Identification and quantification of pigments in plant leaves using thin layer chromatography-Raman spectroscopy (TLC-Raman).

Carotenoids are yellow, orange, and red pigments commonly found in plants. In leaves, these molecules are essential for photosynthesis, but they also play a major role in plant growth and development. Efficiently monitoring concentrations of specific carotenoids in plant tissues could help to explain plant responses to environmental stressors, infection and disease, fertilization, and other conditions. Previously, Raman methods have been used to demonstrate a correlation between plant fitness and the carotenoid content of leaves. Due to solvatochromatic effects and structural similarities within the carotenoid family, current Raman spectroscopy techniques struggle to assign signals to specific carotenoids with certainty, complicating the determination of amounts of individual carotenoids present in a sample. In this work, we use thin layer chromatography-Raman spectroscopy, or TLC-Raman, to identify and quantify carotenoids extracted from tomato leaves. These quick and accurate methods could be applied to study the relationship between pigment content and a number of factors affecting plant health.

Classical vs. quantum plasmon-induced molecular transformations at metallic nanojunctions.

Chemical transformations near plasmonic metals have attracted increasing attention in the past few years. Specifically, reactions occurring within plasmonic nanojunctions that can be detected via surface and tip-enhanced Raman (SER and TER) scattering were the focus of numerous reports. In this context, even though the transition between localized and nonlocal (quantum) plasmons at nanojunctions is documented, its implications on plasmonic chemistry remain poorly understood. We explore the latter through AFM-TER-current measurements. We use two molecules: i) 4-mercaptobenzonitrile (MBN) that reports on the (non)local fields and ii) 4-nitrothiophenol (NTP) that features defined signatures of its neutral/anionic forms and dimer product, 4,4'-dimercaptoazobenzene (DMAB). The transition from classical to quantum plasmons is established through our optical measurements: It is marked by molecular charging and optical rectification. Simultaneously recorded force and current measurements support our assignments. In the case of NTP, we observe the parent and DMAB product beneath the probe in the classical regime. Further reducing the gap leads to the collapse of DMAB to form NTP anions. The process is reversible: Anions subsequently recombine into DMAB. Our results have significant implications for AFM-based TER measurements and their analysis, beyond the scope of this work. In effect, when precise control over the junction is not possible (e.g., in SER and ambient TER), both classical and quantum plasmons need to be considered in the analysis of plasmonic reactions.

Raman and Surface-Enhanced Raman Scattering Detection in Flowing Solutions for Complex Mixture Analysis.

Raman scattering provides a chemical-specific and label-free method for identifying and quantifying molecules in flowing solutions. This review provides a comprehensive examination of the application of Raman spectroscopy and surface-enhanced Raman scattering (SERS) to flowing liquid samples. We summarize developments in online and at-line detection using Raman and SERS analysis, including the design of microfluidic devices, the development of unique SERS substrates, novel sampling interfaces, and coupling these approaches to fluid-based chemical separations (e.g., chromatography and electrophoresis). The article highlights the challenges and limitations associated with these techniques and provides examples of their applications in a variety of fields, including chemistry, biology, and environmental science. Overall, this review demonstrates the utility of Raman and SERS for analysis of complex mixtures and highlights the potential for further development and optimization of these techniques. Expected final online publication date for the Annual Review of Analytical Chemistry, Volume 17 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

A 3D printed sheath flow interface for surface enhanced Raman spectroscopy (SERS) detection in flow.

