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Microbiome Apr 2019Metagenomic next-generation sequencing (mNGS) experiments involving small amounts of nucleic acid input are highly susceptible to erroneous conclusions resulting from...
Metagenomic next-generation sequencing (mNGS) experiments involving small amounts of nucleic acid input are highly susceptible to erroneous conclusions resulting from unintentional sequencing of occult contaminants, especially those derived from molecular biology reagents. Recent work suggests that, for any given microbe detected by mNGS, an inverse linear relationship between microbial sequencing reads and sample mass implicates that microbe as a contaminant. By associating sequencing read output with the mass of a spike-in control, we demonstrate that contaminant nucleic acid can be quantified in order to identify the mass contributions of each constituent. In an experiment using a high-resolution (n = 96) dilution series of HeLa RNA spanning 3-logs of RNA mass input, we identified a complex set of contaminants totaling 9.1 ± 2.0 attograms. Given the competition between contamination and the true microbiome in ultra-low biomass samples such as respiratory fluid, quantification of the contamination within a given batch of biological samples can be used to determine a minimum mass input below which sequencing results may be distorted. Rather than completely censoring contaminant taxa from downstream analyses, we propose here a statistical approach that allows separation of the true microbial components from the actual contribution due to contamination. We demonstrate this approach using a batch of n = 97 human serum samples and note that despite E. coli contamination throughout the dataset, we are able to identify a patient sample with significantly more E. coli than expected from contamination alone. Importantly, our method assumes no prior understanding of possible contaminants, does not rely on any prior collection of environmental or reagent-only sequencing samples, and does not censor potentially clinically relevant taxa, thus making it a generalized approach to any kind of metagenomic sequencing, for any purpose, clinical or otherwise.
Topics: DNA, Bacterial; Escherichia coli; High-Throughput Nucleotide Sequencing; Humans; Metagenomics; Sequence Analysis, DNA
PubMed: 30992055
DOI: 10.1186/s40168-019-0678-6 -
Rapid Communications in Mass... Mar 2019A multi-collector inductively coupled plasma (MC-ICP) mass spectrometer coupled to a UV ns-laser ablation (LA) system was used to measure uranium isotopic ratios ( U/ U,...
Determination of the isotopic composition of single sub-micrometer-sized uranium particles by laser ablation coupled with multi-collector inductively coupled plasma mass spectrometry.
RATIONALE
A multi-collector inductively coupled plasma (MC-ICP) mass spectrometer coupled to a UV ns-laser ablation (LA) system was used to measure uranium isotopic ratios ( U/ U, U/ U and U/ U) in single uranium particles of various sizes and isotopic compositions, including home-made sub-micrometric natural uranium particles of narrow size distribution (415 ± 60 nm).
METHODS
The LA-ICP mass spectrometer was operated in wet plasma conditions thanks to simultaneous injection of the laser aerosol and water vapor through a desolvating nebulizer. The isotopic ratios were corrected for mass bias and gain factors between detectors. The U/ U ratios were also corrected for the presence of U hydrides and tailing of the U peak.
RESULTS
U/ U ratios were successfully measured in micrometer-sized particles from the NBS U050 certified standard material with a U/ U ratio of ~5 × 10 . The analysis of 77 natural uranium sub-μm-sized particles yielded a very good trueness with respect to the expected U/ U and U/ U ratios, while the measurement errors for single particles ranged from -2.7% to +2.1% for U/ U and from -17% to +33% for the U/ U ratios. Their relative combined standard uncertainties ranged from 3.3% to 32.8% and from 0.4% to 4.0% for U/ U and U/ U ratios, respectively. In addition, extremely low detection limits, in the attogram range, were achieved.
CONCLUSIONS
This study demonstrates that coupling of a ns-laser ablation system with a MC-ICP mass spectrometer allows measurements of the isotopic composition in natural uranium particles of a few hundreds of nm with very good trueness, average combined standard uncertainties of ~1% for U/ U ratios and 12% for U/ U ratios, and detections limits of a few ag for minor isotopes.
PubMed: 30496616
DOI: 10.1002/rcm.8366 -
ACS Applied Materials & Interfaces Dec 2018Thin-film probes have been developed for the reversible detection and separation of picric acid (PA) with extreme sensitivity in aqueous media. The free radical...
Thin-film probes have been developed for the reversible detection and separation of picric acid (PA) with extreme sensitivity in aqueous media. The free radical copolymerization of dimethylacrylamide (DMA), benzophenone acrylamide (BPAM), and glycidyl methacrylate (GMA) with a feed ratio of 95:1:4 yielded [p(DMA- co-BPAM- co-GMA)] (P1). P1 was transformed to the final polymeric probe, P2, by a subsequent ring-opening reaction between N-(pyren-1-yl-methyl)propan-1-amine (Py-PA) with the epoxide unit of P1. P2 exhibited rapid and selective sensing properties toward PA in aqueous media via turn-off fluorescence emission. The detection sensitivity was tuned precisely by varying the pH of the solution. After the immobilization of P2 on a quartz slide by spin-coating, followed by exposure to UV light, the resulting film exhibited an attogram-level detection limit toward PA. The photoinduced electron transfer together with an energy-transfer process between PA and the pyrene units of P2 were maximized by the strong π-π stacking of pyrene units of P2, which, in turn, induced rapid exciton energy diffusion. Furthermore, the separation of PA from the mixture of the various nitroaromatic compounds by the P2 film was achieved. Whereas the detection process of PA was reversible and repeatable over multiple cycles, the P2 film could be recycled.
