The reference workload for each drill was calculated across all sessions, using bootstrapping. The bootstrap mean workload and 95% confidence intervals (CI) were then calculated for session 1, sessions 1–2, and continued for remaining sessions , and were compared with those of the reference workload. The minimum sessions to anticipate workload for each drill was identified when the first normative value fell within ±5% or ±10% of the reference workload 95% CI. The minimum sessions were then tested to determine the accuracy to which workload could be anticipated. Three to four sessions were needed to anticipate workload within ±5%, while 2–3 sessions were needed to anticipate workload within ±10%. External workload was anticipated in 0–55% of future sessions using an error range of ±5%, and in 58–89% of sessions using an error range of ±10%. External workload during 5 vs. 5 games-based drills can be anticipated in most sessions using normative values established during a short-term monitoring period with an error range of ±10%.This study determined whether external workload could be anticipated during 5 vs. 5 games-based drills in basketball. Thirteen semi-professional, male basketball players were monitored during 5 vs. 5 training drills across the season. External workload was determined using PlayerLoad™ (AU∙min Basketball is a highly intermittent team sport, with players performing repeated bouts of high-intensity activity interspersed with periods of low- to moderate-intensity activity ,2. AlthoMonitoring players during both training and games is preferred to understand the complete external workloads encountered. However, monitoring external workload is more common in training than games possibly due to player preferences and restrictions in some leagues prohibiting use of wearable technologies during games . It is aGames-based drills are an effective training strategy frequently used by basketball coaches ,9. In faMicrosensor monitoring systems encompassing tri-axial accelerometers, gyroscopes, magnetometers, or a combination of these instruments , are comThirteen players  from the same semi-professional, male basketball team volunteered to participate in this study. The team was registered in the Queensland Basketball League, which is a second-tier, Australian basketball competition. Prior to study commencement, all participants were screened for injuries or health conditions that prevented safe participation in the study. The purpose of the study as well as associated risks and benefits of participation were explained to participants before providing personal and guardian written informed consent (if under 18 years). All procedures were approved by the Central Queensland University Human Research Ethics Committee (reference number: 2019-001).An observational study design was utilized whereby players were monitored during all training sessions across the 16-week, in-season phase (May–August) of the 2019 competitive season. During the season, up to 3 training sessions (2 ± 1 training sessions) were held per week (Monday–Sunday) with games played between Friday–Sunday. A total of 26 on-court, team training sessions were held across the season.Prior to commencement of the first training session, anthropometric data were collected on each player including stature using a portable stadiometer  and body mass using electronic scales . For all training sessions, players were fitted with microsensors  held on the posterior, upper surface of the torso in specially-designed neoprene shirts. All players were familiar with and accustomed to the equipment used in the present study.During each training session and following a standardized warm-up, games-based drills were conducted as part of the normal training regime determined by the coaching staff. Three distinct 5 vs. 5 drills were frequently performed by the team and thus were selected for investigation, including: (1) 5 vs. 5 played on a half-court, (2) 5 vs. 5 played on a full-court, and (3) 5 vs. 5 vs. 5 played on a full-court. Each of these drills were performed toward the end of each training session at similar time-points  following standardized shooting drills and initial games-based drills with less players . The games-based drills investigated along with associated rules and conditions are presented in x), coronal (y), and sagittal (z) planes multiplied by a scaling factor of 0.01 [−1) to account for the varied durations of each drill across the season . External workload was determined via PlayerLoad™ (PL) , which is a proprietary measure derived from the tri-axial accelerometer sampling at 100 Hz. PL represents the accumulated load calculated as the square root of the sum of the squared rate of change in acceleration across the transverse  external workload for each drill. Participation in drills were not consistent for each player across all training sessions. Further, the sample size of the study was not large. Hence we performed nonparametric bootstrapping (1000 replications) to better estimate mean workload values and a bias-corrected 95% confidence interval (CI) for each mean workload value ,23. A boThe second step involved testing the accuracy of the anticipated external workload in each drill across subsequent training sessions. We compared the 95% CI of each mean workload identified in the first step of our analyses for each drill to the 95% CI of the bootstrap mean workload experienced in each subsequent individual session. In this regard, if the 95% CI for workload in each drill in a specific session was within the acceptable error range  of the 95% CI of the workload corresponding to the minimum number of sessions identified, workload for that session was adjudged to be accurately anticipated. All statistical analyses were conducted using Stata/MP 16.0 for Windows .−1, 95% CI = 5.28, 5.85. The minimum number of sessions needed to anticipate the workload per session within ±5% for 5 vs. 5 half-court games-based drills was 3 , while the minimum number of sessions needed to anticipate workload within ±10% was 2 . Using the acceptable error range of ±5%, mean workload from sessions 1–3 did not accurately anticipate the experienced workload in any (0%) remaining sessions. Using the acceptable error range of ±10%, mean workload from sessions 1–2 accurately anticipated the experienced workload in 58% of remaining sessions , while the minimum number of sessions needed to anticipate workload within ±10% was 3 . Using the acceptable error range of ±5%, mean workload from sessions 1–4 accurately anticipated the experienced workload in 27% of remaining sessions. Using the acceptable error range of ±10%, mean workload from sessions 1–3 accurately anticipated the experienced workload in 67% of remaining sessions . Using the acceptable error range of ±5%, mean workload from sessions 1–3 accurately anticipated the experienced workload in 55% of remaining sessions. Using the acceptable error range of ±10%, mean workload from sessions 1–3 accurately anticipated the experienced workload in 89% of remaining sessions  between prescribed and experienced heart rate-derived workloads in elite field hockey players over a 5-week mesocycle. Meanwhile, Lacome, Simpson, Broad, and Buchheit [The ability to anticipate workload has only been investigated using internal workload measures and in team sports other than basketball. In this regard, Perrotta, Taunton, Koehle, White, and Warburton  reportedBuchheit  reportedBuchheit . Consequ−1) to account for the varied durations of each drill across sessions. In this sense, variation in drill duration likely influenced the external workload intensities experienced by players, with basketball players shown to accomplish higher training intensities when external workload is sampled across shorter durations [We showed normative external workloads could be established using only 3–4 training sessions when using an acceptable error range of ±5% of the 95% CI for the mean workload obtained across the entire season. However, our data demonstrate relatively poor accuracy (0–55%) when using an acceptable error range of ±5% to anticipate external workload during 5 vs. 5 games-based drills. The session-to-session variation in external workload using this error range was likely due to the workload management strategies implemented by coaching staff  , as wellurations . ConsequIn contrast, we showed normative external workloads could be established using only 2–3 training sessions when using an acceptable error range of ±10% of the 95% CI for the mean workload obtained across the entire season. Consequently, the more liberal acceptable error range of ±10% yielded greater accuracy (58–89%) in anticipating the external workload experienced during 5 vs. 5 games-based drills. Accuracy may however be dependent upon the drill used as the number of sessions accurately anticipated was just over half (58%) for 5 vs. 5 half-court drills, which increased to 67% and 89% for 5 vs. 5 and 5 vs. 5 vs. 5 full-court drills, respectively. The greater accuracy in anticipating external workload in full-court games-based drills might be due to completion of more consistent running demands during transitions across the court than half-court games-based drills, which evoke more play sequences close to the basket underpinned by variable movement patterns  and tech−1; full-court: 17.9 ± 4.6 AU·min−1) and Montgomery et al. [−1) reported higher relative external workloads than we measured during 5 vs. 5 games-based drills in professional, adult and elite, junior, male players, respectively. However, variations in accelerometer placement across studies likely accounts for these differences given past studies placed the accelerometers on the hips [−1 and 1.0 ± 0.1 AU·min−1 during half- and full-court 5 vs. 5 games-based drills in professional, male players. Despite these data being approximately tenfold lower than the external workloads we reported, differences in the monitoring system, algorithm, and sampling rate used by Vazquez-Guerrero et al. [The present data add to the limited body of knowledge surrounding the external workloads experienced during various 5 vs. 5 games-based drills using microsensors. The mean external workloads we observed vary from results reported in previous studies. Specifically, Schelling and Torres   and RPE  than continuous games-based drills. Consequently, the inclusion of short break periods permits coaches to reduce the demands imposed on players if desired by lowering the work:rest ratio encountered [Our study also quantified the external workload experienced during a novel 5 vs. 5 vs. 5 games-based drill. This drill is practically advantageous for basketball coaching staff as it allows implementation of short break periods, which resemble the intermittent nature of basketball game-play. Furthermore, as previously shown by Conte, Favero, Niederhausen, Capranica, and Tessitore , intermiountered .Although our study provides important information regarding the use of microsensors to quantify and anticipate external workload during 5 vs. 5 games-based drills in basketball, some limitations should be considered. (1) The occurrence of the included 5 vs. 5 games-based drills could not be controlled, resulting in varied sample sizes, in addition to a relatively small sample size, for analysis per drill. Nevertheless, the varied sample sizes is a consequence of conducting applied studies in actual training scenarios, which strengthens the ecological validity of our outcomes. Further, we used bootstrapping, a resampling technique, in an attempt to better estimate the parameters of workload. (2) Where drills were performed more than once in a session, only the first occurrence of the drill was utilized for analysis to control for the confounding influence of fatigue on workloads. Consequently, the inability to anticipate the external workload we observed only applies to the first occurrence of each drill in each session. (3) Drill length was not controlled for across sessions. Future research is encouraged to investigate the potential relationship between drill length and intensity during different games-based drills in basketball. (4) Given the observational nature of the present study, the timing of the included 5 vs. 5 games-based drills within each session could not be controlled. Consequently, the 5 vs. 5 games-based drills were administered ~40–56 min into each session on average. Further research is encouraged to determine whether external workload can be more accurately anticipated during games-based drills when administered at other timepoints during training sessions. (5) Player activity prior to and following each training session was not controlled. However, team training schedules remained consistent across the season, likely allowing for similar recovery and preparation leading into each session. Furthermore, we assessed the ability to anticipate external workload during games-based drills across the season in a natural team training environment to enhance the translation of findings to practical settings. (6) Acceptable error ranges of ±5% and ±10% were included in this exploratory analysis; however, some coaching staff may be more conservative or liberal in their approach, which warrants further research using alternative acceptable error ranges.Normative values obtained across 3–4 sessions showed limited accuracy (0–55%) in anticipating external workload using an acceptable error range of ±5% during 5 vs. 5 games-based drills in semi-professional, male basketball players. However, normative values obtained across 2–3 sessions accurately anticipated external workload across most sessions (58–89%) using an acceptable error range of ±10% during the same drills. Consequently, external workload can be anticipated in most instances during 5 vs. 5 games-based drills using normative values established during a short-term monitoring period using an acceptable error range of ±10%.Our findings have important implications for basketball coaching staff when implementing microsensor monitoring systems in practice. The ability to anticipate external workload using a small sample of sessions to establish normative values allows for the short-term uptake of microsensor monitoring systems, reducing the extensive financial cost, labor, and expertise associated with the long-term monitoring of players across the entire season. In addition, the normative external workloads we provided for different 5 vs. 5 games-based drills in basketball should be considered by coaches when developing training sessions across the season. The novel 5 vs. 5 vs. 5 drill examined in this study may provide a useful training stimulus for coaches when seeking to incorporate short rest periods between active play and up to 15 players in a large team environment, which is important given bench players can complete less training demands than starting players in basketball settings .


