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

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

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.

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.

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.

Photothermal ablation of inflammatory breast cancer tumor emboli using plasmonic gold nanostars.

Inflammatory breast cancer (IBC) is rare, but it is the most aggressive subtype of breast cancer. IBC has a unique presentation of diffuse tumor cell clusters called tumor emboli in the dermis of the chest wall that block lymph vessels causing a painful, erythematous, and edematous breast. Lack of effective therapeutic treatments has caused mortality rates of this cancer to reach 20%-30% in case of women with stage III-IV disease. Plasmonic nanoparticles, via photothermal ablation, are emerging as lead candidates in next-generation cancer treatment for site-specific cell death. Plasmonic gold nanostars (GNS) have an extremely large two-photon luminescence cross-section that allows real-time imaging through multiphoton microscopy, as well as superior photothermal conversion efficiency with highly concentrated heating due to its tip-enhanced plasmonic effect. To effectively study the use of GNS as a clinically plausible treatment of IBC, accurate three-dimensional (3D) preclinical models are needed. Here, we demonstrate a unique in vitro preclinical model that mimics the tumor emboli structures assumed by IBC in vivo using IBC cell lines SUM149 and SUM190. Furthermore, we demonstrate that GNS are endocytosed into multiple cancer cell lines irrespective of receptor status or drug resistance and that these nanoparticles penetrate the tumor embolic core in 3D culture, allowing effective photothermal ablation of the IBC tumor emboli. These results not only provide an avenue for optimizing the diagnostic and therapeutic application of GNS in the treatment of IBC but also support the continuous development of 3D in vitro models for investigating the efficacy of photothermal therapy as well as to further evaluate photothermal therapy in an IBC in vivo model.

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.

Reversible Gating of Plasmonic Coupling for Optical Signal Amplification.

Amplification of optical signals is useful for a wide variety of applications, ranging from data signal transmission to chemical sensing and biomedical diagnostics. One such application in chemical sensing is surface-enhanced Raman scattering (SERS), an important technique for increasing the Raman signal using the plasmonic effect of enhanced electromagnetic fields associated with metallic nanostructures. One of the most important limitations of SERS-based amplification is the difficulty to reproducibly control the SERS signal. Here, we describe the design and implementation of a unique hybrid system capable of producing reversible gating of plasmonic coupling for Raman signal amplification. The hybrid system is composed of two subsystems: (1) colloidal magneto-plasmonic nanoparticles for SERS enhancement and (2) a micromagnet substrate with an externally applied magnetic field to modulate the colloidal nanoparticles. For this proof of concept demonstration, the nanoparticles were labeled with a Raman-active dye, and it was shown that the detected SERS signal could be reproducibly modulated by controlling the externally applied magnetic field. The developed system provides a simple, robust, inexpensive, and reusable device for SERS signal modulation. These properties will open up new possibilities for optical signal amplification and gating as well for high-throughput, reproducible SERS detection.

Tunable and amplified Raman gold nanoprobes for effective tracking (TARGET): in vivo sensing and imaging.

We describe the development of a highly tunable, physiologically stable, and ultra-bright Raman probe, named as TARGET (Tunable and Amplified Raman Gold Nanoprobes for Effective Tracking), for in vitro and in vivo surface-enhanced Raman scattering (SERS) applications. The TARGET structure consists of a gold core inside a larger gold shell with a tunable interstitial gap similar to a "nanorattle" structure. The combination of galvanic replacement and the seed mediated growth method was employed to load Raman reporter molecules and subsequently close the pores to prevent leaking and degradation of reporters under physiologically extreme conditions. Precise tuning of the core-shell gap width, core size, and shell thickness allows us to modulate the plasmonic effect and achieve a maximum electric-field (E-field) intensity. The interstitial gap of TARGET nanoprobes can be designed to exhibit a plasmon absorption band at 785 nm, which is in resonance with the dye absorption maximum and lies in the "tissue optical window", resulting in ultra-bright SERS signals for in vivo studies. The results of in vivo measurements of TARGETs in laboratory mice illustrated the usefulness of these nanoprobes for medical sensing and imaging.

Tracking mesenchymal stromal cells using an ultra-bright TAT-functionalized plasmonic-active nanoplatform.

High-resolution tracking of stem cells remains a challenging task. An ultra-bright contrast agent with extended intracellular retention is suitable for in vivo high-resolution tracking of stem cells following the implantation. Here, a plasmonic-active nanoplatform was developed for tracking mesenchymal stromal cells (MSCs) in mice. The nanoplatform consisted of TAT peptide-functionalized gold nanostars (TAT-GNS) that emit ultra-bright two-photon photoluminescence capable of tracking MSCs under high-resolution optical imaging. In vitro experiment showed TAT-GNS-labeled MSCs retained a similar differentiability to that of non-labeled MSCs controls. Due to their star shape, TAT-GNS exhibited greater intracellular retention than that of commercial Q-Tracker. In vivo imaging of TAT-GNS-labeled MSCs five days following intra-arterial injections in mice kidneys showed possible MSCs implantation in juxta-glomerular (JG) regions, but non-specifically in glomeruli and afferent arterioles as well. With future design to optimize GNS labeling specificity and clearance, plasmonic-active nanoplatforms may be a useful intracellular tracking tool for stem cell research. An ultra-bright intracellular contrast agent is developed using TAT peptide-functionalized gold nanostars (TAT-GNS). It poses minimal influence on the stem cell differentiability. It exhibits stronger two-photon photoluminescence and superior labeling efficiency than commercial Q-Tracker. Following renal implantation, some TAT-GNS-labeled MSCs permeate blood vessels and migrate to the juxta-glomerular region.