PubMed: 30398831
DOI: 10.1021/acsami.8b15369 -
ACS Sensors Aug 2018A novel conjugated cationic polyfluorene (polyelectrolyte) derivative, PFBT, was developed by means of simple and cost-effective oxidative coupling polymerization...
A novel conjugated cationic polyfluorene (polyelectrolyte) derivative, PFBT, was developed by means of simple and cost-effective oxidative coupling polymerization method. PFBT displayed dual state emission in dimethyl sulfoxide (DMSO) as well as in water, a characteristic phenomenon of polyfluorene homopolymers, and tested for nitroexplosive analytes detection to observe a remarkable fluorescence quenching response for picric acid (PA) in the both solvents. The polymer PFBT demonstrated substantial selectivity and ultrasensitivity toward nitroexplosive PA in both the solvents (DMSO and HO) with exceptional quenching constant values of 2.69 × 10 and 2.18 × 10 M and a ultralow limit of detection of 92.7 nM (21.23 ppb) and 0.19 nM (43.53 ppt) in respective solvents. Furthermore, economical portable test strip devices were prepared for easy and fast on-site PA sensing, which can detect up to 0.22 ag level of PA. PA sensing in vapor phase was also established, that could detect up to 42.6 ppb level of PA vapors. Interestingly, the mechanism of sensing in DMSO solvent was attributed to substantial inner filter effect and photoinduced electron transfer, while in HO the sensing occurs via possible resonance energy transfer and photoinduced electron transfer, which is exceptional and not reported earlier for a single probe.
Topics: Cations; Dimethyl Sulfoxide; Fluorenes; Fluorescence Resonance Energy Transfer; Fresh Water; Gases; Limit of Detection; Picrates; Polymers; Water
PubMed: 30039698
DOI: 10.1021/acssensors.8b00093 -
Talanta May 2018Interferometric imaging biosensors are powerful and convenient tools for confirming the existence of DNA monolayer films on silicon microarray platforms. However, their...
Interferometric imaging biosensors are powerful and convenient tools for confirming the existence of DNA monolayer films on silicon microarray platforms. However, their accuracy and sensitivity need further improvement because DNA molecules contribute to an inconspicuous interferometric signal both in thickness and size. Such weaknesses result in poor performance of these biosensors for low DNA content analyses and point mutation tests. In this paper, an interferometric imaging biosensor with weighted spectrum analysis is presented to confirm DNA monolayer films. The interferometric signal of DNA molecules can be extracted and then quantitative detection results for DNA microarrays can be reconstructed. With the proposed strategy, the relative error of thickness detection was reduced from 88.94% to merely 4.15%. The mass sensitivity per unit area of the proposed biosensor reached 20 attograms (ag). Therefore, the sample consumption per unit area of the target DNA content was only 62.5 zeptomoles (zm), with the volume of 0.25 picolitres (pL). Compared with the fluorescence resonance energy transfer (FRET), the measurement veracity of the interferometric imaging biosensor with weighted spectrum analysis is free to the changes in spotting concentration and DNA length. The detection range was more than 1µm. Moreover, single nucleotide mismatch could be pointed out combined with specific DNA ligation. A mutation experiment for lung cancer detection proved the high selectivity and accurate analysis capability of the presented biosensor.
Topics: Algorithms; Biosensing Techniques; DNA; DNA Mutational Analysis; Fluorescence Resonance Energy Transfer; Humans; Interferometry; Lung Neoplasms; Point Mutation; Reproducibility of Results
PubMed: 29426505
DOI: 10.1016/j.talanta.2017.12.066 -
Environmental Science & Technology Feb 2018Quantifying metal and nanoparticle (NP) biouptake and distribution on an individual cellular basis has previously been impossible, given available techniques which...
Quantifying metal and nanoparticle (NP) biouptake and distribution on an individual cellular basis has previously been impossible, given available techniques which provide qualitative data that are laborious to acquire and prone to artifacts. Quantifying metal and metal NP uptake and loss processes in environmental organisms will lead to mechanistic understanding of biouptake and improved understanding of potential hazards and risks of metals and NPs. In this work, we present a new technique, single cell inductively coupled plasma mass spectrometry (SC-ICP-MS), which allows quantification of metal concentrations on an individual cell basis down to the attogram (ag) per cell level. We present data validating the novel method, along with the mass of metal per cell. Finally, we use SC-ICP-MS, with ancillary cell counting methods, to quantify the biouptake and strong sorption and distribution of both dissolved Au and Au NPs in a freshwater alga (Cyptomonas ovate). The data suggests differences between dissolved and NP uptake and loss. In the case of NPs, there was a dose and time dependent uptake, but individual cellular variations; at the highest realistic exposure conditions used in this study up to 40-50% of cells contained NPs, while 50-60% of cells did not.
