Analysis of moxibustion’s smoke and thermal effects on plasma effector substances in KOA model rats using surface-enhanced Raman spectroscopy

Preface to the special issue dedicated to Professor Richard P. Van Duyne (1945–2019)

Using a simple two step fabrication process substrates with a large and uniform Raman enhancement, based on flexible free standing nanopillars can be manufactured over large areas using readily available silicon processing equipment.

Editorial on the special issue of <i>JRS</i> on International Year of Light (IYL)

Surgery is the mainstay treatment of brain tumors, however complete resection is rarely achieved, especially when dealing with grade IV glioblastoma (GBM). GBM is the most lethal brain tumor worldwide with an average survival not longer than 15 months. A reason of this dismal outcome is the lack of intraoperative visualization techniques for the objective identification of true tumor borders and infiltrating tumor cells. Hence, the improvement of GBM visualization during surgical operation is the motivation of this project. Recent developments of handheld fiber optic probes and lasers for low cost systems, together with sensitivity enhancement techniques such as surface enhanced Raman scattering (SERS), have ruled Raman spectroscopy as one of the most promising technologies for surgical guidance. This technique could overcome the limitations of current intraoperative modalities such as neuronavigation, magnetic resonance imaging and fluorescence guided surgery. For increased sensitivity, metallic nanostructures are preferred because they strongly interact with light, due to surface plasmon resonance (SPR), and produce a much higher level of amplification compared to flat surfaces. Among the nanostructures, gold nanoparticles (GNPs) have found wide application in SERS based imaging studies for their higher biocompatibility and versatile functionalization. When properly engineered, visualization can be targeted on tumor-specific biomolecules providing an accurate mapping of tumor spread. For optimized intraoperative visualization of GBM, detection can be tuned to the near-infrared (NIR) window by acting both on the size and shape of GNPs. This enables to overcome the interfering autofluorescence and to reach a deeper tissue penetration. However, surface chemistry becomes essential to create SERS tags for a fast, sensitive and specific detection of tumor cells. Due to the absence of comprehensive studies on the impact of GNPs surface functionalization on SERS based imaging, this thesis elucidates the effect of Raman reporter, inert protective polyethylene glycol (PEG) and anti-epidermal growth factor receptor (EGFR) antibody on colloidal stability, cellular binding specificity, detection sensitivity and speed. EGFR was the target of choice because its gene amplification is the most common molecular hallmark in about 60% of GBM and protein overexpression on cell cytoplasmic membrane makes it easily accessible. Based on the findings, GNPs with dense surface coverage of Raman reporter or PEG produced the maximum imaging sensitivity and binding specificity, respectively. However, GNPs with dense Raman reporter surface coverage were liable to non-specific binding and colloidal aggregation. Conversely, GNPs with dense PEG surface coverage owned the highest stability but underwent more than 90% reduction of SERS sensitivity. Higher integration time (from 0.05 to 0.5 sec) or shorter working distance (from 16.5 to 0.3 mm) to counteract the decreased SERS sensitivity were not considered in view of the final application in vivo. GNPs with surface coverage made of 50% Raman reporter and 50% PEG owned the optimal mixture for an immediate Raman detection of in vitro human GBM cells (LN229wtEGFR, BS153 and U87MG) while minimizing non-specific binding on EGFR-negative cells (IMA2.1). Further, SERS signal was comparable independently on the different EGFR expression level or the presence of EGFRvIII in BS153. The latter is the constitutively active receptor whose presence is associated to the lack of response during fluorescence based visualization of GBM. It was also shown that excess of Raman reporter did not add any significant contribution to SERS sensitivity. Similarly, the conjugation efficiency decreased by 35% through the addition of 10 times the concentration of antibody, compared to the lower concentration. This excess quantity of antibody showed no improvement of binding affinity of GNPs to tumor cells. Because the blood brain barrier (BBB) limits the therapeutic access to brain tumor, the ability of GNPs to cross an in vitro BBB made of a monoculture of human endothelial cells (hCMEC/D3) is crucial for successful intraoperative visualization. About 0.1% of GNPs, with specific ratio of immobilized functionalities, was able to cross the cell monolayer preserving its integrity and eliciting no cytotoxic effects. Similar results were obtained in vivo. By providing an in-depth investigation, this work stresses the significance of identifying the appropriate surface chemistry to improve the biomedical potential of GNPs in SERS based imaging applications. At the same time, it provides an in vitro demonstration that SERS based imaging can be implemented intraoperatively for immediate visualization of GBM offering an adequate alternative for the detection of those GBM or low-grade brain tumors that show variable or no response to fluorescence guided surgery, the current state-of-art for GBM intraoperative visualization.

