Whole community and functional gene changes of biofilms on marine plastic debris in response to ocean acidification.

Plastics are accumulating in the world's oceans, while ocean waters are becoming acidified by increased CO2. We compared metagenome of biofilms on tethered plastic bottles in subtidal waters off Japan naturally enriched in CO2, compared to normal ambient CO2 levels. Extending from an earlier amplicon study of bacteria, we used metagenomics to provide direct insights into changes in the full range of functional genes and the entire taxonomic tree of life in the context of the changing plastisphere. We found changes in the taxonomic community composition of all branches of life. This included a large increase in diatom relative abundance across the treatments but a decrease in diatom diversity. Network complexity among families decreased with acidification, showing overall simplification of biofilm integration. With acidification, there was decreased prevalence of genes associated with cell-cell interactions and antibiotic resistance, decreased detoxification genes, and increased stress tolerance genes. There were few nutrient cycling gene changes, suggesting that the role of plastisphere biofilms in nutrient processes within an acidified ocean may not change greatly. Our results suggest that as ocean CO2 increases, the plastisphere will undergo broad-ranging changes in both functional and taxonomic composition, especially the ecologically important diatom group, with possible wider implications for ocean ecology.

Circulating tumor DNA sequencing in colorectal cancer patients treated with first-line chemotherapy with anti-EGFR.

Circulating tumor DNA (ctDNA) may reveal dynamic tumor status during therapy. We conducted serial ctDNA analysis to investigate potential association with clinical outcome in metastatic colorectal cancer (mCRC) patients receiving chemotherapy. Tissue KRAS/NRAS wild-type mCRC patients were enrolled and treated with first-line cetuximab-containing chemotherapy. ctDNA isolated from plasma were analyzed by next generation sequencing (NGS) with 16 targeted gene panel. Among 93 patients, 84 (90.3%) had at least 1 somatic mutation in baseline ctDNA samples (average 2.74). Five patients with KRAS or NRAS hotspot mutation in the ctDNA showed significantly worse progression-free survival (PFS) (p = 0.029). Changes in average variant allele frequency (VAF) in ctDNA showed significant correlation with tumor size change at the time of first response evaluation (p = 0.020) and progressive disease (PD) (p = 0.042). Patients whose average VAF decreased below cutoff (< 1%) at the first evaluation showed significantly better PFS (p < 0.001), and the average VAF change further discriminated the PFS in the patients in partial response (p = 0.018). At the time of PD, 54 new mutations including KRAS and MAP2K1 emerged in ctDNA. ctDNA sequencing can provide mutation profile that could better reflect tumor mutation status and predict treatment outcome.

Metagenomic analysis reveals rapid development of soil biota on fresh volcanic ash.

Little is known of the earliest stages of soil biota development of volcanic ash, and how rapidly it can proceed. We investigated the potential for soil biota development during the first 3 years, using outdoor mesocosms of sterile, freshly fallen volcanic ash from the Sakurajima volcano, Japan. Mesocosms were positioned in a range of climates across Japan and compared over 3 years, against the developed soils of surrounding natural ecosystems. DNA was extracted from mesocosms and community composition assessed using 16S rRNA gene sequences. Metagenome sequences were obtained using shotgun metagenome sequencing. While at 12 months there was insufficient DNA for sequencing, by 24 months and 36 months, the ash-soil metagenomes already showed a similar diversity of functional genes to the developed soils, with a similar range of functions. In a surprising contrast with our hypotheses, we found that the developing ash-soil community already showed a similar gene function diversity, phylum diversity and overall relative abundances of kingdoms of life when compared to developed forest soils. The ash mesocosms also did not show any increased relative abundance of genes associated with autotrophy (rbc, coxL), nor increased relative abundance of genes that are associated with acquisition of nutrients from abiotic sources (nifH). Although gene identities and taxonomic affinities in the developing ash-soils are to some extent distinct from the natural vegetation soils, it is surprising that so many of the key components of a soil community develop already by the 24-month stage. In this system, however, rapid development may be facilitated by the relatively moderate pH of the Sakurajima ash, proximity of our mesocosms to propagule sources, and the rapid establishment of a productive bryophyte and lichen layer on the surface. Ash from other volcanoes richer in acids or more distant from propagule sources could show a different pattern and slower soil biota development.

