3D-printed phantoms for characterizing SERS nanoparticle detectability in turbid media.

Recent advances in plasmonic nanoparticle synthesis have enabled extremely high per-particle surface-enhanced Raman scattering (SERS) efficiencies. This has led to the development of SERS tags for in vivo applications (e.g. tumor targeting and detection), providing high sensitivity and fingerprint-like molecular specificity. While the SERS enhancement factor is a major contributor to SERS tag performance, in practice the throughput and excitation-collection geometry of the optical system can significantly impact detectability. Test methods to objectively quantify SERS particle performance under realistic conditions are necessary to facilitate clinical translation. Towards this goal, we have developed 3D-printed phantoms with tunable, biologically-relevant optical properties. Phantoms were designed to include 1 mm-diameter channels at different depths, which can be filled with SERS tag solutions. The effects of channel depth and particle concentration on the detectability of three different SERS tags were evaluated using 785 nm laser excitation at the maximum permissible exposure for skin. Two of these tags were commercially available, featuring gold nanorods as the SERS particle, while the third tag was prepared in-house using silver-coated gold nanostars. Our findings revealed that the measured SERS intensity of tags in solution is not always a reliable predictor of detectability when applied in a turbid medium such as tissue. The phantoms developed in this work can be used to assess the suitability of specific SERS tags and instruments for their intended clinical applications and provide a means of optimizing new SERS device-tag combination products.

Plasmonic SERS biosensing nanochips for DNA detection.

The development of rapid, cost-effective DNA detection methods for molecular diagnostics at the point-of-care (POC) has been receiving increasing interest. This article reviews several DNA detection techniques based on plasmonic-active nanochip platforms developed in our laboratory over the last 5 years, including the molecular sentinel-on-chip (MSC), the multiplex MSC, and the inverse molecular sentinel-on-chip (iMS-on-Chip). DNA probes were used as the recognition elements, and surface-enhanced Raman scattering (SERS) was used as the signal detection method. Sensing mechanisms were based on hybridization of target sequences and DNA probes, resulting in a distance change between SERS reporters and the nanochip's plasmonic-active surface. As the field intensity of the surface plasmon decays exponentially as a function of distance, the distance change in turn affects SERS signal intensity, thus indicating the presence and capture of the target sequences. Our techniques were single-step DNA detection techniques. Target sequences were detected by simple delivery of sample solutions onto DNA probe-functionalized nanochips and measuring the SERS signal after appropriate incubation times. Target sequence labeling or washing to remove unreacted components was not required, making the techniques simple, easy-to-use, and cost-effective. The usefulness of the nanochip platform-based techniques for medical diagnostics was illustrated by the detection of host genetic biomarkers for respiratory viral infection and of the dengue virus gene.

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.

Fano resonance in a gold nanosphere with a J-aggregate coating.

We present a facile method to induce J-aggregate formation on gold nanospheres in colloidal solution using polyvinylsulfate. The nanoparticle J-aggregate complex results in an absorption spectrum with a split lineshape due to plasmon-exciton coupling, i.e. via the formation of upper and lower plexcitonic branches. The use of nanoparticles with different plasmon resonances alters the position of the upper plexcitonic band while the lower band remains at the same wavelength. This splitting is investigated theoretically, and shown analytically to arise from Fano resonance between the plasmon of the gold nanoparticles and exciton of the J-aggregates. A theoretical simulation of a J-aggregate coated and uncoated gold nanosphere produces an absorption spectrum that shows good agreement with the experimentally measured spectra.

Plasmonics-based SERS nanobiosensor for homogeneous nucleic acid detection.

Developing a simple and efficient nucleic acid detection technology is essential for clinical diagnostics. Here, we describe a new conceptually simple and selective "turn on" plasmonics-based nanobiosensor, which integrates non-enzymatic DNA strand-displacement hybridization for specific nucleic acid target identification with surface-enhanced Raman scattering (SERS) detection. This SERS nanobiosensor is a target label-free, and rapid nanoparticle-based biosensing system using a homogeneous assay format that offers a simple and efficient tool for nucleic acid diagnostics. Our results showed that the nanobiosensor provided a limit of detection of ~0.1nM (200amol) in the current bioassay system, and exhibited high specificity for single nucleotide mismatch discrimination. FROM THE CLINICAL EDITOR: Surface-enhanced Raman scattering (SERS) is a sensitive technique that enhances Raman scattering by molecules adsorbed on rough metal surfaces. The enhancement means that the technique may even detect single molecules. In this article, the authors describe a simple and efficient nucleic acid detection technology using SERS, with "OFF-to-ON" signal switch upon nucleic acid target identification and capture, which provides high sensitivity and specificity for single nucleotide mismatch discrimination. This new technology will be most welcomed in clinical diagnostics.

