A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice.

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15-1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing

Decontamination Options for Bacillus anthracis-Contaminated Drinking Water Determined from Spore Surrogate Studies ▿

Five parameters were evaluated with surrogates of Bacillus anthracis spores to determine effective decontamination alternatives for use in a contaminated drinking water supply. The parameters were as follows: (i) type of Bacillus spore surrogate (B. thuringiensis or B. atrophaeus), (ii) spore concentration in suspension (102 and 106 spores/ml), (iii) chemical characteristics of the decontaminant (sodium dichloro-S-triazinetrione dihydrate [Dichlor], hydrogen peroxide, potassium peroxymonosulfate [Oxone], sodium hypochlorite, and VirkonS), (iv) decontaminant concentration (0.01% to 5%), and (v) exposure time to decontaminant (10 min to 1 h). Results from 138 suspension tests with appropriate controls are reported. Hydrogen peroxide at a concentration of 5% and Dichlor or sodium hypochlorite at a concentration of 2% were highly effective at spore inactivation regardless of spore type tested, spore exposure time, or spore concentration evaluated. This is the first reported study of Dichlor as an effective decontaminant for B. anthracis spore surrogates. Dichlor's desirable characteristics of high oxidation potential, high level of free chlorine, and a more neutral pH than that of other oxidizers evaluated appear to make it an excellent alternative. All three oxidizers were effective against B. atrophaeus spores in meeting the EPA biocide standard of greater than a 6-log kill after a 10-min exposure time and at lower concentrations than typically reported for biocide use. Solutions of 5% VirkonS and Oxone were less effective as decontaminants than other options evaluated in this study and did not meet the EPA's efficacy standard for a biocide, although they were found to be as effective for concentrations of 102 spores/ml. Differences in methods and procedures reported by other investigators make quantitative comparisons among studies difficult

Comprehensive volatile metabolic fingerprinting of bacterial and fungal pathogen groups

The identification of pathogen-specific volatile metabolic "fingerprints" could lead to the rapid identification of disease-causing organisms either directly from <i>ex vivo</i> patient bio-specimens or from <i>in vitro</i> cultures. In the present study, we have evaluated the volatile metabolites produced by 100 clinical isolates belonging to ten distinct pathogen groups that, in aggregate, account for 90 % of bloodstream infections, 90 % of urinary tract infections, and 80 % of infections encountered in the intensive care unit setting. Headspace volatile metabolites produced in vitro were concentrated using headspace solid-phase microextraction and analyzed via two-dimensional gas chromatography time-of-flight mass spectrometry (HS-SPME-GC×GC-TOFMS). A total of 811 volatile metabolites were detected across all samples, of which 203 were: 1) detected in 9 or 10 (of 10) isolates belonging to one or more pathogen groups, and 2) significantly more abundant in cultures relative to sterile media. Network analysis revealed a distinct metabolic fingerprint associated with each pathogen group, and analysis via Random Forest (RF) using leave-one-out cross-validation (LOOCV) resulted in a 95 % accuracy for the differentiation between groups. The present findings support the results of prior studies that have reported on the differential production of volatile metabolites across pathogenic bacteria and fungi, and provide additional insight through the inclusion of pathogen groups that have seldom been studied previously, including <i>Acinetobacter</i> spp., coagulase-negative <i>Staphylococcus</i>, and <i>Proteus mirabilis</i>, as well as the utilization of HS-SPME-GC×GC-TOFMS for improved sensitivity and resolution relative to traditional gas chromatography-based techniques

Importance du type d'absorbant pour l'échantillonnage des volatils : application sur les cultures bactériennes et l'haleine

Les composés organiques volatils bactériens (VOC) sont considérés comme des biomarqueurs sensibles et spécifiques pour le phénotypage bactérien dans les biofluides humains (haleine, sang, urine, etc.) et dans les milieux de culture. La possibilité d'utiliser les VOCs pour l'identification bactérienne ouvre de nouvelles possibilités pour la mise au point de techniques de diagnostiques plus efficaces. Outre les différences biologiques des environnements in-vivo et in-vitro, il est essentiel d'utiliser la même technique d’échantillonnage pour la caractérisation et la validation de biomarqueurs. Dans cette étude, la chromatographie gazeuse bidimensionnelle couplée à la spectrométrie de masse (GC×GC-MS) a été utilisée pour comparer et évaluer différents adsorbants de tubes de désorption thermique pour l’échantillonnage des VOCs. Plus précisément, les paramètres suivants ont été évalués pour chaque adsorbant: sensibilité, sélectivité, reproductibilité et linéarité. Cinq adsorbants différents (Carbopack Y, X, B, Carboxen 1000 et Tenax), utilisés individuellement ou en combinaison, ont été testés sur un mélange de standards (15 composés). Les meilleures sensibilité et reproductibilité ont été obtenues pour les tubes conditionnés avec du Tenax. Les deux tubes de désorption thermique les plus performants, Tenax et Carbopack Y + X + Carboxen 1000, ont également été évalués sur des cultures de E. coli, S. aureus et P. aeruginosa. Ces deux types de tubes ont pu distinguer les 3 types de culture bactérienne, mais une amélioration de la sensibilité et de la reproductibilité a été obtenue avec les tubes Tenax. Une comparaison similaire sur les performances des tubes a été effectuée sur des échantillons d'haleine

Importance of sorbent material selection for VOCs sampling: application on bacterial cultures and breath

Bacterial volatile organic compounds (VOCs) have been considered as sensitive and specific biomarkers for bacterial phenotyping in both human biofluids (breath, blood, urine, etc.) and culture media. The possibility of using VOCs markers for bacterial identification would open a new frontier for developing more efficient diagnostic techniques of infections. Besides the biological differences in in vivo/in vitro environments, the importance of using the same sampling technique and sorbent phase is crucial for the translation and validation of biomarker discovery. In the present contribution, GC×GC-MS was exploited to compare and evaluate different adsorption materials for thermal desorption tubes for VOCs sampling. Specifically, the following parameters were evaluated: sensitivity, selectivity, reproducibility and linear range. Five different adsorbent materials (Carbopack Y, X, B, Carboxen 1000 and Tenax), packed singularly or in combination, were tested on a standard mixture (15 compounds). The tubes packed with Tenax showed the best reproducibility (max 14% RSD) and sensitivity, with ~24 average fold increase compared to Carbopack Y+X+Carboxen 1000, which was second in terms of sensitivity. The two better performing thermal desorption tubes, Tenax and Carbopack Y+X+Carboxen 1000, was also evaluated on E. coli, S. aureus, and P. aeruginosa cultures. Both tubes were able to discriminate between the 3 culture types, but improved sensitivity and reproducibility were obtained with Tenax tubes. A similar comparison on tube performances was carried out on breath samples

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice.

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15-1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing