERS imaging as well as photothermal therapy (PTT) and photodynamic therapy (PDT).

Development of accessible strategies for efficient detection of nucleic acid biomarkers is a major unmet need for applications ranging from cancer screening to agricultural biotechnology and biofuel development. MicroRNAs (miRNAs) have great promise as a new important class of biomarkers for early detection of various cancers; however, these small molecules have not been adopted into early diagnostics for clinical practice because of challenges adapting complex laboratory techniques into accessible clinical tests. In a blinded study, the surface-enhanced Raman scattering (SERS)-based plasmonics-active nanoprobes described herein, referred to as inverse molecular sentinels (iMS), demonstrated diagnostic accuracy for in vitro identification of endoscopic biopsy samples as tumor, Barrett’s esophagus or normal tissue via miRNA detection. The iMS nanoprobe technology can be designed to detect a wide range of nucleic acids for a variety of applications. In addition to medical applications, the knowledge over gene expression dynamics and location in plants is crucial for applications ranging from basic biological research to agricultural biotechnology. However, current methods are unable to provide in vivo dynamic detection of genomic targets in plants, due to the complex sample preparation needed by current methods for nucleic acids detection, which disrupt spatial and temporal resolution. We have developed a multimodal technique utilizing iMS nanoprobes for in vivo imaging and biosensing of microRNA biotargets within whole plants. This work lays the foundations for in vivo functional imaging of RNA biotargets in plants with previously unmet spatial and temporal resolution.

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. Gold nanostars (AuNS) with tip-enhanced plasmonics have become one of the most promising platforms in photothermal therapy (PTT) as they exhibit superior photon-to-heat conversion efficiency and can be delivered specifically to tumors. We have demonstrated that AuNS are endocytosed into multiple cancer cell lines irrespective of receptor status or drug resistance and allow for the effective photothermal ablation of tumor cells. Additionally, we demonstrate a unique in vitro preclinical model that mimics the tumor structures assumed by inflammatory breast cancer (IBC) in vivo. IBC has a unique presentation of diffuse tumor cell clusters called tumor emboli. AuNS are able to penetrate the tumor embolic core in 3D culture, allowing effective photothermal ablation of the IBC tumor emboli.

Additionally, we have furthered the development of the gold nanostar treatment platform by developing a theranostic nanoconstruct that consist of Raman-labeled gold nanostars coated with a silica shell that is loaded with photosensitizer molecules for PDT. The outer surface of the nanoconstruct was functionalized for targeting to allow for specific treatment of folate positive breast cancer. 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 demonstrated the effectiveness of the nanoconstruct for targeted PDT.