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.     

Noble‐Metal‐Free Materials for Surface‐Enhanced Raman Spectroscopy Detection

Surface‐enhanced Raman spectroscopy (SERS) is an attractive tool for the sensing of molecules in the fields of chemical and biochemical analysis as it enables the sensitive detection of molecular fingerprint information even at the single‐molecule level. In addition to traditional coinage metals in SERS analysis, recent research on noble‐metal‐free materials has also yielded highly sensitive SERS activity. This Minireview presents the recent development of noble‐metal‐free materials as SERS substrates and their potential applications, especially semiconductors and emerging graphene‐based nanostructures. Rather than providing an exhaustive review of this field, possible contributions from semiconductor substrates, characteristics of graphene enhanced Raman scattering, as well as effect factors such as surface plasmon resonance, structure and defects of the nanostructures that are considered essential for SERS activity are emphasized. The intention is to illustrate, through these examples, that the promise of noble‐metal‐free materials for enhancing detection sensitivity can further fuel the development of SERS‐related applications.     

Long‐Term Stable Silver Subsurface Ion‐Exchanged Glasses for SERS Applications

We report on the formation of silver subsurface ion‐exchanged metal oxide (silver SIMO) glasses and their surface‐enhanced Raman scattering (SERS) activity. The samples were prepared by a combined thermal and chemical three‐step methodology and characterized by transmission electron microscopy (TEM), atomic force microscopy (AFM), environmental electron scanning microscopy (ESEM), and UV/Vis spectroscopy. This unique method provides SERS substrates with protection against contamination and strong, reliable and reproducible SERS enhancement. The Raman enhancement factors of the long‐term stable SIMO glasses were estimated to approximately 10⁷.     

DNA Origami Directed Assembly of Gold Bowtie Nanoantennas for Single‐Molecule Surface‐Enhanced Raman Scattering

Metallic bowtie nanoarchitectures can produce dramatic electric field enhancement, which is advantageous in single‐molecule analysis and optical information processing. Plasmonic bowtie nanostructures were successfully constructed using a DNA origami‐based bottom‐up assembly strategy, which enables precise control over the geometrical configuration of the bowtie with an approximate 5 nm gap. A single Raman probe was accurately positioned at the gap of the bowtie. Single‐molecule surface‐enhanced Raman scattering (SM‐SERS) of individual nanostructures, including ones containing an alkyne group, was observed. The design achieved repeatable local field enhancement of several orders of magnitude. This method opens the door on a novel strategy for the fabrication of metal bowtie structures and SM‐SERS, which can be utilized in the design of highly‐sensitive photonic devices.     

Self‐Assembled Au Nanoparticles as Substrates for Surface‐Enhanced Vibrational Spectroscopy: Optimization and Electrochemical Stability

Three‐dimensional nanostructured metallic substrates for enhanced vibrational spectroscopy are fabricated by self‐assembly. Nanostructures consisting of one to 20 depositions of 13 nm‐diameter Au nanoparticles (NPs) on Au films are prepared and characterized by means of AFM and UV/Vis reflection–absorption spectroscopy. Surface‐enhanced polarization modulation infrared reflection–absorption spectroscopy (PM‐IRRAS) is observed from Au NPs modified by the probe molecule 4‐hydroxythiophenol. The limitation of this kind of substrate for surface‐enhanced PM‐IRRAS is discussed. The surface‐enhanced Raman scattering (SERS) from the same probe molecule is also observed and the effect of the number of Au‐NP depositions on the SERS efficiency is studied. The SERS signal from the probe molecule maximizes after 11 Au‐NP depositions, and the absolute SERS intensities from different batches are reproducible within 20 %. In situ electrochemical SERS measurements show that these substrates are stable within the potential window between ?800 and +200 mV (vs. Ag/AgCl/sat. Cl^?).     

