The Compact Integration of Multiple Exonuclease III-Assisted Cyclic Amplification Units for High-Efficiency Ratiometric Electrochemiluminescence Detection of MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA) exhibits multiresistance to a plethora of antibiotics, therefore, accurate detection methods must be employed for timely identification to facilitate effective infection control measures. Herein, we construct a high-efficiency ratiometric electrochemiluminescent (ECL) biosensor that integrates multiple exonuclease (Exo) III-assisted cyclic amplification units for rapid detection of trace amounts of MRSA. The target bacteria selectively bind to the aptamer, triggering the release of two single-stranded DNAs. One released DNA strand initiates the opening of a hairpin probe, inducing exonuclease cleavage to generate a single strand that can form a T-shaped structure with the double strand connecting the oxidation-reduction (O-R) emitter of N-(4-aminobutyl)-N-ethylisoluminol gold (ABEI-Au). Consequently, ABEI-Au is released upon Exo III cleavage. The other strand unwinds the hairpin DNA structure on the surface of the reduction-oxidation (R-O) emitter ZIF-8@CdS, facilitating the subsequent release of a specific single strand through Exo III cleavage. This process effectively anchors the cathode-emitting material to the electrode. The Fe(III) metal-organogel (Fe-MOG) is selected as a substrate, in which the catalytic reduction of hydrogen peroxide by Fe(III) active centers accelerates the generation of reactive oxygen species and enhances signals from both ABEI-Au and ZIF-8@CdS. In this way, the two emitters cooperate to achieve bacterial detection at the single-cell level, and a good linear range is obtained in the range of 100-107 CFU/mL. Moreover, the sensor exhibited excellent performance in detecting MRSA across various authentic samples and accurately quantifying MRSA levels in serum samples, demonstrating its immense potential in addressing clinical bacterial detection challenges.

Spectrally tunable humic acid-based carbon dots: a simple platform for metronidazole and ornidazole sensing in multiple real samples.

Humic acid-based carbon dots (HACDs) have excellent properties and are widely used in environmental detection, bioimaging, and optoelectronic materials. Herein, we investigated the structure-activity relationship between the morphology and optical properties of HACDs, and reported on a novel strategy for metronidazole (MNZ) and ornidazole (ONZ) sensing in multiple real samples. It was found that the average particle size decreased from 3.28 to 2.44 nm, optimal emission wavelength was blue-shifted from 500 to 440 nm, and the quantum yield (QY) improved from 5 to 23% with the temperature increasing from 110 to 400 °C. Under the oxidation of hydrogen peroxide (H2O2) and potassium permanganate (KMnO4), the UV-vis spectra of HACD aqueous solution showed time-dependent behavior, and the fluorescence emission of HACDs achieved spectrally tunable multi-color luminescence in the temporal dimension. The surface of HACDs contained a large number of hydroxyl (-OH) and carboxyl (-COOH) fluorophores, resulting in excellent pH sensing. Meanwhile, the synthesized HACDs revealed sensitive response to MNZ and ONZ with the limit of detection (LOD) of 60 nM and 50 nM in aqueous solutions, which had also been successfully applied in various actual samples such as lake water, honey, eggs, and milk with satisfactory results because of the inner filter effect (IFE). Our research is advantageous to enhance the potential applications of HACDs in advanced analytical systems.

Three-Dimensional Surface-Enhanced Raman Scattering Platform with Hotspots Built by a Nano-mower for Rapid Detection of MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA) has become one of the greatest threats to human health due to its strong drug resistance, wide distribution range, and high infection rate. Rapid identification of MRSA strains is very important for accurate diagnosis and early treatment of MRSA infections. Here, we introduced an Exo III-assisted nanomotor mower to build 3D hotspots for rapid detection of MRSA by surface-enhanced Raman scattering (SERS). As the bacteria bound to the aptamer, two trigger chains were released from the double-stranded structure, and the nano-mowers were activated by opening a hairpin probe on gold nanoparticles (AuNPs). With the continued cleavage of Exo III and cyclic release of the trigger chain, multiple hairpin DNAs on the AuNPs were cleaved to increase the motor power. The resulting nano-mower continued slicing protective DNA from larger AuNPs, exposing the AuNPs. Without the protection of DNA, Mg2+ in the buffer induced spontaneous aggregation of the AuNPs, and a large number of hotspots were formed for SERS measurements. Under optimal conditions, MRSA can be detected within 40 min, and the concentration of MRSA showed a good linear relationship with the SERS intensity at 1342 cm-1, with a limit of detection as low as 1 CFU/mL and a wide linear range (100 to 107 CFU/mL). This strategy creates a rapid bacterial detection method that performs well on actual samples utilizing portable Raman spectroscopy instruments, with potential applications in food detection, water detection, clinical treatment, and diagnosis.

