Split‐GFP: SERS Enhancers in Plasmonic Nanocluster Probes

The assembly of plasmonic metal nanoparticles into hot spot surface‐enhanced Raman scattering (SERS) nanocluster probes is a powerful, yet challenging approach for ultrasensitive biosensing. Scaffolding strategies based on self‐complementary peptides and proteins are of increasing interest for these assemblies, but the electronic and the photonic properties of such hybrid nanoclusters remain difficult to predict and optimize. Here, split‐green fluorescence protein (sGFP) fragments are used as molecular glue and the GFP chromophore is used as a Raman reporter to assemble a variety of gold nanoparticle (AuNP) clusters and explore their plasmonic properties by numerical modeling. It is shown that GFP seeding of plasmonic nanogaps in AuNP/GFP hybrid nanoclusters increases near‐field dipolar couplings between AuNPs and provides SERS enhancement factors above 10⁸. Among the different nanoclusters studied, AuNP/GFP chains allow near‐infrared SERS detection of the GFP chromophore imidazolinone/exocyclic C?C vibrational mode with theoretical enhancement factors of 10⁸–10⁹. For larger AuNP/GFP assemblies, the presence of non‐GFP seeded nanogaps between tightly packed nanoparticles reduces near‐field enhancements at Raman active hot spots, indicating that excessive clustering can decrease SERS amplifications. This study provides rationales to optimize the controlled assembly of hot spot SERS nanoprobes for remote biosensing using Raman reporters that act as molecular glue between plasmonic nanoparticles.     

SERS‐Active‐Charged Microgels for Size‐ and Charge‐Selective Molecular Analysis of Complex Biological Samples

Surface‐enhanced Raman scattering (SERS) provides a dramatic increase of Raman intensity for molecules adsorbed on nanogap‐rich metal nanostructures, serving as a promising tool for molecular analysis. However, surface contamination caused by protein adsorption and low surface concentration of small target molecules reduce the sensitivity, which severely restricts the use of SERS in many applications. Here, charged microgels containing agglomerates of gold nanoparticles (Au NPs) are designed using droplet‐based microfluidics to provide a reliable SERS substrate with molecular selectivity and high sensitivity. The limiting mesh size of hydrogel enables the autonomous exclusion of large proteins and the charged matrix concentrates oppositely charged small molecules through electrostatic attraction. As nanogaps among Au NPs in the agglomerates enhance Raman intensity, Raman spectrum of the adsorbed molecules is selectively measured with high sensitivity in the absence of interruption from adhesive proteins. Therefore, the SERS‐active‐charged microgels can be used for direct analysis of pristine biological samples without the pretreatment steps of separation and concentration, which are commonly a prerequisite for Raman analysis. For the purpose of demonstration, a direct detection of fipronil sulfone with partial negative charges, a metabolite of toxic insecticide, dissolved in eggs using the positively charged microgels without any pretreatment of the samples, is shown.     

Correct Spectral Conversion between Surface‐Enhanced Raman and Plasmon Resonance Scattering from Nanoparticle Dimers for Single‐Molecule Detection

Simultaneous measurement of surface‐enhanced Raman scattering (SERS) and localized surface plasmon resonance (LSPR) in nanoparticle dimers presents outstanding opportunities in molecular identification and in the elucidation of physical properties, such as the size, distance, and deformation of target species. SERS–LSPR instrumentation exists and has been used under limited conditions, but the extraction of SERS and LSPR readouts from a single measurement is still a challenge. Herein, the extraction of LSPR spectra from SERS signals is reported and a tool for measuring the interparticle distance from Raman enhancement data by the standardization of the SERS signal is proposed. The SERS nanoruler mechanism incorporates two important aspects (the LSPR scattering peak shift and the Raman shift for measuring interparticle distance), and signifies their exact one‐to‐one correspondence after spectral correction. The developed methodology is applied to calculate the interparticle distance between nanoparticle dimers from SERS signals, to detect and quantify DNA at the single‐molecule level in a base‐pair‐specific manner. It is also shown that the SERS nanoruler concept can be used in structural analysis for the specific detection of the interaction of immunoglobulin G (IgG) with its target from bianalyte Raman signals with identical shaping at single‐molecule resolution. The SERS profile shaping approach not only offers a new detection mechanism for single molecules, but also has excellent potential for studying protein interactions and the intracellular detection of mRNA.     