DNA bioassay-on-chip using SERS detection for dengue diagnosis.

A novel DNA bioassay-on-chip using surface-enhanced Raman scattering (SERS) on a bimetallic nanowave chip is presented. In this bioassay, SERS signals were measured after a single reaction on the chip's surface without any washing step, making it simple-to-use and reducing the reagent cost. Using the technique, specific oligonucleotide sequences of the dengue virus 4 were detected.

Label-free DNA biosensor based on SERS Molecular Sentinel on Nanowave chip.

Development of a rapid, cost-effective, label-free biosensor for DNA detection is important for many applications in clinical diagnosis, homeland defense, and environment monitoring. A unique label-free DNA biosensor based on Molecular Sentinel (MS) immobilized on a plasmonic 'Nanowave' chip, which is also referred to as a metal film over nanosphere (MFON), is presented. Its sensing mechanism is based upon the decrease of the surface-enhanced Raman scattering (SERS) intensity when Raman label tagged at one end of MS is physically separated from the MFON's surface upon DNA hybridization. This method is label-free as the target does not have to be labeled. The MFON fabrication is relatively simple and low-cost with high reproducibility based on depositing a thin shell of gold over close-packed arrays of nanospheres. The sensing process involves a single hybridization step between the DNA target sequences and the complementary MS probes on the Nanowave chip without requiring secondary hybridization or posthybridization washing, thus resulting in rapid assay time and low reagent usage. The usefulness and potential application of the biosensor for medical diagnostics is demonstrated by detecting the human radical S-adenosyl methionine domain containing 2 (RSAD2) gene, a common inflammation biomarker.

Quantitative surface-enhanced resonant Raman scattering multiplexing of biocompatible gold nanostars for in vitro and ex vivo detection.

Surface-enhanced Raman scattering (SERS)-active plasmonic nanomaterials have become a promising agent for molecular imaging and multiplex detection. To produce strong SERS intensity while retaining the nonaggregated state and biocompatibility needed for bioapplications, we integrated near-infrared (NIR) responsive plasmonic gold nanostars with resonant dyes for resonant SERS (SERRS). The SERRS on nanostars was several orders of magnitude greater than signals from SERRS on nanospheres and nonresonant SERS on nanostars. For the first time, we demonstrated quantitative multiplex detection using four unique nanostar SERRS probes in both in vitro solutions and ex vivo tissue samples under NIR excitation. With further optimization, in vivo tracking of multiple SERRS probes is possible.

Cell-penetrating peptide enhanced intracellular Raman imaging and photodynamic therapy.

We present the application of a theranostic system combining Raman imaging and the photodynamic therapy (PDT) effect. The theranostic nanoplatform was created by loading the photosensitizer, protoporphyrin IX, onto a Raman-labeled gold nanostar. A cell-penetrating peptide, TAT, enhanced intracellular accumulation of the nanoparticles in order to improve their delivery and efficacy. The plasmonic gold nanostar platform was designed to increase the Raman signal via the surface-enhanced resonance Raman scattering (SERRS) effect. Theranostic SERS imaging and photodynamic therapy using this construct were demonstrated on BT-549 breast cancer cells. The TAT peptide allowed for effective Raman imaging and photosensitization with the nanoparticle construct after a 1 h incubation period. In the absence of the TAT peptide, nanoparticle accumulation in the cells was not sufficient to be observed by Raman imaging or to produce any photosensitization effect after this short incubation period. There was no cytotoxic effect observed after nanoparticle incubation, prior to light activation of the photosensitizer. This report shows the first application of combined SERS imaging and photosensitization from a theranostic nanoparticle construct.

Plasmonic nanoprobes for intracellular sensing and imaging.

Recent 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.

TAT peptide-functionalized gold nanostars: enhanced intracellular delivery and efficient NIR photothermal therapy using ultralow irradiance.

Gold nanoparticles have great potential in plasmonic photothermal therapy (photothermolysis), but their intracellular delivery and photothermolysis efficiency have yet to be optimized. We show that TAT-peptide-functionalized gold nanostars (NS) enter cells significantly more than bare or PEGylated NS. The cellular uptake mechanism involves actin-driven lipid raft-mediated macropinocytosis, where particles primarily accumulate in macropinosomes but may also leak out into the cytoplasm. After 4-h incubation of TAT-NS on BT549 breast cancer cells, photothermolysis was accomplished using 850 nm pulsed laser under 0.2 W/cm(2) irradiation, below the maximal permissible exposure of skin. These results demonstrate the enhanced intracellular delivery and efficient photothermolysis of TAT-NS, promising agents in cancer therapy.

Evaluation of standardized performance test methods for biomedical Raman spectroscopy (Erratum).

The erratum corrects an error in Fig. 4 of the published article.

