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.

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.

Comparison of Gap-Enhanced Raman Tags and Nanoparticle Aggregates with Polarization Dependent Super-Resolution Spectral SERS Imaging.

Strongly confined electric fields resulting from nanogaps within nanoparticle aggregates give rise to significant enhancement of surface-enhanced Raman scattering (SERS). Nanometer differences in gap sizes lead to drastically different confined field strengths; so much attention has been focused on the development and understanding of nanostructures with controlled gap sizes. In this work, we report a novel petal gap-enhanced Raman tag (GERT) consisting of a bipyramid core and a nitrothiophenol (NTP) spacer to support the growth of hundreds of small petals and compare its SERS emission and localization to a traditional bipyramid aggregate. To do this, we use super resolution spectral SERS imaging that simultaneously captures the SERS images and spectra while varying the incident laser polarization. Intensity fluctuations inherent of SERS enabled super resolution algorithms to be applied, which revealed subdiffraction limited differences in the localization with respect to polarization direction for both particles. Interestingly, however, only the traditional bipyramid aggregates experienced a strong polarization dependence in their SERS intensity and in the plasmon-induced conversion of NTP to dimercaptoazobenzene (DMAB), which was localized with nanometer precision to regions of intense electromagnetic fields. The lack of polarization dependence (validated through electromagnetic simulations) and surface reactions from the bipyramid-GERTs suggests that the emissions arising from the bipyramid-GERTs are less influenced by confined fields.

Digital surface enhanced Raman spectroscopy for quantifiable single molecule detection in flow.

Surface enhanced Raman scattering (SERS) provides a label free method of analyzing molecules from diverse and complex signals, potentially with single molecule sensitivity. The chemical specificity inherent in the SERS spectrum can identify molecules; however signal variability arising from the diversity of plasmonic environments can limit quantification, particularly at low concentrations. Here we show that digitizing, or counting SERS events, can decrease the limit of detection in flowing solutions enabling quantification of single molecules. By using multivariate curve resolution and establishing a score threshold, each individual spectrum can be classified as containing an event or not. This binary "yes/no" can then be quantified, and a linear region can be established. This method was shown to lower the limit of detection to the lowest physical limit, and lowered the limit of detection by an order of magnitude from the traditional, intensity based LOD calculations.

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.

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.

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.

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.

Super-resolution Surface-Enhanced Raman Scattering Imaging of Single Particles in Cells.

The ability to locate and identify molecular interactions in cells has significant importance for understanding protein function and molecular biology. Functionalized metallic nanoparticles have been used as probes for protein tracking and drug delivery because of their ability to carry therapeutic agents and readily functionalized surfaces. In this work, we present a super-resolution surface-enhanced Raman scattering (SERS) approach for imaging and tracking membrane receptors interacting with peptide-functionalized gold nanostars (AuNS). The αvß3 integrin receptors in colon cancer cells are successfully targeted and imaged using AuNS with the high-affinity amino acid sequence arginine-glycine-aspartic acid-phenylalanine-cysteine (RGDFC) attached. The RGDFC peptide interaction with the integrin receptor provides a bright and fluctuating SERS signal that can be analyzed with localization microscopy algorithms. Additionally, the observed SERS spectrum is used to confirm protein-peptide interaction. Experiments with functionalized and bare AuNS illustrate specific and nonspecific binding events. Specific binding is monitored with a localization precision of ∼6 nm. The observed spatial resolution is associated with tight binding, which was confirmed by the slower diffusion coefficient measured from 4.4 × 10-11 cm2/s for the AuNS-RGDFC compared to 7.8 × 10-10 cm2/s for the bare AuNS. Super-resolution SERS images at different focal planes show evidence of internalized particles and suggest insights into protein orientation on the surface of cells. Our work demonstrates super-resolution SERS imaging to probe membrane receptor interactions in cells, providing chemical information and spatial resolution with potential for diverse applications in life science and biomedicine.