ZnGa2O4 Nanorod Arrays Decorated with Ag Nanoparticles as Surface‐Enhanced Raman‐Scattering Substrates for Melamine Detection

The availability of sensitive, reproducible, and stable substrates is critically important for surface‐enhanced Raman spectroscopy (SERS)‐based applications, but it presently remains a challenge. In this work, well‐aligned zinc gallate (ZnGa₂O₄) nanorod arrays grown on a Si substrate by chemical vapor deposition were used as templates to fabricate SERS substrates by deposition of Ag nanoparticles onto the ZnGa₂O₄ nanorod surfaces. The coverage of the Ag nanoparticles on the ZnGa₂O₄ nanorod surfaces was easily controlled by varying the amount of AgNO₃. SERS measurements showed that the number density of Ag nanoparticles on the ZnGa₂O₄ nanorod surfaces had a great effect on SERS activity. The SERS signals collected by point‐to‐point and SERS mapping images showed that as‐prepared SERS substrates exhibited good spatial uniformity and reproducibility. Detection of melamine molecules at low concentrations (1.0×10^?7 M ) was used as an example to show the possible application of such a substrate. In addition, the effect of benzoic acid on the detection of melamine was also investigated. It was found that the SERS signal intensity of melamine decreased greatly as the concentration of benzoic acid was increased.     

Single‐Molecule Spectroscopy,Imaging, and Photocontrol: Foundations for Super‐Resolution Microscopy (Nobel Lecture)

The initial steps toward optical detection and spectroscopy of single molecules in condensed matter arose out of the study of inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral signatures relating to the fluctuations of the number of molecules in resonance led to the attainment of the single‐molecule limit in 1989 using frequency‐modulation laser spectroscopy. In the early 90s, many fascinating physical effects were observed for individual molecules, and the imaging of single molecules as well as observations of spectral diffusion, optical switching and the ability to select different single molecules in the same focal volume simply by tuning the pumping laser frequency provided important forerunners of the later super‐resolution microscopy with single molecules. In the room temperature regime, imaging of single copies of the green fluorescent protein also uncovered surprises, especially the blinking and photoinduced recovery of emitters, which stimulated further development of photoswitchable fluorescent protein labels. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic observation and localization of individual fluorophores is a key ingredient to imaging beyond the optical diffraction limit. Combining this with active control of the number of emitting molecules in the pumped volume led to the super‐resolution imaging of Eric Betzig and others, a new frontier for optical microscopy beyond the diffraction limit. The background leading up to these observations is described and current developments are summarized.     

Novel Bottom‐Up SERS Substrates for Quantitative and Parallelized Analytics

Surface‐enhanced Raman spectroscopy (SERS) is an emerging technology in the field of analytics. Due to the high sensitivity in connection with specific Raman molecular fingerprint information SERS can be used in a variety of analytical, bioanalytical, and biosensing applications. However, for the SERS effect substrates with metal nanostructures are needed. The broad application of this technology is greatly hampered by the lack of reliable and reproducible substrates. Usually the activity of a given substrate has to be determined by time‐consuming experiments such as calibration or ultramicroscopic studies. To use SERS as a standard analytical tool, cheap and reproducible substrates are required, preferably with a characterization technique that does not interfere with the subsequent measurements. Herein we introduce an innovative approach to produce low‐cost and large‐scale reproducible substrates for SERS applications, which allows easy and economical production of micropatterned SERS active surfaces on a large scale. This approach is based on an enzyme‐induced growth of silver nanostructures. The special structural feature of the enzymatically deposited silver nanoparticles prevents the breakdown of SERS activity even at high particle densities (particle density >60 %) that lead to a conductive layer. In contrast to other approaches, this substrate exhibits a relationship between electrical conductivity and the resulting SERS activity of a given spot. This enables the prediction of the SERS activity of the nanostructure ensemble and therewith the controllable and reproducible production of SERS substrates of enzymatic silver nanoparticles on a large scale, utilizing a simple measurement of the electrical conductivity. Furthermore, through a correlation between the conductivity and the SERS activity of the substrates it is possible to quantify SERS measurements with these substrates.     

Differences in Dynamics between Crosslinked and Non‐Crosslinked Hyaluronates Measured by using Fast Field‐Cycling Relaxometry

The dynamic properties of water molecules in gels containing linear and crosslinked hyaluronic acid polymers are investigated by using an integrated approach that includes relaxometry, solid‐state NMR spectroscopy, and scanning electron microscopy. A model‐free analysis of field‐dependent nuclear relaxation is applied to obtain information on mobility and the population of different pools of water molecules in the gels. Differences between linear and crosslinked hyaluronic acid polymers are observed, indicating that crosslinking increases both the fraction and the correlation time of water molecules with slow dynamics.     