Development and Validation of a Next-Generation Sequencing-Based Multigene Assay to Predict the Prognosis of Estrogen Receptor-Positive, HER2-Negative Breast Cancer.

PURPOSE: Multigene assays provide useful prognostic information regarding hormone receptor (HR)-positive breast cancer. Next-generation sequencing (NGS)-based platforms have numerous advantages including reproducibility and adaptability in local laboratories. This study aimed to develop and validate an NGS-based multigene assay to predict the distant recurrence risk. EXPERIMENTAL DESIGN: In total, 179 genes including 30 reference genes highly correlated with the 21-gene recurrence score (RS) algorithm were selected from public databases. Targeted RNA-sequencing was performed using 250 and 93 archived breast cancer samples with a known RS in the training and verification sets, respectively, to develop the algorithm and NGS-Prognostic Score (NGS-PS). The assay was validated in 413 independent samples with long-term follow-up data on distant metastasis. RESULTS: In the verification set, the NGS-PS and 21-gene RS displayed 91.4% concurrence (85/93 samples). In the validation cohort of 413 samples, area under the receiver operating characteristic curve plotted using NGS-PS values classified for distant recurrence was 0.76. The best NGS-PS cut-off value predicting distant metastasis was 20. Furthermore, 269 and 144 patients were classified as low- and high-risk patients in accordance with the cut-off. Five- and 10-year estimates of distant metastasis-free survival (DMFS) for low- versus high-risk groups were 97.0% versus 77.8% and 93.2% versus 64.4%, respectively. The age-related HR for distant recurrence without chemotherapy was 9.73 (95% CI, 3.59-26.40) and 3.19 (95% CI, 1.40-7.29) for patients aged ≤50 and >50 years, respectively. CONCLUSIONS: The newly developed and validated NGS-based multigene assay can predict the distant recurrence risk in ER-positive, HER2-negative breast cancer.

Tumor Mutation Burden and Prognosis in Patients with Colorectal Cancer Treated with Adjuvant Fluoropyrimidine and Oxaliplatin.

PURPOSE: Recent sequencing studies revealed that a subset of colorectal cancer harbors a significantly higher number of somatic mutations. These hypermutated tumors show distinct clinicopathologic features. However, the prognostic impact of the hypermutated tumors is not clearly established. EXPERIMENTAL DESIGN: We analyzed tumor mutation burden (TMB) from targeted next-generation sequencing data of 40 major genes in 516 patients with colorectal cancer. TMB was defined as total number of nonsynonymous mutations per tumor. Cutoff value for TMB-high was chosen by which best discriminated relapse-free survival (RFS) using the Contal and O'Quigley method. RESULTS: In the TCGA data, mutation count of the selected 40 genes reflected the whole exome mutation burden (Pearson correlation = 0.873, P < 0.001). In our patient cohort, 8 or more mutations in the 40 genes was defined as TMB-high, which best discriminated RFS. A total of 55 patients (10.7%) had TMB-high. TMB-high tumors were more frequently found in a proximal location (63.6%) and had a higher proportion of N0 disease (30.9%) and MSI-H (49.1%) compared with TMB-low. Most importantly, TMB-high was associated with better 5-year RFS compared with TMB-low (96.3% vs. 79.8%, P = 0.005). Although there was significant overlap between TMB-high and MSI-H, MSI-H status was not significantly associated with RFS. Multivariate analysis revealed TMB-high as an independent positive prognostic factor for RFS [adjusted HR, 0.16 (95% confidence interval, 0.04-0.66), P = 0.011]. CONCLUSIONS: TMB-high is associated with better prognosis in patients with colorectal cancer treated with curative surgery followed by adjuvant fluoropyrimidine and oxaliplatin chemotherapy.