Surface enhanced Raman spectroscopy (SERS) is an effective technique for detecting molecules in aqueous solutions due to its insensitivity to water, which makes it especially useful for biological samples. Utilizing SERS in flow can aid in a variety of applications such as metabolomics, pharmaceuticals, and diagnostics. The ability to 3D print complex objects enables rapid dissemination of prototypes. A 3D printed flow cell for sheath flow SERS detection has been developed that can incorporate a variety of planar substrates. The 3D printed flow cell incorporates hydrodynamic focusing, a sheath flow, that confines the analyte near the SERS substrate. Since the SERS signal obtained relies on the interaction between analyte molecules and nanostructures, sheath flow increases the detection efficiency and eliminates many issues associated with SERS detection in solution. This device was optimized by analyzing both molecules and particles with and without using sheath flow for SERS detection. Our results show that the flow rates can be optimized to increase the SERS signal obtained from a variety of analytes, and that the signal was increased when using sheath flow. This 3D printed flow cell offers a straightforward method to disseminate this technology and to facilitate online SERS detection.

Early Diagnosis of Fibromyalgia Using Surface-Enhanced Raman Spectroscopy Combined with Chemometrics.

Fibromyalgia (FM) is a chronic muscle pain disorder that shares several clinical features with other related rheumatologic disorders. This study investigates the feasibility of using surface-enhanced Raman spectroscopy (SERS) with gold nanoparticles (AuNPs) as a fingerprinting approach to diagnose FM and other rheumatic diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), osteoarthritis (OA), and chronic low back pain (CLBP). Blood samples were obtained on protein saver cards from FM (n = 83), non-FM (n = 54), and healthy (NC, n = 9) subjects. A semi-permeable membrane filtration method was used to obtain low-molecular-weight fraction (LMF) serum of the blood samples. SERS measurement conditions were standardized to enhance the LMF signal. An OPLS-DA algorithm created using the spectral region 750 to 1720 cm-1 enabled the classification of the spectra into their corresponding FM and non-FM classes (Rcv > 0.99) with 100% accuracy, sensitivity, and specificity. The OPLS-DA regression plot indicated that spectral regions associated with amino acids were responsible for discrimination patterns and can be potentially used as spectral biomarkers to differentiate FM and other rheumatic diseases. This exploratory work suggests that the AuNP SERS method in combination with OPLS-DA analysis has great potential for the label-free diagnosis of FM.

Accurate Quantification and Imaging of Cellular Uptake Using Single-Particle Surface-Enhanced Raman Scattering.

Understanding the uptake, distribution, and stability of gold nanoparticles (NPs) in cells is of fundamental importance in nanoparticle sensors and therapeutic development. Single nanoparticle imaging with surface-enhanced Raman spectroscopy (SERS) measurements in cells is complicated by aggregation-dependent SERS signals, particle inhomogeneity, and limited single-particle brightness. In this work, we assess the single-particle SERS signals of various gold nanoparticle shapes and the role of silica encapsulation on SERS signals to develop a quantitative probe for single-particle level Raman imaging in living cells. We observe that silica-encapsulated gap-enhanced Raman tags (GERTs) provide an optimized probe that can be quantifiable per voxel in SERS maps of cells. This approach is validated by single-particle inductively coupled mass spectrometry (spICP-MS) measurements of NPs in cell lysate post-imaging. spICP-MS also provides a means of measuring the tag stability. This analytical approach can be used not only to quantitatively assess nanoparticle uptake on the cellular level (as in previous digital SERS methods) but also to reliably image the subcellular distribution and to assess the stability of NPs in cells.

Bleach Cleaning of Commercially Available Gold Nanopillar Arrays for Surface-Enhanced Raman Spectroscopy (SERS).

Surface-enhanced Raman spectroscopy (SERS) is a highly sensitive technique that can assist in trace analysis for biomedical, diagnostic, and environmental applications. However, a major limitation of SERS is surface contamination of the substrates used, which can complicate the spectral reproducibility, limits of detection, and detection of unknown analytes. This is especially prevalent with commercially available substrates as shipping under a controlled and clean environment is difficult. Here we report a method using dilute bleach solutions to remove surface contamination from commercially available substrates consisting of gold-coated nanopillar arrays that maintains functionality. The results show that this method can be used to remove background signals associated with typical surface contamination in commercially available substrates as well as remove thiolated self-assembled monolayers (SAMs). Results indicate the bleach oxidizes the surface contaminants, which can then be easily washed away. Although the metallic surface also becomes oxidized in this process, the surface can be reduced without loss of SERS activity. The SERS intensity of SAMs improved following bleach treatment across all concentrations studied.