Multistage nucleic acid amplification induced nano-aggregation for 3D hotspots-improved SERS detection of circulating miRNAs.

Circulating miRNAs in the blood can regulate disease development and thus indicate disease states via their various expression levels. For these reasons, circulating miRNAs constitute useful biomarkers, and an approach to the accurate detection of circulating miRNAs is attractive in the diagnosis and treatment of diseases. However, methods for clinical detecting of circulating miRNA that take both sensitivity and practicality into account are still needed. Therefore, we aimed herein to solve some inherent problems in the actual detection using a robust surface-enhanced Raman scattering (SERS) platform with integrated nucleic acid amplification and nanoparticle aggregation to construct 3D hotspots for improving performance of analyzing circulating miRNAs. After target recognition and initial signal amplification by DNAzyme, we observed that release triggered an open hairpin DNA on gold nanoparticles (AuNPs), which then promote AuNP aggregation, causing the accumulation of a large number of hotspots in three-dimention. The SERS biosensor achieved a better performance than the sandwich-type separation detection, with a low detection limit (0.37 fM) and a broad linear range (1 fM-10 nM) in liquids. This SERS platform can be used as a powerful tool for the detection of circulating miRNAs, and it can be used to improve the sensitivity and accuracy of various clinical-disease diagnoses.

Reversible Ratiometric Electrochemiluminescence Biosensor Based on DNAzyme Regulated Resonance Energy Transfer for Myocardial miRNA Detection.

Myocardial miRNAs in peripheral blood are closely related to the pathogenic process of myocardial infarction. Rapid identification and accurate quantification of myocardial miRNAs are of great significance to clinical interventions for treating cardiovascular lesions. Therefore, a ratiometric electrochemiluminescence (ECL) biosensor integrating DNAzyme with a resonance energy transfer (RET) system was designed to detect myocardial miRNA. The dual-signal system was composed of rA marked substrate strand functionalized CdTe quantum dots (QDs) as reductive-oxidative (R-O) emitters and Cy5-labeled strand-functionalized Ru(bpy)32+-filled silica nanoparticles (RuSi NPs) as oxidative-reductive (O-R) emitters. In the presence of target miRNA, DNAzyme was activated to cut substrate strands on the CdTe QDs and release triggers for opening hairpin probes. Then, the Cy5 molecule-labeled hairpin DNA on the RuSi NPs was opened to introduce Cy5 molecules and RuSi NPs into the system. The R-O ECL was quenched by ECL-RET between CdTe QDs and Cy5 molecules and the O-R ECL was introduced by the RuSi NPs. In this way, based on the simultaneous changing of the ECL signal, the dual-potential dynamic signal ratiometric ECL sensing platform was developed. By measuring the ratio of O-R ECL signal to R-O ECL signal, the concentration of miRNA-499 was accurately quantified in the range of 10 fM to 10 nM, and the detection limit was as low as 2.44 fM (S/N = 3). This DNAzyme guided dual-potential ratiometric ECL method provides a sensitive and reliable method for myocardial miRNA detection, and it has great potential in clinical diagnosis and treatment.

Band-pass filter-assisted ratiometric fluorescent nanoprobe composed of N-(2-aminoethyl-1,8-naphthalimide)-functionalized gold nanoclusters for the determination of alkaline phosphatase using digital image analysis.