Association of pathway mutation with survival after recurrence in colorectal cancer patients treated with adjuvant fluoropyrimidine and oxaliplatin chemotherapy.

BACKGROUND: Although the prognostic biomarkers associated with colorectal cancer (CRC) survival are well known, there are limited data on the association between the molecular characteristics and survival after recurrence (SAR). The purpose of this study was to assess the association between pathway mutations and SAR. METHODS: Of the 516 patients with stage III or high risk stage II CRC patients treated with surgery and adjuvant chemotherapy, 87 who had distant recurrence were included in the present study. We analyzed the association between SAR and mutations of 40 genes included in the five critical pathways of CRC (WNT, P53, RTK-RAS, TGF-ß, and PI3K). RESULTS: Mutation of genes within the WNT, P53, RTK-RAS, TGF-ß, and PI3K pathways were shown in 69(79.3%), 60(69.0%), 57(65.5%), 21(24.1%), and 19(21.8%) patients, respectively. Patients with TGF-ß pathway mutation were younger and had higher incidence of mucinous adenocarcinoma (MAC) histology and microsatellite instability-high. TGF-ß pathway mutation (median SAR of 21.6 vs. 44.4 months, p = 0.021) and MAC (20.0 vs. 44.4 months, p = 0.003) were associated with poor SAR, and receiving curative resection after recurrence was associated with favorable SAR (Not reached vs. 23.6 months, p <  0.001). Mutations in genes within other critical pathways were not associated with SAR. When MAC was excluded as a covariate, multivariate analysis revealed TGF-ß pathway mutation and curative resection after distant recurrence as an independent prognostic factor for SAR. The impact of TGF-ß pathway mutations were predicted using the PROVEAN, SIFT, and PolyPhen-2. Among 25 mutations, 23(92.0%)-24(96.0%) mutations were predicted to be damaging mutation. CONCLUSIONS: Mutation in genes within TGF-ß pathway may have negative prognostic role for SAR in CRC. Other pathway mutations were not associated with SAR.

Enzymatic construction of shRNA library from oligonucleotide library.

BACKGROUND: Short hairpin RNAs (shRNAs) expressed from vectors have been used as an effective means of exploiting the RNA interference (RNAi) pathway in mammalian cells. Genome-scale screening with shRNA libraries has been used to investigate the relationship between genotypes and phenotypes on a large scale. Although several methods have been developed to construct shRNA libraries, their broad application has been limited by the high cost of constructing these libraries. OBJECTIVE: We develop a new method that efficiently constructs a shRNA library at low cost, using treatments with several enzymes and an oligonucleotide library. METHODS: The library of shRNA expression cassettes, which were cloned into a lentiviral plasmid, was produced through several enzymatic reactions, starting from a library of 20,000 different short oligonucleotides produced by microarray-based oligonucleotide synthesis. RESULTS: The NGS sequence analysis of the library shows that 99.8% of them (19,956 from 20,000 sequences) were contained in the library: 63.2% of them represent the correct sequences and the rest showed one or two base pair differences from the expected sequences. CONCLUSION: Considering the ease of our method, shRNA libraries of new genomes and of specific populations of genes can be prepared in a short period of time for genome-scale RNAi library screening.

Environmental filtering of bacterial functional diversity along an aridity gradient.