The purpose of this minireview is to build a bridge between two research fields: surface-enhanced resonant Raman spectroscopy (SERRS) under near-single-molecule conditions and the branch of plasmonics treating strong coupling between plasmons and molecular excitons. SERRS enables single-molecule spectroscopy owing to its significant enhancement at SERRS hotspots (HSs), localized at gaps or junctions between plasmonic nanoparticle aggregates. SERRS is SERS (surface enhanced Raman spectroscopy) under a resonant Raman excitation condition. The origin of the Raman enhancement in SERRS is electromagnetic coupling between plasmons and molecular excitons at HSs. It has been reported that the coupling energy at HSs reaches the strong coupling region, meaning that they are potential platforms for applications of single molecular excitons modified by strong coupling. In this review, we discuss recent progress related to electronic strong coupling in near-single-molecule SERRS: collective (e.g., vibrational) strong coupling is out of the scope of this minireview. First, we explain the relationship between the electromagnetic enhancement factor and coupling energy. Second, we introduce three theoretical methods for obtaining evidence of strong coupling at HSs. Third, we discuss a method for reproducing enhanced and modified molecular Raman and fluorescence spectra at HSs using the coupling energy. Finally, we propose the use of two experimental methods of absorption spectroscopy at HSs for modifying molecular electronic dynamics by strong coupling and comment on future applications of SERRS HSs to photophysics and photochemistry.

Raman Spectroscopy is a time-honored, non-invasive method for analyzing and identifying the molecular composition of materials. However, unenhanced Raman Spectroscopy has extremely low sensitivity which limits its sensing capability. SERS brings rough nano-metallic surfaces in contact with the material molecules to enormously enhance the Raman signals. The sensitivity of SERS can be exploited in probe applications where the spectrometer needs to be brought near the specimen. For example, a long optical fiber coupled to a SERS device can be used to characterize and identify easy-to-reach cancerous tissues in organisms. Unfortunately, background signals in a long fiber can easily mask any signal returning from the end of the probe. A classical solution is to inject nanoparticles and use multiple optical fibers (one to deliver the excitation light and one or more to return the scattered Raman light). However, the coupling between the fibers is poor, reducing the signal strength, and reproducibility between locations, and removal of the injected nanoparticles present difficulties. This work intends to address those challenges by designing and fabricating a low-cost handheld SERS device. The development of this SERS device is broadly split into (a) fabrication of a probe suitable low-cost SERS substrate (b) design and fabrication of the handheld SERS device optics. The method used for the fabrication and characterization of the SERS layer low-cost substrate involved sandpaper imprint patterning of silver nanoparticles. This was accomplished at low cost with inexpensive equipment, readily available materials, and with no chemical or lithographic steps. The handheld SERS optics incorporated a solid-state laser, diffractive optics, the low-cost SERS substrate at one end of a GRIN lens, and a short-tube pathway to the Raman spectrometer. The response of the optical system and imprinted SERS layer was tested to obtain the Raman spectrum from 1nmol to 1mmol Rhodamine 6G suspension. This yielded good Raman scattering results. The developed device was made with a SERS substrate fabrication method which is a low-cost alternative and with no lithography or chemical synthesis. This SERS portable design is also suitable for bio-probes or remote sampling without the disadvantages associated with injected clouds substrates and multiple collection fiber systems.