Silica-coated gold nanostars for combined surface-enhanced Raman scattering (SERS) detection and singlet-oxygen generation: a potential nanoplatform for theranostics.

This paper reports the synthesis and characterization of surface-enhanced Raman scattering (SERS) label-tagged gold nanostars, coated with a silica shell containing methylene blue photosensitizing drug for singlet-oxygen generation. To our knowledge, this is the first report of nanocomposites possessing a combined capability for SERS detection and singlet-oxygen generation for photodynamic therapy. The gold nanostars were tuned for maximal absorption in the near-infrared (NIR) spectral region and tagged with a NIR dye for surface-enhanced resonance Raman scattering (SERRS). Silica coating was used to encapsulate the photosensitizer methylene blue in a shell around the nanoparticles. Upon 785 nm excitation, SERS from the Raman dye is observed, while excitation at 633 nm shows fluorescence from methylene blue. Methylene-blue-encapsulated nanoparticles show a significant increase in singlet-oxygen generation as compared to nanoparticles synthesized without methylene blue. This increased singlet-oxygen generation shows a cytotoxic effect on BT549 breast cancer cells upon laser irradiation. The combination of SERS detection (diagnostic) and singlet-oxygen generation (therapeutic) into a single platform provides a potential theranostic agent.

Evaluation of standardized performance test methods for biomedical Raman spectroscopy.

SIGNIFICANCE: Raman spectroscopy has emerged as a promising technique for a variety of biomedical applications. The unique ability to provide molecular specific information offers insight to the underlying biochemical changes that result in disease states such as cancer. However, one of the hurdles to successful clinical translation is a lack of international standards for calibration and performance assessment of modern Raman systems used to interrogate biological tissue. AIM: To facilitate progress in the clinical translation of Raman-based devices and assist the scientific community in reaching a consensus regarding best practices for performance testing. APPROACH: We reviewed the current literature and available standards documents to identify methods commonly used for bench testing of Raman devices (e.g., relative intensity correction, wavenumber calibration, noise, resolution, and sensitivity). Additionally, a novel 3D-printed turbid phantom was used to assess depth sensitivity. These approaches were implemented on three fiberoptic-probe-based Raman systems with different technical specifications. RESULTS: While traditional approaches demonstrated fundamental differences due to detectors, spectrometers, and data processing routines, results from the turbid phantom illustrated the impact of illumination-collection geometry on measurement quality. CONCLUSIONS: Specifications alone are necessary but not sufficient to predict in vivo performance, highlighting the need for phantom-based test methods in the standardized evaluation of Raman devices.

Accurate in vivo tumor detection using plasmonic-enhanced shifted-excitation Raman difference spectroscopy (SERDS).

For the majority of cancer patients, surgery is the primary method of treatment. In these cases, accurately removing the entire tumor without harming surrounding tissue is critical; however, due to the lack of intraoperative imaging techniques, surgeons rely on visual and physical inspection to identify tumors. Surface-enhanced Raman scattering (SERS) is emerging as a non-invasive optical alternative for intraoperative tumor identification, with high accuracy and stability. However, Raman detection requires dark rooms to work, which is not consistent with surgical settings. Methods: Herein, we used SERS nanoprobes combined with shifted-excitation Raman difference spectroscopy (SERDS) detection, to accurately detect tumors in xenograft murine model. Results: We demonstrate for the first time the use of SERDS for in vivo tumor detection in a murine model under ambient light conditions. We compare traditional Raman detection with SERDS, showing that our method can improve sensitivity and accuracy for this task. Conclusion: Our results show that this method can be used to improve the accuracy and robustness of in vivo Raman/SERS biomedical application, aiding the process of clinical translation of these technologies.

Experimental investigation of parameters influencing plasmonic nanoparticle-mediated bubble generation with nanosecond laser pulses.

Plasmonic nanoparticles (PNPs) continue to see increasing use in biophotonics for a variety of applications, including cancer detection and treatment. Several PNP-based approaches involve the generation of highly transient nanobubbles due to pulsed laser-induced vaporization and cavitation. While much effort has been devoted to elucidating the mechanisms behind bubble generation with spherical gold nano particles, the effects of particle shape on bubble generation thresholds are not well understood, especially in the nanosecond pulse regime. Our study aims to compare the bubble generation thresholds of gold nanospheres, gold nanorods, and silica-core gold nanoshells with different sizes, resonances, and surface coatings. Bubble generation is detected using a multimodality microscopy platform for simultaneous, nanosecond resolution pump-probe imaging, integrated scattering response, and acoustic transient detection. Nanoshells and large (40-nm width) nanorods were found to have the lowest thresholds for bubble generation, and in some cases they generated bubbles at radiant exposures below standard laser safety limits for skin exposure. This has important implications for both safety and performance of techniques employing pulsed lasers and PNPs.