From the lab to the field: handheld surface enhanced Raman spectroscopy (SERS) detection of viral proteins.

Translating sensors from the lab benchtop to a readily available point-of-need setting is desirable for many fields, including medicine, agriculture, and industry. However, this transition generally suffers from loss of sensitivity, high background signals, and other issues which can impair reproducibility. Here we adapt a label-free surface-enhanced Raman spectroscopy (SERS) sensor for SARS-CoV-2 antigens from a lab-based assay to a handheld device. Utilizing a peptide capture molecule, which we previously employed for a surface-based assay, we optimize a simpler and more cost-efficient nanoparticle-based assay. This new assay allows for the direct detection of these viral antigens by SERS, now with the advantages of robustness and portability. We highlight considerations for nanoparticle modification conditions and warn against methods which can interfere with accurate detection. The comparison of these two assays will help guide further development of SERS-based sensors into devices that can be easily used in point-of-care settings, such as by emergency room nurses, farmers, or quality control technicians.

Cancer Cell Targeting, Magnetic Sorting, and SERS Detection through Cell Surface Receptors.

Integrins are cellular surface receptors responsible for the activation of many cellular pathways in cancer. These integrin proteins can be specifically targeted by small peptide sequences that offer the potential for the differentiation of cellular subpopulations by using magnetically assisted cellular sorting techniques. By adding a gold shell to the magnetic nanoparticles, these integrin-peptide interactions can be differentiated by surface-enhanced Raman spectroscopy (SERS), providing a quick and reliable method for on-target binding. In this paper, we demonstrate the ability to differentiate the peptide-protein interactions of the small peptides CDPGYIGSR and cyclic RGDfC functionalized on gold-coated magnetic nanoparticles with the integrins they are known to bind to using their SERS signal. SW480 and SW620 colorectal cancer cells known to have the integrins of interest were then magnetically sorted using these functionalized nanoparticles, suggesting differentiation between the sorted populations and integrin populations among the two cell lines.

Multiscale analysis of Benjamin Franklin's innovations in American paper money.

Benjamin Franklin was a preeminent proponent of the new colonial and Continental paper monetary system in 18th-century America. He established a network of printers, designing and printing money notes at the same time. Franklin recognized the necessity of paper money in breaking American dependence on the British trading system, and he helped print Continental money to finance the American War of Independence. We use a unique combination of nondistractive, microdestructive, and advanced atomic-level imaging methods, including Raman, Infrared, electron energy loss spectroscopy, X-ray diffraction, X-ray fluorescence, and aberration-corrected scanning transmission electron microscopy, to analyze pre-Federal American paper money from the Rare Books and Special Collections of the Hesburgh Library at the University of Notre Dame. We investigate and compare the chemical compositions of the paper fibers, the inks, and fillers made of special crystals in the bills printed by Franklin's printing network, other colonial printers, and counterfeit money. Our results reveal previously unknown ways that Franklin developed to safeguard printed money notes against counterfeiting. Franklin used natural graphite pigments to print money and developed durable "money paper" with colored fibers and translucent muscovite fillers, along with his own unique designs of "nature-printed" patterns and paper watermarks. These features and inventions made pre-Federal American paper currency an archetype for developing paper money for centuries to come. Our multiscale analysis also provides essential information for the preservation of historical paper money.

Manifold Learning Enables Interpretable Analysis of Raman Spectra from Extracellular Vesicle and Other Mixtures.