Studying how metagenome composition and diversity varies along environmental gradients may improve understanding of the general principles of community and ecosystem structuring. We studied soil bacterial metagenomes along a precipitation gradient on the eastern Tibetan Plateau, varying between 500 mm and 60 mm mean annual precipitation (MAP). We found that lower MAP was strongly associated with reduced functional diversity of bacterial genes. It appears that extreme environmental conditions associated with aridity constrain the diversity of functional strategies present in soil biota - analogous to broad scale patterns found in plant functional diversity along environmental gradients. In terms of specific functions, more extreme arid conditions were also associated with increased relative abundance of genes related to dormancy and osmoprotectants. Decreased relative abundance of genes related to antibiotic resistance and virulence in more arid conditions suggests reduced intensity of biotic interaction under extreme physiological conditions. These trends parallel those seen in earlier, more preliminary comparisons of metagenomes across biomes.

From the High Arctic to the Equator: Do Soil Metagenomes Differ According to Our Expectations?

Comparing the functional gene composition of soils at opposite extremes of environmental gradients may allow testing of hypotheses about community and ecosystem function. Here, we were interested in comparing how tropical microbial ecosystems differ from those of polar climates. We sampled several sites in the equatorial rainforest of Malaysia and Brunei, and the high Arctic of Svalbard, Canada, and Greenland, comparing the composition and the functional attributes of soil biota between the two extremes of latitude, using shotgun metagenomic Illumina HiSeq2000 sequencing. Based upon "classical" views of how tropical and higher latitude ecosystems differ, we made a series of predictions as to how various gene function categories would differ in relative abundance between tropical and polar environments. Results showed that in some respects our predictions were correct: the polar samples had higher relative abundance of dormancy related genes, and lower relative abundance of genes associated with respiration, and with metabolism of aromatic compounds. The network complexity of the Arctic was also lower than the tropics. However, in various other respects, the pattern was not as predicted; there were no differences in relative abundance of stress response genes or in genes associated with secondary metabolism. Conversely, CRISPR genes, phage-related genes, and virulence disease and defense genes, were unexpectedly more abundant in the Arctic, suggesting more intense biotic interaction. Also, eukaryote diversity and bacterial diversity were higher in the Arctic of Svalbard compared to tropical Brunei, which is consistent with what may expected from amplicon studies in terms of the higher pH of the Svalbard soil. Our results in some respects confirm expectations of how tropical versus polar nature may differ, and in other respects challenge them.

Bacterial strategies along nutrient and time gradients, revealed by metagenomic analysis of laboratory microcosms.

There is considerable interest in the functional basis of ecological strategies amongst bacteria. We used laboratory microcosms based on culturing of elutant from soil to study the effects of varying initial nutrient concentration, and time succession, on the community metagenome. We found a distinct set of nutrient-related or time-related changes in the functional metagenome. For example, a high nutrient (copiotrophic) strategy was associated with greater abundance of genes related to cell division and cell cycle, while a low nutrient (oligotrophic) strategy had greater abundance of genes related to carbohydrate metabolism and virulence, disease and defense. We also found time-related changes in the functional metagenome, revealing a distinct 'r'-related strategy with greater abundance of genes related to regulation and cell signaling, and a 'K' strategy rich in motility and chemotaxis-related genes. These different gene-based strategies may help to explain how so many bacterial OTUs coexist in nature, and the functional principles dominating natural communities. In terms of diversity, both the OTU richness and the richness of species assignment of functional genes showed linear correlations with functional gene richness, supporting the hypothesis that greater taxonomic diversity is associated with greater functional diversity, with possible implications for ecosystem stability.

