iSERS: from nanotag design to protein assays and ex vivo imaging.

Proteins are an eminently important class of ubiquitous biomacromolecules with diverse biological functions, and numerous techniques for their detection, quantification, and localisation have been developed. Many of these methods exploit the selectivity arising from molecular recognition of proteins/antigens by immunoglobulins. The combination of surface-enhanced Raman scattering (SERS) with such "immuno"-techniques to immuno-SERS (iSERS) is the central topic of this review, which is focused on colloidal SERS nanotags, i.e., molecularly functionalised noble metal nanoparticles conjugated to antibodies, for their use in protein assays and ex vivo imaging. After contrasting the fundamental differences between label-free SERS and iSERS, including a balanced description of the advantages and drawbacks of the latter, we describe the usual workflow of iSERS experiments. Milestones in the development of the iSERS technology are summarised from a historical perspective. By highlighting selected examples from the literature, we illustrate the conceptual progress that has been achieved in the fields of iSERS-based protein assays and ex vivo imaging. Finally, we attempt to predict what is necessary to fully exploit the transformative potential of the iSERS technology by stimulating the transition from research in academic labs into applications for the benefit of our society.

Contact Electrification as an Emerging Strategy for Controlling the Performance of Metal Nanoparticle Catalysts.

Contact electrification (CE) is an emerging strategy for controlling the performance of metal nanoparticle (NP) catalysts. The underlying physical principle of this control is the sensitivity of the Fermi level to metal-metal contacts. This change in electronic structure has a direct impact on surface properties and chemical reactivity. The concept article briefly introduces the basic theory of CE and its relationship to catalytic performance. We then highlight selected recent examples of advances in the preparation of hybrid metal NP assemblies, experimental techniques for characterizing CE, and finally applications of CE for altering catalytic performance.

Photoswitching of arylazopyrazoles upon S1 (nπ*) excitation studied by transient absorption spectroscopy and ab initio molecular dynamics.

Arylazopyrazoles (AAPs) are an important class of molecular photoswitches with high photostationary states (PSS) and long thermal lifetimes. The ultrafast photoisomerization of four water-soluble arylazopyrazoles, all of them featuring an ortho-dimethylated pyrazole ring, is studied by narrowband femtosecond transient absorption spectroscopy and ab initio molecular dynamics simulations. Upon S1 (nπ*) photoexcitation of the planar E-isomers (E-AAPs), excited-state bi-exponential decays with time constants τ1 in the 220-440 fs range and τ2 in the 1.4-1.8 ps range are observed, comparable to those reported for azobenzene (AB). This is indicative of the same photoisomerization mechanism as has been reported for ABs. In contrast to the planar E-AAPs, a twisted E-AAP with two methyl groups in ortho-position of the phenyl ring displays faster initial photoswitching with τ1 = 170 ± 10 fs and τ2 = 1.6 ± 0.1 ps. Our static DFT calculations and ab initio molecular dynamics simulations of E-AAPs on the S0 and S1 potential energy surfaces suggest that twisted E-isomer azo photoswitches exhibit faster initial photoisomerization dynamics out of the Franck-Condon region due to a weaker π-coordination of the central CNNC unit to the aromatic ligands.

Curvature dependent onset of quantum tunneling in subnanometer gaps.

The quantum tunneling in subnanometer gap sizes in gold dimers is studied in order to account for the dependency of the onset of quantum tunneling on the dimer's radius and accordingly the gap wall's curvature, realized in experiments. Several nanodimers both nanowires and nanospheres with various radii and gap sizes are modelled and simulated based on the quantum corrected model, determining the onset of the quantum tunneling. Results show that the onset of quantum tunneling is both dependent on the gap size as well as on the dimer's radius. As larger dimers result in larger effective conductivity volumes, the influence of the quantum tunneling begins in larger gap sizes in larger dimers.

Tuning the Electronic Properties of Platinum in Hybrid-Nanoparticle Assemblies for use in Hydrogen Evolution Reaction.

Platinum is the best electrocatalyst for the hydrogen evolution reaction (HER). Here, we demonstrate that by contact electrification of Pt nanoparticle satellites on a gold or silver core, the Fermi level of Pt can be tuned. The electronic properties of Pt in such hybrid nanocatalysts were experimentally characterized by X-ray photoelectron spectroscopy (XPS) and surface-enhanced Raman scattering (SERS) with the probe molecule 2,6-dimethyl phenyl isocyanide (2,6-DMPI). Our experimental findings are corroborated by a hybridization model and density functional theory (DFT) calculations. Finally, we demonstrate that tuning of the Fermi level of Pt results in reduced or increased overpotentials in water splitting.