Extracellular vesicles (EVs) have emerged as promising diagnostic and therapeutic candidates in many biomedical applications. However, EV research continues to rely heavily on in vitro cell cultures for EV production, where the exogenous EVs present in fetal bovine (FBS) or other required serum supplementation can be difficult to remove entirely. Despite this and other potential applications involving EV mixtures, there are currently no rapid, robust, inexpensive, and label-free methods for determining the relative concentrations of different EV subpopulations within a sample. In this study, we demonstrate that surface-enhanced Raman spectroscopy (SERS) can biochemically fingerprint fetal bovine serum-derived and bioreactor-produced EVs, and after applying a novel manifold learning technique to the acquired spectra, enables the quantitative detection of the relative amounts of different EV populations within an unknown sample. We first developed this method using known ratios of Rhodamine B to Rhodamine 6G, then using known ratios of FBS EVs to breast cancer EVs from a bioreactor culture. In addition to quantifying EV mixtures, the proposed deep learning architecture provides some knowledge discovery capabilities which we demonstrate by applying it to dynamic Raman spectra of a chemical milling process. This label-free characterization and analytical approach should translate well to other EV SERS applications, such as monitoring the integrity of semipermeable membranes within EV bioreactors, ensuring the quality or potency of diagnostic or therapeutic EVs, determining relative amounts of EVs produced in complex co-culture systems, as well as many Raman spectroscopy applications.

Chemical conjugation to differentiate monosaccharides by Raman and surface enhanced Raman spectroscopy.

Sugars play important roles in numerous biological processes, from providing energy to modifying proteins to alter their function. Glycosylation, the attachment of a sugar residue to a protein, is the most common post translational modification. Identifying the glycans on a protein is a useful tool both for pharmaceutical development as well as probing the proteome and glycome further. Sugars, however, are difficult analytes to probe due to their isomeric nature. In this work, Raman spectroscopy and surface enhanced Raman spectroscopy (SERS) are used to identify different monosaccharide species based on the vibrational modes of these isomeric analytes. The weak scattering of the sugars was overcome through conjugation with phenylboronic acid to provide a larger Raman scattering cross section and induce slight changes in the observed spectra associated with the structure of the monosaccharides. Spontaneous Raman, SERS in flow, and static SERS detection were performed in order to discriminate between arabinose, fructose, galactose, glucose, mannose, and ribose, as well as provide a method for identification and quantification for these sugar conjugates.

Multipolar Raman Scattering vs Interfacial Nanochemistry: Case of 4-Mercaptopyridine on Gold.

Caution needs to be exercised in associating changes in plasmon-enhanced Raman spectra with chemical transformations. This is demonstrated through a detailed analysis of tip-enhanced Raman (TER) scattering from 4-mercaptopyridine (MPY) on gold. The substrate used consists of gold nanoplates atop a gold surface featuring heterogeneous grooves, all coated with a monolayer of MPY. The brightest spectra across the substrate exhibit features that can only be recovered by considering the generalized polarizability of oriented MPY molecules. The complex TER spectra we observe do not mark interfacial chemistry but rather multipolar TER scattering driven by local field gradients.

A Wide-Field Imaging Approach for Simultaneous Super-Resolution Surface-Enhanced Raman Scattering Bioimaging and Spectroscopy.

High spatial resolution imaging and chemical-specific detection in living organisms is important in a wide range of fields from medicine to catalysis. In this work, we characterize a wide-field surface-enhanced Raman scattering (SERS) imaging approach capable of simultaneously capturing images and SERS spectra from nanoparticle SERS tags in cancer cells. By passing the image through a transmission diffraction grating before it reaches an array detector, we record the image and wavelength dispersed signal simultaneously on the camera sensor. Optimization of the experiment provides an approach with better spectral resolution and more rapid acquisition than liquid crystal tunable filters commonly used for wide-field SERS imaging. Intensity fluctuations inherent to SERS enabled localization algorithms to be applied to both the spatial and spectral domain, providing super-resolution SERS images that are correlated with improved peak positions identified in the spectrum of the SERS tag. The detected Raman signal is shown to be sensitive to the focal plane, providing three-dimensional (3D) sectioning abilities for the detected nanoparticles. Our work demonstrates spectrally resolved super-resolution SERS imaging that has the potential to be applied to complex physical and biological imaging applications.

