Hsin-Neng Wang

Multiplex detection of breast cancer biomarkers using plasmonic molecular sentinel nanoprobes

We have demonstrated for the first time the feasibility of multiplex detection using the surface-enhanced Raman scattering-based molecular sentinel (MS) technology in a homogeneous solution. Two MS nanoprobes tagged with different Raman labels were used to detect the presence of the erbB-2 and ki-67 breast cancer biomarkers. The multiplexing capability of the MS technique was demonstrated by mixing the two MS nanoprobes and tested in the presence of single or multiple DNA targets.

Plasmonic nanoprobes: from chemical sensing to medical diagnostics and therapy

This article provides an overview of the development and applications of plasmonics-active nanoprobes in our laboratory for chemical sensing, medical diagnostics and therapy. Molecular Sentinel nanoprobes provide a unique tool for DNA/RNA biomarker detection both in a homogeneous solution or on a chip platform for medical diagnostics. The possibility of combining spectral selectivity and high sensitivity of the surface-enhanced Raman scattering (SERS) process with the inherent molecular specificity of nanoprobes provides an important multiplex diagnostic modality. Gold nanostars can provide an excellent multi-modality platform, combining two-photon luminescence with photothermal therapy as well as Raman imaging with photodynamic therapy. Several examples of optical detection using SERS and photonics-based treatments are presented to illustrate the usefulness and potential of the plasmonic nanoprobes for theranostics, which seamlessly combines diagnostics and therapy.

Plasmonic nanoprobes for intracellular sensing and imaging

AbstractRecent advances in integrating nanotechnology and optical microscopy offer great potential in intracellular applications with improved molecular information and higher resolution. Continuous efforts in designing nanoparticles with strong and tunable plasmon resonance have led to new developments in biosensing and bioimaging, using surface-enhanced Raman scattering and two-photon photoluminescence. We provide an overview of the nanoprobe design updates, such as controlling the nanoparticle shape for optimal plasmon peak position; optical sensing and imaging strategies for intracellular nanoparticle detection; and addressing practical challenges in cellular applications of nanoprobes, including the use of targeting agents and control of nanoparticle aggregation. FigurePlasmonic nanoprobe characterization (TEM, simulation) and applications in pH sensing, SERS mapping, and TPL imaging

Applications of carbon nanotubes for cancer research

Carbon nanotubes have many unique properties such as high surface area, hollow cavities, and excellent mechanical and electrical properties. Interfacing carbon nanotubes with biological systems could lead to significant applications in various disease diagnoses. Significant progress in interfacing carbon nanotubes with biological materials has been made in key areas such as aqueous solubility, chemical and biological functionalization for biocompatibility and specificity, and electronic sensing of proteins. In addition, the bioconjugated nanotubes combined with the sensitive nanotube-based electronic devices would enable sensitive biosensors toward medical diagnostics. Furthermore, recent findings of improved cell membrane permeability for carbon nanotubes would also expand medical applications to therapeutics using carbon nanotubes as carriers in gene delivery systems. This article reviews the current trends in biological functionalization of carbon nanotubes and their potential applications for breast cancer diagnostics. The article also reports the applications of confocal microscopy for use in understanding the interactions of biological materials such as antibodies on carbon nanotubes that are specific to surface receptors in breast cancer cells. Furthermore, a nanotube-field-effect transistor is demonstrated for electronic sensing of antibodies that are specific to surface receptors in cancer cells.

Characterization of nanoprobe uptake in single cells: spatial and temporal tracking via SERS labeling and modulation of surface charge.

UNLABELLED A critical aspect for use of nanoprobes in biomedical research and clinical applications involves fundamental spatial and temporal characterization of their uptake and distribution in cells. Raman spectroscopy and two-dimensional Raman imaging were used to identify and locate nanoprobes in single cells using surface-enhanced Raman scattering detection. To study the efficiency of cellular uptake, silver nanoparticles functionalized with three different positive-, negative-, and neutrally charged Raman labels were co-incubated with cell cultures and internalized 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. These results indicate that the functionalized nanoparticle construct has potential for sensing and delivery in single living cells and that use of surface-enhanced Raman scattering for tracking and detection is a practical and advantageous alternative to traditional fluorescence methods. FROM THE CLINICAL EDITOR Cell labeling and tracking methods are commonly required in biomedical research. This paper presents specific functionalized nanoparticle constructs with potential for sensing and delivery in single living cells. The use of surface-enhanced Raman scattering enables tracking and detection of these cells as a practical alternative to traditional fluorescence methods.