Untargeted Tumor Metabolomics with Liquid Chromatography-Surface-Enhanced Raman Spectroscopy.

Metabolomics is a powerful systems biology approach that monitors changes in biomolecule concentrations to diagnose and monitor health and disease. However, leading metabolomics technologies, such as NMR and mass spectrometry (MS), access only a small portion of the metabolome. Now an approach is presented that uses the high sensitivity and chemical specificity of surface-enhanced Raman scattering (SERS) for online detection of metabolites from tumor lysates following liquid chromatography (LC). The results demonstrate that this LC-SERS approach has metabolite detection capabilities comparable to the state-of-art LC-MS but suggest a selectivity for the detection of a different subset of metabolites. Analysis of replicate LC-SERS experiments exhibit reproducible metabolite patterns that can be converted into barcodes, which can differentiate different tumor models. Our work demonstrates the potential of LC-SERS technology for metabolomics-based diagnosis and treatment of cancer.

The impact of optically rectified fields on plasmonic electrocatalysis.

Studies have shown that the excitation of plasmon resonances on nanostructured materials can drive catalytic processes. Plasmon resonances can be tuned across the solar spectrum, thus offering intriguing possibilities for their application in plasmonic catalysis. Previous work carried out by our group has indicated that nanostructures with tight junctions can create direct current (DC) electric fields. These fields arise from an optical rectification of the plasmon resonance on the plasmonic surface, and our group has shown that these fields modulate photocatalytic activity. This work looks to shed further light on the impact that optically rectified fields can have on catalytic reactions. Cyclic voltammetry shows that the electrochemical reduction and oxidation potentials of a 2 mM CuSO4 solution occur at ∼100 mV lower overpotential on an optically excited Ag nanodendrite electrode. Stark spectroscopy of the nitriles absorbed to these surfaces indicate photo-associated changes in the surface potential across the Ag nanodendrites. Localized areas evince photo-induced changes in the surface potential upwards of 300 mV. These results provide evidence of optically rectified fields altering the electrochemical reactivity on plasmonic surfaces and suggest that optimizing this nonlinear phenomenon may improve plasmonic photocatalysts.

Protein corona-resistant SERS tags for live cell detection of integrin receptors.

Chemical signals are conveyed to cells through ligand-receptor binding, triggering cascades of biochemical reactions and resulting in pivotal cellular functions. These binding events are important in understanding membrane signaling and drug interactions. To probe ligand-receptor binding, surface enhanced Raman scattering (SERS) tags are a promising tool. SERS tags are plasmonic nanostructures functionalized with a protective coating, a Raman reporter molecule, and a biorecognition element. In biological fluids, native proteins have affinity for bare nanoparticles and form a protein corona. SERS tags have a protective shell which eliminates this complication. It is important to analyze ligand-receptor binding with SERS tags in live cells since cell fixatives alter protein structure, leading to spectral changes and data misinterpretation. In this study, we synthesized a novel SERS tag by creating a mixed monolayer of the small cyclic arginine-glycine-aspartic acid-phenylalanine-cysteine (RGDFC) peptide and 4-mercaptobenzonitrile (MBN) on the surface of spherical gold nanoparticles (Au NP). Au-RGDFC-MBN NP showed resistance to PC formation and were successfully detected in both fixed and living human metastatic colon cancer cells.

Online Liquid Chromatography-Sheath-Flow Surface Enhanced Raman Detection of Phosphorylated Carbohydrates.

Online detection and quantification of three phosphorylated carbohydrate molecules: glucose 1-phosphate, glucose 6-phosphate, and fructose 6-phosphate was achieved by coupling sheath-flow surface enhanced Raman spectroscopy (SERS) to liquid chromatography. The presence of an alkanethiol (hexanethiol) self-assembled monolayer adsorbed to a silver SERS-active substrate helps retain and concentrate the analytes of interest at the SERS substrate to improve the detection sensitivity significantly. Mixtures of 2 µM of phosphorylated carbohydrates in pure water as well as in cell culture media were successfully separated by HPLC, with identification using the sheath-flow SERS detector. The quantification of each analyte was achieved using partial least-squares (PLS) regression analysis and acetonitrile in the mobile phases as an internal standard. These results illustrate the utility of sheath-flow SERS for molecular specific detection in complex biological samples appropriate for metabolomics and other applications.