The high precision in visualizing the true extent of tumor spread afforded by the newest generations of SERRS nanoparticles will increase the accuracy with which cancer can be diagnosed, resected or destroyed"

Surface-enhanced Raman spectroscopy for studying the interaction of N-propyl substituted imidazole compound with salmon sperm DNA.

Residual pesticides in fruits and vegetables have become one of the major food safety concerns around the world. At present, routine analytical methods used for the determination of pesticide residue on the surface of fruits and vegetables are destructive, complex, time-consuming, high cost and not environmentally friendly. In this study, a novel Surface Enhanced Raman Spectroscopy (SERS) method with silver colloid was developed for fast and sensitive nondestructive detection of residual pesticides in fruits and vegetables by using a self-developed Raman system. SERS technology is a combination of Raman spectroscopy and nanotechnology. SERS can greatly enhance the Raman signal intensity, achieve single-molecule detection, and has a simple sample pre-treatment characteristic of high sensitivity and no damage; in recent years it has begun to be used in food safety testing research. In this study a rapid and sensitive method was developed to identify and analyze mixed pesticides of chlorpyrifos, deltamethrin and acetamiprid in apple samples by SERS. Silver colloid was used for SERS measurement by hydroxylamine hydrochloride reduced. The advantages of this method are seen in its fast preparation at room temperature, good reproducibility and immediate applicability. Raman spectrum is highly interfered by noise signals and fluorescence background, which make it too complex to get good result. In this study the noise signals and fluorescence background were removed by Savitzky-Golay filter and min-max signal adaptive zooming method. Under optimal conditions, pesticide residues in apple samples can be detected by SERS at 0.005 μg/cm2 and 0.002 μg/cm2 for individual acetamiprid and thiram, respectively. When mixing the two pesticides at low concentrations, their characteristic peaks can still be identified from the SERS spectrum of the mixture. Based on the synthesized material and its application in SERS operation, the method represents an ultrasensitive SERS performance in apple samples detection without sample pre-treatment, which indicates that it could be served as a useful means in monitoring pesticide residues.

This study aims to examine the development of research related to Graphene-Based Surfaced-Enhanced Raman Scattering (SERS) through a bibliometric approach to computational mapping analysis using VOSviewer. The acquisition of article data was obtained from the Google Scholar database using the publish or perish reference manager application. The keywords used to guide the process of searching for the title and abstract of the article were "Graphene, SER, surface enhanced raman scattering, nanoparticle". A total of 920 articles were obtained which were considered related to the topic of this research. The study period used as study material is Google Scholar indexed articles for the last 10 years (2012 to 2022). The results showed that the Graphene-Based Surfaced-Enhanced Raman Scattering (SERS) research can be separated into 4 terms:Raman Spectroscopy, Graphene, Nanoparticle and Surface. The term “Raman Spectroscopy” is associated with 189 links with total link strength 1539 The term “Graphene” has 198 links with total link strength 2036 the term “Nanoparticle” has 199 links with total link strength in 2739 and the term “Surface” has 189 links with total link strength 1651. The results of the analysis of the development of Graphene-Based Surfaced-Enhanced Raman Scattering (SERS) in the last 10 years show an increase. However, in 2020-2021, there was a slight decrease from 136 in 2020 to 135 in 2021. The increase in research occurred from 2014 - 2020 (49, 63, 80, 99, 105,116 and 136 publications per year respectively). While the popular Graphene-Based Surfaced-Enhanced Raman Scattering (SERS) research was carried out in 2020, there were 136 studies. From the results of research on article data using VOS viewer on Graphene-Based Surfaced-Enhanced Raman Scattering (SERS) and its relationship to the problem area, the results show that there has been an increase over the last 10 years. This study can be an initial consideration for future researchers who will conduct research related to this research topic.

