Availability of a rapid, accurate, and reliable point-of-care (POC) device for detection of infectious agents and pandemic pathogens, such as swine-origin influenza A (H1N1) virus, is crucial for effective patient management and outbreak prevention. Due to its ease of use, rapid processing, and minimal power and laboratory equipment requirements, the lateral-flow (immuno)assay (LFA) has gained much attention in recent years as a possible solution. However, since the sensitivity of LFA has been shown to be inferior to that of the gold standards of pathogen detection, namely cell culture and real-time PCR, LFA remains an ineffective POC assay for preventing pandemic outbreaks. A practical solution for increasing the sensitivity of LFA is to concentrate the target agent in a solution prior to the detection step. In this study, an aqueous two-phase micellar system comprised of the nonionic surfactant Triton X-114 was investigated for concentrating a model virus, namely bacteriophage M13 (M13), prior to LFA. The volume ratio of the two coexisting micellar phases was manipulated to concentrate M13 in the top, micelle-poor phase. The concentration step effectively improved the M13 detection limit of the assay by tenfold from 5 × 108 plaque forming units (pfu)/mL to 5 × 107 pfu/mL. In the future, the volume ratio can be further manipulated to yield a greater concentration of a target virus and further decrease the detection limits of the LFA.FigureA schematic representation of concentrating viruses with an aqueous two-phase micellar system containing Triton X-114 surfactant prior to the detection of the virus through the lateral-flow immunoassayElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-010-4213-7) contains supplementary material, which is available to authorized users.

Transferrin (Tf) conjugates of CRM107 are currently being tested in clinical trials for treatment of malignant gliomas. However, the rapid cellular recycling of Tf limits its efficiency as a drug carrier. We have developed a mathematical model of the Tf/TfR trafficking cycle and have identified the Tf iron release rate as a previously unreported factor governing the degree of Tf cellular association. The release of iron from Tf is inhibited by replacing the synergistic carbonate anion with oxalate. Trafficking patterns for oxalate Tf and native Tf are compared by measuring their cellular association with HeLa cells. The amount of Tf associated with the cells is an average of 51% greater for oxalate Tf than for native Tf over a two hour period at Tf concentrations of 0.1 nM and 1 nM. Importantly, diphtheria toxin (DT) conjugates of oxalate Tf are more cytotoxic against HeLa cells than conjugates of native Tf. Conjugate IC 50 values were determined to be 0.06 nM for the oxalate Tf conjugate vs. 0.22 nM for the native Tf conjugate. Thus, we show that inhibition of Tf iron release improves the efficacy of Tf as a drug carrier through increased association with cells expressing TfR.

Pyrosequencing is a bioluminometric DNA sequencing technique that measures the release of pyrophosphate during DNA synthesis. The amount of pyrophosphate is proportionally converted into visible light by a cascade of enzymatic reactions. Pyrosequencing has heretofore been used for generating short sequence reads (1-100 nucleotides) because certain factors limit the system's ability to perform longer reads accurately. In this study, we have characterized the main read length limiting factors in both three-enzyme and four-enzyme Pyrosequencing systems. A new simulation model was developed to simulate the read length of both systems based on the inhibitory factors in the chemical equations governing each enzymatic cascade. Our results indicate that nonsynchronized extension limits the obtained read length, albeit to a different extent for each system. In the four-enzyme system, nonsynchronized extension due mainly to a decrease in apyrase's efficiency in degrading excess nucleotides proves to be the main limiting factor of read length. Replacing apyrase with a washing step for removal of excess nucleotide proves to be essential in improving the read length of Pyrosequencing. The main limiting factor of the three-enzyme system is shown to be loss of DNA fragments during the washing step. If this loss is minimized to 0.1% per washing cycle, the read length of Pyrosequencing would be well beyond 300 bases.

The concentration of biomarkers, such as DNA, prior to a subsequent detection step may facilitate the early detection of cancer, which could significantly increase chances for survival. In this study, the partitioning behavior of mammalian genomic DNA fragments in a two-phase aqueous micellar system was investigated using both experiment and theory. The micellar system was generated using the nonionic surfactant Triton X-114 and phosphate-buffered saline (PBS). Partition coefficients were measured under a variety of conditions and compared with our theoretical predictions. With this comparison, we demonstrated that the partitioning behavior of DNA fragments in this system is primarily driven by repulsive, steric, excluded-volume interactions that operate between the micelles and the DNA fragments, but is limited by the entrainment of micelle-poor, DNA-rich domains in the macroscopic micelle-rich phase. Furthermore, the volume ratio, that is, the volume of the top, micelle-poor phase divided by that of the bottom, micelle-rich phase, was manipulated to concentrate DNA fragments in the top phase. Specifically, by decreasing the volume ratio from 1 to 1/10, we demonstrated proof-of-principle that the concentration of DNA fragments in the top phase could be increased two- to nine-fold in a predictive manner.