Monitoring and Characterization of Milk Fouling on Stainless Steel Using a High-Pressure High-Temperature Quartz Crystal Microbalance with Dissipation.

Fouling at interfaces deteriorates the efficiency and hygiene of processes within numerous industrial sectors, including the oil and gas, biomedical device, and food industries. In the food industry, the fouling of a complex food matrix to a heated stainless steel surface reduces production efficiency by increasing heating resistance, pumping requirements, and the frequency of cleaning operations. In this work, quartz crystal microbalance with dissipation (QCM-D) was used to study the interface formed by the fouling of milk on a stainless steel surface at different flow rates and protein concentrations at high temperatures (135 °C). Subsequently, the QCM-D response was recorded during the cleaning of the foulant. Two phases of fouling were identified. During phase-1, the fouling rate was dependent on the flow rate, while the fouling rate during phase-2 was dependent on the flow rate and protein concentration. During cleaning, foulants deposited at the higher flow rate swelled more than those deposited at the lower flow rate. The composition of the fouling deposits consisted of both protein and mineral species. Two crystalline phases of calcium phosphate, ß-tricalcium phosphate and hydroxyapatite, were identified at both flow rates. Stratification in topography was observed across the surface of the QCM-D sensor with a brittle and cracked structure for deposits formed at 0.2 mL/min and a smooth and close-packed structure for deposits formed at 0.1 mL/min. These stratifications in the composition and topography were correlated to differences in the reaction time and flow dynamics at different flow rates. This high-temperature application of QCM-D to complex food systems illuminates the initial interaction between proteins and minerals and a stainless steel surface, which might otherwise be undetectable in low-temperature applications of QCM-D or at larger bench and industrial scales. The methods and results presented here have implications for optimizing processing scenarios that limit fouling formation while also enhancing removal during cleaning.

High-Throughput Surface-Enhanced Raman Scattering for Screening Chemical Sensor Candidates Enabled by Bipolar Electrochemistry.

A variety of hydrothermal or electrochemical methods have been explored to prepare noble metal nanostructures as surface-enhanced Raman scattering (SERS) substrates. However, most of those metallic nanoarrays are structurally homogeneous, which makes it laborious to select the high-performance substrates for particular Raman sensing purposes. Here, a high-throughput SERS imaging strategy is demonstrated for the first time for screening chemical sensors with sub-nanomolar sensitivities. Bipolar electrochemistry was applied to generate Au or Au-Ag gradient nanoarrays with diverse chemical compositions, morphologies, and particle dimensions ranging from several nanometers to micrometers. The selected "hot-spots" on the Au-Ag alloy nanoarray exhibited a 660-fold enhancement in SERS intensity compared to those on the pure Au gradient nanoarray. The SERS screening of 4-aminothiophenol, 4-nitrothiophenol, and 4-mercaptobenzoic acid was carried out that provided a limit of detection (LOD) between 1 and 5 pM. The distinctive LODs among three thiophenolic Raman probes are ascribed to the differences in the affinity of the probe to the alloy, orientation of the metal-ligand monolayer, or plasmonic environment of the nanoarray surface. As a continuous, rapid, and cost-effective manner to fabricate transitional nanostructures and screen out SERS responsive sites, this method not only facilitates controllable synthesis of noble metal nanoarrays but has the potential to provide an alternative tool for ultrasensitive chemical sensing on a wide range of bimetallic substrates.

Determination of lentiviral titer by surface enhanced Raman scattering.

