Machine learning-assisted label-free colorectal cancer diagnosis using plasmonic needle-endoscopy system.

Early and accurate detection of colorectal cancer (CRC) is critical for improving patient outcomes. Existing diagnostic techniques are often invasive and carry risks of complications. Herein, we introduce a plasmonic gold nanopolyhedron (AuNH)-coated needle-based surface-enhanced Raman scattering (SERS) sensor, integrated with endoscopy, for direct mucus sampling and label-free detection of CRC. The thin and flexible stainless-steel needle is coated with polymerized dopamine, which serves as an adhesive layer and simultaneously initiates the nucleation of gold nanoparticle (AuNP) seeds on the needle surface. The AuNP seeds are further grown through a surface-directed reduction using Au ions-hydroxylamine hydrochloride solution, resulting in the formation of dense AuNHs. The formation mechanism of AuNHs and the layered structure of the plasmonic needle-based SERS (PNS) sensor are thoroughly analyzed. Furthermore, a strong field enhancement of the PNS sensor is observed, amplified around the edges of the polyhedral shapes and at nanogap sites between AuNHs. The feasibility of the PNS sensor combined with endoscopy system is further investigated using mouse models for direct colonic mucus sampling and verifying noninvasive label-free classification of CRC from normal controls. A logistic regression-based machine learning method is employed and successfully differentiates CRC and normal mice, achieving 100% sensitivity, 93.33% specificity, and 96.67% accuracy. Moreover, Raman profiling of metabolites and their correlations with Raman signals of mucus samples are analyzed using the Pearson correlation coefficient, offering insights for identifying potential cancer biomarkers. The developed PNS-assisted endoscopy technology is expected to advance the early screening and diagnosis approach of CRC in the future.

Molybdenum Disulfide-Assisted Spontaneous Formation of Multistacked Gold Nanoparticles for Deep Learning-Integrated Surface-Enhanced Raman Scattering.

Several fabrication methods have been developed for label-free detection in various fields. However, fabricating high-density and highly ordered nanoscale architectures by using soluble processes remains a challenge. Herein, we report a biosensing platform that integrates deep learning with surface-enhanced Raman scattering (SERS), featuring large-area, close-packed three-dimensional (3D) architectures of molybdenum disulfide (MoS2)-assisted gold nanoparticles (AuNPs) for the on-site screening of coronavirus disease (COVID-19) using human tears. Some AuNPs are spontaneously synthesized without a reducing agent because the electrons induced on the semiconductor surface reduce gold ions when the Fermi level of MoS2 and the gold electrolyte reach equilibrium. With the addition of polyvinylpyrrolidone, a two-dimensional large-area MoS2 layer assisted in the formation of close-packed 3D multistacked AuNP structures, resembling electroless plating. This platform, with a convolutional neural network-based deep learning model, achieved outstanding SERS performance at subterascale levels despite the microlevel irradiation power and millisecond-level acquisition time and accurately assessed susceptibility to COVID-19. These results suggest that our platform has the potential for rapid, low-damage, and high-throughput label-free detection of exceedingly low analyte concentrations.

Cancer screening through surface-enhanced Raman spectroscopy fingerprinting analysis of urinary metabolites using surface-carbonized silver nanowires on a filter membrane.

BACKGROUND: Label-free surface-enhanced Raman spectroscopy (SERS)-based metabolic profiling has great potential for early cancer diagnosis, but further advancements in analytical methods and clinical evidence studies are required for clinical applications. To improve the cancer diagnostic accuracy of label-free SERS spectral analysis of complex biological fluids, it is necessary to obtain specifically enhanced SERS signals of cancer-related metabolites present at low concentrations. RESULTS: This study presents a novel 3D SERS sensor, comprising a surface-carbonized silver nanowire (AgNW)-stacked filter membrane, alongside an optimized urine/methanol/chloroform extraction technique, which specifically changes the molecular adsorption and orientation of aromatic metabolites onto SERS substrates. By analyzing the pretreated urine samples on the surface-carbonized AgNW 3D SERS sensor, distinct and highly enhanced SERS peaks derived from semi-polar aromatic metabolites were observed for pancreatic cancer and prostate cancer samples compared with normal controls. Urine metabolite analysis using SERS fingerprinting successfully differentiated pancreatic cancer and prostate cancer groups from normal control group: normal control (n = 56), pancreatic cancer (n = 40), and prostate cancer (n = 39). SIGNIFICANCE AND NOVELTY: We confirmed the clinical feasibility of performing fingerprint analysis of urinary metabolites based on the surface-carbonized AgNW 3D SERS sensor and methanol/chloroform extraction for noninvasive cancer screening. This technology holds potential for large-scale screening owing to its high accuracy, and cost effective, simple and rapid detection method.