Association between mutations of critical pathway genes and survival outcomes according to the tumor location in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) develops through the alteration of several critical pathways. This study was aimed at evaluating the influence of critical pathways on survival outcomes for patients with CRC. METHODS: Targeted next-generation sequencing of 40 genes included in the 5 critical pathways of CRC (WNT, P53, RTK-RAS, phosphatidylinositol-4,5-bisphosphate 3-kinase [PI3K], and transforming growth factor ß [TGF-ß]) was performed for 516 patients with stage III or high-risk stage II CRC treated with surgery followed by adjuvant fluoropyrimidine and oxaliplatin chemotherapy. The associations between critical pathway mutations and relapse-free survival (RFS) and overall survival were analyzed. The associations were further analyzed according to the tumor location. RESULTS: The mutation rates for the WNT, P53, RTK-RAS, PI3K, and TGF-ß pathways were 84.5%, 69.0%, 60.7%, 30.0%, and 28.9%, respectively. A mutation in the PI3K pathway was associated with longer RFS (adjusted hazard ratio [HR], 0.59; 95% confidence interval [CI], 0.36-0.99), whereas a mutation in the RTK-RAS pathway was associated with shorter RFS (adjusted HR, 1.60; 95% CI, 1.01-2.52). Proximal tumors showed a higher mutation rate than distal tumors, and the mutation profile was different according to the tumor location. The mutation rates of Kirsten rat sarcoma viral oncogene homolog (KRAS), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA), and B-Raf proto-oncogene serine/threonine kinase (BRAF) were higher in proximal tumors, and the mutation rates of adenomatous polyposis coli (APC), tumor protein 53 (TP53), and neuroblastoma RAS viral oncogene homolog (NRAS) were higher in distal tumors. The better RFS with the PI3K pathway mutation was significant only for proximal tumors, and the worse RFS with the RTK-RAS pathway mutation was significant only for distal tumors. CONCLUSIONS: A PI3K pathway mutation was associated with better RFS for CRC patients treated with adjuvant chemotherapy, and an RTK-RAS pathway mutation was associated with worse RFS. The significance of the prognostic impact differed according to the tumor location. Cancer 2017;123:3513-23. © 2017 American Cancer Society.

Effect of experimental soil disturbance and recovery on structure and function of soil community: a metagenomic and metagenetic approach.

There has been little study of effects of disturbance on soil biota combining closely controlled experimental conditions and DNA-based methods. We sampled pots of soil at varying times following an initial simulated mass mortality event. Soil DNA was extracted at intervals up to 24 weeks after the event, and shotgun metagenomes sequenced using NextSeq. Compared to initial conditions, we found: consistent, sequential changes in functional metagenome and community structure over time, indicating successional niche differentiation amongst soil biota. As predicted, early successional systems had greater abundance of genes associated with motility, but fewer genes relating to DNA/RNA/protein metabolism, cell division and cell cycle. Contrary to predictions, there were no significant differences in cell signaling, virulence and defense-related genes. Also, stress related genes were less abundant in later succession. The early successional system had lower taxonomic diversity but higher functional gene diversity. Over time, community characteristics changed progressively, but by the end of the experiment had not returned to the 'original' state of the system before disturbance. Results indicated a predictable sequence of gene functions and taxa following disturbance, analogous to ecosystem succession for large organisms. It is unclear if and when the system would return to its pre-disturbance state.

Development of Soil Bacterial Communities in Volcanic Ash Microcosms in a Range of Climates.

There is considerable interest in understanding the processes of microbial development in volcanic ash. We tested the predictions that there would be (1) a distinctive bacterial community associated with soil development on volcanic ash, including groups previously implicated in weathering studies; (2) a slower increase in bacterial abundance and soil C and N accumulation in cooler climates; and (3) a distinct communities developing on the same substrate in different climates. We set up an experiment, taking freshly fallen, sterilized volcanic ash from Sakurajima volcano, Japan. Pots of ash were positioned in multiple locations, with mean annual temperature (MAT) ranging from 18.6 to -3 °C. Within 12 months, bacteria were detectable by qPCR in all pots. By 24 months, bacterial copy numbers had increased by 10-100 times relative to a year before. C and N content approximately doubled between 12 and 24 months. HiSeq and MiSeq sequencing of the 16S rRNA gene revealed a distinctive bacterial community, different from developed vegetated soils in the same areas, for example in containing an abundance of unclassified bacterial groups. Community composition also differed between the ash pots at different sites, while showing no pattern in relation to MAT. Contrary to our predictions, the bacterial abundance did not show any relation to MAT. It also did not correlate to pH or N, and only C was statistically significant. It appears that bacterial community development on volcanic ash can be a rapid process not closely sensitive to temperature, involving distinct communities from developed soils.