A smartphone-based dual-wavelength digital imaging platform containing red (539-695 nm) and blue (389-511 nm) band-pass filters was developed for point-of-care (POC) testing of alkaline phosphatase (ALP) activity. The platform was based on dual-emitting fluorescent nanohybrids (AuNC@NAN), the ratiometric probe, which had a fluorescence "on-off-on-off" response. The probe comprised red-emitting gold nanoclusters (AuNCs) acting as the signal report units and blue-emitting N-(2-aminoethyl-1,8-naphthalimide) (NAN) acting as an internal reference. The different responses of the ratiometric probes resulted in a continuous color-multiplexing change from pink-red to dark-purple upon exposure to ALP. The dual-wavelength digital imaging platform was employed to acquire images of AuNC or NAN fluorescence signals without the influence of background light. Unlike the classical one-time digital imaging mode, the accurate red (R) and blue (B) channel values of the generated images can help to directly judge or eliminate the disturbance from unavoidable interfering factors. The R/B values were successfully employed for determining the ALP activity at a range 2.0 to 35.0 mU·mL-1 with the detection limit of 1.04 mU·mL-1. Such sensing imaging platform is also successful in determining ALP activity in human serum with 94.9-105% recoveries and relative standard deviation in the range 4.2-5.6%. A novel dual-wavelength smartphone-based digital imaging platform was proposed for simultaneous readout of the reporting and internal reference signals from dual-emitting ratiometric fluorescence probes, which allowed us to the accurate, reliable, and highly sensitive assay of ALP activity in complex samples.

A Multitargeted Electrochemiluminescent Biosensor Coupling DNAzyme with Cascading Amplification for Analyzing Myocardial miRNAs.

Several circulating miRNAs are associated with the pathogenic process of acute myocardial infarction (AMI). Thus, analyzing myocardial miRNAs in the circulatory system is important for the diagnosis and treatment of AMI, especially for early-stage diagnosis. Based on the characteristics of myocardial miRNAs, an ultrasensitive and multitargeted electrochemiluminescence (ECL) sensing platform was developed with a versatile probe that can couple DNAzyme with hybridization chain reaction amplification. The target miRNA and auxiliary chains form a circular unit that shears the versatile probe hairpin, and the products subsequently trigger cascading amplification; a long strand of dsDNA is then generated with many C-rich sequences that can undergo in situ reductions to generate ECL luminophore silver clusters. Using this strategy, three myocardial miRNAs are successfully detected with a detection limit as low as 29.6 aM (S/N = 3). Notably, our method can detect myocardial miRNA groups composed of multiple related circulating miRNAs with high selectivity over interfering miRNAs in blood. This is extremely important for solving the problem of diverse and low abundance of infarct-associated miRNAs. Our strategy pioneers a new idea of miRNA detection, and given its versatility and sensitivity, it is promising for the diagnosis of multigene-regulated cardiovascular diseases.

Recent progress of SERS optical nanosensors for miRNA analysis.

This review focuses on emerging applications of surface-enhanced Raman spectroscopy (SERS) optical nanosensors for miRNA analysis, in which the key enhancement factors of the SERS signal, i.e. SERS-active substrates, SERS nanoprobes and nano-assembly strategy, are emphasized. This article includes many nanomaterials for miRNA analysis by the SERS technique. We summarize these reported nanomaterials mainly according to their function in the miRNA assay biosensor. We also briefly summarize the research progress of these nanomaterials in SERS detection of intracellular miRNA. Finally, we discussed the prospect and limitations of SERS nanosensors for analyzing miRNA.

Hemin-Bridged MOF Interface with Double Amplification of G-Quadruplex Payload and DNAzyme Catalysis: Ultrasensitive Lasting Chemiluminescence MicroRNA Imaging.

