A ratiometric SERS aptasensor based on catalytic hairpin self-assembly mediated cyclic signal amplification strategy for the reliable determination of E. coli O157:H7.

A ratiometric SERS aptasensor based on catalytic hairpin self-assembly (CHA) mediated cyclic signal amplification strategy was developed for the rapid and reliable determination of Escherichia coli O157:H7. The recognition probe was synthesized by modifying magnetic beads with blocked aptamers, and the SERS probe was constructed by functionalizing gold nanoparticles (Au NPs) with hairpin structured DNA and 4-mercaptobenzonitrile (4-MBN). The recognition probe captured E. coli O157:H7 specifically and released the blocker DNA, which activated the CHA reaction on the SERS probe and turned on the SERS signal of 6-carboxyl-x-rhodamine (ROX). Meanwhile, 4-MBN was used as an internal reference to calibrate the matrix interference. Thus, sensitive and reliable determination and quantification of E. coli O157:H7 was established using the ratio of the SERS signal intensities of ROX to 4-MBN. This aptasensor enabled detection of 2.44 × 102 CFU/mL of E. coli O157:H7 in approximately 3 h without pre-culture and DNA extraction. In addition, good reliability and excellent reproducibility were observed for the determination of E. coli O157:H7 in spiked water and milk samples. This study offered a new solution for the design of rapid, sensitive, and reliable SERS aptasensors.

Recent advances of sensing strategies for the detection of ß-glucuronidase activity.

ß-Glucuronidase (ß-GLU), a kind of hydrolase, is widely distributed in mammalian tissues, body fluids, and microbiota. Abnormal changes of ß-GLU activity are often correlated with the occurrence of diseases and deterioration of water quality. Therefore, detection of ß-GLU activity is of great significance in biomedicine and environmental health such as cancer diagnosis and water monitoring. However, the conventional ß-GLU activity assay suffers from the limitations of low sensitivity, poor accuracy, and complex procedure. With the development of analytical chemistry, many advances have been made in the detection of ß-GLU activity in recent years. The sensors for ß-GLU activity detection which have the advantages of rapid and reliable detection have been attracting increased attentions. In this paper, the principles, performances, and limitations of these ß-GLU sensors, including colorimetric sensing, fluorescent sensing, electrochemical sensing for the determination of ß-GLU activity, have been summarized and discussed. Moreover, the challenges and research trends of ß-GLU activity assay are proposed.

Reliable and Rapid Detection and Quantification of Enrofloxacin Using a Ratiometric SERS Aptasensor.

Reliable detection and quantification of antibiotic residues in food using surface-enhanced Raman spectroscopy remain challenging, since the intensities of SERS signals are vulnerable to matrix and experimental factors. In this work, a ratiometric SERS aptasensor using 6-Carboxyl-X-Rhodamine (ROX)-labeled aptamers and 4-mercaptobenzonitrile (4-MBN)-functionalized gold nanoparticles (Au NPs) as SERS probes was established for the reliable and rapid detection and quantification of enrofloxacin. In the presence of enrofloxacin, the conformational transform of aptamers took place, and the distance between ROX and Au NP increased, which resulted in a decrease in the SERS signal intensity of ROX. Meanwhile, the intensity of the SERS signal of 4-MBN was used as an internal standard. Reliable determination of enrofloxacin was realized using the ratio of the SERS signal intensities of ROX to 4-MBN. Under optimal conditions, the developed ratiometric SERS aptasensor provided a wide linear range from 5 nM to 1 µM, with a correlation coefficient (R2) of 0.98 and a limit of detection (LOD) of 0.12 nM (0.043 ppb). In addition, the developed ratiometric SERS aptasensor was successfully applied for the determination of enrofloxacin in fish and chicken meat, with recovery values of 93.6-112.0%. Therefore, the established ratiometric SERS aptasensor is sensitive, reliable, time-efficient, and has the potential to be applied in the on-site detection of enrofloxacin in complex matrices.

Recent Advances in Metal Organic Frameworks Based Surface Enhanced Raman Scattering Substrates: Synthesis and Applications.