Elucidating Protein/Ligand Recognition with Combined Surface Plasmon Resonance and Surface Enhanced Raman Spectroscopy.

The ability to distinguish between specific and nonspecific binding is important for assessing the interactions between protein receptors and ligands. Surface plasmon resonance (SPR) spectroscopy is an advanced tool to measure binding events, yet the ability to distinguish between specific and nonspecific binding remains a limitation. To address this problem, we use SPR spectroscopy correlated with surface enhanced Raman scattering (SERS). The chemical information present in SERS spectra provides insight into the molecular interactions between functionalized nanoparticles and proteins, which are not detectable by SPR alone. Using a custom instrument with the Kretschmann configuration, we successfully demonstrate simultaneous affinity and the chemical characterization of streptavidin-functionalized gold nanoparticles (STV-NPs) binding to biotin immobilized on a gold film in both air and flowing phosphate buffered saline (PBS). The SPR performance is consistent with that of previous reports. The association constant (KA) for streptavidin/biotin and STV-NPs/biotin interactions observed (2 ± 1 × 107 M-1 and 2.4 ± 0.3 × 1010 M-1, respectively) agree with literature values and show a strong avidity effect associated with the STV-NPs. The SERS scattering from STV-NPs is excited by the surface plasmon polariton and collected from an objective lens mounted over the fluidic channel. The SERS spectra are recorded simultaneously with the SPR sensorgram, and the detected Raman bands provide chemical insight into the binding event. Multivariate curve resolution analysis of the spectra can differentiate specific from nonspecific binding. This label-free, real time, and surface sensitive detection method provides chemical information to protein/ligand binding affinity measurements.

Probing Membrane Receptor-Ligand Specificity with Surface- and Tip- Enhanced Raman Scattering.

The specific interaction between a ligand and a protein is a key component in minimizing off-target effects in drug discovery. Investigating these interactions with membrane protein receptors can be quite challenging. In this report, we show how spectral variance observed in surface-enhanced Raman scattering (SERS) and tip-enhanced Raman scattering (TERS) can be correlated with ligand specificity in affinity-based assays. Variations in the enhanced Raman spectra of three peptide ligands (i.e., cyclic-RGDFC, cyclic-isoDGRFC, and CisoDGRC), which have different binding affinity to αvß3 integrin, are reported from isolated proteins and from receptors in intact cancer cell membranes. The SERS signal from the purified proteins provides basis spectra to analyze the signals in cells. Differences in the spectral variance within the SERS and TERS data for each ligand indicate larger variance for nonspecific ligand-receptor interactions. The SERS and TERS results are correlated with single particle tracking experiments of the ligand-functionalized nanoparticles with purified receptors on glass surfaces and living cells. These results demonstrate the ability to elucidate protein-ligand recognition using the observed vibrational spectra and provide perspective on binding specificity for small-molecule ligands in intact cell membranes, demonstrating a new approach for investigating drug specificity.

Detection of electron tunneling across plasmonic nanoparticle-film junctions using nitrile vibrations.