Staphylococcus aureus (S. aureus) is an opportunistic pathogen capable of causing serious health implications in susceptible individuals once it invades the host’s protective barriers. Methicillin-susceptible S. aureus (MSSA) often receives lesser attention although it has been frequently associated with serious infections in human. We aim to investigate the genomic features of a highly virulent yet pan susceptible MSSA strain (coded as HS-MSSA) which caused concurrent bacteraemia in a dengue patient, ultimately resulted in sepsis death of the patient. Whole genome sequence analysis was performed. The draft genome of HS-MSSA is approximately 2.78 Mb (GC content = 32.7%) comprising of 2637 predicted coding sequences. In silico genotyping of the HS-MSSA strain revealed a novel combined genotype (t091/ST2990). The HS-MSSA carries a SaPIn1-like pathogenicity island that harbours the staphylococcal enterotoxin and enterotoxin-like genes (sec3 and selL). The strain-specific β-lactamase (blaZ)-bearing plasmid region was identified in HS-MSSA. Core genome phylogeny showed that the HS-MSSA strain shared a common ancestry with the European MRSA clone. We report herein the genomic features of an MSSA lineage with novel genotype previously not reported elsewhere. Staphylococcus aureus (S. aureus) is a Gram-positive round-shaped bacterium commonly found colonizing the skin and mucous membranes of humans. Approximately 15% of the human population carry S. aureus in the nasal cavity, specifically in the anterior nares1. S. aureus is an opportunistic pathogen capable of causing serious health implications in susceptible hosts when it enters the bloodstream or internal tissues. Potentially serious infections caused by S. aureus include bacteraemia, pneumonia, endocarditis, and osteomyelitis2. In fact, the nasal carriage of S. aureus has shown a strong association with the aetiology of bacteraemia in susceptible hosts3. Bacteraemia caused by invasive S. aureus often results in greater mortality rate due to its ability to secrete a wide spectrum of virulence factors that contribute to the evasion of host immune responses4.S. aureus infections are often associated with healthcare facilities due to the breaching of the skin barrier with surgical devices or implants, although it is not uncommon to have community acquired infections5. Antimicrobial resistance (AMR) among the healthcare-associated S. aureus infections has been an important global public health issue. Since the emergence of methicillin-resistant S. aureus (MRSA) in the 1960s, this organism has been notorious for its ability to cause severe forms of infection in hospital settings and has eventually spread to the community after 30 years of evolution, most probably due to the transfer of the AMR gene cassette SCCmec to local methicillin-susceptible lineages6. This evolutionary theory for the emergence of MRSA lineage has recently been strengthened by whole genome sequence analysis7.The severe consequences of S. aureus (MSSA) infection has persisted in both healthcare settings and the community, amidst the increasing global prevalence of MRSA8. In fact, a slow epidemic shift was observed in S. aureus infections worldwide during the past two decades. In the United States (US), multiple clinical studies have recorded an increase in the prevalence of MSSA and a simultaneous decrease in MRSA incidences since 200510. Recent surveillance in the US has seen an increase in bloodstream infections caused by community acquired MSSA from 2012 to 201711. A similar observation was recorded in Europe, whereby a decrease in MRSA bacteraemia was observed since 2005, accompanied by a continuous increase in MSSA bacteraemia12.Despite receiving less attention compared to its drug-resistant counterpart, the methicillin-susceptible 15. More often than not, invasive MSSA causes greater illness severity in patients suffering from bacteraemia due to its ability to express multiple virulence factors17. Community-onset MSSA bacteraemia has documented a higher mortality rate compared to that of MRSA in many regions18. These observations showed that while much attention has been directed to MRSA infections in recent years, the often neglected MSSA infections have caused a greater and increasing disease burden worldwide.Genotyping studies from different geographical regions often revealed a greater genetic diversity among the MSSA populations compared to the MRSAThe ability of the diverse MSSA lineages to express a multitude of virulence genes and the possibility of developing multi-drug resistance phenotypes due to horizontal gene transfer have posed a great risk to public health. Nonetheless, the genetic drives that are responsible for the emergence and persistence of such virulent MSSA lineages remain obscured. In this study, we aimed to investigate the genomic features of an invasive MSSA strain which caused a fatal sepsis case in human. The patient was initially diagnosed with dengue fever upon admission to the hospital, but later developed pneumonia and severe sepsis rapidly and succumbed to multiple organs failure within 24 h post-admission.19.The community acquired MSSA strain (coded as HS-MSSA) was isolated from the blood culture of a patient in 2017. The clinical presentations as well as the AMR and virulence characteristics related to the infection have been previously described20. The HS-MSSA genomic DNA was subsequently subjected to next-generation sequencing via Illumina Genome AnalyzerIIx  by a commercial sequencing vendor. Paired-end 2 × 150 bp reads were generated using Illumina chemistry. The quality of the sequence reads was assessed and assembled de novo using the CLC Genomics Workbench version 5.1  with default parameters. Contigs with an average coverage of 30 and above were selected to represent the bacterial draft genome for further analyses. Reordering of the HS-MSSA contigs was achieved via genomic mapping with the reference genome S. aureus subspecies aureus NCTC 8325  GenBank accession number: NC007795) by using Mauve software version 2.4.021. The subset of the contigs that were mapped to the complete chromosome of the S. aureus NCTC 8325 was identified as the chromosomal region of the HS-MSSA strain. Genomic annotation was performed using the webserver Rapid Annotations using Subsystems Technology 22. The viewing and preliminary sequence-based comparison between the annotated HS-MSSA and reference genomes were done using SEED Viewer version 2.0 (http://pubseed.theseed.org/)23, and further validated by using Mauve.The genomic DNA of the HS-MSSA strain was extracted using DNeasy Blood & Tissue Kit  and the quality of the extracted genomic DNA was evaluated based on spectrophotometric measurementshttps://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify genetically similar S. aureus strains (taxonomy ID: 46170) from the complete and draft genomes database curated in the NCBI GenBank. The BLAST search was performed using the discontinuous megablast algorithm with default parameters. Multiple genomes alignment and visualization were performed using Mauve and BLAST Ring Image Generator (BRIG) software24. The genotype of the HS-MSSA strain was inferred in-silico by using web-based servers. The conventional seven-gene multi-locus sequence typing (MLST), whole genome MLST (wgMLST), and ribosomal MLST (rMLST) predictions made use of the S. aureus database in PubMLST (https://pubmlst.org/saureus/) sited at the University of Oxford25. The online analysis tools available from the Center for Genomic Epidemiology (CGE) server (http://www.genomicepidemiology.org/) were used to predict the staphylococcal protein A (spa) type, presence of SCCmec elements, plasmid elements, and pathogenicity genes in the HS-MSSA genome29. Phage-associated genes and genomic regions in the HS-MSSA genome was identified using PHAge Search Tool Enhanced Release server 31. The genomic islands were predicted using IslandViewer 4 (http://www.pathogenomics.sfu.ca/islandviewer/)32. All genomic predictions were completed using default parameters, validated by cross-examination with RAST-annotation output, and manually interrogated using NCBI BLAST nucleotide search tool.The chromosomal region of the HS-MSSA was subjected to a microbial genome search using the NCBI Basic Local Alignment Search Tool  was used to identify AMR genes in the assembled HS-MSSA genome33. The AMR genes selection criteria were set to perfect (100% identity) and strict (> 95% identity) hits only to the curated reference sequences in the Comprehensive Antibiotic Resistance Database . Prediction of partial genes was accepted as the HS-MSSA genome examined in this study resembles an incomplete genome. The RGI predictions were cross-checked with ResFinder version 3.2 (https://cge.cbs.dtu.dk/services/ResFinder/). AMR genes prediction results obtained using default parameters34. All predicted AMR genes were validated by cross-examination with RAST-annotation output and manually interrogated using NCBI BLAST.The online tool, Resistance Genes Identifier version 5.1.0  was used to identify relevant single nucleotide polymorphism (SNP) sites for core genome phylogenetic analysis35. Complete and draft genomes of 20 global S. aureus strains were selected for core genome SNP-based (cgSNP) phylogenetic analysis based on considerations including high genetic identity , geographical distribution, type of organisms (MSSA and MRSA), and MLST clonal complexes. The selected S. aureus genomes were retrieved from the NCBI GenBank and PubMLST databases and the strains information is tabulated and appended as Supplementary Table S. aureus NCTC 8325 was used as the reference genome for multiple alignments. The multiple genomes sequence alignment generated by the REALPHY tool was subsequently used to construct a phylogenetic tree. An unrooted tree was inferred via the approximate maximum likelihood (ML) method using FastTree2 software36, and visualized using FigTree version 1.4.3 (https://github.com/rambaut/figtree/releases)37. 