Here, we report a double-amplified sensing platform for ultrasensitive chemiluminescence (CL) miRNA detection in real patients' blood in which a hemin-bridged metal-organic framework (MOF) is employed as a functional interface to boost the payload and catalysis of G-quadruplex (G4) DNAzymes. Hemin is here used as the organic ligand for the MOF synthesis, which endows the MOF with an intrinsic peroxidase-like catalytic activity. Most importantly, the MOF surface provides a large amount of binding sites for polymeric G4 DNAzymes that are produced by miRNA-triggered rolling circle amplification reactions, and meanwhile, the interfaced G4 DNAzymes on MOFs (G4/MOFzymes) display an about 100-fold higher catalytic activity than those in solution. By using the G4/MOFzyme catalysts in the luminol/H2O2 CL system, the amplification detection of two acute myocardial infarction (AMI)-related miRNAs (low to 1 fM seen with naked eyes) is achieved in human serum with a smartphone as a portable imaging detector, which provides a facile methodology for point-of-care (POC) diagnosis of AMI. Compared with previous smartphone-based counterparts not requiring sophisticated equipment, this new facile methodology shows both 6 orders of magnitude higher sensitivity and an ∼50-fold longer duration for CL miRNA imaging. These unique features allow our developed G4/MOFzymes to be further employed as a novel luminescent ink for printing commonly used patterns.

Target-Catalyzed Self-Growing Spherical Nucleic Acid Enzyme (SNAzyme) as a Double Amplifier for Ultrasensitive Chemiluminescence MicroRNA Detection.

Portable chemiluminescence (CL) imaging with a smartphone has shown a great promise for point-of-care testing of diseases, especially for acute myocardial infarction (AMI), which may occur abruptly. A challenge remains how to improve the imaging sensitivity that usually is several orders of magnitude lower than those of counterpart methodologies using the sophisticated equipment. Toward this goal, here, we report the target-triggered in situ growth of AuNP@hairpin-DNA nanoprobes into spherical nucleic acid enzymes (SNAzymes), which serve as both nanolabels and amplifiers for portable CL imaging of microRNAs (miRNAs) with an ultrahigh sensitivity comparable to that of the instrumental measurement under same conditions. A G-quadruplex (G4) DNA dense layer is dynamically produced on the gold nanocore via a DNAzyme machine-driven hairpin cleaving and captures the cofactor hemin to form the SNAzymes with higher peroxidase activity and stronger nuclease resistance than the commonly used G4 DNAzymes. The matured SNAzymes are then utilized as catalytic labels in a luminol-artesunate CL system for miRNA imaging with a smartphone as the portable detector. In this way, two AMI-related miRNAs, miRNA-499 and miRNA-133a, are successfully detected in real patients' serum with a naked eye-visualized CL change at 10 fM, showing a 5 order of magnitude improvement on the sensitivity of visualizing the same disease markers in clinical circulating blood as compared to the reported strategy. In addition, a good selectivity of our developed CL imaging platform is demonstrated. These unique features make it promising to employ this portable imaging platform for clinical AMI diagnosis.

Target-Induced Payload Amplification for Spherical Nucleic Acid Enzyme (SNAzyme)-Catalyzed Electrochemiluminescence Detection of Circulating microRNAs.

A high level of circulating myocardial microRNAs (miRNAs) is commonly considered as one indicator of acute myocardial infarction (AMI) for early stage diagnosis, and hence, it is of particular significance to develop ultrasensitive methodologies for detecting these miRNAs in circulating blood. Here we build an electrochemiluminescence (ECL) sensing platform for circulating miRNAs utilizing AuNPs@G-quadruplex (G4) spherical nucleic acid enzyme (SNAzyme) as the nanocatalyst, which shows good stability, strong nuclease resistance, and improved catalytic performance toward a luminol-H2O2 ECL system than the commonly used G4 DNAzyme. Target miRNA is employed to open the probe hairpin DNA to trigger the cascade amplification and then produce a long dsDNA chain with many sticky linkers that capture the SNAzyme nanocatalyst onto the electrode. In this way, two AMI-related miRNAs are detected accurately. The detection limit is 0.4 fM (S/N = 3), and the platform shows high selectivity in circulating blood. Given the good controllability, our ECL biosensors are promising for developing clinical diagnostic platforms for multiple indicators.

Spherical Nucleic Acid Enzyme (SNAzyme) Boosted Chemiluminescence miRNA Imaging Using a Smartphone.