The significant electric field enhancements that occur in plasmonic nanogap junctions are instrumental in boosting the performance of spectroscopy, optoelectronics and catalysis. Electron tunneling, associated with quantum effects in small junctions, is reported to limit the electric field enhancement. However, observing and quantitatively determining how tunneling alters the electric fields within small gaps is challenging due to the nanoscale dimensions and heterogeneity present experimentally. Here, we report the use of a nitrile probe placed in the nanoparticle-film gap junctions to demonstrate that the change in the nitrile stretching band associated with the vibrational Stark effect can be directly correlated with the local electric field environment modulated by gap size variations. The emergence of Stark shifts correlates with plasmon resonance shifts associated with electron tunneling across the gap junction. Time dependent changes in the nitrile band with extended illumination further support a build up of charge associated with optical rectification in the coupled plasmon system. Computational models agree with our experimental observations that the frequency shifts arise from a vibrational Stark effect. Large local electric fields associated with the smallest gap junctions give rise to significant Stark shifts. These results indicate that nitrile Stark probes can measure the local field strengths in plasmonic junctions and monitor the subtle changes in the local electric fields resulting from electron tunneling.

Bioanalytical applications of surface-enhanced Raman spectroscopy: de novo molecular identification.

Surface enhanced Raman scattering (SERS) has become a powerful technique for trace analysis of biomolecules. The use of SERS-tags has evolved into clinical diagnostics, the enhancement of the intrinsic signal of biomolecules on SERS active materials shows tremendous promise for the analysis of biomolecules and potential biomedical assays. The detection of the de novo signal from a wide range of biomolecules has been reported to date. In this review, we examine different classes of biomolecules for the signals observed and experimental details that enable their detection. In particular, we survey nucleic acids, amino acids, peptides, proteins, metabolites, and pathogens. The signals observed show that the interaction of the biomolecule with the enhancing nanostructure has a significant influence on the observed spectrum. Additional experiments demonstrate that internal standards can correct for intensity fluctuations and provide quantitative analysis. Experimental methods that control the interaction at the surface are providing for reproducible SERS signals. Results suggest that combining advances in methodology with the development of libraries for SERS spectra may enable the characterization of biomolecules complementary to other existing methods.

Sensing Glucose in Urine and Serum and Hydrogen Peroxide in Living Cells by Use of a Novel Boronate Nanoprobe Based on Surface-Enhanced Raman Spectroscopy.

Hydrogen peroxide (H2O2) is known as a key molecule in a variety of biological processes, as well as a crucial byproduct in many enzymatic reactions. Therefore, being able to selectively and sensitively detect H2O2 is not only important in monitoring, estimating, and decoding H2O2 relevant physiological pathways but also very helpful in developing enzymatic-based biosensors for other analytes of interest. Herein, we report a plasmonic probe for H2O2 based on 3-mercaptophenylboronic acid (3-MPBA) modified gold nanoparticles (AuNPs) which is coupled with surface-enhanced Raman scattering (SERS) to yield a limit of detection (LOD) of 70 nM. Our probe quantifies both exogenous and endogenous H2O2 levels in living cells and can further be coupled with glucose oxidase (GOx) to achieve quantitative and selective detection of glucose in artificial urine and human serum.

Selective Detection of RGD-Integrin Binding in Cancer Cells Using Tip Enhanced Raman Scattering Microscopy.

Ligand-receptor interactions play important roles in many biological processes. Cyclic arginine-glycine-aspartic acid (RGD) containing peptides are known to mimic the binding domain of extracellular matrix protein fibronectin and selectively bind to a subset of integrin receptors. Here we report the tip enhanced Raman scattering (TERS) detection of RGD-functionalized nanoparticles bound to integrins produces a Raman scattering signal specific to the bound protein. These results demonstrate that this method can detect and differentiate between two different integrins (α5ß1 and αvß3) bound to RGD-conjugated gold nanoparticles both on surfaces and in a cancer cell membrane. In situ measurements of RGD nanoparticles bound to purified α5ß1 and αvß3 receptors attached to a glass surface provide reference spectra for a multivariate regression model. The TERS spectra observed from nanoparticles bound to cell membranes are analyzed using this regression model and the identity of the receptor can be determined. The ability to distinguish between receptors in the cell membrane provides a new tool to chemically characterize ligand-receptor recognition at molecular level and provide chemical perspective on the molecular recognition of membrane receptors.