100 bootstrap replicates were performed to support the phylogenetic inferences made in this study for practical reasons. A bootstrap analysis with 100–500 replicates is sufficient to produce qualitatively comparable support values with more than 99.5% correlation with the reference values in ML trees with higher numbers of bootstrap replicates38.The online tool, Reference sequence Alignment based Phylogeny builder version 1.12  for the central STs of the major clonal complexes  and other frequently encountered clonal complexes  were used to construct a phylogenetic tree. Multiple alignments of the concatenated sequences were performed by Molecular Evolutionary Genetics Analysis (MEGA) X39. Next, the aligned sequences were subjected to a statistical selection of best-fit phylogenetic model using jModelTest version 2.1.640. An unrooted tree was subsequently inferred via the ML method using MEGA X software.The concatenated allelic sequences of the seven housekeeping genes  and 65 RNAs were predicted in the HS-MSSA draft genome. The general features of the HS-MSSA draft genome are summarized in Table The size of the HS-MSSA draft genome is approximately 2.78 Mb (GC content = 32.7%), of which 2,740,810 bp belongs to the genomic region that corresponds to the chromosome of S. aureus chromosomal regions. Microbial genome BLAST analysis showed that the HS-MSSA chromosome shares greatest genetic homology  with the MRSA strains isolated in the United States of America (USA). More than half of the MRSA strains that are genetically similar to the HS-MSSA were obtained from a clonal cluster of community acquired MRSA that caused skin and soft tissue infections (SSTIs) in a military base in the USA41. Similar genetic homology was observed between the MRSA USA300 clones and HS-MSSA, albeit at slightly lower scores. The two USA300 clones that showed greatest genetic homology with HS-MSSA are USA300_TCH1516 (NC_010079) and USA300_SUR1 (NZ_CP009423), both being community acquired MRSA strains isolated from USA and Republic of Suriname in South America respectively. A circular genome map was constructed to illustrate the genetic resemblance of HS-MSSA to the representative MRSA clones from the North and South America (MRSA strains 2148.C01 (NZ_CP017094) and USA300_SUR1), and an MSSA strain KT/314250 (NZ_AOCP00000000) isolated in Malaysia  that caused an outbreak of SSTIs shared common MLST type (ST8) with the community acquired USA300 clone from Texas, USA43. The complete genome analysis revealed that the South American outbreak strains were genetic variants of the North American USA300 clone45. Previous genomic studies have proven that USA300 clones are clonally related to the European nosocomial MRSA strain COL (NC_002951) emerged from a methicillin-susceptible ancestral strain via the acquisition of methicillin resistance determinant following the introduction of methicillin in 195946. Therefore, the MRSA clones USA300 and COL are most likely sharing a common MSSA ancestry but evolved separately through different MRSA lineages47. Accordingly, the close genomic resemblance of the HS-MSSA strain to these MRSA clones suggests that this MSSA lineage in Malaysia could have been the less evolved (with reference to AMR) descendant of the common MSSA ancestry with the MRSA clones in the USA and Europe. This inference was made on the basis that chromosomal recombination occurs in S. aureus at low frequencies, except for regions associated with virulence and pathogenicity48. The descendants of this MSSA ancestral strain have spread widely over different continents and persisted in the human populations over the years, causing community acquired SSTIs and invasive infections in the susceptible hosts.The great genomic resemblance between the HS-MSSA strain and the MRSA clones from the American continent indicates the possible common ancestry between these lineages, despite the wide geographical distance where the strains were originated. The MRSA clones originated from the USA that showed high genetic homology with HS-MSSA were all community acquired, capable of causing SSTIs and sepsis, and were largely resistant to chlorhexidinespa type t304 and MLST type ST6 (clone t304/ST6) which caused recurrent outbreaks in a hospital in Denmark since 201149. This MRSA clone t304/ST6 was detected and predominated among the MRSA strains isolated in a teaching hospital in Oman within the same period50. However, this clone remains rarely reported outside these two geographical regions. The finding of similar plasmids in the HS-MSSA strain in Malaysia and MRSA clone in European region suggests possible horizontal gene transfer event, which could have occurred due to chance events after the divergence from the common MSSA ancestral strain. One other hypothesis is that the HS-MSSA strain could have evolved from an MSSA lineage carrying this plasmid, of which some of its descendants might have diverged to another evolutionary path via the acquisition of SCCmec gene cassette. The latter hypothesis is more plausible as this plasmid carries the penicillin-resistant blaZ gene. Based on an earlier study using Staphylococcus species mainly from the bovine origin, the authors demonstrated that plasmid-borne blaZ is often species-specific and lack conjugative transfer mechanisms, thus making horizontal gene transfer an extremely rare event51. Therefore, the presence of this blaZ-bearing plasmid in the HS-MSSA strain supports our inference on the common ancestry of this local MSSA with the MRSA clones in European regions.Detailed examination of the 15 contigs that were not included in the HS-MSSA chromosomal region revealed a 19,763 bp region (contig-33) identified as a plasmid-associated genomic region. BLAST analysis of contig-33 revealed the closest plasmid homolog as pl1_M2024 (CP047022), at 100% query coverage with 99% nucleotide identity . The pl1_M2024 is a complete plasmid retrieved from the nosocomial MRSA clone with S. aureus by rMLST. The 7-loci allelic profile for HS-MSSA is 1-1-1-1-330-1-10, arranged in the order arcC-aroE-glpF-gmk-pta-tpi-yqiL. All loci MLST (wgMLST) results showed 1358 exact matches to sequence type ST2990 (Supplementary Table S. aureus strain 128A deposited in the database (PubMLST ID: 5521) shares the same sequence type as HS-MSSA and was also isolated in Malaysia, earlier in 2014. S. aureus 128A was isolated from the infected wound of an elderly diabetic patient (66 years old) examined in a tertiary hospital in the capital Kuala Lumpur. The strain was found susceptible to oxacillin, methicillin, and vancomycin upon investigation. The S. aureus 128A was confirmed as community acquired MSSA which causes wound infection. The spa type of HS-MSSA is t091 (spa repeat succession 07-23-21-17-34-12-23-02-12-23). Therefore, the combined genotype of the HS-MSSA strain is noted as t091/ST2990 (spa type/MLST type).In silico MLST analyses showed that HS-MSSA belongs to the sequence type ST2990, with its identity confirmed as S. aureus strain 128A which shares the identical sequence type (ST2990), the HS-MSSA strain examined in this study was isolated from a diabetic patient who was admitted to the hospital due to dengue fever19. However, instead of causing SSTI (infected diabetic wound) as strain 128A did, the HS-MSSA was more invasive and caused a bloodstream infection that resulted in severe sepsis. The common STs of S. aureus in Malaysia, especially the most studied MRSA strains, mainly comprised of the pandemic clone ST239, with ST22 clone being increasingly documented in the past decade54. Similar to the global epidemiology of MSSA, the few studies that examined the MSSA genotypes in Malaysia documented a higher genetic diversity compared to the MRSA populations in this region57. MRSA with spa type t091 has been reported in Malaysia, but S. aureus strain with genotype t091/ST2990 has not been previously documented58. Nonetheless, the isolation of ST2990 strains from different geographical regions suggests that this MSSA genotype may already be widely disseminated in Malaysia. Although S. aureus of CC1 complex  does not predominate in Malaysia, the persistence of these genotypes throughout the years may indicate their evolutionary success58.Similar to the mec (SCCmec) elements were not detected in the HS-MSSA genome based on SCCmecFinder analysis at 90% identity threshold and a minimum length of 60%. The absence of SCCmec elements in the HS-MSSA genome is reflected in the methicillin-susceptible phenotype of this strain. The SCCmec genomic cassette is a mobile genomic region with two important gene complexes, mec-gene complex encoding methicillin resistance and cassette chromosome recombinase (ccr)-gene complex encoding site-specific recombinase(s) for the mobility of this DNA region59. The presence of SCCmec region in the bacterial chromosome is a signature of MRSA strains and the acquisition of which results in the emergence of new MRSA lineage from an ancestral MSSA strain.The staphylococcal chromosome cassette mec elements provides a selective advantage to S. aureus, it is not uncommon for it to spontaneously excised from the MRSA in human hosts60. Partial excision is sufficient to result in methicillin-susceptible phenotype, and most often such MSSA strains showed great genetic homology to its MRSA counterpart, although less prevalent61. Given the genomic resemblance of the HS-MSSA strain to the global MRSA clones, we decided to investigate whether such an excision event has occurred in this local MSSA strain. In order to identify possible remnants of SCCmec cassette or its homologs in the genome, the database search was repeated at 60% identity threshold with 30% minimum query cover length. The search result remains the same, i.e. intact or partial mec genes and the whole cassette was not detected, but a 1.5 kb DNA region associated with the insertion sequence-like IS1272 in SCCmec type I was detected in contig-4, albeit with considerable genetic variation (94% query coverage and 82% nucleotide identity). BLAST analysis of this 1.5 kb region revealed the presence of a pseudogene of IS1182 family transposase in S. aureus . In short, none of the SCCmec elements was detected except for the 1.5 kb DNA region homologous to IS1272 commonly found in the mec-gene complex in MRSA that may also present infrequently in MSSA62.While the acquisition of SCC19. Genomic sequence analysis revealed a low number of AMR-conferring genes compared to virulence genes, and a majority of which are involved in bacterial efflux systems . Cross-examination with virulence genes analysis showed that the HS-MSSA strain carries multiple genes encoding for bacterial adherence, enzyme production, host immune evasion, various prominent staphylococcal toxins, a type VII secretion system, and intact regulatory operons for virulence expression . The virulence analysis of the HS-MSSA strain is reported in detail in our earlier publication19  in the MRSA represses the transcription of the agr operon, thus halting the production of the virulence factors that are regulated by this operon. Therefore, the ability of the HS-MSSA to cause severe sepsis in the human host could be related to the lack of SCCmec genes cassette in its genome, thus the expression of its multiple virulence factors was not impeded.The pathogenicity genes analysis showed that the HS-MSSA harbours 536 CDSs which matched to known lukFS-PV). When mapped to the reference genome S. aureus