Topics: Fresh Water; Gold; Mass Spectrometry; Metal Nanoparticles; Particle Size
PubMed: 29400052
DOI: 10.1021/acs.est.7b04968 -
Analytical Chemistry Jan 2018In this contribution, completely different from traditional probes based on molecular host-guest interaction, crystal level interaction was proposed as an ultrasensitive...
In this contribution, completely different from traditional probes based on molecular host-guest interaction, crystal level interaction was proposed as an ultrasensitive design strategy for molecule detection. In such a strategy, crystal-guest interaction triggered rapid crystal disaggregation to gain great signal change, far more efficient than molecule scale coupling. Highly ordered pyrene methanol (PM) crystal was self-assembled with hydrogen bond chain and π-π stacking force. Its {011} surface can be destabilized by methamphetamine hydrochlorate (MA, heavily used illicit drug) selectively and efficiently. The detection limit was improved to an amazing attogram level (5.4 ag/mL, 29.1 fM), 9 orders of magnitude lower than the best reported host-guest result.
PubMed: 29237262
DOI: 10.1021/acs.analchem.7b04783 -
Angewandte Chemie (International Ed. in... Nov 2017Current trends in nanoengineering are bringing along new structures of diverse chemical compositions that need to be meticulously defined in order to ensure their...
Current trends in nanoengineering are bringing along new structures of diverse chemical compositions that need to be meticulously defined in order to ensure their correct operation. Few methods can provide the sensitivity required to carry out measurements on individual nano-objects without tedious sample pre-treatment or data analysis. In the present study, we introduce a pathway for the elemental identification of single nanoparticles (NPs) that avoids suspension in liquid media by means of optical trapping and laser-induced plasma spectroscopy. We demonstrate spectroscopic detection and identification of individual 25(±3.7) to 70(±10.5) nm in diameter Cu NPs stably trapped in air featuring masses down to 73±35 attograms. We found an increase in the absolute number of photons produced as size of the particles decreased; pointing towards a more efficient excitation of ensembles of only ca. 7×10 Cu atoms in the onset plasma.
PubMed: 28877398
DOI: 10.1002/anie.201708870 -
Analytical Chemistry Sep 2017The characterization of the aerosol (size, composition, and concentration) generated by Laser Ablation is of great interest due to its impact on the analytical...
The characterization of the aerosol (size, composition, and concentration) generated by Laser Ablation is of great interest due to its impact on the analytical performances when coupled to Inductively Coupled Plasma Mass Spectrometry (ICPMS). The capabilities of High Resolution ICPMS as a direct tool to characterize nanoparticles produced by femtosecond Laser Ablation of pure copper are presented. An analytical protocol, similar to the "single particle ICPMS" technique used to characterize the size distribution of nanoparticles in solution, was developed in order to observe the signals of individual particles produced by a single ablation shot. A Visual Basic for Applications (VBA) data processing was developed to count and sort the particles as a function of their size and thus determine the particle size distribution. To check the reliability of the method, the results were compared to a more conventional technique, namely, Electrical Low Pressure Impaction (ELPI) for 4000 shots. Detection limit for the particles produced by the laser ablation of a copper foil is of a few attograms corresponding to a nanoparticle of 14 nm. The direct online determination of particle size by ICPMS gave similar results than ELPI for copper particles ejected during the ablation shot by shot at a fixed spot, from 1 to 100 shots. Particles larger than 159 nm represented less than 1% of the aerosol whose distribution was centered on 25-51 nm.
PubMed: 28689407
DOI: 10.1021/acs.analchem.7b01041 -
Scientific Reports Apr 2017Using doubly-clamped silicon (Si) microbeam resonators, we demonstrate sub-attogram per Hertz (ag/Hz) mass sensitivity, which is extremely high sensitivity achieved by...
Using doubly-clamped silicon (Si) microbeam resonators, we demonstrate sub-attogram per Hertz (ag/Hz) mass sensitivity, which is extremely high sensitivity achieved by micro-scale MEMS mass sensors. We also characterize unusual buckling phenomena of the resonators. The thin-film based resonator is composed of a Si microbeam surrounded by silicon nitride (SiN) anchors, which significantly improve performance by providing fixation on the microbeam and stabilizing oscillating motion. Here, we introduce two fabrication techniques to further improve the mass sensitivity. First, we minimize surface stress by depositing a sacrificial SiN layer, which prevents damage on the Si microbeam. Second, we modify anchor structure to find optimal design that allows the microbeam to oscillate in quasi-one dimensional mode while achieving high quality factor. Mass loading is conducted by depositing Au/Ti thin films on the local area of the microbeam surface. Using sequential mass loading, we test effects of changing beam dimensions, position of mass loading, and distribution of a metal film on the mass sensitivity. In addition, we demonstrate that microbeams suffer local micro-buckling and global buckling by excessive mass loading, which are induced by two different mechanisms. We also find that the critical buckling length is increased by additional support from the anchors.
PubMed: 28429793
DOI: 10.1038/srep46660