Rolling‐Circle Amplification Detection of Thrombin Using Surface‐Enhanced Raman Spectroscopy with Core‐Shell Nanoparticle Probe

An ultrasensitive surface‐enhanced Raman spectroscopy (SERS) sensor based on rolling‐circle amplification (RCA)‐increased “hot‐spot” was developed for the detection of thrombin. The sensor contains a SERS gold nanoparticle@Raman label@SiO₂ core‐shell nanoparticle probe in which the Raman reporter molecules are sandwiched between a gold nanoparticle core and a thin silica shell by a layer‐by‐layer method. Thrombin aptamer sequences were immobilized onto the magnetic beads (MBs) through hybridization with their complementary strand. In the presence of thrombin, the aptamer sequence was released; this allowed the remaining single‐stranded DNA (ssDNA) to act as primer and initiate in situ RCA reaction to produce long ssDNAs. Then, a large number of SERS probes were attached on the long ssDNA templates, causing thousands of SERS probes to be involved in each biomolecular recognition event. This SERS method achieved the detection of thrombin in the range from 1.0×10^?12 to 1.0×10^?8 M and a detection limit of 4.2×10^?13 M , and showed good performance in real serum samples.     

Plasmonic Hotspots in Air: An Omnidirectional Three‐Dimensional Platform for Stand‐Off In‐Air SERS Sensing of Airborne Species

Molecular‐level airborne sensing is critical for early prevention of disasters, diseases, and terrorism. Currently, most 2D surface‐enhanced Raman spectroscopy (SERS) substrates used for air sensing have only one functional surface and exhibit poor SERS‐active depth. “Aerosolized plasmonic colloidosomes” (APCs) are introduced as airborne plasmonic hotspots for direct in‐air SERS measurements. APCs function as a macroscale 3D and omnidirectional plasmonic cloud that receives laser irradiation and emits signals in all directions. Importantly, it brings about an effective plasmonic hotspot in a length scale of approximately 2.3 cm, which affords 100‐fold higher tolerance to laser misalignment along the z‐axis compared with 2D SERS substrates. APCs exhibit an extraordinary omnidirectional property and demonstrate consistent SERS performance that is independent of the laser and analyte introductory pathway. Furthermore, the first in‐air SERS detection is demonstrated in stand‐off conditions at a distance of 200 cm, highlighting the applicability of 3D omnidirectional plasmonic clouds for remote airborne sensing in threatening or inaccessible areas.     

Cryogenic Correlative Single‐Particle Photoluminescence Spectroscopy and Electron Tomography for Investigation of Nanomaterials

Cryogenic single‐particle photoluminescence (PL) spectroscopy has been used with great success to directly observe the heterogeneous photophysical states present in a population of luminescent particles. Cryogenic electron tomography provides complementary nanometer scale structural information to PL spectroscopy, but the two techniques have not been correlated due to technical challenges. Here, we present a method for correlating single‐particle information from these two powerful microscopy modalities. We simultaneously observe PL brightness, emission spectrum, and in‐plane excitation dipole orientation of CdSSe/ZnS quantum dots suspended in vitreous ice. Stable and fluctuating emitters were observed, as well as a surprising splitting of the PL spectrum into two bands with an average energy separation of 80 meV. In some cases, the onset of the splitting corresponded to changes in the in‐plane excitation dipole orientation. These dynamics were assigned to structures of individual quantum dots and the excitation dipoles were visualized in the context of structural features.     