Metal-organic frameworks (MOFs) are supramolecular nanomaterials, in which metal ions or clusters are connected by organic ligands to form crystalline lattices with highly ordered periodic porous network structure. MOFs have been widely applied in various fields, such as catalyst, sample preparation, and sensing. In recent years, MOFs based surface enhanced Raman scattering (SERS) substrates have attracted much attention since MOFs can largely improve the performance of metallic SERS substrates toward target enrichment and signal enhancement. MOFs have been exploited in SERS analysis to tackle some challenges that bare metal substrates cannot achieve. Combination of MOFs and SERS improved the sensitivity of traditional SERS analysis and extended the application scope of SERS. With the increasing exploration of MOFs based SERS substrates, there is a great demand to review the advances in these researches. Herein, this review concentrated on summarizing the preparation and applications of MOFs based SERS substrates. Representative researches were discussed to better understand the property of MOFs based SERS substrates. The advantages of MOFs based SERS substrates were highlighted, as well as their limitations. In addition, the challenges, opportunities, and future trends in MOFs based SERS analysis were tentatively discussed.

CARM1-mediated methylation of protein arginine methyltransferase 5 represses human γ-globin gene expression in erythroleukemia cells.

Protein arginine methyltransferase 5 (PRMT5) is a member of the arginine methyltransferase protein family that critically mediates the symmetric dimethylation of Arg-3 at histone H4 (H4R3me2s) and is involved in many key cellular processes, including hematopoiesis. However, the post-translational modifications (PTMs) of PRMT5 that may affect its biological functions remain less well-understood. In this study, using MS analyses, we found that PRMT5 itself is methylated in human erythroleukemia Lys-562 cells. Biochemical assays revealed that coactivator-associated arginine methyltransferase 1 (CARM1) interacts directly with and methylates PRMT5 at Arg-505 both in vivo and in vitro. Substitutions at Arg-505 significantly reduced PRMT5's methyltransferase activity, decreased H4R3me2s enrichment at the γ-globin gene promoter, and increased the expression of the γ-globin gene in Lys-562 cells. Moreover, CARM1 knockdown consistently reduced PRMT5 activity and activated γ-globin gene expression. Importantly, we show that CARM1-mediated methylation of PRMT5 is essential for the intracellular homodimerization of PRMT5 to its active form. These results thus reveal a critical PTM of PRMT5 that represses human γ-globin gene expression. We conclude that CARM1-mediated asymmetric methylation of PRMT5 is critical for its dimerization and methyltransferase activity leading to the repression of γ-globin expression. Given PRMT5's crucial role in diverse cellular processes, these findings may inform strategies for manipulating its methyltransferase activity for managing hemoglobinopathy or cancer.

Ratiometric Surface Enhanced Raman Scattering Immunosorbent Assay of Allergenic Proteins via Covalent Organic Framework Composite Material Based Nanozyme Tag Triggered Raman Signal "Turn-on" and Amplification.

The exploration of nanomaterials with mimic enzyme activity (named nanozyme) has gained extensive attention in the fields of advanced analytical chemistry and materials science. Herein, the gold nanoparticles doped covalent organic frameworks (COFs) were prepared, which exhibited not only excellent mimic nitroreductase activity but also robust stability. By replacing the traditional natural enzyme tag in an enzyme-linked immunosorbent assay (ELISA), we employed the proposed nanozyme to label the detecting antibody. According to the catalytic properties of the nanozyme, 4-nitrothiophenol (4-NTP) was introduced as the substrate, which can be transformed to 4-aminothiophenol (4-ATP) in the presence of NaBH4. In a surface enhanced Raman scattering (SERS) assay, 4-ATP was capable of functioning as a powerful bridge to connect the gold nanostars (with excellent SERS performance) by both the Au-S bond and electrostatic force to further produce a Raman "hot spot". Meanwhile, the Raman signal of 4-nitrothiophenol at 1573 cm-1 was weakened, and a new signal at 1591 cm-1 generated by 4-ATP was turned on, leading to the generation of a ratiometric SERS signal. Based on this performance, a ratiometric nanozyme-linked immunosorbent assay (NELISA) strategy was developed delicately, which was applied to detect ß-lactoglobulin (allergenic protein) by monitoring the ratiometric signal of I1591/I1573 with a limit of detection (LOD) of 0.01 ng/mL. The linear range is 25.65-6.2 × 104 ng/mL, covering more than 3 orders of magnitude. The developed method showed many advantages such as low-cost, higher recovery, and lower cross-reactivity, providing new insight into the application of SERS technology for trace target analysis.