In Situ Monitoring of Palladium-Catalyzed Chemical Reactions by Nanogap-Enhanced Raman Scattering using Single Pd Cube Dimers.

The central dilemma in label-free in situ surface-enhanced Raman scattering (SERS) for monitoring of heterogeneously catalyzed reactions is the need of plasmonically active nanostructures for signal enhancement. Here, we show that the assembly of catalytically active transition-metal nanoparticles into dimers boosts their intrinsically insufficient plasmonic activity at the monomer level by several orders of magnitude, thereby enabling the in situ SERS monitoring of various important heterogeneously catalyzed reactions at the single-dimer level. Specifically, we demonstrate that Pd nanocubes (NCs), which alone are not sufficiently plasmonically active as monomers, can act as a monometallic yet bifunctional platform with both catalytic and satisfactory plasmonic activity via controlled assembly into single dimers with an ∼1 nm gap. Computer simulations reveal that the highest enhancement factors (EFs) occur at the corners of the gap, which has important implications for the SERS-based detection of catalytic conversions: it is sufficient for molecules to come in contact with the "hot spot corners", and it is not required that they diffuse deeply into the gap. For the widely employed Pd-catalyzed Suzuki-Miyaura cross-coupling reaction, we demonstrate that such Pd NC dimers can be employed for in situ kinetic SERS monitoring, using a whole series of aryl halides as educts. Our generic approach based on the controlled assembly into dimers can easily be extended to other transition-metal nanostructures.

Rapid and Sensitive SERS-Based Lateral Flow Test for SARS-CoV2-Specific IgM/IgG Antibodies.

As an immune response to COVID-19 infection, patients develop SARS-CoV-2-specific IgM/IgG antibodies. Here, we compare the performance of a conventional lateral flow assay (LFA) with a surface-enhanced Raman scattering (SERS)-based LFA test for the detection of SARS-CoV-2-specific IgM/IgG in sera of COVID-19 patients. Sensitive detection of IgM might enable early serological diagnosis of acute infections. Rapid detection in serum using a custom-built SERS reader is at least an order of magnitude more sensitive than the conventional LFAs with naked-eye detection. For absolute quantification and the determination of the limit of detection (LOD), a set of reference measurements using purified (total) IgM in buffer was performed. In this purified system, the sensitivity of SERS detection is even 7 orders of magnitude higher: the LOD for SERS was ca. 100 fg/mL compared to ca. 1 µg/mL for the naked-eye detection. This outlines the high potential of SERS-based LFAs in point-of-care testing once the interference of serum components with the gold conjugates and the nitrocellulose membrane is minimized.

A fresh look at the structure of aromatic thiols on Au surfaces from theory and experiment.

A detailed study of the adsorption structure of self-assembled monolayers of 4-nitrothiophenol on the Au(111) surface was performed from a theoretical perspective via first-principles density functional theory calculations and experimentally by Raman and vibrational sum frequency spectroscopy (vSFS) with an emphasis on the molecular orientation. Simulations-including an explicit van der Waals (vdW) description-for different adsorbate structures, namely, for (3×3), (2 × 2), and (3 × 3) surface unit cells, reveal a significant tilting of the molecules toward the surface with decreasing coverage from 75° down to 32° tilt angle. vSFS suggests a tilt angle of 50°, which agrees well with the one calculated for a structure with a coverage of 0.25. Furthermore, calculated vibrational eigenvectors and spectra allowed us to identify characteristic in-plane (NO2 scissoring) and out-of-plane (C-H wagging) modes and to predict their strength in the spectrum in dependence of the adsorption geometry. We additionally performed calculations for biphenylthiol and terphenylthiol to assess the impact of multiple aromatic rings and found that vdW interactions are significantly increasing with this number, as evidenced by the absorption energy and the molecule adopting a more upright-standing geometry.

Spectral Screening of the Energy of Hot Holes over a Particle Plasmon Resonance.

Plasmonic hot carriers have been recently identified as key elements for photocatalysis at visible wavelengths. The possibility to transfer energy between metal plasmonic nanoparticles and nearby molecules depends not only on carrier generation and collection efficiencies but also on their energy at the metal-molecule interface. Here an energy screening study was performed by monitoring the aniline electro-polymerization reaction via an illuminated 80 nm gold nanoparticle. Our results show that plasmon excitation reduces the energy required to start the polymerization reaction as much as 0.24 eV. Three possible photocatalytic mechanisms were explored: the enhanced near field of the illuminated particle, the temperature increase at the metal-liquid interface, and the excited electron-hole pairs. This last phenomenon is found to be the one contributing most prominently to the observed energy reduction.