Immobilization of silver nanoparticles into POEGMA polymer brushes as SERS‐active substrates

Surface‐enhanced Raman scattering (SERS) has attracted a great deal of interest during the past four decades and emerged as an ultrasensitive optical technique for chemical and biomedical analysis. It is widely accepted that the facile fabrication of SERS substrates with high activity and good reproducibility is of crucial importance for their applications. Herein, we report on a fast and robust method for the synthesis and immobilization of silver nanoparticles (AgNPs) into poly(oligo(ethylene glycol) methacrylate) (POEGMA) brushes under mild conditions without using any reducing agents. POEGMA brushes of different chain lengths were synthesized directly on silicon wafers by surface‐initiated atom transfer radical polymerization with various reaction time. X‐ray photoelectron spectroscopy and field emission scanning electron microscope measurements indicated that the AgNPs were firmly and homogeneously embedded into POEGMA brushes. The resulting POEGMA–AgNP hybrid films were employed as SERS substrates for the detection of 4‐aminothiophenol, giving rise to an enhancement factor of up to 1.9 × 10⁶. The influence of the POEGMA's chain length on SERS performance was also investigated. Copyright © 2016 John Wiley & Sons, Ltd.     

Carbon‐Nanotube‐Based Hybrid Materials: Nanopeapods

This review article focuses on the structures and properties of novel hybrid nanocarbon materials, which are created by incorporating atoms and molecules into the hollow spaces of carbon nanotubes (CNTs); thus they are called nanopeapods. After dealing with synthesis procedures, we discuss the structures and electronic properties of the hybrid materials based on high‐resolution transmission electron microscopy (HRTEM), electron energy‐loss spectroscopy (EELS), X‐ray and electron diffraction, scanning tunneling microscopy (STM), and field‐effect transistor transport measurements. Utilization of the low‐dimensional nanosized spaces of CNTs to produce novel low‐dimensional nanocluster, nanowire, and nanotube materials is also discussed.     

Probing Surface Enhancement Effect of Molecules On/Between Silver Nanoparticles in Surface Enhanced Raman Scattering

To explore the mechanisms in Surface‐enhanced Raman Scattering (SERS) measurements, silver nanoparticles (AgNPs) were first prepared by a silver mirror reaction to form different particle sizes and different distributions on glass substrates. After the resulting surfaces were probed with molecules of p‐Amino‐thiophenol (pATP), p‐Nitrothiophenol (pNTP), and p‐Mercaptobenzoic acid (pMBA) individually, the substrates were placed into reaction solutions to grow additional AgNPs. In this way, probe molecules could be trapped between two nanoparticles, possibly having the so‐called “hot spot” effect. To examine the variations of morphologies of AgNPs in each of the steps, the substrates were examined by field‐emission scanning electron microscope (FE‐SEM). The morphologies also were correlated with the SERS signals. Two bands in the SERS spectra of probe molecules were selected as indications of the enhancements from electromagnetic (EM) effect and charge‐transfer (CT). Results indicate that the SERS signals from the EM effect were increased ca. 5 times after growing additional AgNPs on the molecule‐modified AgNPs substrates. The SERS signals from CT effect were increased two orders of magnitude after growing additional AgNPs. The increase of enhancement for molecules between AgNPs was caused mostly by CT effect. Based on the effect of particle size and distribution of the AgNPs, the EM effect was strongly influenced by the particle size of the AgNPs, while the CT effect was less sensitive to the variation of the morphologies of the AgNPs.     

Photocatalytically Powered Matchlike Nanomotor for Light‐Guided Active SERS Sensing

Surface enhanced Raman spectroscopy (SERS) is a powerful optical sensing technique that can detect analytes of extremely low concentrations. However, the presence of enough SERS probes in the detection area and a close contact between analytes and SERS probes are critical for efficient acquisition of a SERS signal. Presented here is a light‐powered micro/nanomotor (MNM) that can serve as an active SERS probe. The matchlike AgNW@SiO₂ core–shell structure of the nanomotors work as SERS probes based on the shell‐isolated enhanced Raman mechanism. The AgCl tail serves as photocatalytic nanoengine, providing a self‐propulsion force by light‐induced self‐diffusiophoresis. The phototactic behavior was utilized to achieve enrichment of the nanomotor‐based SERS probes for on‐demand biochemical sensing. The results demonstrate the possibility of using photocatalytic nanomotors as active SERS probes for remote, light‐controlled, and smart biochemical sensing on the micro/nanoscale.     

