A Rapid, Accurate, Single Molecule Counting Method Detects Clostridium difficile Toxin B in Stool Samples.

We describe a new rapid and accurate immunoassay-based technology capable of counting single target molecules using digital imaging without magnification. Using the technology, we developed a rapid test for Clostridium difficile toxin B, which is responsible for the pathology underlying potentially fatal C. difficile infections (CDI). There are currently no tests for CDI that are rapid, sensitive, and specific. The MultiPath C. difficile toxin B test images and counts complexes of target-specific magnetic and fluorescent particles that have been tethered together by toxin B molecules in minimally processed stool samples. The performance characteristics of the 30 minute test include a limit of detection of 45 pg/mL, dynamic range covering 4-5 orders of magnitude, and coefficient of variation of less than 10%. The MultiPath test detected all toxinotypes and ribotypes tested, including the one most commonly occurring in the US and EU; shows no cross reactivity with relevant bacterial species; and is robust to potential interferants commonly present in stool samples. On a training set of 320 clinical stool samples, the MultiPath C. difficile toxin B test showed 97.0% sensitivity (95% CI, 91.4-99.4%); 98.3% specificity (95% CI, 96.8-99.2%); and 98.2% accuracy (95% CI, 96.7-99.0%) compared to the cellular cytotoxicity neutralization assay (CCNA) reference method. Based on these compelling performance characteristics, we believe the MultiPath technology can address the lack of rapid, sensitive, specific, and easy-to-use diagnostic tests for C. difficile.

Portable, Constriction-Expansion Blood Plasma Separation and Polymerization-Based Malaria Detection.

A portable, microfluidic blood plasma separation device is presented featuring a constriction-expansion design, which produces 100.0% purity for undiluted blood at 9% yield. This level of purity represents an improvement of at least 1 order of magnitude with increased yield compared to that achieved previously using passive separation. The system features high flow rates, 5-30 µL/min plasma collection, with minimal clogging and biofouling. The simple, portable blood plasma separation design is hand-driven and can easily be incorporated with microfluidic or laboratory scale diagnostic assays. The separation system was applied to a paper-based diagnostic test for malaria that produced an amplified color change in the presence of Plasmodium falciparum histidine-rich protein 2 at a concentration well below clinical relevancy for undiluted whole blood.

Evaluating the sensitivity of hybridization-based epigenotyping using a methyl binding domain protein.

Hypermethylation of CpG islands in gene promoter regions has been shown to be a predictive biomarker for certain diseases. Most current methods for methylation profiling are not well-suited for clinical analysis. Here, we report the development of an inexpensive device and an epigenotyping assay with a format conducive to multiplexed analysis.

Uncertainty in techno-economic estimates of cellulosic ethanol production due to experimental measurement uncertainty.

BACKGROUND: Cost-effective production of lignocellulosic biofuels remains a major financial and technical challenge at the industrial scale. A critical tool in biofuels process development is the techno-economic (TE) model, which calculates biofuel production costs using a process model and an economic model. The process model solves mass and energy balances for each unit, and the economic model estimates capital and operating costs from the process model based on economic assumptions. The process model inputs include experimental data on the feedstock composition and intermediate product yields for each unit. These experimental yield data are calculated from primary measurements. Uncertainty in these primary measurements is propagated to the calculated yields, to the process model, and ultimately to the economic model. Thus, outputs of the TE model have a minimum uncertainty associated with the uncertainty in the primary measurements. RESULTS: We calculate the uncertainty in the Minimum Ethanol Selling Price (MESP) estimate for lignocellulosic ethanol production via a biochemical conversion process: dilute sulfuric acid pretreatment of corn stover followed by enzymatic hydrolysis and co-fermentation of the resulting sugars to ethanol. We perform a sensitivity analysis on the TE model and identify the feedstock composition and conversion yields from three unit operations (xylose from pretreatment, glucose from enzymatic hydrolysis, and ethanol from fermentation) as the most important variables. The uncertainty in the pretreatment xylose yield arises from multiple measurements, whereas the glucose and ethanol yields from enzymatic hydrolysis and fermentation, respectively, are dominated by a single measurement: the fraction of insoluble solids (fIS) in the biomass slurries. CONCLUSIONS: We calculate a $0.15/gal uncertainty in MESP from the TE model due to uncertainties in primary measurements. This result sets a lower bound on the error bars of the TE model predictions. This analysis highlights the primary measurements that merit further development to reduce the uncertainty associated with their use in TE models. While we develop and apply this mathematical framework to a specific biorefinery scenario here, this analysis can be readily adapted to other types of biorefining processes and provides a general framework for propagating uncertainty due to analytical measurements through a TE model.

Particle morphology characterization and manipulation in biomass slurries and the effect on rheological properties and enzymatic conversion.

An improved understanding of how particle size distribution relates to enzymatic hydrolysis performance and rheological properties could enable enhanced biochemical conversion of lignocellulosic feedstocks. Particle size distribution can change as a result of either physical or chemical manipulation of a biomass sample. In this study, we employed image processing techniques to measure slurry particle size distribution and validated the results by showing that they are comparable to those from laser diffraction and sieving. Particle size and chemical changes of biomass slurries were manipulated independently and the resulting yield stress and enzymatic digestibility of slurries with different size distributions were measured. Interestingly, reducing particle size by mechanical means from about 1 mm to 100 µm did not reduce the yield stress of the slurries over a broad range of concentrations or increase the digestibility of the biomass over the range of size reduction studied here. This is in stark contrast to the increase in digestibility and decrease in yield stress when particle size is reduced by dilute-acid pretreatment over similar size ranges.