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.
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.
Although nanotechnology has led to important advances in in vitro diagnostics, the development of nanosensors for in vivo molecular 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 “smart tattoo” nanosensor used in this study employs the “inverse molecular sentinel” (iMS) detection scheme, which is a label-free homogeneous biosensing system based on a non-enzymatic DNA strand-displacement process and conformational change of stem-loop (hairpin) oligonucleotide probes upon target binding. In this study, plasmonics‐active nanostar was utilized as an efficient in vivo SERS sensing platform due to their tunable absorption bands in the near infrared region of the “tissue optical window. The results of this study illustrate the usefulness of SERS iMS nanosensors as an implantable skin‐based in vivo biosensing platform, providing a foundation for developments in continuous health status sensing, disease biomarker monitoring, and other clinical translation applications.
The use of plasmonic nanoparticles (PNPs) in biophotonics has been steadily increasing, with a wide variety of emerging applications including detection and treatment of cancers. PNPs have unique, tunable optical properties arising from their surface plasmon resonance (SPR), and can be functionalized to achieve molecular targeting in vivo. Highly transient nanobubbles generated around PNPs due to pulsed laser-induced vaporization and cavitation may significantly impact diagnostic and therapeutic procedures. While much effort has been devoted to elucidating the mechanisms behind PNP nanobubble generation, there has not been a comprehensive study on the effect of nanoparticle size on nanobubble formation thresholds, and whether particle photodegradation occurs at these levels. Herein, we evaluate the melting and bubble generation thresholds of commercially available gold nanospheres in the 20-100 nm diameter range when exposed to nanosecond laser pulses. The 532-nm laser pulses are generated by a second-harmonic Q-switched Nd:YAG laser, providing output near the SPR of the gold nanospheres. Bubble generation is detected using a multimodality microscopy platform developed for simultaneous, nanosecond resolution pump-probe imaging, scattering response monitoring, and acoustic transient detection. PNP damage was further evaluated using transmission electron microscopy and spectrophotometry. The results of this study provide insights into damage thresholds as well as the concurrent and rapid optical, thermal, acoustic, and mechanical phenomena involved. This work will provide a foundation for improved understanding of a variety of PNPs and inform development of standard test methods for safety and efficacy evaluation of pulsed laser-PNP interactions in biophotonics.
The development of rapid, easy-to-use and highly sensitive DNA detection methods has received increasing interest for medical diagnostics and research purposes. Our laboratory has developed several chip-based DNA biosensors including molecular sentinel-on-chip (MSC), multiplex MSC, and inverse molecular sentinel-on-chip (iMS-on-Chip). These sensors use surface-enhanced Raman scattering (SERS) plasmonic chips, functionalized with DNA probes for single-step DNA detection. The sensing mechanisms is based on the hybridization of target sequences and DNA probes, resulting in a displacement of a SERS reporter from the chip surface. This distance increase results in change in SERS signal intensity from the reporter, thus indicating the capture, and therefore the presence, of the target nucleic acid sequence. The nucleic acid probes and the SERS chip, which compose the sensing platform, were designed for single-step DNA detection. The target sequences are detected by delivery of a sample solutions on a functionalized chip and characterization of the SERS signals, after 1 - 2 hr incubation. These techniques avoid labeling of the target sequence or washing to remove unreacted components, making them easy-to-use and cost effective. The use of SERS chip for medical diagnostics was demonstrated by detecting genetic biomarkers for respiratory viral infection and the DNA of dengue virus 4.
The development of rapid, easy-to-use, cost-effective, high accuracy, and high sensitive DNA detection methods for molecular diagnostics has been receiving increasing interest. Over the last five years, our laboratory has developed several chip-based DNA detection techniques including the molecular sentinel-on-chip (MSC), the multiplex MSC, and the inverse molecular sentinel-on-chip (iMS-on-Chip). In these techniques, plasmonic surface-enhanced Raman scattering (SERS) Nanowave chips were functionalized with DNA probes for single-step DNA detection. Sensing mechanisms were based on hybridization of target sequences and DNA probes, resulting in a distance change between SERS reporters and the Nanowave chip’s gold surface. This distance change resulted in change in SERS 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 Nanowave chips and SERS signals were measured after 1h - 2h incubation. 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 techniques for medical diagnostics was illustrated by the detection of genetic biomarkers for respiratory viral infection and of dengue virus 4 DNA.
The development of sensitive and selective biosensing techniques is of great interest for clinical diagnostics. Here, we describe the development and application of a surface enhanced Raman scattering (SERS) sensing technology, referred to as "inverse Molecular Sentinel (iMS)" nanoprobes, for the detection of nucleic acid biomarkers in biological samples. This iMS nanoprobe involves the use of plasmonic-active nanostars as the sensing platform for a homogenous assay for multiplexed detection of nucleic acid biomarkers, including DNA, RNA and microRNA (miRNA). The "OFF-to-ON" signal switch is based on a non-enzymatic strand-displacement process and the conformational change of stem-loop (hairpin) oligonucleotide probes upon target binding. Here, we demonstrate the development of iMS nanoprobes for the detection of DNA sequences as well as a modified design of the nanoprobe for the detection of short (22-nt) microRNA sequences. The application of 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 nucleic acid biomarkers, including short miRNAs molecules.