Local and Remote Charge‐Transfer‐Enhanced Raman Scattering on One‐Dimensional Transition‐Metal Oxides

The one‐dimensional (1D) transition‐metal oxide MoO₃ belt is synthesized and characterized with X‐ray diffraction, scanning electron microscopy, and Raman spectroscopy. Charge‐transfer‐(CT) enhanced Raman scattering of 4‐mercaptobenzoic acid (4‐MBA) on a 1D MoO₃ belt was investigated experimentally and theoretically. The chemical enhancement of surface‐enhanced Raman scattering (SERS) of 4‐MBA on the MoO₃ belt by CT is in the order of 10³. The SERS of 4‐MBA was investigated theoretically by using a quantum chemical method. The remote SERS of 4‐MBA along the 1D MoO₃ belt (the light excitation to one side of the MoO₃ belt, and the SERS spectrum is collected on the other side of the MoO₃ belt) is also shown experimentally, which provides potential applications of SERS. The incident polarization dependence of remote SERS spectra has also been investigated experimentally.     

Sub-wavelength Raman spectroscopy on isolated silver islands

Surface Enhanced Raman Spectroscopy (SERS) is a valuable analytical tool for the investigation of molecules adsorbed on roughened noble metal surfaces. The shape, size, and surrounding of the metal protrusions play an important role in the Raman scattering enhancement. By combining scanning near-field optical microscopy (SNOM) with Raman spectroscopy the spatial resolution suffices for investigating isolated silver islands on SERS active substrates. We demonstrate an optical resolution below 70 nm for recording spectra on specifically prepared and fully characterized SERS substrates. For a quantitative evaluation of the SERS signal the spatial distribution of Rhodamine 6G (R6G) deposited on the SERS substrate was determined by friction force measurements. By comparing the Raman intensities of the SERS substrates with those of unmetallized support plates absolute SERS enhancement factors at specific locations on top and in the vicinity of the silver islands were determined directly.     

Characterization of aluminum–organic‐stabilized platinum–colloid networks with electron and photon spectroscopies

We have measured and interpreted the ultraviolet (HeI) and X‐ray photoelectron spectra and the metastable impact electron spectra (MIES) from aluminum–organic‐stabilized platinum–colloids and colloid networks, deposited on silicon substrates and characterized by X‐ray photoelectron spectroscopy, scanning Auger electron microscopy and transmission electron microscopy. MIES, in particular, gives information on the electronic structure of the spacer molecules interconnecting the colloids. In addition, changes in the electronic structure of the platinum clusters that are induced by different spacer molecules were identified by means of X‐ray absorption near‐edge structure measurements at the platinum L_III‐edge of these materials. This combination of techniques was also employed to follow the chemical changes that occur upon heating of the network in situ. It turns out that the thermal decomposition of the network is driven by the disintegration of the spacer molecules. Moreover, less sintering of the colloidal particles occurs in the networked systems than in unconnected particles. Most of the networked platinum–particles are still present in their original shape even after the destruction of spacer molecules. This observation could be linked to the encapsulation of these platinum particles into an (Al? O) protecting shell. Copyright © 2003 John Wiley & Sons, Ltd.     

Tailoring Size and Coverage Density of Silver Nanoparticles on Monodispersed Polymer Spheres as Highly Sensitive SERS Substrates

Silver nanoparticles (AgNPs) were deposited onto the monodispersed carboxylic polystyrene (CPS) spheres by an improved in situ reduction method. The size and coverage density of the AgNPs on the surface of CPS spheres could be easily tailored by tuning the concentrations of carboxylic functional groups and silver precursor. The morphologies and structures of the resulting CPS/Ag hybrid particles were studied by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X‐ray diffraction (XRD), UV‐Vis‐NIR spectrometer and X‐ray photoelectron spectroscopy (XPS), etc. The surface enhanced Raman scattering (SERS) performances of the resulting uniform CPS/Ag hybrid particles were investigated using 4‐aminobenzenethiol (4‐ABT) as the probe molecule. The optimized CPS/Ag hybrid particles show high enhancement factor (EF) of 2.71×10⁷, low limit of detection (LOD) of 10^?10 m and good reproducibility with relative standard deviation (RSD) of 9.64 %. The good SERS improvement properties demonstrate these hybrid particles could be employed as simple and effective substrates in the SERS spectroscopy.