NCTC 8325, the PR2 showed great genetic similarity with a 72 kb prophage on NCTC 8325. The PR3 was identified on contig-11, closely associated with genes encoding for β-hemolysin (hlb), cytolytic pore-forming protein (lukGH), extracellular adherence protein (eap), and staphylococcal complement inhibitor (scn). A total of six genomic islands (GI1-GI6) were identified in the HS-MSSA chromosome by using S. aureus NCTC 8325 as the reference 67. An array of staphylococcal virulence genes mainly associated with bacterial adherence and stress response were found in GI4, while GI5 consisted of mainly hypothetical proteins.Three prophage regions were identified in the HS-MSSA chromosome. Phage region 1 and 3 (PR1 and PR3) are incomplete prophages, while phage region 2 (PR2) is an intact prophage spanning a 64.1 kb genomic region , sharing of similar mobile genomic regions , same geographical origin (KT/314250), same clonal complex (MSSA476), and representative MSSA and MRSA genomes (MSSA476 and COL) from European region . The HS-MSSA genome showed ≥ 94% genome coverage and ≥ 99.16% nucleotide identity  with all selected S. aureus genomes. The ST22 reference genome HO 5096 0412 (NC_017763) from a clonal complex (CC22) less closely related to other S. aureus strains was used as an outgroup in constructing the ML tree.Representative 67. Together with another MRSA strain M2024 (NZ_CP047021) which caused recurrent clonal outbreaks in Denmark49, the three S. aureus strains formed a loose cluster with relatively divergent evolutionary paths and geographical origins (Cluster I). Interestingly, the MSSA476 from the United Kingdom that shares a common clonal complex (CC1) with HS-MSSA formed another distinctive cluster (II) although both are community acquired MSSA capable of causing bacteraemia in human hosts and carried the strain-specific plasmid-borne blaZ gene70. Similarly, the MSSA strain KT/314250 which was also isolated in Malaysia formed a separate clade distinctive from the HS-MSSA lineage but is tightly clustered with the MSSA476 strain. The representative S. aureus strains from Europe (NCTC 8325 and COL) and MRSA clones from America formed a third cluster (III) more distantly related to the HS-MSSA strain.The phylogenetic analysis results revealed that the HS-MSSA strain formed a distinctive clade by itself, sharing a more recent common ancestry with the MRSA clones in Europe and Japan (M2024 and N315)  with HS-MSSA only appeared on scientific records in 2014. Based on the limited strain data available in the PubMLST database for the MSSA strain 128A, we hypothesize that the ST2990 strains in Malaysia showed variable clinical manifestations, ranging from non-invasive SSTI to severe bacteraemia. The HS-MSSA lineage has probably accumulated sufficient adaptive features through the acquisition of mobile genetic elements, giving rise to its unusual virulence in causing severe invasive infection in human host19. Moreover, the close genetic proximity with the MRSA clones and its genome plasticity also suggests that the HS-MSSA lineage could provide a stable genetic environment to allow the acquisition of genetic elements that confer drug resistance6. Therefore, continuous molecular surveillance to detect highly virulent MSSA in clinical settings is essential to allow timely management of invasive staphylococcal infections and the development of potential anti-virulence therapy.The phylogenetic relationships of the HS-MSSA strain with the global S. aureus strains selected for cgSNP-based phylogenetic analysis, we observed that the HS-MSSA strain genotyped as CC1/ST2990 was not found on the same clade as other CC1 strains. In order to investigate the accuracy of this phylogenetic clustering, representative genomes of the central STs for the major CCs were retrieved from the PubMLST database for further analysis71. Overall, the clustering of the STs corresponded with the CC grouping based on the 7-loci MLST scheme  which involves the assessment of 1351 loci (for S. aureus) and the whole genome MLST (wgMLST) are the more appropriate approaches for inferring phylogenetic relationship of genetically clonal strains. Previous studies using different bacterial species have observed a high concordance between cgMLST and wgMLST with SNP-based analysis, generating phylogenetic trees with great statistical similarity and almost equivalent clustering of bacterial strains73. Therefore, the current clustering of ST2990 into CC1 needs to be re-assessed by the scientific community. Nonetheless, more ST2990 genomes should be included for cgSNP-based analysis in the future to verify this notion. The present study is limited by only one ST2990 (HS-MSSA) genome that was available on publicly accessible genomic databases at the time of writing.Our observations support the previous notion that the current grouping of We report herein a novel genotype of MSSA (t091/ST2990) of community origin that caused severe sepsis in the human host. Upon examination, the HS-MSSA genome harbours only very few AMR genes, which was reflected in its pan-susceptible phenotype. The HS-MSSA strain carries multiple virulence genes in its genome, including prominent staphylococcal toxin genes that are associated with mobile genetic elements (prophages and SaPI). The HS-MSSA strain shares a common ancestry with the MRSA clone originated from the European region, but instead of developing drug resistance, it has evolved through a different path via the acquisition of multiple virulence and pathogenicity factors. Due to the limited epidemiological data available for this novel genotype (t091/ST2990), we could not conclude that this MSSA lineage has emerged locally or the HS-MSSA strain was just a sporadic strain imported from other regions without molecular typing data. Although the patient reported no history of recent travels upon admission, we cannot rule out the possibility of local transmission. Therefore, increased effort in molecular surveillance is essential to allow the development of appropriate treatment strategies to reduce the mortality risk of severe invasive infections caused by this organism.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.


Graphene has become the focus of extensive research efforts and it can now be produced in wafer-scale. For the development of next generation graphene-based electronic components, electrical characterization of graphene is imperative and requires the measurement of work function, sheet resistance, carrier concentration and mobility in both macro-, micro- and nano-scale. Moreover, commercial applications of graphene require fast and large-area mapping of electrical properties, rather than obtaining a single point value, which should be ideally achieved by a contactless measurement technique. We demonstrate a comprehensive methodology for measurements of the electrical properties of graphene that ranges from nano- to macro- scales, while balancing the acquisition time and maintaining the robust quality control and reproducibility between contact and contactless methods. The electrical characterisation is achieved by using a combination of techniques, including magneto-transport in the van der Pauw geometry, THz time-domain spectroscopy mapping and calibrated Kelvin probe force microscopy. The results exhibit excellent agreement between the different techniques. Moreover, we highlight the need for standardized electrical measurements in highly controlled environmental conditions and the application of appropriate weighting functions. One of the most promising methods is the use of CVD to grow graphene onto various metallic substrates. In CVD growth, graphene is grown on the surface of the metal after hydrocarbons decompose2. The most promising metal substrate used until now is Cu3, however graphene is currently grown also on Ni(111)6, Ru(0001)7, Pt(111)9, and Ir(111)11. Subsequently to the growth process, for graphene to be use in electronic applications, it needs to be transferred on an insulating substrate , which is a transparent and flexible substrate). The already happening now use of graphene in RF electronics12, integrated circuits13 and optoelectronics14 has triggered impressive progress in large-scale production, however the community still lacks standardised electrical measurements to extract useful parameters such as carrier concentration, mobility and sheet resistance, all of those are often presented as figures-of-merit of the graphene quality. Currently, the most widely used method for electrical characterisation involves magneto-transport measurements in lithographically patterned Hall bars to extract carrier concentration, mobility and resistance. However, this method is not suitable for high throughput characterisation and often the measured graphene is significantly altered due to the microfabrication processes, in which case the electrical properties of the pristine transferred graphene are different from the ones measured. An alternative method for mapping the sheet resistance of graphene films is using micro four-point probes (M4PP)16. Recently the use of microwave cavities18 and Terahertz time-domain Spectroscopy (THz-TDS)23 measurements provided a non-contact method of measuring the conductivity of graphene films. Despite the attractiveness of these techniques, fluctuations in ambient conditions (such as humidity) affect the electrical properties of graphene25, resulting in discriminations between measurements at different laboratories.The unique properties of graphene26. This method was originally introduced by van der Pauw in 1958 to measure the resistivity and Hall coefficient of arbitrary shape semiconductors28.In contrast to the conventional Hall effect, where device fabrication is required for carrier concentration and mobility measurements, the van der Pauw (vdP) method only requires thin uniform samples with relatively small Ohmic contacts31.THz-TDS has recently been demonstrated to be an excellent tool to characterize the quality and conductivity of graphene deposited on large areas with suitable choices of substrates and models. THz-TDS can provide a map of the absolute value of conductivity/resistivity in a non-contact and non-destructive manner. The measurement is fast and can cover the entire area of a sample and can be used in all stages of a material development since the prototyping up to manufacturing scale32 is commonly used for measuring the local work function and distribution of graphene layers with nanometre resolution. Nevertheless, this technique requires ultra-high vacuum to operate and the surface must be completely clean, meaning that high temperature annealing (~400 °C) is used prior the measurement. Both conditions pose a significant drawback