Rapid concentration detection and differentiation of bacteria in skimmed milk using surface enhanced Raman scattering mapping on 4-mercaptophenylboronic acid functionalized silver dendrites.

A novel method was developed to rapidly concentrate, detect, and differentiate bacteria in skimmed milk using surface enhanced Raman scattering (SERS) mapping on 4-mercaptophenylboronic acid (4-MPBA) functionalized silver (Ag) dendrites. The 4-MPBA functionalized Ag dendritic SERS substrate was used to capture the bacterial cells and enhance the bacterial signal. Salmonella, a significantly important food pathogen, was used as the representative strain to optimize and evaluate the developed method. The capture efficiency for Salmonella enterica subsp enterica BAA1045 (SE1045) was 84.92 ± 3.25% at 106 CFU/mL and as high as 99.65 ± 3.58% at 103 CFU/mL. Four different strains, two gram-negative and two gram-positive, can be clearly distinguished by their SERS spectra using principle component analysis. A mapping technique was utilized to automatically collect 400 spectra over an area of 60 µm × 60 µm to construct a visual image for a sensitive and statistically reliable detection within 30 min. Using this method, we were able to detect as low as 103 CFU/mL bacterial cells in 50 mM NH4HCO3 solution and 102 CFU/mL cells in both 1% casein and skimmed milk. Our results demonstrate the feasibility of using SERS mapping method coupled with 4-MPBA functionalized Ag dendrites for rapid and sensitive bacteria detection in complex liquid samples. Graphical Abstract A novel SERS mapping method based on 4-mercaptophenylboronic acid functionalized silver (Ag) dendrites was developed to rapidly concentrate, detect, and differentiate bacteria.

Label-free mapping of single bacterial cells using surface-enhanced Raman spectroscopy.

Here we presented a simple, rapid and label-free surface-enhanced Raman spectroscopy (SERS) based mapping method for the detection and discrimination of Salmonella enterica and Escherichia coli on silver dendrites. The sample preparation was first optimized to maximize sensitivity. The mapping method was then used to scan through the bacterial cells adsorbed on the surface of silver dendrites. The intrinsic and distinct SERS signals of bacterial cells were used as the basis for label-free detection and discrimination. The results show the developed method is able to detect single bacterial cells adsorbed on the silver dendrites with a limit of detection as low as 10(4) CFU mL(-1), which is two orders of magnitude lower than the traditional SERS method under the same experimental condition. The time needed for collecting a 225 points map was approximately 24 minutes. Moreover, the developed SERS mapping method can realize simultaneous detection and identification of Salmonella enterica subsp. enterica BAA1045 and Escherichia coli BL21 from a mixture sample using principle component analysis. Our results demonstrate the great potential of the label-free SERS mapping method to detect, identify and quantify bacteria and bacterial mixtures simultaneously.

Characterization of Lactococcus lactis response to ampicillin and ciprofloxacin using surface-enhanced Raman spectroscopy.

Decades of antibiotic use or misuse has resulted in antibiotic resistance in lactic acid bacteria, a group of common culture starters and probiotic microorganisms. This has urged researchers to study how lactic acid bacteria respond to antibiotics, so as to have a better strategy to identify and predict the antibiotic-resistant bacteria. This study aimed to characterize the biochemical profiles of Lactococcus lactis responding to antibiotics using surface-enhanced Raman spectroscopy (SERS). Lactococcus lactis exposed to antibiotics was mixed with 50-nm gold nanoparticles for subsequent SERS measurements. The SERS spectra analyzed by principal component analysis showed no significant change after 30 min of antibiotic treatment, whereas distinct changes were clearly observed after 60 and 90 min of antibiotic treatment. Different antibiotics induced different spectral changes, and these changes revealed the detailed biochemical information of cellular responses. This study demonstrates that the SERS method developed not only senses the changes in the bacterial cell wall, but also reveals details of the biochemical profiles, which help us to understand how lactic acid bacteria respond to antibiotics, as well as to set a base for the detection of antibiotic susceptibility of bacteria by SERS.

Recent Progress on Toxicity and Detection Methods of Polychlorinated Biphenyls in Environment and Foodstuffs.