Hybrid Structures for Surface‐Enhanced Raman Scattering: DNA Origami/Gold Nanoparticle Dimer/Graphene

A combination of three innovative materials within one hybrid structure to explore the synergistic interaction of their individual properties is presented. The unique electronic, mechanical, and thermal properties of graphene are combined with the plasmonic properties of gold nanoparticle (AuNP) dimers, which are assembled using DNA origami nanostructures. This novel hybrid structure is characterized by means of correlated atomic force microscopy and surface‐enhanced Raman scattering (SERS). It is demonstrated that strong interactions between graphene and AuNPs result in superior SERS performance of the hybrid structure compared to their individual components. This is particularly evident in efficient fluorescence quenching, reduced background, and a decrease of the photobleaching rate up to one order of magnitude. The versatility of DNA origami structures to serve as interface for complex and precise arrangements of nanoparticles and other functional entities provides the basis to further exploit the potential of the here presented DNA origami–AuNP dimer–graphene hybrid structures.     

Graphene Oxide‐Assisted and DNA‐Modulated SERS of AuCu Alloy for the Fabrication of Apurinic/Apyrimidinic Endonuclease 1 Biosensor

Fabrication of high‐performance surface‐enhanced Raman scattering (SERS) biosensors relies on the coordination of SERS substrates and sensing strategies. Herein, a SERS active AuCu alloy with a starfish‐like structure is prepared using a surfactant‐free method. By covering the anisotropic AuCu alloy with graphene oxide (GO), enhanced SERS activity is obtained owing to graphene‐enhanced Raman scattering and assembly of Raman reporters. Besides, stability of SERS is promoted based on the protection of GO to the AuCu alloy. Meanwhile, it is found that SERS activity of AuCu/GO can be regulated by DNA. The regulation is sequence and length dual‐dependent, and short polyT reveals the strongest ability of enhancing the SERS activity. Relying on this phenomenon, a SERS biosensor is designed to quantify apurinic/apyrimidinic endonuclease 1 (APE1). Because of the APE1‐induced cycling amplification, the biosensor is able to detect APE1 sensitively and selectively. In addition, APE1 in human serum is analyzed by the SERS biosensor and enzyme‐linked immunosorbent assay (ELISA). The data from the SERS method are superior to that from ELISA, indicating great potential of this biosensor in clinical applications.     

Volume‐Enhanced Raman Scattering Detection of Viruses

Virus detection and analysis are of critical importance in biological fields and medicine. Surface‐enhanced Raman scattering (SERS) has shown great promise in small molecule and even single molecule detection, and can provide fingerprint signals of molecules. Despite the powerful detection capabilities of SERS, the size discrepancy between the SERS “hot spots” (generally, <10 nm) and viruses (usually, sub‐100 nm) yields poor detection reliability of viruses. Inspired by the concept of molecular imprinting, a volume‐enhanced Raman scattering (VERS) substrate composed of hollow nanocones at the bottom of microbowls (HNCMB) is developed. The hollow nanocones of the resulting VERS substrates serve a twofold purpose: 1) extending the region of Raman signal enhancement from the nanocone surface (e.g., surface “hot spots”) to the hollow area within the cone (e.g., volume “hot spots”)—a novel method of Raman signal enhancement, and 2) directing analyte such as viruses of a wide range of sizes to those VERS “hot spots” while simultaneously increasing the surface area contributing to SERS. Using HNCMB VERS substrates, greatly improved Raman signals of single viruses are demonstrated, an achievement with important implications in disease diagnostics and monitoring, biomedical fields, as well as in clinical treatment.     