when rapid characterisation is needed. An alternative, complementary technique that can provide valuable information about local electrical properties, such as surface potential, doping and work function as well as providing essential information about layer distribution down to ~20 nm spatial resolution is frequency-modulated Kelvin probe force microscopy (FM-KPFM)34. KPFM has already demonstrated great potential for graphene characterisation, identification of graphene layers as well as measuring the surface potential of the samples with nanometre resolution40.An important property of graphene is its work function 41. They will become part of Good Practice Guides for the measurement of electrical properties of graphene and will be proposed as an input to normative bodies, towards the development of documentary standards. In this work, we demonstrate appropriate measurement techniques to characterise the most important electrical properties of graphene from macroscale down to nanoscale and establish the validity of the measurements through correlation in an inter-laboratory study and applying a weighting correction function between the results obtained from THz-TDS and van der Pauw methods.In this paper, we propose and demonstrate a standardised procedure for measuring and mapping the electrical properties of scalable graphene using a combination of contact and contactless complementary methods, such as van der Pauw magneto-transport, THz-TDS conductivity mapping and Kelvin probe force microscopy. The methodologies here described are currently being developed within the GRACE projectIntensity-Z curves for each pixel from the stack of images. The system is operated in ambient air and does not require any sample preparation. For these measurements, individual images with high magnification (100 × objective) were captured and then stitched together to generate a high resolution image of a larger area42.Confocal laser scanning microscopy (CLSM) is a powerful optical technique that enables rapid structural characterisation of large-area graphene and graphene nanostructures by pushing the spatial resolution beyond the optical diffraction limit and stacking individual images of different focal planes. CLSM was performed using an Olympus LEXT OLS4100 system equipped with 5×, 10×, 20×, 50× and 100× objectives  and with further 1× to 8× optical zoom. Therefore, images of a field-of-view ranging from 2560 μm to 16 μm can be captured. The system uses a 405 nm wavelength laser, which scans in X-Y directions using an electromagnetic micro-electromechanical system (MEMS) scanner and a high-precision galvano-mirror. The optical images  are generated using a photomultiplier which captures the reflected light. In this confocal setup, in-focus reflected light is only allowed to pass through the circular confocal pinhole, therefore eliminating flare from out-of-focus areas. Due to the shallow depth-of-field of the CLSM setup, the objective is moved vertically to capture images of different focal planes. Using this Z-stacking technique, a single output of the 2D reflected intensity map is extracted by determining the maximum brightness value of the calculated f0 ≈ 300 kHz, with an AC voltage of much lower frequency fmod ≈ 3 kHz of 5 V amplitude also applied, to induce a frequency shift of f0 ± fmod. The side lobes (monitored by a PID feedback loop) generated by this shift are minimized by applying a DC compensation voltage. By measuring this DC voltage at each pixel, a surface potential map [i.e. contact potential difference . Since FM-KPFM measures the surface potential (difference in work function between the tip and the sample), it provides a qualitative result, highly dependent on the tip status. In our approach we demonstrate a method to appropriately calibrate the work function of the scanning tip (24). Then the same HOPG reference sample is measured using FM-KPFM, and the tip is calibrated using FM-KPFM uses the force gradient mentclass2pt{minimRsTHz) is based on measuring the difference of the THz reflectivity of the film on top of the substrate and the reflectivity of the bare substrate. In transmission geometry, the conductivity is extracted by comparing the signal of the sample with the thin film and the signal of the bare substrate in transmission mode.The extraction of the resistivity of a thin film on a substrate using THz-TDS in reflection geometry  of the sample, passing current and measuring voltages in different configurationsBy applying current and measuring the voltage drop at the opposite sides and repeating the operation by rotating the sample by 90° Fig. , 8 resisUsing the values from Eq. , the resTherefore, the sheet resistance VH) is measured between the remaining two corners  and mobility (μ) can be calculated using the following equations from the Hall coefficient RH:Finally, the carrier concentration  response of the AC biased sample is measured. In both VH measurements, the voltages are measured using a lock-in amplifier (LIA) referenced to the first harmonic of To measure the magneto-transport properties in vdP geometry, an Arduino microcontroller is used to trigger solid state relays, allowing current to pass and voltage measurements to be done at the different configurations as shown in Table ing Eqs.  and 8)..\documenρ(P) is the local sheet resistivity at point P, 47 to identify the general form of ρ(P) itself. The zero-order approximation for 47), with a value of about 3.4 in the centre and vanishing on the square edges. Therefore, the resistivity near the centre affects The sheet resistance values In the following, we present and discuss results obtained on a batch of 6 samples of CVD graphene transferred on a single quartz substrate and diced into chips.Figure 25. The summarized results of work function measurements of the different samples are shown in Fig. When a single-atomic layer such as graphene is measured, surface contamination may lead to incorrect results. When graphene is transferred on a target substrate, the top surface is covered with polymer residues. In our measurements, the residues are removed prior to the measurement using contact-mode atomic force microscopy (AFM) to expose a clean surface. The results in ambient are shown in Fig. Figure entclass1pt{minima48, and they can be related to the presence of microscopic defects, such as cracks, and to the presence of growth domain. The measurements are affected by edge effects that span about 1 mm from the edge due to the size of the THz beam, which is estimated to be 0.5 mm in diameter. Most of the features show up as spots or islands of significantly higher sheet resistance, indicating a low quality of the graphene deposited in that particular area . Other features show up as two distinct sheet resistance areas separated by a varying size transition zone (e.g. samples 3 and 39).The features observed in these maps have been reported in the literature in25. Additional advantage of the vdP method is that using the current setup both ambient and vacuum measurements can be performed and thus the effect of the environmental exposure on the electrical properties of graphene can be studied.Following the THz-TDS sheet resistance mapping, the magneto-transport properties of six samples were measured using the home-build vdP setup described in Section 2.4. VdP can be considered as a complementary method to THz-TDS measurements as the later one only provided information about the sheet resistance/conductance and can only be performed in ambient conditions. As both carrier concentration and mobility are essential parameters for the material performance, their measurements provide crucial information about the quality of the graphene layer25. Therefore, using the dedicated measurement setup, the magneto-transport measurements in the van der Pauw geometry were also performed in vacuum. Prior the measurement, the samples were annealed (170 °C) in vacuum  Fig. , such asers Fig. .Figure 6In order to compare the results of vdP and THz-TDS methods, the following equations were used to calculate the relative percent differences on resistance:wing Eq. ) demonstA visual explanation of the correction can be given for sample S3 as an example in Fig. The characteristics of each technique are summarised in Table 41 will become an important part to contribute to the standards development work of the normative bodies, through the initiation of and dissemination of new written standards for the electrical characterisation of graphene based on the Good Practice Guides developed within the project.In conclusion, we introduce a procedure for performing electrical measurements of CVD graphene from macro- to nano-scale using a variety of contact and contactless techniques. The sheet resistance, carrier concentration and mobility were measured using macroscale magneto-transport measurements in the vdP geometry, both in vacuum and ambient conditions to extract the pristine electrical properties of graphene. Upon annealing in vacuum, the carrier concentration has significantly decreased due to desorption of atmospheric molecules (such as water), while the carrier mobility and sheet resistance have significantly increased. The measurements were compared to the sheet resistance maps obtained using microscale THz-TDS mapping, where a weighted average function was applied. For most of the samples, the difference in the sheet resistance as obtained by vdP and THz-TDS was below 5%, demonstrating the good agreement between the different methods. A method for calibrated KPFM measurements of graphene samples is demonstrated, allowing high resolution mapping of the work function on the nanoscale and highlighting the impact of polymer residues on the surface. This work highlights the importance of using a standardized methodology for electrical measurements using a variety of complimentary techniques to obtain a complete understanding of the sample on a variety of lateral scales. The development of these presented metrology and methodologies within the GRACE project3 containing solution. Distilled water was used to clean the film several times after it was transferred onto a quartz substrate. Finally, the PMMA layer was removed by immersion in acetone. In this work we use 4″ CVD graphene sample, which was diced into 10 × 10 mm2 squares. Several diced chips were chosen in random and studied by a number of methods as discussed.The mono-layer graphene growth was carried out in a cold-walled chemical vapour deposition (CVD) reactor (Aixtron BM) using Cu foil as the catalyst. Following the growth, PMMA was spin-coated onto the graphene-covered Cu foil, acting as a sacrificial support layer. The Cu foil was then etched using a FeClSupplementary Information.