Multiplex detection of disease biomarkers using SERS molecular sentinel-on-chip

AbstractDeveloping techniques for multiplex detection of disease biomarkers is important for clinical diagnosis. In this work, we have demonstrated for the first time the feasibility of multiplex detection of genetic disease biomarkers using the surface-enhanced Raman scattering (SERS)-based molecular sentinel-on-chip (MSC) diagnostic technology. The molecular sentinel (MS) sensing mechanism is based upon the decrease of SERS intensity when Raman labels tagged at 3′-ends of MS nanoprobes are physically displaced from the nanowave chip’s surface upon DNA hybridization. The use of bimetallic layer (silver and gold) for the nanowave fabrication was investigated. SERS measurements were performed immediately following a single hybridization reaction between the target single-stranded DNA sequences and the complementary MS nanoprobes immobilized on the nanowave chip without requiring target labeling (i.e., label-free), secondary hybridization, or post-hybridization washing, thus shortening the assay time and reducing cost. Two nucleic acid transcripts, interferon alpha-inducible protein 27 and interferon-induced protein 44-like, are used as model systems for the multiplex detection concept demonstration. These two genes are well known for their critical role in host immune response to viral infection and can be used as molecular signature for viral infection diagnosis. The results indicate the potential of the MSC technology for nucleic acid biomarker multiplex detection. FigureScheme of two-multiplex detection of complementary target ssDNA sequences using SERS-based molecular sentinel-on-chip diagnostic technology

Plasmonic Coupling Interference (PCI) Nanoprobes for Nucleic Acid Detection

A label-free approach using plasmonic coupling interference (PCI) nanoprobes for nucleic acid detection using surface-enhanced Raman scattering (SERS) is described. To induce a strong plasmonic coupling effect, a nanonetwork of silver nanoparticles with the Raman label located between adjacent nanoparticles is assembled by Raman-labeled DNA-locked nucleic acid (LNA) duplexes. The PCI method then utilizes specific nucleic acid sequences of interest as competitor elements for the Raman-labeled DNA strands to interfere the formation of nanonetworks in a competitive binding process. As a result, the plasmonic coupling effect induced through the formation of the nanonetworks is significantly diminished, resulting in a reduced SERS signal. The potential of the PCI technique for biomedical applications is illustrated by detecting single-nucleotide polymorphism (SNP) and microRNA sequences involved in breast cancers. The results of this study could lead to the development of nucleic acid diagnostic tools for biomedical diagnostics and biosensing applications using SERS detection.

In vivo detection of SERS-encoded plasmonic nanostars in human skin grafts and live animal models

Surface-enhanced Raman scattering (SERS)-active plasmonic nanomaterials have become a promising agent for molecular imaging and multiplex detection. Among the wide variety of plasmonics-active nanoparticles, gold nanostars offer unique plasmon properties that efficiently induce strong SERS signals. Furthermore, nanostars, with their small core size and multiple long thin branches, exhibit high absorption cross sections that are tunable in the near-infrared region of the tissue optical window, rendering them efficient for in vivo spectroscopic detection. This study investigated the use of SERS-encoded gold nanostars for in vivo detection. Ex vivo measurements were performed using human skin grafts to investigate the detection of SERS-encoded nanostars through tissue. We also integrated gold nanostars into a biocompatible scaffold to aid in performing in vivo spectroscopic analyses. In this study, for the first time, we demonstrate in vivo SERS detection of gold nanostars using small animal (rat) as well as large animal (pig) models. The results of this study establish the usefulness and potential of SERS-encoded gold nanostars for future use in long-term in vivo analyte sensing.

Development of plasmonics-active SERS substrates on a wafer scale for chemical and biological sensing applications

This paper describes the fabrication of highly efficient plasmonics-active SERS substrates - having metallic nanowire structures with pointed geometries and sub-5 nm gap between the metallic nanowires enabling concentration of high EM fields in these regions - on a wafer-scale by a reproducible process that is compatible with large-scale development of these substrates. These SERS substrates were employed for detecting chemical and biological molecules of interest.

Surface-enhanced Raman scattering nanosensors for in vivo detection of nucleic acid targets in a large animal model

Although nanotechnology has led to important advances in in vitro diagnostics, the development of nanosensors for in vivo detection remains very challenging. Here, we demonstrated the proof-of-principle of in vivo detection of nucleic acid targets using a promising type of surface-enhanced Raman scattering (SERS) nanosensor implanted in the skin of a large animal model (pig). The in vivo nanosensor used in this study involves the “inverse molecular sentinel” detection scheme using plasmonics-active nanostars, which have tunable absorption bands in the near infrared region of the “tissue optical window”, rendering them efficient as an optical sensing platform for in vivo optical detection. Ex vivo measurements were also performed using human skin grafts to demonstrate the detection of SERS nanosensors through tissue. In this study, a new core–shell nanorattle probe with Raman reporters trapped between the core and shell was utilized as an internal standard system for self-calibration. These results illustrate the usefulness and translational potential of the SERS nanosensor for in vivo biosensing.