One-Stop Plasmonic Nanocube-Excited SERS Immunoassay Platform of Multiple Cardiac Biomarkers for Rapid Screening and Progressive Tracing of Acute Myocardial Infarction.

Rapid and precise acute myocardial infarction (AMI) diagnosis is essential for preventing patient death. In addition, the complementary roles of creatine kinase muscle brain (CK-MB) and cardiac troponin I (cTnI) cardiac biomarkers in the early and late stages of AMI demand their simultaneous detection, which is difficult to implement using conventional fluorescence and electrochemical technologies. Here, a nanotechnology-based one-stop immuno-surface-enhanced Raman scattering (SERS) detection platform is reported for multiple cardiac indicators for the rapid screening and progressive tracing of AMI events. Optimal SERS is achieved using optical property-based, excitation wavelength-optimized, and high-yield anisotropic plasmonic gold nanocubes. Optimal immunoassay reaction efficiencies are achieved by increasing immobilized antibodies. Multiple simultaneous detection strategies are implemented by incorporating two different Raman reports with narrow wavenumbers corresponding to two indicators and by establishing a computational SERS mapping process to accurately detect their concentrations, irrespective of multiple enzymes in the human serum. The SERS platform precisely estimated AMI onset and progressive timing in human serum and made rapid AMI identification feasible using a portable Raman spectrometer. This integrated platform is hypothesized to significantly contribute to emergency medicine and forensic science by providing timely treatment and observation.

3D plasmonic coral nanoarchitecture paper for label-free human urine sensing and deep learning-assisted cancer screening.

Practical human biofluid sensing requires a sensor device to differentiate patients from the normal group with high sensitivity and specificity. Label-free molecular identification from human biofluids allows direct classification of abnormal samples, providing insights for disease diagnosis and finding of new biomarkers. Here, we introduce a label-free surface-enhanced Raman scattering sensor based on a three-dimensional plasmonic coral nanoarchitecture (3D-PCN), which has strong electromagnetic field enhancement through multiple hot spots. The 3D-PCN was synthesized on a paper substrate via direct one-step gold reduction, forming a coral-like nanoarchitecture with high absorption property for biofluids. This was fabricated as a urine test strip and then integrated with a handheld Raman system to develop an on-site urine diagnostic platform. The developed platform successfully classified the human prostate and pancreatic cancer urines in a label-free method supported by two types of deep learning networks, with high clinical sensitivity and specificity. Our technology has the potential to be utilized not only for urinary cancer diagnosis but also for various human biofluid sensing systems as a future point-of-care testing platform.

In-situ fabrication of 3D interior hotspots templated with a protein@Au core-shell structure for label-free and on-site SERS detection of viral diseases.

Nanoscale plasmonic hotspots play a critical role in the enhancement of molecular Raman signals, enabling the sensitive and reliable trace analysis of biomedical molecules via surface-enhanced Raman spectroscopy (SERS). However, effective and label-free SERS diagnoses in practical fields remain challenging because of clinical samples' random adsorption and size mismatch with the nanoscale hotspots. Herein, we suggest a novel SERS strategy for interior hotspots templated with protein@Au core-shell nanostructures prepared via electrochemical one-pot Au deposition. The cytochrome c and lysates of SARS-CoV-2 (SLs) embedded in the interior hotspots were successfully functionalized to confine the electric fields and generate their optical fingerprint signals, respectively. Highly linear quantitative sensitivity was observed with the limit-of-detection value of 10-1 PFU/mL. The feasibility of detecting the targets in a bodily fluidic environment was also confirmed using the proposed templates with SLs in human saliva and nasopharyngeal swabs. These interior hotspots templated with the target analytes are highly desirable for early and on-site SERS diagnoses of infectious diseases without any labeling processes.

Design and Control of Lightweight Bionic Arm Driven by Soft Twisted and Coiled Artificial Muscles.