Nucleic acid-based molecular diagnostics at the point-of-care (POC) and in resource-limited settings is still a challenge. We present a sensitive yet simple DNA detection method with single nucleotide polymorphism (SNP) identification capability. The detection scheme involves sandwich hybridization of magnetic beads conjugated with capture probes, target sequences, and ultrabright surface-enhanced Raman Scattering (SERS) nanorattles conjugated with reporter probes. Upon hybridization, the sandwich probes are concentrated at the detection focus controlled by a magnetic system for SERS measurements. The ultrabright SERS nanorattles, consisting 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 ultrasensitive signal detection. Specific DNA sequences of the malaria parasite Plasmodium falciparum and dengue virus 1 (DENV1) were used as the model marker system. Detection limit of approximately 100 attomoles was achieved. Single nucleotide polymorphism (SNP) discrimination of wild type malaria DNA and mutant malaria DNA, which confers resistance to artemisinin drugs, was also demonstrated. The results demonstrate the molecular diagnostic potential of the nanorattle-based method to both detect and genotype infectious pathogens. The method's simplicity makes it a suitable candidate for molecular diagnosis at the POC and in resource-limited settings.
Demonstrated herein is a simple method for the induction of J-aggregate formation in a colloidal solution of gold nanoparticles through the use of pseudoisocyanine (PIC) and polyvinyl sulfate. The plasmon-exciton coupling of the nanoparticle J-aggregate complex results in a split lineshape absorption spectrum with upper and lower plexcitonic branches. The use of nanoparticles with various plasmon resonances causes a shift in the upper plexcitonic band while the lower plexcitonic band remains at the same wavelength.
Surface-enhanced Raman scattering (SERS) provides a non-destructive sensing method with high sensitivity and multiplex detection capability since SERS takes advantage of high enhancement from surface plasmon resonance and unique “fingerprint” spectral signature using Raman spectroscopy. Our group has developed a unique plasmonics-active nanoparticle, gold nanostars, with tunable plasmonics in near-infrared (NIR) “tissue optical window” without using toxic surfactant. We present their applications for chemical and biological sensing with SERS method as well as theoretical studies to investigate and confirm experimentally measured SERS results.
Cancer has become one of most significant death reasons and causes approximately 7.9 million human deaths worldwide each year. The challenge to detect cancer at an early stage makes cancer-related biomarkers sensing attract more and more research interest and efforts. Surface-enhanced Raman scattering (SERS) provides a promising method for various biomarkers (DNA, RNA, protein, et al.) detection due to its high sensitivity, specificity and capability for multiple analytes detection. Raman spectroscopy is a non-destructive photon-scattering technique, which provides molecule-specific information on molecular vibrational energy levels. SERS takes advantage of plasmonic effects and can enhance Raman signal up to 1015 at “hot spots”. Due to its excellent sensitivity, SERS has been capable of achieving single-molecule detection limit. Local pH environment has been identified to be a potential biomarker for cancer diagnosis since solid cancer contains highly acidic environments. A near-infrared (NIR) SERS nanoprobe based on gold nanostars for pH sensing is developed for future cancer detection. Near-infrared (NIR) light is more suitable for in vivo applications because of its low attenuation rate and tissue auto fluorescence. SERS spectrum of pH reporter under various pH environments is monitored and used for pH sensing. Furthermore, density functional theory (DFT) calculation is performed to investigate Raman spectra changes with pH at the molecular level. The study demonstrates that SERS is a sensitive tool to monitor minor molecular structural changes due to local pH environment for cancer detection.
Nanotechnology provides tremendous biomedical opportunities for cancer diagnosis, imaging, and therapy. In contrast to
conventional chemotherapeutic agents where their actual target delivery cannot be easily imaged, integrating imaging
and therapeutic properties into one platform facilitates the understanding of pharmacokinetic profiles, and enables
monitoring of the therapeutic process in each individual. Such a concept dubbed “theranostics” potentiates translational
research and improves precision medicine. One particular challenging application of theranostics involves imaging and
controlled delivery of nanoplatforms across blood-brain-barrier (BBB) into brain tissues. Typically, the BBB hinders
paracellular flux of drug molecules into brain parenchyma. BBB disrupting agents (e.g. mannitol, focused ultrasound),
however, suffer from poor spatial confinement. It has been a challenge to design a nanoplatform not only acts as a
contrast agent but also improves the BBB permeation. In this study, we demonstrated the feasibility of plasmonic gold
nanoparticles as both high-resolution optical contrast agent and focalized tumor BBB permeation-inducing agent. We
specifically examined the microscopic distribution of nanoparticles in tumor brain animal models. We observed that
most nanoparticles accumulated at the tumor periphery or perivascular spaces. Nanoparticles were present in both
endothelial cells and interstitial matrices. This study also demonstrated a novel photothermal-induced BBB permeation.
Fine-tuning the irradiating energy induced gentle disruption of the vascular integrity, causing short-term extravasation of
nanomaterials but without hemorrhage. We conclude that our gold nanoparticles are a powerful biocompatible contrast
agent capable of inducing focal BBB permeation, and therefore envision a strong potential of plasmonic gold
nanoparticle in future brain tumor imaging and therapy.
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