The impact of tropical forest logging and oil palm agriculture on the soil microbiome.

Selective logging and forest conversion to oil palm agriculture are rapidly altering tropical forests. However, functional responses of the soil microbiome to these land-use changes are poorly understood. Using 16S rRNA gene and shotgun metagenomic sequencing, we compared composition and functional attributes of soil biota between unlogged, once-logged and twice-logged rainforest, and areas converted to oil palm plantations in Sabah, Borneo. Although there was no significant effect of logging history, we found a significant difference between the taxonomic and functional composition of both primary and logged forests and oil palm. Oil palm had greater abundances of genes associated with DNA, RNA, protein metabolism and other core metabolic functions, but conversely, lower abundance of genes associated with secondary metabolism and cell-cell interactions, indicating less importance of antagonism or mutualism in the more oligotrophic oil palm environment. Overall, these results show a striking difference in taxonomic composition and functional gene diversity of soil microorganisms between oil palm and forest, but no significant difference between primary forest and forest areas with differing logging history. This reinforces the view that logged forest retains most features and functions of the original soil community. However, networks based on strong correlations between taxonomy and functions showed that network complexity is unexpectedly increased due to both logging and oil palm agriculture, which suggests a pervasive effect of both land-use changes on the interaction of soil microbes.

Microbiome Variation Across Amphibian Skin Regions: Implications for Chytridiomycosis Mitigation Efforts.

Cutaneous bacteria may play an important role in the resistance of amphibians to the pathogenic fungus Batrachochytrium dendrobatidis (Bd). Microbial communities resident on hosts' skin show topographical diversity mapping to skin features, as demonstrated by studies of the human microbiome. We examined skin microbiomes of wild and captive fire-bellied toads (Bombina orientalis) for differences across their body surface. We found that bacterial communities differed between ventral and dorsal skin. Wild toads showed slightly higher bacterial richness and diversity in the dorsal compared to the ventral region. On the other hand, captive toads hosted a higher richness and diversity of bacteria on their ventral than their dorsal skin. Microbial community composition and relative abundance of major bacterial taxonomic groups also differed between ventral and dorsal skin in all populations. Furthermore, microbiome diversity patterns varied as a function of their Bd infection status in wild toads. Bacterial richness and diversity was greater, and microbial community structure more complex, in wild than captive toads. The results suggest that bacterial community structure is influenced by microhabitats associated with skin regions. These local communities may be differentially modified when interacting with environmental bacteria and Bd. A better understanding of microbiome variation across skin regions will be needed to assess how the skin microbiota affects the abilities of amphibian hosts to resist Bd infection, especially in captive breeding programs.

Predictable communities of soil bacteria in relation to nutrient concentration and successional stage in a laboratory culture experiment.

It is difficult to understand the processes that structure immensely complex bacterial communities in the soil environment, necessitating a simplifying experimental approach. Here, we set up a microcosm culturing experiment with soil bacteria, at a range of nutrient concentrations, and compared these over time to understand the relationship between soil bacterial community structure and time/nutrient concentration. DNA from each replicate was analysed using HiSeq2000 Illumina sequencing of the 16S rRNA gene. We found that each nutrient treatment, and each time point during the experiment, produces characteristic bacterial communities that occur predictably between replicates. It is clear that within the context of this experiment, many soil bacteria have distinct niches from one another, in terms of both nutrient concentration, and successional time point since a resource first became available. This fine niche differentiation may in part help to explain the coexistence of a diversity of bacteria in soils. In this experiment, we show that the unimodal relationship between nutrient concentration/time and species diversity often reported in communities of larger organisms is also evident in microbial communities.

Effects of functionalized and raw multi-walled carbon nanotubes on soil bacterial community composition.