Twisted and coiled actuators (TCAs), which are light but capable of producing significant power, were developed in recent times. After their introduction, there have been numerous improvements in performance, including development of techniques such as actuation strain and heating methods. However, the development of robots using TCA is still in its early stages. In this study, a bionic arm driven by TCAs was developed for light and flexible operation. The aim of this study was to gain a foothold in the future of robot development using TCA, which is considered as the appropriate artificial muscle. The main developments were with regard to the design (from actuator design to system design), system configuration for control, and control method. First, a process technology for repeatedly manufacturing TCA, which can be used practically and delivers sufficient performance, was developed. Based on the developed actuator, a joint was designed to move the elbow and hand. The final bionic arm was developed by integrating the TCA, pulley joint, and control system. It moved the elbow up to 100° and allowed the hand to move in three degrees of freedom. Using the control method for each joint, we were able to show the movement by using the hand and elbow.

Organometallic hotspot engineering for ultrasensitive EC-SERS detection of pathogenic bacteria-derived DNAs.

The sensitivity and limit-of-detection (LOD) of the traditional surface-enhanced Raman spectroscopy (SERS) platform suffer from the requirement of precise positioning of small analytes, including DNAs and bacteria, into narrow hotspots. In this study, a novel SERS sensor was developed using electrochemical deposition onto metal nanopillars (ECOMPs) combined with complementary DNAs (cDNAs) for the detection of pathogenic bacteria. Applying a redox potential to AuCl4- ions actively engineered the organometallic hotspots based on the cDNAs in a short time (<10 min) and simultaneously produced SERS signals. Because of the influence of potential-driven morphological properties on the SERS efficiency in the cDNA domains and the resonant coupling of internal fields with the fields confined between adjacent ECOMPs-cDNAs, the optimum growth time was determined to be 5 min. The EC-SERS detection and discrimination of Enterococcus faecium and Staphylococcus aureus were successfully carried out because of the DNA complementarity. Compared with plasmonic metal nanopillars (MPs)-cDNAs, the enhancement factor of the ECOMPs-cDNAs was estimated to be ∼2.0 × 103. A quantitative investigation revealed that a highly linear progression in the target DNA concentration range (0.05-100 nM) and a LOD of ∼0.035 nM were achieved. The specificity of the ECOMPs-cDNAs was validated by cross-hybridization. The platform was also used to assay human whole blood containing 0.1 nM bacterial DNAs. The proposed strategy provides the potential for highly sensitive SERS-based multiplex DNA detection in clinical diagnostics.

Rapid and sensitive multiplex molecular diagnosis of respiratory pathogens using plasmonic isothermal RPA array chip.

The current clinically available multiplex molecular diagnostic technologies are difficult to apply to onsite diagnostic platforms due to their large and sophisticated instrumentation, long amplification times and limited number of simultaneous detections. We developed a plasmonic isothermal recombinase polymerase amplification (RPA) array chip for rapid and sensitive multiplex molecular detection. The 3D plasmonic substrate composed of Au nanoparticles (NPs) on dense Au nanopillars (NPOP) showed highly enhanced plasmon-enhanced fluorescence (PEF) of RPA products with long DNA amplicons (~200 bp). The plasmonic 4-plex RPA array chip successfully detected bacterial DNA within 30 min and viral RNA within 40 min; the sensitivity of the plasmonic RPA array chip was comparable to or 10-fold higher than that of the 4-pelx liquid-phase RPA and 4-plex liquid-phase PCR techniques. Additionally, no cross-reactivity was observed. The 4-plex plasmonic RPA array chip was preliminary evaluated using clinical respiratory viral-positive nasopharyngeal swab samples. This approach enables rapid, sensitive and high-multiplex molecular detection and can be used in the realization of a simplified and miniaturized platform for onsite multiplex molecular diagnostics.

Label-Free SERS Analysis of Urine Using a 3D-Stacked AgNW-Glass Fiber Filter Sensor for the Diagnosis of Pancreatic Cancer and Prostate Cancer.