Polychlorinated biphenyls (PCBs), a class of synthetic organochlorine chemicals, were broadly employed in industrial and commercial applications in the last century due to their good thermal and chemical stability. However, PCBs have a great influence on both individual organism and the entire ecosystem. It has been proven that PCBs pose potential risks to human health with neurotoxicity, carcinogenicity, reproductive toxicity, immunotoxicity, hepatotoxicity, and cardiovascular toxicity. Moreover, PCBs exhibit the long-range transport effect on the global scale and bio-enrichment effect along the food chains. This review mainly encompasses recent progress on the toxicity and detection techniques of PCBs in environment and foodstuffs. First of all, we highlighted the latest improvements and achievements of the classification, source, distribution, and toxicity of PCBs. Then, comprehensive summaries of the current technologies for sample preparation (e.g., SPE, DSPE, SPME and SBSE) and analytical determination (e.g., GC-ECD, GC-MS, GC-HRMS, HPLC-MS/MS and sensing technologies) were given. In the end, the shortcomings and prospects of the pretreatment methods for PCBs analysis as well as the future opportunities and challenges are tentatively discussed.

Label-free quantitative proteomic analysis of the mechanism of salt stress promoting selenium enrichment in Lactobacillus rhamnosus.

Lactobacillus rhamnosus can metabolize selenite into organic selenium and Se0. In this paper, label-free quantitative proteomics was applied to explore the mechanism of salt stress promoting selenium enrichment of L.rhamnosus. 397 proteins were up-regulated and 147 proteins were down-regulated of selenium-enriched L.rhamnosus under salt stress. The differentially expressed proteins (DEPs) were mainly involved in metabolism, membrane transport and genetic information processing. The results of quantitative real-time PCR showed that gene opuA, metN, trxR and ldh of Se-enriched L.rhamnosus with salt stress were significantly up-regulated. However, the expression levels of gene luxS, groEL, dnaK and pgk were down-regulated. It was indicated that L.rhamnosus promoted part of amino acids combining with selenium into selenoamino acids with salt stress. Secondly, sodium chloride stimulated the expression of key enzymes involved in metabolism to provide energy for the process of Se-enrichment. In addition, NaCl induced the expression of enzymes and genes involved in the synthesis of selenoproteins. SIGNIFICANCE: It was indicated that L.rhamnosus promoted part of amino acids combining with selenium into selenoamino acids with salt stress. Secondly, sodium chloride stimulated the expression of key enzymes involved in metabolism to provide energy for the process of Se-enrichment. In addition, NaCl induced the expression of enzymes and genes involved in the synthesis of selenoproteins.

Highly sensitive multiplex detection of foodborne pathogens using a SERS immunosensor combined with novel covalent organic frameworks based biologic interference-free Raman tags.

Rapid and reliable multiplex detection of foodborne pathogens is in great demand for ensuring food safety and preventing foodborne diseases. In this study, we developed a highly sensitive SERS immunosensor for the simultaneous detection of multiple foodborne pathogens. Novel biologic interference-free Raman tags synthesized by using the covalent organic frameworks (COF) TBDP as nanocontainer to load biologic interference-free Raman reporters and specific antibodies for interested targets were used to convert and amplify signals of foodborne pathogens. In addition, lectin functionalized magnetic nanoparticles (MNPs@Con A) which could efficiently bind to the carbohydrate constituents on the surface of pathogens were prepared to capture and isolate multiple pathogens simultaneously. The recognition of the target foodborne pathogen impels the generation of sandwich-like composites of MNPs@Con A/pathogen/TBDP@Raman tags, and these composites could be quickly separated from the sample matrix with the assistance of an external magnet. Besides, a mass of Raman reporters was released by eluting the collected MNPs@Con A/pathogen/TBDP@Raman tags composites. Combined with a portable Raman system, characteristic Raman signals (2271 and 2113 cm-1) of the occupied reporters located at the biologic interference-free region were observed and used for the simultaneous detection of two different foodborne pathogenic strains. And an equal limit of detection of 101 CFU/mL was achieved for each strain. This strategy provides new insight into the application of SERS in the detection of pathogenic bacteria.

Ultrasensitive CRISPR/Cas12a-Driven SERS Biosensor for On-Site Nucleic Acid Detection and Its Application to Milk Authenticity Testing.