Study question Can Surface Enhanced Raman Spectroscopy (SERS) with gold nanoparticles improve the prediction of male infertility ? Summary answer SERS effectively differentiates fertile and infertile sperm samples, showing high sensitivity and specificity in predicting male infertility What is known already Male infertility affects a significant proportion of the global population, with up to 50% of infertility cases attributed to male factors. Conventional semen analysis relies on sperm concentration, motility, and morphology, but many cases remain idiopathic. Raman spectroscopy provides a molecular fingerprint of cells and has been applied to reproductive medicine. The SERS effect enhances Raman signals using metal nanoparticles, allowing a more detailed biochemical analysis of sperm quality. Previous studies indicate that SERS can detect molecular changes linked to sperm DNA integrity and oxidative stress. Study design, size, duration This case-control study evaluated sperm samples from 93 men (45 infertile, 48 fertile controls). Raman spectra were obtained after treating sperm with gold nanoparticles. A Partial Least Squares Discriminant Analysis (PLS-DA) model was applied to classify sperm quality based on spectral variations. The study was conducted over 12 months. Participants/materials, setting, methods Semen samples were collected from 93 men following 2–5 days of sexual abstinence. Sperm was processed with gold nanoparticles for SERS analysis. Raman spectra were collected, and multivariate statistical models were developed to classify fertile and infertile sperm samples. A PLS-DA model was used to assess diagnostic performance, with cross-validation and external validation Main results and the role of chance This study demonstrates the effectiveness of Surface Enhanced Raman Spectroscopy (SERS) in predicting male infertility by analyzing sperm biochemical composition. Key Raman spectral variations were identified, particularly in nucleic acids, phospholipids, and proteins, serving as molecular markers of sperm quality. Using Partial Least Squares Discriminant Analysis (PLS-DA), the model achieved a sensitivity of 85% and specificity of 83% in distinguishing fertile and infertile samples. Receiver Operating Characteristic (ROC) curve analysis showed an area under the curve (AUC) of 0.939, confirming high accuracy. The most significant spectral bands were found at 730 cm⁻¹ (DNA/RNA guanine), 1310 cm⁻¹ (CH3/CH2 lipids), and 1560 cm⁻¹ (proteins in acrosomal vesicles). Multivariate analysis validated low random variation, ensuring the robustness of the SERS methodology. Bivariate and trivariate models showed increased classification accuracy, particularly when DNA methylation and nucleic acid wagging were combined, reaching an 82.8% success rate. These findings highlight the potential of SERS as a non-invasive, highly accurate tool for assessing sperm quality and predicting male infertility. Future research aims to refine classification models and integrate SERS into assisted reproductive technologies (ART) for clinical applications. Limitations, reasons for caution The study was conducted on a single cohort, requiring validation in larger, diverse populations. Instrument variability and sample preparation may influence spectral outcomes. Standardization is needed before clinical implementation Wider implications of the findings SERS provides a promising, non-invasive method for male infertility diagnosis, potentially reducing reliance on conventional semen analysis. Integrating this technology into clinical settings could enhance sperm selection in assisted reproductive techniques Trial registration number No

Surface Enhanced Raman spectroscopy (SERS) provides significant improvements over conventional methods for single and multianalyte quantification. Specifically, the spectroscopic fingerprint provided by Raman scattering allows for a direct multiplexing potential far beyond that of fluorescence and colorimetry. Additionally, SERS generates a comparatively low financial and spatial footprint compared with common fluorescence based systems. Despite the advantages of SERS, it has remained largely an academic pursuit. In the field of biosensing, techniques to apply SERS to molecular diagnostics are constantly under development but, most often, assay protocols are redesigned around the use of SERS as a quantification method and ultimately complicate existing protocols. Our group has sought to rethink common SERS methodologies in order to produce translational technologies capable of allowing SERS to compete in the evolving, yet often inflexible biosensing field. This work will discuss the development of two techniques for quantification of microRNA, a promising biomarker for homeostatic and disease conditions ranging from cancer to HIV. First, an inkjet-printed paper SERS sensor has been developed to allow on-demand production of a customizable and multiplexable single-step lateral flow assay for miRNA quantification. Second, as miRNA concentrations commonly exist in relatively low concentrations, amplification methods (e.g. PCR) are therefore required to facilitate quantification. This work presents a novel miRNA assay alongside a novel technique for quantification of nuclease driven nucleic acid amplification strategies that will allow SERS to be used directly with common amplification strategies for quantification of miRNA and other nucleic acid biomarkers.