As acute myocardial infarction (AMI) has now become a severe death threat to humans and may abruptly occur at home or outdoors where sophisticated equipment is not available, it is of great importance to develop facile methodologies for the point-of-care (POC) diagnosis of AMI. Toward this goal, here we build a sensing platform for chemiluminescence (CL) microRNA (miRNA) imaging with a smartphone as the portable detector, and for the first time we achieve visualization of AMI-related miRNAs in real patients' serum. We first construct a spherical nucleic acid enzyme (termed SNAzyme) derived from a dense layer of G-quadruplex (G4) DNAzyme formed on the gold nanoparticle core, which displays ∼100-fold and higher catalytic activity and improved resistance to nuclease degradation in a real blood sample as compared to those of the G4 DNAzyme itself. These unique features endow the SNAzyme-boosted CL platform with superior imaging performance for analyzing an AMI-related miRNA, miRNA-133a. This miRNA is employed to trigger the target-catalyzed hairpin assembly to produce a sticky dsDNA linker that captures the SNAzyme nanolabel onto the substrate. In this way, miRNA-133a is successfully detected, with a limit of detection of 0.3 pM (S/N = 3) and a high selectivity over other miRNA analogs in patients' blood. Given its unique features in physiological environments, our SNAzyme-boosted imaging platform holds great promise for use in the POC diagnosis of AMI.

Composition-Tunable Hollow Au/Ag SERS Nanoprobes Coupled with Target-Catalyzed Hairpin Assembly for Triple-Amplification Detection of miRNA.

Detecting disease-related biomarkers is of great significance for disease diagnosis and therapy. In this work, we develop an ultrasensitive surface-enhanced Raman scattering (SERS) biosensor for the detection of an acute myocardial infarction-related miRNA (miR-133a) using composition-adjustable hollow Ag/Au nanosphere-based SERS probes coupled with the target-catalyzed hairpin assembly (CHA) strategy. Bimetallic probes displaying high stability and a strong surface plasmon resonance effect were synthesized with a controllable ratio of silver and gold by a galvanic replacement method and then captured by a duplex linker produced in the CHA process to accomplish signal amplification. In this way, the target miR-133a can be detected in a wide linear range with a detection limit of 0.306 fM and high selectivity over other miRNAs expressed in human hearts. Practical applications in human blood samples reveal the strong anti-interference ability and ideal sensitivity of our developed sensing platform in physiological environments, benefiting its potential biomedical applications.

Ultrasensitive Simultaneous Detection of Multiplex Disease-Related Nucleic Acids Using Double-Enhanced Surface-Enhanced Raman Scattering Nanosensors.

Developing ultrasensitive probes holds great significance for simultaneous detection of multiplexed cancer-associated nucleic acids. Bimetallic nanoparticles containing silver may be exploited as nanoprobes for disease detection, which can produce stable and strong surface-enhanced Raman scattering (SERS) signals. However, it remains extremely challenging that such SERS nanoprobes are directly synthesized. Herein gold-silver nanosnowmen, grown via a DNA-mediated approach and attached to thiol-containing Raman dyes, are successfully synthesized. Stable SERS-enhanced gold substrates are also prepared and used as the enriching containers, where the capture DNAs are tethered to sense the target genes jointly enhanced by the SERS nanoprobes in a sandwich hybridization assay. This means detection of the target gene can obtain a limit of detection close to 0.839 fM. Such double-enhanced SERS nanosensors are further employed to simultaneously detect the three types of prostate carcinoma-related genes with high sensitivity and specificity, which meanwhile exhibit robust capacity of resisting disturbance in practical samples. Simultaneous and multiplexed detection of cancer-related genes may provide further biomedical applications with new opportunity.

Cellular environment-responsive intelligent DNA logic circuits for controllable molecular sensing.

Here we report smart molecular logic circuits built on a well-designed H-shaped DNA nanostructure that can recognize cell-simulated bioenvironments and modulate the operations of a DNA nanosensor. By assembling a wild-type ATP aptamer and a parallel G-quadruplex into the H-shaped DNA scaffold, two intrinsic cellular components, ATP and K+, are utilized to activate the logic circuits, enabling fluorescent detection of the target DNA via toehold-mediated strand displacement. In this way, two logic circuits consisting of cascaded "AND-AND" and "OR-AND" gates are achieved, which are responsive to the ATP and/or K+ concentration change outside and inside cells, and therefore control whether or not the downstream DNA sensor works. This work illustrates a novel concept for developing new bioinspired DNA molecular devices for not only programmable molecular sensing but also targeted drug delivery.