An ultrasensitive surface-enhanced Raman scattering (SERS) biosensor driven by CRISPR/Cas12a was proposed for on-site nucleic acid detection. We tactfully modified single-strand DNA (ssDNA) with a target-responsive Prussian blue (PB) nanolabel to form a probe and fastened it in the microplate. Attributed to the specific base pairing and highly efficient trans-cleavage ability of the CRISPR/Cas12a effector, precise target DNA recognition and signal amplification can be achieved, respectively. In the presence of target DNA, trans-cleavage towards the probe was activated, leading to the release of a certain number of PB nanoparticles (NPs). Then, these free PB NPs would be removed. Under alkali treatment, the breakdown of the remaining PB NPs in the microplate was triggered, producing massive ferricyanide anions (Fe(CN)64-), which could exhibit a unique characteristic Raman peak that was located in the "biological Raman-silent region". By mixing the alkali-treated solution with the SERS substrate, Au@Ag core-shell NP, the concentration of the target DNA was finally exhibited as SERS signals with undisturbed background, which can be detected by a portable Raman spectrometer. Importantly, this strategy could display an ultralow detection limit of 224 aM for target DNA. Furthermore, by targeting cow milk as the adulterated ingredient in goat milk, the proposed biosensor was successfully applied to milk authenticity detection.

Rapid and reliable detection and quantification of organophosphorus pesticides using SERS combined with dispersive liquid-liquid microextraction.

Rapid and reliable detection and quantification of pesticide residues in complex matrices by surface enhanced Raman spectroscopy (SERS) remain challenging due to the low level of target molecules and the interference of nontarget components. In this study, SERS was combined with dispersive liquid-liquid microextraction (DLLME) to develop a rapid and reliable method for the detection of organophosphorus pesticides (OPPs). In this method, DLLME was used to extract and enrich two representative OPPs (triazophos and parathion-methyl) from a liquid sample, and a portable Raman spectrometer was used to analyze the separated sediment using homemade gold nanoparticles colloids as enhancing substrates. The results showed that the developed method displayed good sensitivity and stability for the detection and quantification of triazophos and parathion-methyl with R2 ≥ 0.98. The calculated limits of detection (LODs) in the simultaneous detection of triazophos and parathion-methyl were 2.17 × 10-9 M (0.679 ppb) and 2.28 × 10-8 M (5.998 ppb), and the calculated limits of quantification (LOQs) were 7.23 × 10-9 M (2.26 ppb) and 7.62 × 10-8 M (19.098 ppb), respectively. Furthermore, the developed SERS method was successfully applied to the detection of triazophos and parathion-methyl in apple juice with recoveries between 78.07% and 110.87% and relative standard deviations (RSDs) ≤ 2.06%. Therefore, the developed DLLME facilitated liquid SERS method exhibited good sensitivity and stability for the rapid detection and quantification of OPPs and had the potential to be applied to the rapid detection of OPPs in complex matrices.

Recent advances in dual recognition based surface enhanced Raman scattering for pathogenic bacteria detection: A review.

Rapid and reliable detection of pathogenic bacteria at the early stage represents a highly topical research area for food safety and public health. Although culture based method is the gold standard method for bacteria detection, recent techniques have promoted the development of alternative methods, such as surface enhanced Raman scattering (SERS). SERS provides additional advantages of high speed, simultaneous detection and characterization, multiplex analysis, and comparatively low cost. However, conventional SERS methods for bacteria detection are facing limitations of low sensitivity, susceptible to matrix interference, and poor accuracy. In recent years, specific detection of pathogenic bacteria with dual recognition based SERS methods has attracted increasing attentions. These methods include two steps recognition of target bacteria, and integrate the functions of target separation and detection. Considering their merits of excellent specificity, ultrahigh sensitivity, multiplex detection capability, and potential for on-site applications, these methods are promising alternatives for rapid and reliable detection of pathogenic bacteria. Herein, this review aims to summarize the recent advances in dual recognition based SERS methods for specific detection of pathogenic bacteria. Their advantages and limitations are discussed, and further perspectives are tentatively given. This review provides new insights into the application of SERS as a reliable tool for pathogenic bacteria detection.

Thiol radical-based chemical isotope labelling for sterols quantitation through high performance liquid chromatography-tandem mass spectrometry analysis.