Competitive Reaction Pathway for Site‐Selective Conjugation of Raman Dyes to Hotspots on Gold Nanorods for Greatly Enhanced SERS Performance

Common methods to prepare SERS (surface‐enhanced Raman scattering) probes rely on random conjugation of Raman dyes onto metal nanostructures, but most of the Raman dyes are not located at Raman‐intense electromagnetic hotspots thus not contributing to SERS enhancement substantially. Herein, a competitive reaction between transverse gold overgrowth and dye conjugation is described to achieve site selective conjugation of Raman dyes to the hotspots (ends) on gold nanorods (GNRs). The preferential overgrowth on the nanorod side surface creates a barrier to prevent the Raman dyes from binding to the side surface except the ends of the GNRs, where the highest SERS enhancement factors are expected. The SERS enhancement observed from this special structure is dozens of times larger than that from conjugates synthesized by conventional methods. This simple and powerful strategy to prepare SERS probes can be extended to different anisotropic metal nanostructures with electromagnetic hotspots and has immense potential in in‐depth SERS‐based biological imaging and single‐molecule detection.     

Highly Surface‐roughened “Flower‐like” Silver Nanoparticles for Extremely Sensitive Substrates of Surface‐enhanced Raman Scattering

Surface‐enhanced Raman scattering (SERS) is a new optical spectroscopic analysis technique with potential for highly sensitive detection of molecules. Recently, many efforts have been made to find SERS substrates with high sensitivity and reproducibility. In this Research News article, we provide a focused review on the synthesis of monodispersed silver particles with a novel, highly roughened, “flower‐like” morphology by reducing silver nitrate with ascorbic acid in aqueous solutions. The nanometer‐scale surface roughness of the particles can provide several hot spots on a single particle, which significantly increases SERS enhancement. The incident polarization‐dependent SERS of individual particles is also studied. Although the different “hot spots” on a single particle can have a strong polarization dependency, the total Raman signals from an individual particle usually have no obvious polarization dependency. Moreover, these flower‐like silver particles can be measured by SERS with high enhancement several times, which indicates the high stability of the hot spots. Hence, the flower‐like silver particles here can serve as highly sensitive and reproducible SERS substrates.     

High Surface‐Enhanced Raman Scattering Performance of Individual Gold Nanoflowers and Their Application in Live Cell Imaging

Molecular imaging techniques based on surface‐enhanced Raman scattering (SERS) face a lack of reproducibility and reliability, thus hampering its practical application. Flower‐like gold nanoparticles have strong SERS enhancement performance due to having plenty of hot‐spots on their surfaces, and this enhancement is not dependent on the aggregation of the particles. These features make this kind of particle an ideal SERS substrate to improve the reproducibility in SERS imaging. Here, the SERS properties of individual flower‐like gold nanoparticles are systematically investigated. The measurements reveal that the enhancement of a single gold nanoparticle is independent of the polarization of the excitation laser with an enhancement factor as high as 10⁸. After capping with Raman signal molecules and folic acid, the gold nanoflowers show strong Raman signal in the living cells, excellent targeting properties, and a high signal‐to‐noise ratio for SERS imaging.     

Au Dotted Magnetic Network Nanostructure and Its Application for On‐Site Monitoring Femtomolar Level Pesticide

A novel magnetically responsive and surface‐enhanced Raman spectroscopy (SERS) active nanocomposite is designed and prepared by direct grafting of Au nanoparticles onto the surface of magnetic network nanostructure (MNN) with the help of a nontoxic and environmentally friendly reagent of inositol hexakisphosphate shortly named as IP₆. The presence of IP₆ as a stabilizer and a bridging agent could weave Fe₃O₄ nanoparticles (NPs) into magnetic network nanostructure, which is easily dotted with Au nanoparticles (Au NPs). It has been shown firstly that the huge Raman enhancement of Au‐MNN is reached by an external magnetic collection. Au‐MNN presenting the large surface and high detection sensitivity enables it to exhibit multifunctional applications involving sufficient adsorption of dissolved chemical species for enrichment, separation, as well as a Raman amplifier for the analysis of trace pesticide residues at femtomolar level by a portable Raman spectrometer. Therefore, such multifunctional nanocomposites can be developed as a smart and promising nanosystem that integrates SERS approach with an easy assay for concentration by an external magnet for the effective on‐site assessments of agricultural and environmental safety.     