Towards Reliable and Quantitative Surface-Enhanced Raman Scattering (SERS): From Key Parameters to Good Analytical Practice.

Experimental results obtained in different laboratories world-wide by researchers using surface-enhanced Raman scattering (SERS) can differ significantly. We, an international team of scientists with long-standing expertise in SERS, address this issue from our perspective by presenting considerations on reliable and quantitative SERS. The central idea of this joint effort is to highlight key parameters and pitfalls that are often encountered in the literature. To that end, we provide here a series of recommendations on: a) the characterization of solid and colloidal SERS substrates by correlative electron and optical microscopy and spectroscopy, b) on the determination of the SERS enhancement factor (EF), including suitable Raman reporter/probe molecules, and finally on c) good analytical practice. We hope that both newcomers and specialists will benefit from these recommendations to increase the inter-laboratory comparability of experimental SERS results and further establish SERS as an analytical tool.

UV resonance Raman spectroscopy of the supramolecular ligand guanidiniocarbonyl indole (GCI) with 244 nm laser excitation.

Ultraviolet resonance Raman (UVRR) spectroscopy is a powerful vibrational spectroscopic technique for the label-free monitoring of molecular recognition of peptides or proteins with supramolecular ligands such as guanidiniocarbonyl pyrroles (GCPs). The use of UV laser excitation enables Raman binding studies of this class of supramolecular ligands at submillimolar concentrations in aqueous solution and provides a selective signal enhancement of the carboxylate binding site (CBS). A current limitation for the extension of this promising UVRR approach from peptides to proteins as binding partners for GCPs is the UV-excited autofluorescence from aromatic amino acids observed for laser excitation wavelengths >260 nm. These excitation wavelengths are in the electronic resonance with the GCP for achieving both a signal enhancement and the selectivity for monitoring the CBS, but the resulting UVRR spectrum overlaps with the UV-excited autofluorescence from the aromatic binding partners. This necessitates the use of a laser excitation <260 nm for spectrally separating the UVRR spectrum of the supramolecular ligand from the UV-excited autofluorescence of the peptide or protein. Here, we demonstrate the use of UVRR spectroscopy with 244 nm laser excitation for the characterization of GCP as well as guanidiniocarbonyl indole (GCI), a next generation supramolecular ligand for the recognition of carboxylates. For demonstrating the feasibility of the UVRR binding studies without an interference from the disturbing UV-excited autofluorescence, benzoic acid (BA) was chosen as an aromatic binding partner for GCI. We also present the UVRR results from the binding of GCI to the ubiquitous RGD sequence (arginylglycylaspartic acid) as a biologically relevant peptide. In the case of RGD, the more pronounced differences between the UVRR spectra of the free and complexed GCI (1:1 mixture) clearly indicate a stronger binding of GCI to RGD compared with BA. A tentative assignment of the experimentally observed changes upon molecular recognition is based on the results from density functional theory (DFT) calculations.

Vibrational Spectroscopic Characterization of 2-(2,4-Dinitrobenzyl)-pyridine (α-DNBP) in Solution by Polarization-Resolved Spontaneous Raman Scattering and Broadband CARS.

The photochromic molecule 2-(2,4-dinitro-benzyl)-pyridine (α-DNBP) is characterized in solution by a combination of density functional theory employing a polarizable continuum model and polarization-resolved spontaneous and nonlinear Raman spectroscopies. By the comparison of theoretically predicted wavenumber positions and depolarization ratios with the experimental spectra acquired under electronically nonresonant conditions, polarized and depolarized Raman bands are assigned. Specifically, the symmetric stretching vibrations of the two nitro groups in ortho and para positions to the pyridine ring can be experimentally differentiated, mainly because of their different Raman depolarization ratios, which supports our prediction from theory. Compared to the polarization-resolved spontaneous Raman experiments, the vibrational spectroscopic differentiation of the two nitro groups is more pronounced in time-delayed polarization-resolved coherent anti-Stokes Raman scattering experiments. Overall, this linear and nonlinear vibrational spectroscopic characterization of the CH form paves the way for the interpretation of future time-resolved pump/nonlinear Raman probe studies on the ultrafast photoinduced intramolecular proton transfer in α-DNBP involving a nitro group as an intramolecular proton acceptor.

Rapid, Quantitative, and Ultrasensitive Point-of-Care Testing: A Portable SERS Reader for Lateral Flow Assays in Clinical Chemistry.