Metabolomics shows tremendous potential for the early diagnosis and screening of cancer. For clinical application as an effective diagnostic tool, however, improved analytical methods for complex biological fluids are required. Here, we developed a reliable rapid urine analysis system based on surface-enhanced Raman spectroscopy (SERS) using 3D-stacked silver nanowires (AgNWs) on a glass fiber filter (GFF) sensor and applied it to the diagnosis of pancreatic cancer and prostate cancer. Urine samples were pretreated with centrifugation to remove large debris and with calcium ion addition to improve the binding of metabolites to AgNWs. The label-free urine-SERS detection using the AgNW-GFF SERS sensor showed different spectral patterns and distinguishable specific peaks in three groups: normal control (n = 30), pancreatic cancer (n = 22), and prostate cancer (n = 22). Multivariate analyses of SERS spectra using unsupervised principal component analysis and supervised orthogonal partial least-squares discriminant analysis showed excellent discrimination between the pancreatic cancer group and the prostate cancer group as well as between the normal control group and the combined cancer groups. The results demonstrate the great potential of the urine-SERS analysis system using the AgNW-GFF SERS sensor for the noninvasive diagnosis and screening of cancers.

A Wearable Surface-Enhanced Raman Scattering Sensor for Label-Free Molecular Detection.

A wearable surface-enhanced Raman scattering (SERS) sensor has been developed as a patch type to utilize as a molecular sweat sensor. Here, the SERS patch sensor is designed to comprise a sweat-absorbing layer, which is an interface to the human skin, an SERS active layer, and a dermal protecting layer that prevents damage and contaminations. A silk fibroin protein film (SFF) is a basement layer that absorbs aqueous solutions and filtrates molecules larger than the nanopores created in the ß-sheet matrix of the SFF. On the SFF layer, a plasmonic silver nanowire (AgNW) layer is formed to enhance the Raman signal of the molecules that penetrated through the SERS patch in a label-free method. A transparent dermal protecting layer (DP) allows laser penetration to the AgNW layer enabling Raman measurement through the SERS patch without its detachment from the surface. The molecular detection capability and time-dependent absorption properties of the SERS patch are investigated, and then, the feasibility of its use as a wearable drug detection sweat sensor is demonstrated using 2-fluoro-methamphetamine (2-FMA) on the human cadaver skin. It is believed that the developed SERS patch can be utilized as various flexible and wearable biosensors for healthcare monitoring.

A cyclodextrin-decorated plasmonic gold nanosatellite substrate for selective detection of bipyridylium pesticides.

A cyclodextrin-decorated gold nanosatellite (AuNSL) substrate was developed as a surface-enhanced Raman scattering sensor for the selective sensing of bipyridylium pesticides such as paraquat (PQ), diquat (DQ), and difenzoquat (DIF). The AuNSL structure was fabricated via vacuum deposition of gold nanoparticles (AuNPs) on a gold nanopillar substrate, and a large density of hot-spots was formed for Raman signal enhancement. Thiolated ß-cyclodextrin (SH-CD) was surface-modified on the AuNSL as a chemical receptor. The detection limit of PQ, DQ, and DIF on the SH-CD-coated AuNSL (CD-AuNSL) was 0.05 ppm for each, and showed linear correlation in a concentration range of 10 ppm-0.05 ppm. Then, selective bipyridylium pesticide detection was performed by comparing the Raman intensity of each pesticide with and without the washing step. After the washing step, 90% of the PQ, DQ, and DIF Raman signals were maintained on the CD-AuNSL substrate with a uniform selectivity in a mapping area of 200 µm × 200 µm. Furthermore, selective pesticide detection was performed using a ground-apple solution without pretreatment. Raman signals were clearly observed after the washing step and they showed a limit of detection down to a concentration of 0.05 ppm for each pesticide. Principal component analysis (PCA) of the binary and ternary mixtures of PQ, DQ, and DIF showed that each component could be easily identified via the typical Raman fingerprint analysis. The developed CD-AuNSL is expected to be applied for various chemical sensors, especially for pyridine-containing toxic substances in the environment and metabolite biomarkers in biofluids.

Highly sensitive and repeatable DNA-SERS detection system using silver nanowires-glass fiber filter substrate.