Probing the propeller-like loops of DNA G-quadruplexes with looped-out 2-aminopurine for label-free switchable molecular sensing.

We report a new signal readout mechanism for DNA molecular sensing devices using ligand-free fluorogenic G-quadruplexes in which the propeller-like loops are distinguished from the diagonal and lateral loops with incorporated 2-aminopurine (2-AP, a fluorescent analogue of adenine). We study the fluorescence behavior of looped-out 2-AP in duplexes and G-quadruplexes and demonstrate that it shows better fluorescence properties in shorter loops. In particular, 2-AP in the propeller-like loops of parallel or hybrid G-quadruplexes displays a perfect fluorescence emission whereas that in the diagonal and lateral loops does not. This loop-environment-sensitive feature allows 2-AP to probe the propeller-like loops of G-quadruplexes, illustrated by an ion-tuned allosteric G-quadruplex FG9A and a (3 + 1) hybrid human telomeric DNA. In the presence of K+, FG9A folds into a parallel structure where 2-AP is in the propeller-like loops and shows a high fluorescence signal, which can probe K+ concentrations down to 25 µM. Upon addition of Pb2+, the folded FG9A converts into an antiparallel structure which is revealed by a sharp decrease in 2-AP fluorescence, which can easily be reset with EDTA. This process is utilized to reversibly sense Pb2+ with a detection limit of 100 nM. Furthermore, its ability to probe the propeller-like loops may allow 2-AP to identify the folding topologies of unknown G-quadruplexes in human gene regions.

Exonuclease III-boosted cascade reactions for ultrasensitive SERS detection of nucleic acids.

A variety of nucleic acid amplification techniques have been integrated into different detection methods to promote the development of sensitive and convenient analysis of nucleic acids. However, it is still in urgent need to develop amplified nucleic acid biosensors for the analysis of susceptible gene and even distinguishing single-base mismatched DNA in complex biological samples. Benefiting from the achieved detection strategies, here we boost isothermal nucleic acid amplification by resorting to enzyme amplification, and combine this two-stage amplification method with surface-enhanced Raman spectroscopy (SERS) to develop a signal-on nucleic acid detection platform. Due to the high cleavage efficiency of Exonuclease III (Exo III), a large amount of trigger DNA are produced to initiate multiple hybridization chain reaction circles. The product structure tagged with Tamra is then anchored onto the plasmonic SERS substrate and meanwhile enriched. It is demonstrated that this detection platform is sensitive toward the myocardial infarction disease related gene. A detection limit of 1 fM for the gene analysis in a linear relationship in the wide range from 1 fM to 10nM is achieved, better than most of previous counterparts. Meanwhile, our developed detection platform exhibits a high selectivity for the target gene over mismatched analogues. Our strategy provides a robust tool for signal amplification of gene detection even in blood samples.

Three-dimensional hotspots in evaporating nanoparticle sols for ultrahigh Raman scattering: solid-liquid interface effects.

Three-dimensional (3D) hotspots for ultrahigh surface-enhanced Raman scattering (SERS) has been experimentally demonstrated by evaporating a droplet of citrate-Ag sols on both hydrophobic and hydrophilic flat surfaces. Interestingly, the hydrophobic surface increased the Raman enhancement by two orders of magnitude and exhibits a better signal stability than the hydrophilic one. This study highlights the differences between hydrophilic and hydrophobic surfaces in enhanced Raman scattering by the use of extremely diluted rhodamine 6G (R6G) as the SERS reporter. In situ synchrotron-radiation small-angle X-ray scattering (SR-SAXS) was employed to explore the evolution of the 3D geometry of Ag nanoparticles in a single droplet and verify the influence mechanism of these two kinds of surface. The ideal situation of 3D self-assembly of nanoparticles in the evaporation process is a collaborative behaviour, but our results evidenced that a progressive 3D self-assembly of nanoparticles was more preferred due to the interface effects. Our experimental data derived from in situ SR-SAXS reveals that a truly distinct 3D geometry of the Ag particles develops during the evaporation process on both hydrophilic and hydrophobic surfaces. In this type of 3D geometry, the increased uniformity of the interparticle distance induced a sharp peak of the SR-SAXS signal, differing significantly from the dry state. In particular, the fluorosilylated surface reduces the interaction with particles and decreases the electrostatic adsorption on the flat surface, which helps to control the interparticle distance to remain within a small range, produce a larger number of hotspots in 3D space, and amplify the SERS enhancement accordingly.