Microfluidic Surface‐Enhanced Raman Scattering Sensors Based on Nanopillar Forests Realized by an Oxygen‐Plasma‐Stripping‐of‐Photoresist Technique

A novel surface‐enhanced Raman scattering (SERS) sensor is developed for real‐time and highly repeatable detection of trace chemical and biological indicators. The sensor consists of a polydimethylsiloxane (PDMS) microchannel cap and a nanopillar forest‐based open SERS‐active substrate. The nanopillar forests are fabricated based on a new oxygen‐plasma‐stripping‐of‐photoresist technique. The enhancement factor (EF) of the SERS‐active substrate reaches 6.06 × 10⁶, and the EF of the SERS sensor is about 4 times lower due to the influence of the PDMS cap. However, the sensor shows much higher measurement repeatability than the open substrate, and it reduces the sample preparation time from several hours to a few minutes, which makes the device more reliable and facile for trace chemical and biological analysis.     

Hierarchical 3D SERS Substrates Fabricated by Integrating Photolithographic Microstructures and Self‐Assembly of Silver Nanoparticles

Most of the surface‐enhanced Raman scattering (SERS) substrates are 2D planar systems, which limits the SERS active area to a single Cartesian plane. Here, we fabricate 3D SERS substrates with the aim to break the traditional 2D SERS active area limitation, and to extend the SERS hotspots into the third dimension along the z‐axis. Our 3D SERS substrates are tailored with increased SERS hotspots in the z‐direction from tens of nanometers to tens of micrometers, increasing the hotspots in the z‐direction by at least an order of magnitude larger than the confocal volume (～1 μm) of most Raman spectrometers. Various hierarchical 3D SERS‐active microstructures are fabricated by combining 3D laser photolithography with Langmuir‐Blodgett nanoparticle assembly. 3D laser photolithography creates microstructured platforms required to extend the SERS‐active area into 3D, and the self‐assembly of Ag nanoparticles ensures homogeneous coating of SERS‐active Ag nanoparticles over the entire microstructured platforms. Large‐area 3D Raman imaging demonstrates that homogeneous signals can be collected throughout the entire 3D SERS substrates. We vary the morphology, height, and inclination angles of the 3D microstructures, where the inclination angle is found to exhibit strong influence on the SERS signals. We also demonstrate a potential application of this hierarchical 3D SERS substrate in information tagging, storage and encryption as SERS micro‐barcodes, where multiple micro‐barcodes can be created within a single set of microstructures.     

In Situ Two‐Step Photoreduced SERS Materials for On‐Chip Single‐Molecule Spectroscopy with High Reproducibility

A method is developed to synthesize surface‐enhanced Raman scattering (SERS) materials capable of single‐molecule detection, integrated with a microfluidic system. Using a focused laser, silver nanoparticle aggregates as SERS monitors are fabricated in a microfluidic channel through photochemical reduction. After washing out the monitor, the aggregates are irradiated again by the same laser. This key step leads to full reduction of the residual reactants, which generates numerous small silver nanoparticles on the former nanoaggregates. Consequently, the enhancement ability of the SERS monitor is greatly boosted due to the emergence of new “hot spots.” At the same time, the influence of the notorious “memory effect” in microfluidics is substantially suppressed due to the depletion of surface residues. Taking these advantages, two‐step photoreduced SERS materials are able to detect different types of molecules with the concentration down to 10^?13m . Based on a well‐accepted bianalyte approach, it is proved that the detection limit reaches the single‐molecule level. From a practical point of view, the detection reproducibility at different probing concentrations is also investigated. It is found that the effective single‐molecule SERS measurements can be raised up to ≈50%. This microfluidic SERS with high reproducibility and ultrasensitivity will find promising applications in on‐chip single‐molecule spectroscopy.     

Direct Experimental Observation of Facet‐Dependent SERS of Cu2O Polyhedra

Semiconductor‐based surface enhanced Raman scattering (SERS) has attracted great attention due to its excellent spectral reproducibility, high uniformity, and good anti‐interference ability. However, its relatively low SERS sensitivity still hinders its further developments in both performance and applications. Since the SERS is a peculiar surface effect, investigating the facet‐dependent SERS activity of semiconductor nanostructures is crucial to boost their SERS signals. Although the semiconductor facet‐dependent SERS effect is predicted via numerical calculations, convincing experimental evidence is scarce due to complicated and undefined surface conditions. In this work, three facet‐defined ({100}, {110}, and {111} facets) Cu₂O microcrystals (MCs) with clear surface atomic configuration are utilized to investigate the facet‐dependent SERS effect. The results from the Kelvin probe force microscopy measurements on single Cu₂O polyhedron, demonstrate that the facet‐dependent work function plays a crucial role in the interfacial charge transfer process. Comparing with the {110} and {111} facets, the {100} facet possesses the lowest electronic work function, which enables more efficient interfacial charge transfer. The simulation results further confirm that the {100}‐facets can transfer the most electrons from Cu₂O MCs to molecules due to its lowest facet work function, resulting in the largest increment of the molecular polarization.     