This paper describes a new simple DNA detection method based on surface-enhanced Raman scattering (SERS) technology using a silver nanowire stacked-glass fiber filter substrate. In this system, DNA-intercalating dye (EVAGreen) and reference dye (ROX) are used together to improve the repeatability and reliability of the SERS signals. We found that the SERS signal of EVAGreen was reduced by intercalation into DNA amplicons of a polymerase chain reaction on the silver nanowire stacked-glass fiber filter substrate, whereas that of ROX stayed constant. The DNA amplicons could be quantified by correcting the EVAGreen-specific SERS signal intensity with the ROX-specific SERS signal intensity. Multivariate analysis by partial least square methods was also successfully performed. And we further applied it to loop-mediated isothermal amplification with potential use for on-site diagnostics. The sensitivities of the DNA-SERS detection showed about 100 times higher than those of conventional fluorescence-based detection methods. The DNA-SERS detection method can be applied to various isothermal amplification methods, which is expected to realize on-site molecular diagnostics with high sensitivity, repeatability, simplicity, affordability, and convenience.

Encapsulation of 3D plasmonic nanostructures with ultrathin hydrogel skin for rapid and direct detection of toxic small molecules in complex fluids.

Nanogap-rich 3D plasmonic nanostructures provide enhanced molecular Raman fingerprints in a nondestructive and label-free manner. However, the molecular detection of small target molecules in complex fluids is challenging due to nonspecific protein adsorption, which prevents access of the target molecules. Therefore, the molecular detection for complex mixtures usually requires a tedious and time-consuming pretreatment of samples. Herein, we report the encapsulation of 3D plasmonic nanostructures with an ultrathin hydrogel skin for the rapid and direct detection of small molecules in complex mixtures. To demonstrate the proof of concept, we directly detect pesticide dissolved in milk without pretreatment. This detection is enabled by the selective permeation of target molecules into the 3D mesh of the hydrogel skin and the adsorption onto plasmonic hotspots, accompanied by the rejection of large adhesive proteins and colloids. The high sensitivity of nanogap-rich plasmonic nanostructures in a conjunction with the molecular selection of the hydrogel skin enables the fast and reliable detection of tricyclazole in whole milk with a limit of detection as low as 10 ppb within 1 h. We believe that this plasmonic platform is highly adaptable for in situ and on-site detection of small molecules in various complex mixtures including foods, biological fluids, and environmental fluids.

Quasi-3D Plasmonic Nanowell Array for Molecular Enrichment and SERS-Based Detection.

We report on a quasi-three-dimensional (3D) plasmonic nanowell array with high structural uniformity for molecular detection. The quasi-3D plasmonic nanowell array was composed of periodic hexagonal Au nanowells whose surface is densely covered with gold nanoparticles (Au NPs), separated by an ultrathin dielectric interlayer. The uniform array of the Au nanowells was fabricated by nanoimprint lithography and deposition of Au thin film. A self-assembled monolayer (SAM) of perfluorodecanethiol (PFDT) was coated on the Au surface, on which Au was further deposited. Interestingly, the PFDT-coated Au nanowells were fully covered with Au NPs with an ultra-high density of 375 µm-2 rather than a smooth film due to the anti-wetting property of the low-energy surface. The plasmonic nanogaps formed among the high-density Au NPs led to a strong near-field enhancement via coupled localized surface plasmon resonance and produced a uniform surface-enhanced Raman spectroscopy (SERS) response with a small relative standard deviation of 5.3%. Importantly, the highly uniform nanostructure, featured by the nanoimprint lithography and 3D growth of densely-packed Au NPs, minimizes the spatial variation of Raman intensity, potentially providing quantitative analysis. Moreover, analyte molecules were highly concentrated and selectively deposited in nanowells when a water droplet containing the analyte was evaporated on the plasmonic substrate. The analyte formed a relatively thick overcoat in the nanowells near the triple line due to the coffee-ring effects. Combining 3D plasmonic nanowell substrates with molecular enrichments, highly sensitive detection of lactic acid was demonstrated. Given its combination of high sensitivity and signal uniformity, the quasi-3D plasmonic nanowell substrate is expected to provide a superior molecular detection platform for biosensing applications.

Tethered molecular redox capacitors for nanoconfinement-assisted electrochemical signal amplification.