As a bio/chemical sensing technique, surface enhanced Raman spectroscopy (SERS) offers sensitivity comparable to that of fluorescence detection while providing highly specific information about the analyte. Although single molecule identification with SERS was demonstrated over a decade ago, today a need exists to develop practical solutions for point-of-sample and point-of-care SERS systems. In recent years, optofluidic SERS has emerged, in which microfluidic functions are integrated to improve the performance of SERS. Advancements in optofluidic SERS are leading towards portable analytical systems, but the devices are currently too expensive and too cumbersome for limited resource settings. Recently, we demonstrated the fabrication of SERS substrates by inkjet printing silver nanostructures onto paper. Using a low-cost commercial inkjet printer, we chemically patterned cellulose paper to form hydrophobic regions, which can control the aqueous sample on the paper microsystem. Additionally, we inkjet-printed silver nanoparticles with micro-scale precision to form SERS-active biosensors. Using these devices, we have been able to achieve detection limits comparable to conventional nanofabricated substrates. Furthermore, we leverage the fluidic properties to enhance the performance of the SERS devices while also enabling unprecedented ease of use. Paper dipsticks concentrate a relatively large sample volume into a small SERS-active detection region at the tip. Likewise, paper swabs collect samples from a large surface area and concentrate the collected molecules into a SERS sensor on the paper. We will summarize the progress in the fabrication and use of these paper-based optofluidic devices, and will describe their use in practical applications for point-of-sample detection.

Raman spectroscopy, which is based on the inelastic scattering of photons by chemical entities, has been successfully utilized for the investigation of adsorbed molecules on surfaces, although the low cross section limits its applications. Surface-enhanced Raman scattering (SERS) has drawn a lot of attention since its discovery in 1974, primarily because it can greatly enhance the normally weak Raman signal and thereby facilitate the convenient identification of the vibrational signatures of molecules in chemical and biological systems. Recently, the observation of single-molecule Raman scattering has further enhanced the Raman detection sensitivity limit and widened the scope of SERS for sensor applications. Although SERS effects can be achieved simply by exploiting the electromagnetic resonance properties of roughened surfaces or nanoparticles of Au or Ag, the fabrication of reliable SERS substrates with uniformly high enhancement factors remains the focus of much research. Spraying Au or Ag colloids on a substrate leads to an extremely high SERS signal at some local ‘hot-junctions’; however, it is not easy to achieve a reliable, stable, and uniform SERS signal spanning a wide dynamical range using this method. Van Duyne and coworkers have used nanosphere lithography, while Liu and Lee exploited soft lithography, in order to fabricate Ag nanoparticle arrays with high SERS activity and improved uniformity. Kall and co-workers have shown theoretically that the effective Raman cross section of a molecule placed between two metal nanoparticles can be enhanced by more than 12 orders of magnitude. Such enhancement is likely to be related to the ‘hot-junctions’ observed in some SERS experiments. Several theoretical groups have also investigated field enhancement for SERS from metal nanoparticle arrays. Specifically, Garcia–Vidal and Pendry proposed that very localized plasmon modes, created by strong electromagnetic coupling between two adjacent metallic objects, dominate the SERS response in an array of nanostructures. The interparticle-coupling-induced enhancement was attributed to the broadening of the plasmon resonance peak because the probability of the resonance covering both the excitation wavelength and the Raman peak increases with its width. They calculated the average enhancement factor over the surfaces of an array of infinitely long Ag nanorods with semicircular cross sections, and showed that significant near-field interaction occurs between adjacent nanorods when the gap between the nanorods reaches half the value of their diameter. Other groups have studied the dependence of the enhancement factor on the gap between adjacent nanoparticles on a SERS active substrate. For example, Gunnarsson et al. investigated SERS on ordered Ag nanoparticle arrays with an interparticle gap above 75 nm. Lee and co-workers were able to achieve the temperature-controlled variation of interparticle gaps between Ag nanoparticles embedded in a polymer membrane. Wei et al. performed SERS on self-organized Au nanoparticle arrays with narrow interparticle gaps, although they have not carried out a detailed investigation of the dependence of the SERS signal on the interparticle gap. Sauer et al. investigated SERS from nanowire arrays embedded in an alumina matrix with interparticle gaps of ∼ 110 nm, but no gap-related enhancement was observed in their experiment. These theoretical and experimental studies indicate that the precise control of gaps between nanostructures on a SERS-active substrate in the sub-10 nm regime, which is extremely difficult to obtain by existing nanofabrication methods, is likely to be critical for the fabrication of substrates with uniformly high enhancement factors, and for understanding collective surface plasmons existing inside the gaps. C O M M U N IC A IO N S