Enlightened by the high specificity and reactivity of thiol radical toward allyl, here, we first established a rapid thiol radical-based chemical isotope-labelling (CIL) strategy coupled with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis for the quantitative profiling of sterols. In this strategy, N-(4-(carbazole-9-yl)-phenyl)-N-maleimide labelled derivative of ethylenedithiol (NCPM-d0-SH) and its deuterated analogue NCPM-d2-SH were employed as a novel pair of CIL reagents to efficiently label sterols. Under lighting condition, the thiol radical obtained from NCPM-d0/d2-SH attacks one allyl hydrogen in the B-ring of sterols to produce a reactive radical intermediate which can quickly react with another thiol radical to form the last labelled derivatives. This labelling reaction can rapidly complete only within 1.5 min. Absorbingly, the NCPM-d0-SH and NCPM-d2-SH labelled derivatives of sterols can produce two specific product ions (PIs) containing different isotope tags at m/z of 431.6 and 433.6 via collision induced dissociation, which were employed to develop the multiple reaction monitoring (MRM) mode-based analysis. According to the specific mass differences with a fixed value, the peak pairs with similar retention times can be easily extracted from the two PIs spectrums and designated as the candidates for the identification of sterols. NCPM-d0-SH and NCPM-d2-SH labelled derivatives of sterols can be readily distinguished from their several ion chromatograms. Thus, sterols from two samples labelled by different isotope tags were ionized at the same conditions and measured respectively, providing excellent identification and precise quantitation by compensating the matrix effect and instrument fluctuation during MS-based analysis. The detection sensitivities of thiol-containing drugs improved by 53.5-560.3-fold due to NCPM-labelling. The limits of detection (LODs) and the limits of quantitation (LOQs) were in the range of 0.15-0.40 µg kg-1 and 0.50-1.30 µg kg-1, respectively. Using the developed method, we quantitatively profiled five sterols in vegetable oils with good applicability. As promising, the proposed thiol radical-based CIL strategy is a potential platform for the quantitation of sterols.

Purification and characterization of a novel phloretin-2'-O-glycosyltransferase favoring phloridzin biosynthesis.

Phloretin-2'-O-glycosyltransferase (P2'GT) catalyzes the last glycosylation step in the biosynthesis of phloridzin that contributes to the flavor, color and health benefits of apples and processed apple products. In this work, a novel P2'GT of Malus x domestica (MdP2'GT) with a specific activity of 46.82 µkat/Kg protein toward phloretin and uridine diphosphate glucose (UDPG) at an optimal temperature of 30 °C and pH 8.0 was purified from the engineered Pichia pastoris broth to homogeneity by anion exchange chromatography, His-Trap affinity chromatography and gel filtration. The purified MdP2'GT was low N-glycosylated and secreted as a stable dimer with a molecular mass of 70.7 kDa in its native form. Importantly, MdP2'GT also exhibited activity towards quercetin and adenosine diphosphate glucose (ADPG), kaempferol and UDPG, quercetin and UDP-galactose, isoliquiritigenin and UDPG, and luteolin and UDPG, producing only one isoquercitrin, astragalin, hyperoside, isoliquiritin, or cynaroside, respectively. This broad spectrum of activities make MdP2'GT a promising biocatalyst for the industrial preparation of the corresponding polyphenol glycosides, preferably for their subsequent isolation and purification. Besides, MdP2'GT displayed the lowest Km and the highest kcat/Km for phloretin and UDPG compared to all previously reported P2'GTs, making MdP2'GT favor phloridzin synthesis the most.

Nanoimprinted Patterned Pillar Substrates for Surface-Enhanced Raman Scattering Applications.

A pragmatic method to deposit silver nanoparticles on polydopamine-coated nanoimprinted pillars for use as surface-enhanced Raman scattering (SERS) substrates was developed. Pillar arrays consisting of poly(methyl methacrylate) (PMMA) that ranged in diameter from 300 to 500 nm were fabricated using nanoimprint lithography. The arrays had periodicities from 0.6 to 4.0 µm. A polydopamine layer was coated on the pillars in order to facilitate the reduction of silver ions to create silver nucleation sites during the electroless deposition of sliver nanoparticles. The size and density of silver nanoparticles were controlled by adjusting the growth time for the optimization of the SERS performance. The size of the surface-adhered nanoparticles ranged between 75 and 175 nm, and the average particle density was ∼30 particles per µm(2). These functionalized arrays had a high sensitivity and excellent signal reproducibility for the SERS-based detection of 4-methoxybenzoic acid. The substrates were also able to allow the SERS-based differentiation of three types of bacteriophages (λ, T3, and T7).