Nanostructured materials offer the potential to drive future developments and applications of electrochemical devices, but are underutilized because their nanoscale cavities can impose mass transfer limitations that constrain electrochemical signal generation. Here, we report a new signal-generating mechanism that employs a molecular redox capacitor to enable nanostructured electrodes to amplify electrochemical signals even without an enhanced reactant mass transfer. The surface-tethered molecular redox capacitor engages diffusible reactants and products in redox-cycling reactions with the electrode. Such redox-cycling reactions are facilitated by the nanostructure that increases the probabilities of both reactant-electrode and product-redox-capacitor encounters (i.e., the nanoconfinement effect), resulting in substantial signal amplification. Using redox-capacitor-tethered Au nanopillar electrodes, we demonstrate improved sensitivity for measuring pyocyanin (bacterial metabolite). This study paves a new way of using nanostructured materials in electrochemical applications by engineering the reaction pathway within the nanoscale cavities of the materials.

3D nanoporous plasmonic chips for extremely sensitive NO2 detection.

The detection of toxic gas molecules using the surface-enhanced Raman spectroscopy (SERS) technique is very challenging due to the low affinity of gas molecules. Here, we report extremely sensitive SERS-based NO2 gas sensors based on 3D nanoporous Au nanostructures with a high affinity for NO2 gas molecules and high density of hotspots.

Development of a DNA isolation device using poly(3,4-dihydroxy-L-phenylalanine)-coated swab for on-site molecular diagnostics.

For on-site molecular diagnostics, a pre-treatment step for isolation of nucleic acid from clinical samples on site is desired. However, conventional commercialized silica-based nucleic acid isolation kits require repetitive pipetting and a centrifugation or permanent magnet for buffer exchange. In this study, we developed a poly(3,4-dihydroxy-L-phenylalanine) (L-DOPA)-coated swab that can absorb and desorb DNA depending on pH of buffers and a portable integrated DNA isolation device that comprises integrated chambers containing DNA isolation buffers. The poly(L-DOPA)-coated swab interacts with each buffer by passing through the membrane between the integrated chambers. Our device involves a simple operation and does not require any large equipment or skilled experts. By connecting the device with an automated polymerase chain reaction system, an isothermal amplification system, or a non-amplified DNA detection method, on-site molecular diagnosis of various diseases can be quickly realized.

Highly Sensitive and Selective Nanogap-Enhanced SERS Sensing Platform.

This paper reports a highly sensitive and selective surface-enhanced Raman spectroscopy (SERS) sensing platform. We used a simple fabrication method to generate plasmonic hotspots through a direct maskless plasma etching of a polymer surface and the surface tension-driven assembly of high aspect ratio Ag/polymer nanopillars. These collapsed plasmonic nanopillars produced an enhanced near-field interaction via coupled localized surface plasmon resonance. The high density of the small nanogaps yielded a high plasmonic detection performance, with an average SERS enhancement factor of 1.5 × 107. More importantly, we demonstrated that the encapsulation of plasmonic nanostructures within nanofiltration membranes allowed the selective filtration of small molecules based on the degree of membrane swelling in organic solvents and molecular size. Nanofiltration membrane-encapsulated SERS substrates do not require pretreatments. Therefore, they provide a simple and fast detection of toxic molecules using portable Raman spectroscopy.

Compact Integration of TiO2 Nanoparticles into the Cross-Points of 3D Vertically Stacked Ag Nanowires for Plasmon-Enhanced Photocatalysis.

The compact integration of semiconductor TiO2 nanoparticles (NPs) into the 3D crossed region of stacked plasmonic Ag nanowires (NWs) enhanced the photocatalytic activities through synergistic effects between the strong localized surface plasmon resonance (LSPR) excitation at the 3D cross-points of the Ag NWs and the efficient hot electron transfer at the interface between the Ag NWs and the TiO2 NPs. This paper explored new hybrid nanostructures based on the selective assembly of TiO2 NPs onto 3D cross-points of vertically stacked Ag NWs. The assembled TiO2 NPs directly contacted the 3D Ag NWs; therefore, charge separation occurred efficiently at the interface between the Ag NWs and the TiO2 NPs. The composite nanomaterials exhibited high extinction across the ultraviolet-visible range, rendering the nanomaterials high-performance photocatalysts across the full (ultraviolet-visible) and the visible spectral regions. Theoretical simulations clearly revealed that the local plasmonic field was highly enhanced at the 3D crossed regions of the vertically stacked Ag NWs. A Raman spectroscopic analysis of probe dye molecules under photodegradation conditions clearly revealed that the nanogap in the 3D crossed region was crucial for facilitating plasmon-enhanced photocatalysis and plasmon-enhanced spectroscopy.