Bright Surface‐Enhanced Raman Scattering with Fluorescence Quenching from Silica Encapsulated J‐Aggregate Coated Gold Nanoparticles

Plexitonic nanoparticles offer variable optical properties through tunable excitations, in addition to electric field enhancements that far exceed molecular resonators. This study demonstrates a way to design an ultrabright surface‐enhanced Raman spectroscopy (SERS) signal while simultaneously quenching the fluorescence background through silica encapsulation of the semiconductor–metal composite nanoparticles. Using a multistep approach, a J‐aggregate‐forming organic dye is assembled on the surface of gold nanoparticles using a cationic linker. Excitonic resonance of the J‐aggregate–metal system shows an enhanced SERS signal at an appropriate excitation wavelength. Further encapsulation of the decorated particles in silica shows a significant reduction in the fluorescence signal of the Raman spectra (5× reduction) and an increase in Raman scattering (7× enhancement) when compared to phospholipid encapsulation. This reduction in fluorescence is important for maximizing the useful SERS enhancement from the particle, which shows a signal increase on the order of 10⁴ times greater than J‐aggregated dye in solution and 24 times greater than Oxonica S421 SERS tag. The silica layer also serves to promote colloidal stability. The combination of reduced fluorescence background, enhanced SERS intensity, and temporal stability makes these particles highly distinguishable with potential to enable high‐throughput applications such as SERS flow cytometry.     

A Droplet‐Based High‐Throughput SERS Platform on a Droplet‐Guiding‐Track‐Engraved Superhydrophobic Substrate

A novel droplet‐based surface‐enhanced Raman scattering (SERS) sensor for high‐throughput real‐time SERS monitoring is presented. The developed sensors are based on a droplet‐guiding‐track‐engraved superhydrophobic substrate covered with hierarchical SERS‐active Ag dendrites. The droplet‐guiding track with a droplet stopper is designed to manipulate the movement of a droplet on the superhydrophobic substrate. The superhydrophobic Ag dendritic substrates are fabricated through a galvanic displacement reaction and subsequent self‐assembled monolayer coating. The optimal galvanic reaction time to fabricate a SERS‐active Ag dendritic substrate for effective SERS detection is determined, with the optimized substrate exhibiting an enhancement factor of 6.3 × 10⁵. The height of the droplet stopper is optimized to control droplet motion, including moving and stopping. Based on the manipulation of individual droplets, the optimized droplet‐based real‐time SERS sensor shows high resistance to surface contaminants, and droplets containing rhodamine 6G, Nile blue A, and malachite green are successively controlled and detected without spectral interference. This noble droplet‐based SERS sensor reduces sample preparation time to a few seconds and increased detection rate to 0.5 µ L s^?1 through the simple operation mechanism of the sensor. Accordingly, our sensor enables high‐throughput real‐time molecular detection of various target analytes for real‐time chemical and biological monitoring.     

Reaction Kinetics‐Mediated Control over Silver Nanogap Shells as Surface‐Enhanced Raman Scattering Nanoprobes for Detection of Alzheimer's Disease Biomarkers

It is very challenging to accurately quantify the amounts of amyloid peptides Aβ40 and Aβ42, which are Alzheimer's disease (AD) biomarkers, in blood owing to their low levels. This has driven the development of sensitive and noninvasive sensing methods for the early diagnosis of AD. Here, an approach for the synthesis of Ag nanogap shells (AgNGSs) is reported as surface‐enhanced Raman scattering (SERS) colloidal nanoprobes for the sensitive, selective, and multiplexed detection of Aβ40 and Aβ42 in blood. Raman label chemicals used for SERS signal generation modulate the reaction rate for AgNGSs production through the formation of an Ag‐thiolate lamella structure, enabling the control of nanogaps at one nanometer resolution. The AgNGSs embedded with the Raman label chemicals emit their unique SERS signals with a huge intensity enhancement of up to 10⁷ and long‐term stability. The AgNGS nanoprobes, conjugated with an antibody specific to Aβ40 or Aβ42, are able to detect these AD biomarkers in a multiplexed manner in human serum based on the AgNGS SERS signals. Detection is possible for amounts as low as 0.25 pg mL^?1. The AgNGS nanoprobe‐based sandwich assay has a detection dynamic range two orders of magnitude wider than that of a conventional enzyme‐linked immunosorbent assay.     