Several bacteria evolve in spore if the environmental conditions get to adverse e.g. for nutrient deprivation. The bacteria of genus Bacillus are Gram-positive aerobic bacteria and they are able to produce endospores (physiological inactive and resistant form), usually dispersed as aerosols. Endospores can survive for long time, until the conditions get back favourable. The genera Bacillus include pathogens, as Bacillus anthracis, used in the past as biological weapon. Prompt, accurate and sensitive detection is crucial for its control as pathogens or bioterrorism attacks. In case of the contamination with spores of B. anthracis, the time is essential to assure success in rescue. So, in this context, the early and fast analytical techniques, that need no or negligible sample preparation, is strongly required. Raman spectroscopy, and in particular Surface Enhanced Raman Spectroscopy (SERS), that can amplify nonlinearly the inherently weak Raman signal by several orders of magnitude, have become recognized and versatile analytical techniques also in microorganisms detection. These techniques can be used as sensitive tools for the detection and classification of biological threats, they can provide the chemical fingerprint of samples without complex and time-consuming pre-treatment samples preparation. Furthermore the development of in-field portable compact Raman platforms allows for using SERS for routine analysis. In the framework of the RAMBO (Rapid Air particle Monitoring against BiOlogical threats) project the feasibility of the SERS technique for the rapid identification and classification of few units of Bacillus spp (B. atrophaeus and B. thuringiensis) spores was investigated. B. atrophaeus and B. thuringiensis are harmless but genetically similar to the deadly B. anthracis. The RAMBO project purpose is the development of an advanced sensor with high performances, capable of detecting few spores or bacilli, with high selectivity and reliability, by means of two sensing techniques: SERS for early warning of bioagents dispersed in air or in water, and Polymerase Chain Reaction (PCR) technique for final recognition and validation. SERS and PCR will work in a microfluidic chip. In order to bind selectively the endospores, specific peptide receptors for B. thuringiensis have been selected to functionalize SERS substrates. To characterize the substrates, with and without spores to assess the effective immobilization of target, microscopy inspections, by optical microscope and Scanning Electron Microscope (SEM), were also carried out. The results show up the poor selectively of these peptides for B. thuringiensis, used as target, compared with the non specific Bacillus control. The performance of the system seems to be quite similar for both of them: the data processing by Principal Component Analysis and the following clustering analysis suggest the presence of indistinct answers for any bound endospore on the surface, and this is confirmed by microscope inspection. It could decrease the discrimination power of the sensor. Despite of such poor receptor selectivity, the SERS spectra of B. thuringiensis endospores show characteristic signals that can be related to DNA fragments or, much more probably, to the peptidoglycan (component of the external coat). This spectral feature could be used to detect the presence of B. thuringiensis endospores.