The online version of this article (10.1007/s10853-020-04662-y) contains supplementary material, which is available to authorized users. Chemicadelivery  or the adelivery , 14.π–π conjugation, leading to the loss of mechanical strength, as well as reduced electrical and thermal conductivity [3 times lower than pristine graphene [Most existing covalent functionalisation of graphene family nanomaterials is based on grafting molecules through oxygen-containing functional groups of graphene oxide (GO), followed by chemical or thermal reduction to obtain reduced graphene oxide (rGO) , 16. Grauctivity , 19. Youuctivity , and evegraphene . GO-baseπ system which forms attractive hydrophobic and π–π interactions with aromatic molecules [Functionalisation of pristine graphene can be achieved by both covalent and non-covalent interactions . Pristinolecules , 22. Priolecules , 24, pyrolecules , triphenolecules , and corolecules , as wellolecules . Althougolecules  and condolecules  of the psp2 carbons on the basal plane using diazonium salts [π system and so degrades the product’s conductivity due to the conversion of sp2 carbons to sp3 carbons [Covalent modification of pristine graphene can be achieved through free radical addition to um salts –37 or beum salts  to form um salts –47. This carbons , 33, 48, carbons .3 is traditionally used. Benzoic acid derivatives with polyphosphoric acid and phosphorus pentoxide have been used for milder graphene acylations [Edge-selective covalent functionalisation minimises chemical reactions on graphene’s basal plane and the attendant deterioration of engineering properties. Ball milling graphite in the presence of gases or gas mixtures has been reported to produce edge-specific functionalisation . The tecylations , 56, 57.ylations –60. Otheylations . Furtherylations .The current work extends the top-down method of electrophilic aromatic substitution to create five distinct types of edge-modified graphene with no intermediate carbonyl moiety. Our modifications significantly modified the graphene nanoparticles’ dispersibility. The synthetic utility of the directly attached reactive moieties was demonstrated by creating a “glycographene” through radical addition of allyl mannoside to edge-thiolated graphene. Chemical modifications were confirmed by FT-IR and XPS. Edge localisation was visualised on modified CVD graphene samples by scanning electron microscopy of gold nanoparticles attached to thiol groups, epifluorescence microscopy of a fluorescently tagged lectin–glycographene bioconjugate, and quenched stochastic optical reconstruction microscopy (qSTORM) –66, a su2 g−1 and 300 m2 g−1, respectively, corresponding to 4–5 graphene layers for C750 and 8–9 layers for C300. The batches used in this work had BET specific surface areas of 794.9 ± 1.6 m2 g−1 and 268 m2 g−1. The M25 grade has a nominal lateral dimension of 25 µm. The specific surface area specified by the manufacturer is 120–150 m2 g−1, corresponding to about 17–22 graphene layers; the batch used in this work had a BET specific surface area of 55 m2 g−1. On receipt, the GNPs were portioned into 50-ml Falcon tubes and sealed with Parafilm.Graphene/graphite nanoplatelets  were purchased from XG Sciences . The C750 and C300 grades have nominal specific surface areas of 750 m2 sheets to a silicon wafer covered with 300 nm thermal oxide. Before use, CVD graphene was stored in a covered Petri dish under ambient conditions.Monolayer graphene produced by chemical vapour deposition (CVD) on copper was provided by 2-DTech  and transferred as ~ 1 cmN-methyl-2-pyrrolidone (NMP) at 37 Hz for 48 h in an ultrasonic bath (Elmasonic PH750EL). Remaining graphite was removed by cetrifugation (3 × 20 min at 4000 rpm). The supernatant was a graphene dispersion (ca. 0.4 g l−1). Graphene reaction dispersions were vacuum filtered through 0.02-µm Whatman Anodisc membrane filters to create graphene laminates. For analysis, samples were dried as laminates on the filter membrane or re-dispersed in water by sonication and freeze-dried . For long-term storage, the samples were kept in a desiccator at room temperature.Graphene dispersions were also prepared by ultrasonic exfoliation of natural graphite flakes . Following the method of Hernandez , 2 g graN,N-dimethylformamide  and methanol were purchased from Fisher Scientific, UK. Sodium borohydride (≥ 96%), iodine (≥ 99.8%) and triphenylphosphine (99%) were purchased from Sigma-Aldrich. Raney nickel catalyst (50% in water) and chlorosulphonic acid  were purchased from Merck Chemicals. All chemicals were used as supplied. Water was purified to a resistivity of 18.2 MΩ cm at 25 °C (Milli-Q).SnakeSkin dialysis tubing  was purchased from Thermo Scientific. Disposable folded capillary cells for Zetasizer Nano Series were purchased from Malvern.3−, Scheme Sulphonated graphene (G–SO3) in 30 ml of toluene and sonication under N2 atmosphere for 15 min. Then, the reaction flask was connected to a condenser with nitrogen flow. 2.5 g of triphenylphosphine and 200 mg iodine were added into the mixture that was left stirring at 80 °C for 21 h. The product was filtered using vacuum filtration and 0.45-μm HV membrane filter and washed with toluene, acetone, 0.1 M sodium thiosulphate solution, and deionised water, followed by freeze-drying.Thiolated graphene  were bound to thiolated CVD graphene. The nanoparticle solution was diluted tenfold in phosphate-buffered saline . A sample of modified CVD graphene was immersed in 10 ml of the diluted nanoparticle suspension overnight at ambient temperature. The sample was rinsed with water and acetone and dried in a nitrogen gas stream and stored at room temperature.Gold nanoparticles  in a glass vial wrapped in aluminium foil. The CVD glycographene sample was added to the vial and incubated at 37 °C for 2 h. The sample was rinsed with water and dried in a nitrogen gas stream.Glycographene Scheme  was made−1 using a Renishaw inVia, with a 633-nm excitation laser set to 10% (0.89 mW) power or a 514-nm laser (0.176 mW). The energy resolution was 0.3 cm−1. Raman shifts were calibrated by setting the G (E2g) peak from silicon to 1581 cm−1 [−1 every 0.5 µm . Data were processed using WiRE 4.2 software to zero the baseline and remove cosmic rays. Nonlinear fits (SI) were applied to the peaks following the methods of Puech et al. [D band (i.e. ID/IG) using the Tuinstra–Koenig relationship (D band (i.e. AD/AG) using the relationship from Cançado et al. (LD) was estimated using the expression from Lucchese et al. (Eq. S4) [Raman spectra were recorded from 100 to 3200 cm581 cm−1 , 69. The581 cm−1 . Raman mh et al. . Domain  Eq. S1) , the rel Eq. S2) , and the Eq. S3) . The ave(Eq. S4) .−8 mbar during measurements. Peaks were fit using CasaXPS with Shirley background correction. The C 1s energy was calibrated by fixing the binding energy for the sp2 component to 284.5 eV. The C 1s peak was fit as five components summarised in Table sp3:sp2 C ratio were from Monte Carlo simulations within CasaXPS. Fits to 2p1/2 and 2p3/2 peaks were constrained to have identical FWHM values and an area ratio of 1:2.X-ray photoelectron spectra (XPS) were acquired using a Kratos Axis Ultra with an Al Kα source 1486.6 eV) operated at 15 kV and 10 mA. The pressure of the vacuum chamber was below 5 × 10486.6 eV −1, 32 scans, 4 cm−1) were acquired using a Nicolet 5700 FT-IR spectrometer in air. Background spectra were recorded every 10 min.FT-IR samples were prepared by mixing approximately 0.2 mg modified GNPs with 300 mg KBr  using an agate mortar and pestle then pressed at 10 tons from a hydraulic press for 5 min to obtain a sample disc. Transmission spectra (4000–400 cmx–y stage (PRIOR HLD117) and a PRIOR ProScan III controller. The sample was illuminated by laser beams delivered to the back of the microscope using an optical fibre. A vibration motor was attached to the fibre to remove coherent artefacts in the final image due to laser speckle. Light from the sample was collected by the lens and incident on a Hamamatsu ORCA Flash v2 sCMOS camera. Up to 20000 individual images were acquired for each area with an exposure time of 10 ms. External and internal edges of thiolated CVD graphene were labelled with BODIPY FL L-cystine  and Alexa Fluor 647 maleimide . The graphene samples were immersed in 10 mL deionised water to which 10 mM dye stock in DMSO was added for 2 h at room temperature. After coupling, the samples were washed with water and acetone, dried under a nitrogen stream, and kept at 4 °C and away from light. Image data were processed using ImageJ with the ThunderSTORM plugin [STORM images were taken using a previously described custom-built STORM system , consistM plugin .Epifluorescence images were collected on an Olympus BX51 upright microscope using UPlanFLN objectives and captured using a Coolsnap camera (Photometrics) through MetaVue software (Molecular Devices). A specific bandpass filter set for FITC  was used.2/Si were mounted on 12.5-mm aluminium stubs. No surface coatings were applied.Scanning electron microscopy (SEM) images were acquired using an FEI Quanta 650 FEG-SEM operating at 1–5 kV. CVD graphene samples on SiOSpecific surface area was determined from the Brunauer–Emmett–Teller (BET) method using a Micromeritics Gemini V surface area and pore size analyser.−1 from 30 to 800 °C in a N2 atmosphere.Thermogravimetric analysis (TGA) was performed on a TA Instruments Q500 thermogravimetric analyser. 1–3 mg of graphene sample was heated at 10 °C min−1) in deionised water were prepared and placed in disposable foldable capillary cells for testing. The measurement was repeated 6 times for each type of functionalised graphene.Zeta potential was determined using a Malvern Zetasizer Nano series. Aqueous graphene suspensions (0.05 mg mlk0) to pristine and edge-modified CVD graphene samples were carried out in a two-electrode set-up described Valota et al. [Estimates of the interfacial electron transfer rate  at 550 °C. The micropipette was filled with 5 mM potassium ferricyanide  in 6 M LiCl aqueous solution . A high concentration of LiCl was used to prevent droplet evaporation . The fil64− adsorption on highly oriented pyrolytic graphic (HOPG) lowered electron transfer rates [k0 values [−6 cm2 s−1 was used based on previous measurements using the Randles–Ševčík equation [A camera with a microscope objective  and light source were positioned parallel to the sample surface . Droplet deposition was monitored and recorded using Infinity Analyze software (v.4.2) . A second camera above the sample provided low-resolution, live imaging of the sample to identify areas for deposition. After depositing a droplet, cyclic voltammetry was run using NOVA software (v. 1.11). An uncertainty of 24.4 mV was assigned according to the step size during the scan. Cyclic voltammograms (CVs) were conducted for seven scan rates in nine separate droplets. A set of measurements on a single droplet typically required ca. 30 min, an important consideration in light of the observations by Patel et al. that Fe(CN)er rates . Typical0 values . A diffuequation . Some elequation , 83.3−, Scheme Graphene/graphite nanoplatelets (GNPs) and monolayer CVD graphene were treated with chlorosulphonic acid and then hydrolysed with base to produce edge-modified graphene sulphonate  than the thiol S 2p peaks (165.1 ± 0.2 eV and 164.0 ± 0.2 eV). The S 2p spectrum for G–SH showed that the reduction