Unravelling the relationship between Raman enhancement and photocatalytic activity on single anisotropic Au microplates.

Anisotropic noble-metal structures are attracting increasing attention because of interesting size- and shape-dependent properties and have emerging applications in the fields of optics and catalysis. However, it remains a significant challenge to overcome chemical contributions and acquire molecular insight into the relationship between Raman enhancement and photocatalytic activity. This study gives visualized experimental evidence of the anisotropic spatial distribution of Raman signals and photocatalytic activity at the level of single nanometer-thin Au microtriangles and microhexagons. Theoretical simulations indicate an anisotropic spatial distribution and sharpness-dependent strength of the electric-field enhancement. Analysis by using statistical surface-enhanced Raman scattering (SERS) supports this view, that is, Raman enhancement is on the order of corner>edge>face for a single microplate, but SERS measurements at different depths of focus also imply a concentration-dependent feature of SERS signals, especially at the corners and edges. Similarly, the SERS signals of product molecules in plasmonic photocatalysis also exhibit asymmetrical strengths at different corners of the same microplate. However, by examining the variations in the relative intensities of the SERS peaks, the difference in the photocatalytic activities at the corners, edges, and faces has been successfully calculated and is highly consistent with electric-field simulations, thus indicating that an increased number of molecules adsorbed at specific sites does not necessarily lead to a higher conversion ratio in noble-metal photocatalysis. Our strategy weakens the assumed impact of plasmonic local heating and, to a certain extent, excludes the influence of concentration effects and chemical contributions in noble-metal photocatalysis, thus clearly profiling plasmon-related characteristics. This study also promises a new research direction to understand the enhancement mechanism of SERS-active structures.

Three-dimensional and time-ordered surface-enhanced Raman scattering hotspot matrix.

The "fixed" or "flexible" design of plasmonic hotspots is a frontier area of research in the field of surface-enhanced Raman scattering (SERS). Most reported SERS hotspots have been shown to exist in zero-dimensional point-like, one-dimensional linear, or two-dimensional planar geometries. Here, we demonstrate a novel three-dimensional (3D) hotspot matrix that can hold hotspots between every two adjacent particles in 3D space, simply achieved by evaporating a droplet of citrate-Ag sols on a fluorosilylated silicon wafer. In situ synchrotron-radiation small-angle X-ray scattering (SR-SAXS), combined with dark-field microscopy and in situ micro-UV, was employed to explore the evolution of the 3D geometry and plasmonic properties of Ag nanoparticles in a single droplet. In such a droplet, there is a distinct 3D geometry with minimal polydispersity of particle size and maximal uniformity of interparticle distance, significantly different from the dry state. According to theoretical simulations, the liquid adhesive force promotes a closely packed assembly of particles, and the interparticle distance is not fixed but can be balanced in a small range by the interplay of the van der Waals attraction and electrostatic repulsion experienced by a particle. The "trapping well" for immobilizing particles in 3D space can result in a large number of hotspots in a 3D geometry. Both theoretical and experimental results demonstrate that the 3D hotspots are predictable and time-ordered in the absence of any sample manipulation. Use of the matrix not only produces giant Raman enhancement at least 2 orders of magnitude larger than that of dried substrates, but also provides the structural basis for trapping molecules. Even a single molecule of resonant dye can generate a large SERS signal. With a portable Raman spectrometer, the detection capability is also greatly improved for various analytes with different natures, including pesticides and drugs. This 3D hotspot matrix overcomes the long-standing limitations of SERS for the ultrasensitive characterization of various substrates and analytes and promises to transform SERS into a practical analytical technique.