The design of a portable Raman/SERS-LFA reader with line illumination using a custom-made fiber optic probe for rapid, quantitative, and ultrasensitive point-of-care testing (POCT) is presented. The pregnancy hormone human chorionic gonadotropin (hCG) is detectable in clinical samples within only 2-5 s down to approximately 1.6 mIU mL-1 . This acquisition time is several orders of magnitude shorter than those of existing approaches requiring expensive Raman instrumentation, and the method is 15-times more sensitive than a commercially available lateral flow assay (LFA) as the gold standard. The SERS-LFA technology paves the way for affordable, quantitative, and ultrasensitive POCT with multiplexing potential in real-world applications, ranging from clinical chemistry to food and environmental analysis as well as drug and biowarfare agent testing.

Effect of Antigen Retrieval Methods on Nonspecific Binding of Antibody-Metal Nanoparticle Conjugates on Formalin-Fixed Paraffin-Embedded Tissue.

Immunohistochemical analysis of formalin-fixed paraffin-embedded (FFPE) tissues provides important diagnostic and prognostic information in pathology. Metal nanoparticles (NPs) and, in particular, surface-enhanced Raman scattering (SERS) nanotags as a new class of labeling reagents are promising to be used for multiplexed protein profiling on tissue sections. However, nonspecific binding of NPs onto the tissue specimens greatly hampers their clinical applications. In this study, we found that the antigen retrieval method strongly influences the extent of nonspecific binding of the antibody-SERS NP conjugates to the tissue. Our SERS labels comprised ca. 70 nm Au nanostars coated with ethylene glycol-modified Raman reporter molecules for hydrophilic stabilization and subsequent covalent bioconjugation to antibodies. We systematically investigated the influence of heat- and protease-induced epitope retrieval (HIER and PIER, respectively) on the immunostaining quality of prostate-specific antigen (PSA) on human prostate tissue sections. The best staining results were obtained with PIER. Pretreatment of the tissue sections by HIER led to selective but nonspecific adsorption of the antibody-Au nanostar conjugates onto epithelial cells, while enzymatic treatment within PIER did not. In addition to gold nanostars, also other types of metal NPs with different shapes and sizes (including ca. 20 nm quasi-spherical Au NPs and ca. 60 nm quasi-spherical Au/Ag nanoshells) as well as tissue sections from different organs (including prostate and breast) were tested; in each case the same tendency was observed, i.e., PIER yielded better results than HIER. Therefore, we recommend PIER for future NP-based tissue immunostaining such as immuno-SERS microscopy. Alternatively, for antigens that can only be unmasked by heating, PEGylation of the NPs is recommended to avoid nonspecific binding.

Simultaneous Rayleigh/Mie and Raman/Fluorescence Characterization of Molecularly Functionalized Colloids by Correlative Single-Particle Real-Time Imaging in Suspension.

Many applications of nano- and microparticles require molecular functionalization. Assessing the heterogeneity of a colloidal sample in terms of its molecular functionalization is highly desirable but not accessible by conventional ensemble experiments. Retrieving this information necessitates single-particle experiments which simultaneously detect both functionalized and nonfunctionalized particles via two separate imaging channels. In this contribution, we present an optical setup for performing correlative single-particle imaging using laser light-sheet illumination: the first detection channel records elastic light scattering (Rayleigh/Mie), while the second channel detects inelastic light scattering (Raman) or fluorescence. The instrument is tested with Raman reporter-functionalized SERS-active metal nanoparticles (core/satellite silver nanoparticles, dimers and monomers of gold nanoparticles) and fluorophore-functionalized colloids (fluorescent polymer microparticles, dye-labeled protein on gold nanoparticles).

Ideal Dimers of Gold Nanospheres for Precision Plasmonics: Synthesis and Characterization at the Single-Particle Level for Identification of Higher Order Modes.

Ideal dimers comprising gold nanoparticles with a smooth surface and high sphericity are synthesized by a substrate-based assembly strategy with efficient cetyltrimethylammonium bromide removal. An unprecedented structural and plasmonic uniformity at the single-particle level is observed since inhomogeneities resulting from variations in gap morphology are eliminated. Single ideal dimers are analyzed by polarization-resolved dark-field scattering spectroscopy. Contributions from transverse as well as quadrupolar and octupolar longitudinal plasmon coupling modes can be discriminated because of their orthogonal polarization behavior. The assignment of these higher order coupling modes is supported by computer simulations.

Quantitative Determination of Contribution by Enhanced Local Electric Field, Antenna-Amplified Light Scattering, and Surface Energy Transfer to the Performance of Plasmonic Organic Solar Cells.