Carbon nanotubes (CNTs) are widely used in industry, but their environmental impacts on soil microbial communities are poorly known. In this paper, we compare the effect of both raw and acid treated or functionalized (fCNTs) multi-walled carbon nanotubes (MWCNTs) on soil bacterial communities, applying different concentrations of MWCNTs (0 µg/g, 50 µg/g, 500 µg/g and 5000 µg/g) to a soil microcosm system. Soil DNA was extracted at 0, 2 and 8 weeks and the V3 region of the 16S rRNA gene was PCR-amplified and sequenced using paired-end Illumina bar-coded sequencing. The results show that bacterial diversity was not affected by either type of MWCNT. However, overall soil bacterial community composition, as illustrated by NMDS, was affected only by fMWCNT at high concentrations. This effect, detectable at 2 weeks, remained equally strong by 8 weeks. In the case of fMWCNTs, overall changes in relative abundance of the dominant phyla were also found. The stronger effect of fMWCNTs could be explained by their intrinsically acidic nature, as the soil pH was lower at higher concentrations of fMWCNTs. Overall, this study suggests that fMWCNTs may at least temporarily alter microbial community composition on the timescale of at least weeks to months. It appears, by contrast, that raw MWCNTs do not affect soil microbial community composition.

A high-throughput optomechanical retrieval method for sequence-verified clonal DNA from the NGS platform.

Writing DNA plays a significant role in the fields of synthetic biology, functional genomics and bioengineering. DNA clones on next-generation sequencing (NGS) platforms have the potential to be a rich and cost-effective source of sequence-verified DNAs as a precursor for DNA writing. However, it is still very challenging to retrieve target clonal DNA from high-density NGS platforms. Here we propose an enabling technology called 'Sniper Cloning' that enables the precise mapping of target clone features on NGS platforms and non-contact rapid retrieval of targets for the full utilization of DNA clones. By merging the three cutting-edge technologies of NGS, DNA microarray and our pulse laser retrieval system, Sniper Cloning is a week-long process that produces 5,188 error-free synthetic DNAs in a single run of NGS with a single microarray DNA pool. We believe that this technology has potential as a universal tool for DNA writing in biological sciences.

Tuning and maximizing the single-molecule surface-enhanced Raman scattering from DNA-tethered nanodumbbells.

We extensively study the relationships between single-molecule surface-enhanced Raman scattering (SMSERS) intensity, enhancement factor (EF) distribution over many particles, interparticle distance, particle size/shape/composition and excitation laser wavelength using the single-particle AFM-correlated Raman measurement method and theoretical calculations. Two different single-DNA-tethered Au-Ag core-shell nanodumbbell (GSND) designs with an engineerable nanogap were used in this study: the GSND-I with various interparticle nanogaps from ∼4.8 nm to <1 nm or with no gap and the GSND-II with the fixed interparticle gap size and varying particle size from a 23-30 nm pair to a 50-60 nm pair. From the GSND-I, we learned that synthesizing a <1 nm gap is a key to obtain strong SMSERS signals with a narrow EF value distribution. Importantly, in the case of the GSND-I with <1 nm interparticle gap, an EF value of as high as 5.9 × 10(13) (average value = 1.8 × 10(13)) was obtained and the EF values of analyzed particles were narrowly distributed between 1.9 × 10(12) and 5.9 × 10(13). In the case of the GSND-II probes, a combination of >50 nm Au cores and 514.5 nm laser wavelength that matches well with Ag shell generated stronger SMSERS signals with a more narrow EF distribution than <50 nm Au cores with 514.5 nm laser or the GSND-II structures with 632.8 nm laser. Our results show the usefulness and flexibility of these GSND structures in studying and obtaining SMSERS structures with a narrow distribution of high EF values and that the GSNDs with < 1 nm are promising SERS probes with highly sensitive and quantitative detection capability when optimally designed.