M‐shaped Grating by Nanoimprinting: A Replicable,Large‐Area,Highly Active Plasmonic Surface‐Enhanced Raman Scattering Substrate with Nanogaps

Plasmonic nanostructures separated by nanogaps enable strong electromagnetic‐field confinement on the nanoscale for enhancing light‐matter interactions, which are in great demand in many applications such as surface‐enhanced Raman scattering (SERS). A simple M‐shaped nanograting with narrow V‐shaped grooves is proposed. Both theoretical and experimental studies reveal that the electromagnetic field on the surface of the M grating can be pronouncedly enhanced over that of a grating without such grooves, due to field localization in the nanogaps formed by the narrow V grooves. A technique based on room‐temperature nanoimprinting lithography and anisotropic reactive‐ion etching is developed to fabricate this device, which is cost‐effective, reliable, and suitable for fabricating large‐area nanostructures. As a demonstration of the potential application of this device, the M grating is used as a SERS substrate for probing Rhodamine 6G molecules. Experimentally, an average SERS enhancement factor as high as 5×10⁸ has been achieved, which verifies the greatly enhanced light–matter interaction on the surface of the M grating over that of traditional SERS surfaces.     

Monitoring Early‐Stage Nanoparticle Assembly in Microdroplets by Optical Spectroscopy and SERS

Microfluidic microdroplets have increasingly found application in biomolecular sensing as well as nanomaterials growth. More recently the synthesis of plasmonic nanostructures in microdroplets has led to surface‐enhanced Raman spectroscopy (SERS)‐based sensing applications. However, the study of nanoassembly in microdroplets has previously been hindered by the lack of on‐chip characterization tools, particularly at early timescales. Enabled by a refractive index matching microdroplet formulation, dark‐field spectroscopy is exploited to directly track the formation of nanometer‐spaced gold nanoparticle assemblies in microdroplets. Measurements in flow provide millisecond time resolution through the assembly process, allowing identification of a regime where dimer formation dominates the dark‐field scattering and SERS. Furthurmore, it is shown that small numbers of nanoparticles can be isolated in microdroplets, paving the way for simple high‐yield assembly, isolation, and sorting of few nanoparticle structures.     

High‐Yield Synthesis of Hollow Octahedral Silver Nanocages with Controllable Pack Density and Their High‐Performance Sers Application

Nanoparticle‐assembled octahedral Ag nanocages with sharp edges have been successfully synthesized through a Cu₂O‐based template‐assisted strategy. In the reaction system, Ag nanoparticles can be self‐assembled on the surface of Cu₂O octahedrons, which is accomplished by the reduction of Ag⁺ by NaBH₄ in the presence of sodium citrate as a capping agent. The hollow octahedral Ag nanocages are obtained after removing the inner Cu₂O cores with acetic acid. According to the scanning electron microscopy (SEM) and transmission electron microscopy characterization, the Ag nanocages are weaved by small nanoparticles, the rough surfaces are bestrewed with pores and sharp edges. It is found that the pack density of Ag nanoparticles strongly affects the surface enhanced Raman scattering (SERS) activities. The as‐prepared 1.05‐Ag cages with optimal pack density have suitable interparticle distance and suitable size of pores, which significantly enhance SERS signals. The SERS signals of rhodamine 6G (R6G) molecules can be detected at an ultralow concentration of 10^?14 m when 1.05‐Ag cages are used as substrates. In addition to sensitivity, 1.05‐Ag cages also exhibit good reproducibility. It is expected that the ultrahigh sensitivity will endow the Ag nanocages to become a promising candidate as high‐performance SERS‐based chemical sensor.