Plasmonic metal nanostructures are widely used as subwavelength light concentrators to enhance light harvesting of organic solar cells through two photophysical effects, including enhanced local electric field (ELEF) and antenna-amplified light scattering (AALS), while their adverse quenching effect from surface energy transfer (SET) should be suppressed. In this work, a comprehensive study to unambiguously distinguish and quantitatively determine the specific influence and contribution of each effect on the overall performance of organic solar cells incorporated with Ag@SiO2 core-shell nanoparticles (NPs) is presented. By investigating the photon conversion efficiency (PCE) as a function of the SiO2 shell thickness, a strong competition between the ELEF and SET effects in the performance of the devices with the NPs embedded in the active layers is found, leading to a maximum PCE enhancement of 12.4% at the shell thickness of 5 nm. The results give new insights into the fundamental understanding of the photophysical mechanisms responsible for the performance enhancement of plasmonic organic solar cells and provide important guidelines for designing more-efficient plasmonic solar cells in general.

Rapid and sensitive SERS detection of the cytokine tumor necrosis factor alpha (tnf-α) in a magnetic bead pull-down assay with purified and highly Raman-active gold nanoparticle clusters.

Tumor necrosis factor alpha (TNF-α) is a cytokine with significance in early diagnosis of cardiovascular diseases, obesity and insulin resistance. We demonstrate the proof of concept for a rapid and sensitive detection of TNF-α using a magnetic bead pull-down assay in combination with surface-enhanced Raman scattering (SERS). The use of purified and highly SERS-active small clusters of gold nanoparticles (AuNP) provides the high sensitivity of the assay with a limit of detection of ca. 1 pg/mL. Continuous density gradient centrifugation was employed for separating the very bright silica-encapsulated AuNP dimers and trimers from the significantly weaker AuNP monomers. Negative control experiments with other cytokines (IL-6, IL-8) and bovine serum albumin (BSA) confirm the high specificity of the assay, but indicate also space for future improvements by further reducing non-specific binding between proteins and the SERS nanotags. The multiplexing potential of this SERS-based detection scheme is exemplarily demonstrated by using a set of three spectrally distinct and highly SERS-active AuNP clusters with unique spectral barcodes. Graphical abstract ᅟ.

Site-Specific SERS Assay for Survivin Protein Dimer: From Ensemble Experiments to Correlative Single-Particle Imaging.

An assay for Survivin, a small dimeric protein which functions as modulator of apoptosis and cell division and serves as a promising diagnostic biomarker for different types of cancer, is presented. The assay is based on switching on surface-enhanced Raman scattering (SERS) upon incubation of the Survivin protein dimer with Raman reporter-labeled gold nanoparticles (AuNP). Site-specificity is achieved by complexation of nickel-chelated N-nitrilo-triacetic acid (Ni-NTA) anchors on the particle surface by multiple histidines (His6 -tag) attached to each C-terminus of the centrosymmetric protein dimer. Correlative single-particle analysis using light sheet laser microscopy enables the simultaneous observation of both elastic and inelastic light scattering from the same sample volume. Thereby, the SERS-inactive AuNP-protein monomers can be directly discriminated from the SERS-active AuNP-protein dimers/oligomers. This information, i.e. the percentage of SERS-active AuNP in colloidal suspension, is not accessible from conventional SERS experiments due to ensemble averaging. The presented correlative single-particle approach paves the way for quantitative site-specific SERS assays in which site-specific protein recognition by small chemical and in particular supramolecular ligands can be tested.

Advanced SERS Sensor Based on Capillarity-Assisted Preconcentration through Gold Nanoparticle-Decorated Porous Nanorods.

A preconcentrating surface-enhanced Raman scattering (SERS) sensor for the analysis of liquid-soaked tissue, tiny liquid droplets and thin liquid films without the necessity to collect the analyte is reported. The SERS sensor is based on a block-copolymer membrane containing a spongy-continuous pore system. The sensor's upper side is an array of porous nanorods having tips functionalized with Au nanoparticles. Capillarity in combination with directional evaporation drives the analyte solution in contact with the flat yet nanoporous underside of the SERS sensor through the continuous nanopore system toward the nanorod tips where non-volatile components of the analyte solution precipitate at the Au nanoparticles. The nanorod architecture increases the sensor surface in the detection volume and facilitates analyte preconcentration driven by directional solvent evaporation. The model analyte 5,5'-dithiobis(2-nitrobenzoic acid) can be detected in a 1 × 10-3 m solution ≈300 ms after the sensor is brought into contact with the solution. Moreover, a sensitivity of 0.1 ppm for the detection of the dissolved model analyte is achieved.