Lentiviruses are commonly used to deliver genetic code into host cells for biomedical applications, such as gene therapy, pharmaceuticals, and vaccine development. Knowing the infectious titer of these virus particles is critical for development in these areas. Current methods of determining viral titer often require cell culture, where a cell is infected and the inserted genetic code is expressed in a known number of cells, which can require days or weeks to prepare and analyze samples. To provide a more rapid method of determining viral titer, the use of surface enhanced Raman spectroscopy (SERS) was explored. SERS provides both chemical and structural information by using plasmonic metallic nanostructures to amplify the Raman signal. Two different lentiviruses, one with a vector encoding a GFP gene and the same virus without the GFP gene included, were analyzed by SERS in viral production media at various concentrations. The SERS response was demonstrated to be sensitive to the incorporation of the GFP gene into the viral vector. Chemometric analysis using multivariate curve resolution (MCR) was able to identify a component in the observed SERS spectra that correlated with the concentration of GFP containing virus particles. Using the MCR model and the SERS response, the viral titer of lentivirus encoding for GFP was determined. The viral titer determined by SERS agreed well with expression of the GFP in infected cells. The SERS response using different metals and excitation wavelengths was also explored. Overall, this work demonstrates the utility of SERS for rapid determination of lentiviral titer.

In Vitro Imaging of Lycopene Delivery to Prostate Cancer Cells.

The ability to monitor the uptake and distribution of food nutrients in in vitro cell culture models is key to understanding the efficacy of these nutraceuticals to treat and prevent disease. Lycopene is a carotenoid found in chloroplasts and chromoplasts of tomatoes, providing the familiar red color, and a bioactive that inhibits prostate carcinogenesis. We employed live-cell Raman microscopy to visualize lycopene delivery from tween 80 micelles into PC-3 prostate cancer cells. The tween 80 micelle provides a mimic of natural lipoprotein complexes that deliver lycopene in vivo, overcomes the low aqueous solubility of lycopene and challenges replicating physiological uptake to cells, and provides a stable signal to assess cellular uptake of the nutraceutical formulation. The Raman images indicate subcellular localization of the lycopene within the cells. The lycopene Raman signal is resonantly enhanced at an excitation wavelength of 532 nm, providing a convenient, sensitive, and label-free technique to detect and quantify lycopene uptake in living cells. Analysis of the acquired Raman spectra in the maps determines the concentration of lycopene at each point in the cell. In addition to the expected lycopene Raman signal, Raman scattering from the tween 80 vehicle is also mapped in the cells. The Raman data correlates with scattering features observed in darkfield microscopy images of the cells, which display the cell membrane and other features for reference. Overall, the Raman maps indicate lycopene likely accumulates in lipid membranes of cytoplasmic organelles.

Investigation of SERS Frequency Fluctuations Relevant to Sensing and Catalysis.

The excitation of plasmon resonances on nanoparticles generates locally enhanced electric fields commonly used for sensing applications and energetic charge carriers can drive chemical transformations as photocatalysts. The surface-enhanced Raman scattering (SERS) spectra from mercaptobenzoic acid (MBA) adsorbed to gold nanoparticles (AuNP) and silica encapsulated gold nanoparticles (AuNP@silica) can be used to assess the impact of energetic charge carriers on the observed signal. Measurements were recorded using a traditional point focused Raman spectroscopy and a wide-field spectral imaging approach to assess changes in the spectra of the different particles at increasing power density. The wide-field approach provides an increase in sampling statistics and shows evidence of SERS frequency fluctuations from MBA at low power densities, where it is commonly difficult to record spectra from a point focused spot. The increased spectral resolution of the point spectroscopy measurement provides improved peak identification and the ability to correlate the frequency fluctuations to charged intermediate species. Interestingly, our work suggests that isolated nanoparticles may undergo frequency fluctuations more readily than aggregates.

Super-Resolution SERS Spectral Bioimaging.

Advances in nanotechnology enable the detection of trace molecules from the enhanced Raman signal generated at the surface of plasmonic nanoparticles. We have developed technology to enable super-resolution imaging of plasmonic nanoparticles, where the fluctuations in the surface enhanced Raman scattering (SERS) signal can be analyzed with localization microscopy techniques to provide nanometer spatial resolution of the emitting molecule's location. Additional work now enables the super-resolved SERS image and the corresponding spectrum to be acquired simultaneously. Here we will discuss how this approach can be applied to provide new insights into biological cells.