Sensitive DNA detection and SNP discrimination using ultrabright SERS nanorattles and magnetic beads for malaria diagnostics.

One of the major obstacles to implement nucleic acid-based molecular diagnostics at the point-of-care (POC) and in resource-limited settings is the lack of sensitive and practical DNA detection methods that can be seamlessly integrated into portable platforms. Herein we present a sensitive yet simple DNA detection method using a surface-enhanced Raman scattering (SERS) nanoplatform: the ultrabright SERS nanorattle. The method, referred to as the nanorattle-based method, involves sandwich hybridization of magnetic beads that are loaded with capture probes, target sequences, and ultrabright SERS nanorattles that are loaded with reporter probes. Upon hybridization, a magnet was applied to concentrate the hybridization sandwiches at a detection spot for SERS measurements. The ultrabright SERS nanorattles, composed of a core and a shell with resonance Raman reporters loaded in the gap space between the core and the shell, serve as SERS tags for signal detection. Using this method, a specific DNA sequence of the malaria parasite Plasmodium falciparum could be detected with a detection limit of approximately 100 attomoles. Single nucleotide polymorphism (SNP) discrimination of wild type malaria DNA and mutant malaria DNA, which confers resistance to artemisinin drugs, was also demonstrated. These test models demonstrate the molecular diagnostic potential of the nanorattle-based method to both detect and genotype infectious pathogens. Furthermore, the method's simplicity makes it a suitable candidate for integration into portable platforms for POC and in resource-limited settings applications.

Folate Receptor-Targeted Theranostic Nanoconstruct for Surface-Enhanced Raman Scattering Imaging and Photodynamic Therapy.

We report the synthesis of a folate receptor (FR)-targeted theranostic nanocomposite for surface-enhanced Raman scattering (SERS) imaging and photodynamic therapy (PDT). FR-specific SERS detection and PDT are demonstrated in vitro using two FR-positive cancer cell lines and one FR-negative cancer cell lines.

Multiplexed Detection of MicroRNA Biomarkers Using SERS-Based Inverse Molecular Sentinel (iMS) Nanoprobes.

MicroRNAs (miRNAs) have demonstrated great promise as a novel class of biomarkers for early detection of various cancers, including breast cancer. However, due to technical difficulties in detecting these small molecules, miRNAs have not been adopted into routine clinical practice for early diagnostics. Thus, it is important to develop alternative detection strategies that could offer more advantages over conventional methods. Here, we demonstrate the application of a "turn-on" SERS sensing technology, referred to as "inverse Molecular Sentinel (iMS)" nanoprobes, as a homogeneous assay for multiplexed detection of miRNAs. This SERS nanoprobe involves the use of plasmonic-active nanostars as the sensing platform. The "OFF-to-ON" signal switch is based on a nonenzymatic strand-displacement process and the conformational change of stem-loop (hairpin) oligonucleotide probes upon target binding. This technique was previously used to detect a synthetic DNA sequence of interest. In this study, we modified the design of the nanoprobe to be used for the detection of short (22-nt) miRNA sequences. The demonstration of using iMS nanoprobes to detect miRNAs in real biological samples was performed with total small RNA extracted from breast cancer cell lines. The multiplex capability of the iMS technique was demonstrated using a mixture of the two differently labeled nanoprobes to detect miR-21 and miR-34a miRNA biomarkers for breast cancer. The results of this study demonstrate the feasibility of applying the iMS technique for multiplexed detection of short miRNAs molecules.

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.

Multifunctional gold nanostars for molecular imaging and cancer therapy.

Plasmonics-active gold nanoparticles offer excellent potential in molecular imaging and cancer therapy. Among them, gold nanostars (AuNS) exhibit cross-platform flexibility as multimodal contrast agents for macroscopic X-ray computer tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), as well as nanoprobes for photoacoustic tomography (PAT), two-photon photoluminescence (TPL), and surface-enhanced Raman spectroscopy (SERS). Their surfactant-free surface enables versatile functionalization to enhance cancer targeting, and allow triggered drug release. AuNS can also be used as an efficient platform for drug carrying, photothermal therapy, and photodynamic therapy (PDT). This review paper presents the latest progress regarding AuNS as a promising nanoplatform for cancer nanotheranostics. Future research directions with AuNS for biomedical applications will also be discussed.

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.