of the sulphonate to the thiol was incomplete (–SO3−/–SH = 2.8 in the example shown). Quantification of the XPS survey spectra for G–SO3− and G–SH gave a S:C atomic ratio of about 0.3:100  –89, an etransfer .sp2 carbon in the GNPs after edge modification was deduced from deconvolution of the C 1s peak , caused by a one-phonon defect-assisted electron–defect scattering event from edges, grain boundaries, or internal defects [ID/IG ratios and equally prominent D′ features . Larger GNPs (XG C300 and M25) showed a decreasing ID/IG ratio with increasing GNP size , consistent with the Tuinstra–Koenig relationship [ID/IG caused by the edge modification. A Raman map of sulphonated CVD graphene  showed that the ID/IG ratio was lowered at the edges, demonstrating that the chemical modifications complicate this simple metric for graphene order. This effect has previously been discussed for graphene with its basal plane modified by diazonium salts [areas of the D and G peaks, namely the average domain diameter, LD, were more consistent. Sets of modifications to commercial GNPs and graphene produced by mechanically exfoliating graphite in NMP showed little changes in LD on modification relative to the source materials (Table S6). For CVD graphene, LD consistently dropped by about half and Raman analysis of the GNPs before and after edge modifications  of the edge-modified G–SO3− produced in this study, regardless of whether the GNPs or graphite exfoliated in NMP was used (Table S5). The Raman 2D peak shifts to lower energy with decreasing layer number when the number of layers is below ~ 10 [Sulphonated graphene (SGnP) has been produced by chemical exfoliation of graphite in chlorosulphonic acid. The work of Abdolmaleki et al.  reportedlow ~ 10 , but no low ~ 10 –96.D peak, and a ID2/IG ratio of over 2 and a FWHM of 24 cm−1, indicative of single-layer graphene [ID2/IG ratio falls below 1 for bilayer graphene and drops further as the layer number increases. The spectra taken from the edges of the graphene sheet had a measurable but low ID/IG and lower ID2/IG ratio, consistent with the literature [ID/IG ratio, consistent with the more ragged appearance of the flake by visible light microscopy  and STORM  of the samples over a range of 100–900 °C  between the unmodified samples and the dye-labelled G–SH  × 10−4 cm s−1 . The large uncertainty in the result is likely due to inhomogeneity of the CVD surface . The mean k0 for the drops sitting at the edges of CVD G–SO3− was (3.6 ± 1.7) × 10−4 cm s−1 . Electron transfer rates to the edges of single-layer graphene are expected to be two orders of magnitude greater than the basal plane [k0 reduced to (1.0 ± 0.5) × 10−5 cm s−1 , comparable to unmodified CVD graphene. Some of the decrease must be attributed to the bulky dye slowing edge electron transfer because the value of LD estimated from Raman spectra remained about half the value of unmodified CVD graphene, as it was for G–SH (Table S6). The electron transfer rates for the unfunctionalised CVD graphene and dye-labelled CVD graphene are comparable to those of the basal plane of HOPG exposed to air for > 60 min after exfoliation, based on comparable peak separations from scans at 100 mV s−1 [The al plane . The onl0 mV s−1 .The edge modifications produce marked changes in the graphenes’ dispersibility, consistent with the chemical changes . Sulphonation produces graphene that is water dispersible; reduction to the thiol creates a material dispersible only in toluene. Glycographene is moderately dispersible in water, making it a conceivable candidate for applications in biosensors or biomaterials. Contact angle measurements (Table S4) were consistent with these observations.The edge sulphonation of larger GNPs also produced an unexpected trend. These were presumed to have a smaller amount of edge length for a given mass. Elemental analysis by XPS  showed the opposite trend; however, the largest flakes had the highest S/C ratio. This observation could be an artefact residual, unreacted chlorosulphonic acid trapped between the layers of the GNPs. Consistent with this, graphite has previously been intercalated by tosylate  and meth2) was performed and reduced to aminographene (G–NH2). FT-IR  confirmed the presence of the nitrogen-containing functional groups. G–NO2 showed a distinct, sharp N–O vibration at 1385 cm−1 characteristic of nitrated aromatic systems. This signal disappeared when G–NO2 was reduced to form G–NH2, and distinctive N–H vibrations appeared from 3700 to 3300 cm−1 along with C–N stretches and N–H scissoring bands at lower energies.The nitration of GNPs . The relative amount of the nitro group (Eb = 406.9 ± 0.3 eV) decreases between the G–NO2 sample and its reduced G–NH2 analogue. The largest peak at 400.1 ± 0.2 eV can be assigned to organic nitrogen groups, including amines and nitrogen heterocycles [2 sample. A small contribution at 402.8 ± 0.4 eV was attributed to nitrogen in an intermediate oxidation state, such as a hydroxyl amine. Nitration produced products with a greater heteroatom content (N:C around 0.5:100). Analysis of the G–NH2 was complicated by the presence of by-products from the nitro reduction over Raney nickel.The N 1rocycles –105, butAs a nitration caused too damaging to CVD graphene, epifluorescence of FITC-labelled GNPs M25 aminographene was an alternative method to highlight the specific edge modification in this study . A faint green fluorescence on the edge of GNP  could be a possible dye attachment. Nevertheless, conventional fluorescence microscopy was not suitable to observe the edge nitration of GNPs due to a low fluorophore attachment.sp3:sp2 C ratio, and estimated distance between defects (LD) from Raman are evidence of chemical functionalisation predominantly at the edges and internal defects. This straightforward and scalable method of edge-specific functionalisation creates a versatile platform material for biotechnological and biomedical applications of graphene, rather than graphene oxide or its derivatives. Ongoing and future work on these materials includes measurements of their electronic conductivity and interfacial electron transfer coefficients, applying them in nanoscale theronostics devices, using them as platforms for tissue engineering and regenerative medicine , synthesising MRI contrast agents (e.g. with Gd-DOTA–based side groups), creating multiple reactive groups (e.g. both thiol and amine) for bio-orthogonal functionalisation, and assessing whether the mechanical and electrical properties are maintained upon edge functionalisation.Super-resolution microscopy of fluorophore-labelled edge-modified graphene, the s XPS spectra; elemental analysis based on XPS survey spectra; Raman spectra; schematic for microelectrochemistry and representative measurements; thermogravimetric analysis of pristine and edge-modified GNPs; dispersibility and contact angle measurements; Raman comparison of GNP source for edge sulphonation; Raman and XPS analysis of GNP size on edge sulphonation. In compliance with the University of Manchester’s requirements and those of the funding agencies, these data for this work are available to download from https://data.mendeley.com/datasets/mwrvrcj639/draft?a=20782f0e-4a57-4056-a4e7-c24057beded6Supporting information available: synthesis of nitrographene and aminographene; Raman spectrum analysis methods; representative survey and C 1 (PDF 3805 kb)Below is the link to the electronic supplementary material.


Blank  and experimental  groups of lotus seeds were stored at 25 °C and relative humidity of 40%, 50%, 60% and 70% for about 30 days, respectively. The mildew was observed and the changes of Cw, Aw, together with the production of aflatoxins were measured. Results showed that no mildew was found and aflatoxins were not detected in lotus seeds when they were stored for 30 days at 25 °C and relative humidity of 40%, 50% and 60% with Cw < 12% and Aw < 0.6. While, when the relative humidity was up to 70%, the Cw and Aw values rose quickly, and the Cw exceeded the officially-permitted level (14%). Although no mildew was observed, AFB1 was still detected, increasing the potential risk of lotus seeds regarding aflatoxins. For warranting the quality with economic and safe storage, lotus seeds are suggested to be stored at 25 °C and relative humidity lower than 60% with 12% and 0.6 as the safe Cw and Aw control points, respectively, to prevent medicinal and edible products from mildew and the contamination of aflatoxins.Affected by the inner properties and the external environmental conditions, medicinal and edible lotus seeds are susceptible to mildew with fungal infection under suitable temperature and humidity conditions, leading to the production and contamination of various mycotoxins, along with threats to its quality and safety. In this study, the changes of water content (C Foods including medicinal and edible products, feeds and medicinal plants are vulnerable to fungal conidia and mildew if improperly treated in the processes of growth, harvest, processing, storage and transportation , as a valuable and commonly-used edible and medicinal matrix, not only has the properties of tonifying spleen, stopping diarrhea, stopping bandage, tonifying kidney, nourishing heart and tranquilizing mind, but also is a common food or food additive with wide nutritional and edible functions. However, affected by the nature of their components and the external environmental conditions, lotus seeds are susceptible to mildew with fungal infection under suitable temperature and humidity conditions, especially during the plum rain season, leading to the production and residue of various mycotoxins, such as aflatoxins, ochratoxins, etc. Aflatoxins, mainly containing aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1), and G2 (AFG2) with strong hepatotoxicity and carcinogenic, teratogenic and mutagenic effects , water activity (Aw) and environmental temperature and humidity, to ensure their quality and safety.Various mycotoxins have been found with residue in different kinds of matrices  system. In practice, foods are commonly dried to reduce the water content to prevent them from mildew. In the other hand, salt or sugar is added to decrease the Aw of matrices to inhibit the growth of toxigenic fungi. Therefore, in public view, effective control Cw and Aw could prevent foods from mildew and the production of mycotoxins to ensure their safety.It is generally believed that in the processing, storage and transportation processes, foods, medicinal plants and other matrices should be dried sufficiently to control their water content for inhibiting the growth of mildew or pests. In addition, Aw and Aw changes of lotus seeds under different storage conditions according to the experimental design in Fig. w and Aw and mildew for exploring the optimum water content and water activity control points for the safe storage. These findings will provide powerful supports and valuable references for screening for the reasonable, reliable and effective storage conditions to prevent medicinal and edible foods and other products from mildew and mycotoxins contamination for ensuring their quality and safety.Therefore, through the investigation of the C1 and AFG1, and 0.5 mg/mL of AFB2 and AFG2 was purchased from Pribolab Pte. Ltd. (Singapore). The ToxinFast®-Aflatoxins Test immunoaffinity columns were bought from Huaan Magnech Bio-Tech Co., Ltd . The aflatoxigenic  Aspergillus flavus (A. flavus) lyophilized powder (CGMCC 3.4410) were got from the China General Microbiological Culture Collection Center  and cultured to 1 × 107 conidia/mL suspension for use.The mixed aflatoxins standard solution containing 2.0 mg/mL of AFB2HPO4.12H2O, NaH2PO4, KH2PO4, and tween-20 were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd . Purified Wahaha water from Wahaha Group Co., Ltd  was used for all the experiments.HPLC-grade acetonitrile and methanol were obtained from Thermo Fisher Scientific Inc . NaCl, KCl, NaNelumbo nucifera Gaertn.) samples, produced in Hubei province,  were purchased from Beijing Tong Ren Tang Co., LTD. All the samples were divided into the blank and experimental groups. For the blank groups: 20 g lotus seeds were added into 16 petri dishes, respectively, followed by irradiation for 5 h under an ultraviolet lamp. For the experimental groups: 20 g lotus seeds were added into another 16 petri dishes, respectively. After irradiation for 5 h, 1 mL of A. flavus conidia solution was artificially added onto the surface of lotus seeds and were air-dried. Then, all the petri dishes containing the raw and treated samples were stored in an incubator at 25 °C and relative humidity conditions of 40%, 50%, 60% and 70%, respectively. On the 0, 10th, 20th and 30th day, all the samples were observed by naked eyes regarding mildew. In addition, the samples were treated for the measurement of Aw and Cw, along with the determination of potential aflatoxins by the optimized high performance liquid chromatography coupled with photochemical derivatization and fluorescence detection (HPLC-PCD-FLD) method on the 0, 3rd, 6th, 9th, 12th, 22nd and 32nd day.Lotus seed  of sample was accurately calculated based on the lost weight.The weight loss method recommended in the Chinese Pharmacopoeia (Chinese Pharmacopoeia Commission w) by using a HD-6  intelligent moisture activity measuring instrument. 15 min later, data of Aw were recorded.Five gram lotus seeds out of the above samples stored at 25 °C and different relative humidity  conditions were taken out on the 0, 3rd, 6th, 9th, 12th, 22nd and 32nd day for the measurement of water activity (Av/v) and water was selected as the mobile phase with isocratic elution at a flow rate of 1.2 mL/min. The column temperature was set at 30 °C. The injection volume was 20 μL. The AURA INDUSTRIES PCD reactor  was consisted of a mercury lamp (λ = 254 nm) and a knitted reactor coil of 0.74 mL (15 m × 0.25 mm). The eluate was monitored by using a fluorescence detector at an excitation wavelength of 360 nm and an emission wavelength of 450 nm.Chromatographic separation of four aflatoxins was performed on a MG-III-C18 column  through a Shimadzu LC-20AT HPLC system  consisting of two LC-20 AT pumps, an SIL-20A autosampler, a CTO-20A column oven, a CMB-20A controller, the post-column photochemical derivatization (PCD) reactor and an RF-20AXL fluorescence detector (FLD). Methanol–acetonitrile  solution was obtained by dispersing 0.2599 g NaH2PO4, 6.4204 g Na2HPO4.12H2O and 40 mL of tween-20 in 1960 mL of purified water.Firstly, Phosphate buffer saline (PBS) solution was prepared by dissolving 0.2 g KCl, 8.0 g NaCl, 0.2 g KHv/v) solution. After blending by vortex and ultrasonication for 30 min, the mixture was followed by centrifugation for 5 min at 10,000 r/min and 5 mL of the supernatant was collected and transferred into a 50-mL EP tube containing 40 mL of 1% PBT solution. The solution was mixed with pH being adjusted to 6–8, followed by filtration through a 0.45-µm syringe nylon filter. 40 mL of the filtrate was precisely measured and elutriated through an immunoaffinity column at a flow rate of 1–2 drop(s) per second, then 10 mL of PBS and water were taken, respectively, to wash the column successively, and finally 800 µL of methanol was added to elute the target aflatoxins. 1 mL of the eluent was collected and filtered through a 0.22-µm syringe nylon filter for HPLC-PCD-FLD analysis.Then, 5 g homogenized lotus seed sample powders that have been stored at 25 °C and relative humidity of 40%, 50%, 60% and 70% for 30 days were accurately weighed and placed in a 50-mL centrifuge tube with the addition of 1 g NaCl and 25 mL of methanol–water  versus the injection concentration (x). Then, the contents of aflatoxins in the tested samples were quantified according to the standard curves. All the working solutions were freshly prepared just before being injected into the HPLC analytical system.The mixed standard solution of aflatoxins was diluted to 9 different concentrations of working solutions, and 20 µL of the dilution was injected successively from low concentration to high concentration into the HPLC-PCD-FLD system. The peak area of each aflatoxin was recorded and the standard curve was established by linear regression of the peak area (w and Aw were expressed as mean ± standard deviation (SD) and analyzed by using the Origin 2016 software.All data regarding Cw of lotus seeds stored at 25 °C and different relative humidity conditions of 40%, 50%, 60% and 70% within 32 days was measured and shown in Fig. w values exhibited a fluctuation under different relative humidity conditions within the first 10 days, and then were relatively stable during the next 22 days, which was increased successively from low humidity to high humidity. The higher the relative humidity was, the bigger the Cw value was. The samples stored at relative humidity 70% exhibited the highest Cw values. After storage for 12 days at 25 °C and relative humidity 70%, the Cw value exceeded the prescribed limit (14%) for lotus seeds in the Chinese Pharmacopoeia , and further to prevent this medicinal and edible food from mildew.The Cw) data of lotus seeds stored at 25 °C and different relative humidity conditions of 40%, 50%, 60% and 70% within 32 days were determined and listed in Fig. w values expressed a similar trend with Cw, which presented some fluctuation under different relative humidity conditions within the first 10 days, and then were stable during the next 22 days. After storage for 10 days at 25 °C, the Aw values were increased with increasing the relative humidity from 40% to 70%, which reached the highest values at relative humidity 70%. After storage for 12 days at 25 °C and relative humidity 70%, the Aw values were bigger than 0.7, which exceeded the relative humidity value of 70%. When the relative humidity condition was lower than 70%, the Aw values were all smaller than 0.6, which might be considered as the suitable water activity control point for safe storage of lotus seed in practice. These data illustrate that the increase of relative humidity of storage condition will lead to the enhancement of the water activity of lotus seeds, which may raise the incidence of mildew of this edible and medicinal food. While, from then on, the maximum limit standard, as well as some regulations on Aw for lotus seed and other matrices are lacking. To prevent lotus seeds from mildew and mycotoxins contamination, it is suggested that the relative humidity conditions for the storage environment of lotus seed should be effectively controlled to lower the Aw in an acceptable range (not more than 0.6).The water activity  and experimental samples (inoculated with A. flavus conidia that could produce aflatoxins) stored at 25 °C and relative humidity 40%, 50%, 60% and 70% for 30 days was observed at an interval of 10 days. It could be seen in Fig. The above results have shown that the CyB1, yB2, yG1, yG2) of four aflatoxins at different concentrations (x) were determined and the linear regression equations with their correlation coefficient (R2), as well as the limit of detection (LOD) and quantitation (LOQ) were obtained and listed in Table Under the optimized chromatographic conditions, the HPLC-PCD-FLD chromatogram of four aflatoxins was recorded and shown in Fig. 1 was detected in both the blank and experimental lotus seed samples after they were stored at 25 °C for 30 days, and the concentration in the samples of experimental group reached 6.6 μg/kg, which has exceeded the officially-recommended MRL (5.0 μg/kg).Although no visible mildew was observed on the surface of lotus seeds by naked eyes within 30 days, it was not definite if toxic aflatoxins were produced in the samples. Herein, the lotus samples stored at 25 °C and relative humidity of 40%, 50%, 60% and 70% for 0, 10, 20 and 30 days were collected and prepared for aflatoxins determination using the developed HPLC-PCD-FLD method. The results in Table 1 could still be detected in lotus seeds stored for long time (about 30 days) at 25 °C and high relative humidity (around 70%), especially in the samples without incubation with toxigenic fungi, which would increase the risk incidence of potential safety threatens of lotus seeds, and should be given more attention. Therefore, in practice, the relative humidity of storage environment for lotus seeds should be effectively controlled in less than 70% with the optimum water content and activity control points at 12% and 0.6, respectively, for safe storage to ensure the quality and safety of this edible and medicinal food related products, as well as the health of consumers.These results indicated that lotus seed was a susceptible matrix to mildew and aflatoxins contamination. Although no visible mildew was observed, AFBw and Aw of the matrices for fungal growth, further may result in the occurrence of mildew and mycotoxins contamination . If storage conditions are poorly managed, some fungi species can infect these matrices, leading to mycotoxin contamination. Environmental temperature and humidity conditions are the two key factors for the growth of fungi, which should be in effective control to prevent foods, feeds and medicinal plants from mildew. In common, most fungi will grow and multiply quickly at 20–35 °C and relative humidity more than 70%  and experimental  samples of lotus seeds were stored at 25 °C and different relative humidity conditions to measure the Cw and Aw changes. Our results have shown that both the Cw and Aw values tended to be stable after storage for 10 days, and no obvious mildew was observed on the surface of all tested samples for 30 days. When the relative humidity was up to 70%, the Cw and Aw values were increased quickly, and the Cw value exceeded the official-permitted level (14%), which was in agreement with the reports  starch (Zhang et al. w and Aw conditions were seldom paid special attention and failed to control. Changes of weather and environmental conditions will lead to large humidity fluctuations. When the environmental humidity is above 70% for a long time, the water content and activity of foods will be high, which will easily result in the mildew of TCMs, along with mycotoxins contamination. Our results have exhibited that when the relative humidity was no more than 60%, the Cw and Aw values approximately equal to about 12% and 0.6, respectively, which might be recommended as the optimum water content and water activity control points for safe storage of lotus seeds in practice at 25 °C with the environmental relative humidity less than 60%, to ensure the quality, safety and effectiveness of medicinal and edible products, as well as the health of the consumers.In the practical medicinal and edible foods and TCMs storage process, the experimental temperature and humidity, as well as the C