Pulsed laser damage of gold nanorods in turbid media and its impact on multi-spectral photoacoustic imaging.

Innovative biophotonic modalities such as photoacoustic imaging (PAI) have the potential to provide enhanced sensitivity and molecule-specific detection when used with nanoparticles. However, high peak irradiance levels generated by pulsed lasers can lead to modification of plasmonic nanoparticles. Thus, there is an outstanding need to develop practical methods to effectively predict the onset nanoparticle photomodification as well as a need to better understand the process during PAI. To address this need, we studied pulsed laser damage of gold nanorods (GNRs) using turbid phantoms and a multi-spectral near-infrared PAI system, comparing results with spectrophotometric measurements of non-scattering samples. Transmission electron microscopy and Monte Carlo modeling were also performed to elucidate damage processes. In the phantoms, shifts in PAI-detected spectra indicative of GNR damage were initiated at exposure levels one-third of that seen in non-scattering samples, due to turbidity-induced enhancement of subsurface fluence. For exposures approaching established safety limits, damage was detected at depths of up to 12.5 mm. Typically, GNR damage occurred rapidly, over the course of a few laser pulses. This work advances the development of test methods and numerical models as tools for assessment of nanoparticle damage and its implications, and highlights the importance of considering GNR damage in development of PAI products, even for exposures well below laser safety limits.

Author Correction: Quantitative Evaluation of Nanosecond Pulsed Laser-Induced Photomodification of Plasmonic Gold Nanoparticles.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Quantitative Evaluation of Nanosecond Pulsed Laser-Induced Photomodification of Plasmonic Gold Nanoparticles.

The rapid growth of gold nanoparticle applications in laser therapeutics and diagnostics has brought about the need for establishing innovative standardized test methods for evaluation of safety and performance of these technologies and related medical products. Furthermore, given the incomplete and inconsistent data on nanoparticle photomodification thresholds provided in the literature, further elucidation of processes that impact the safety and effectiveness of laser-nanoparticle combination products is warranted. Therefore, we present a proof-of-concept study on an analytical experimental test methodology including three approaches (transmission electron microscopy, dynamic light scattering, and spectrophotometry) for experimental evaluation of damage thresholds in nanosecond pulsed laser-irradiated gold nanospheres, and compared our results with a theoretical model and prior studies. This thorough evaluation of damage threshold was performed based on irradiation with a 532 nm nanosecond-pulsed laser over a range of nanoparticle diameters from 20 to 100 nm. Experimentally determined damage thresholds were compared to a theoretical heat transfer model of pulsed laser-irradiated nanoparticles and found to be in reasonably good agreement, although some significant discrepancies with prior experimental studies were found. This study and resultant dataset represent an important foundation for developing a standardized test methodology for determination of laser-induced nanoparticle damage thresholds.

Human Adipose-Derived Stem Cells Labeled with Plasmonic Gold Nanostars for Cellular Tracking and Photothermal Cancer Cell Ablation.

BACKGROUND: Gold nanostars are unique nanoplatforms that can be imaged in real time and transform light energy into heat to ablate cells. Adipose-derived stem cells migrate toward tumor niches in response to chemokines. The ability of adipose-derived stem cells to migrate and integrate into tumors makes them ideal vehicles for the targeted delivery of cancer nanotherapeutics. METHODS: To test the labeling efficiency of gold nanostars, undifferentiated adipose-derived stem cells were incubated with gold nanostars and a commercially available nanoparticle (Qtracker), then imaged using two-photon photoluminescence microscopy. The effects of gold nanostars on cell phenotype, proliferation, and viability were assessed with flow cytometry, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide metabolic assay, and trypan blue, respectively. Trilineage differentiation of gold nanostar-labeled adipose-derived stem cells was induced with the appropriate media. Photothermolysis was performed on adipose-derived stem cells cultured alone or in co-culture with SKBR3 cancer cells. RESULTS: Efficient uptake of gold nanostars occurred in adipose-derived stem cells, with persistence of the luminescent signal over 4 days. Labeling efficiency and signal quality were greater than with Qtracker. Gold nanostars did not affect cell phenotype, viability, or proliferation, and exhibited stronger luminescence than Qtracker throughout differentiation. Zones of complete ablation surrounding the gold nanostar-labeled adipose-derived stem cells were observed following photothermolysis in both monoculture and co-culture models. CONCLUSIONS: Gold nanostars effectively label adipose-derived stem cells without altering cell phenotype. Once labeled, photoactivation of gold nanostar-labeled adipose-derived stem cells ablates neighboring cancer cells, demonstrating the potential of adipose-derived stem cells as a vehicle for the delivery of site-specific cancer therapy.