Bioluminescence DNA hybridization assay for Plasmodium falciparum based on the photoprotein aequorin.

A bioluminescence DNA hybridization assay for the detection of Plasmodium falciparum, the most deadly species of malaria, using the photoprotein aequorin as a bioluminescent label has been developed. The current gold standard for the detection of malaria is light microscopy, which can detect down to approximately 50 parasites/microL of blood, but has low-throughput, high costs, and requires high skill, which limit the applicability of the method, especially in the developing regions where malaria detection is mostly needed. The utilization of aequorin as a bioluminescence label offers the advantages of high signal-to-noise ratio and reliable detection down to attomole levels, allowing for the development of highly sensitive and miniaturized high-throughput bioluminescence assays. Herein, we developed a DNA hybridization assay for the detection of P. falciparum based on the competition between the target DNA and the signal generating DNA streptavidin-aequorin for hybridization with the probe DNA. This bioluminescence hybridization assay demonstrated a detection limit of 3 pg/microL and was employed for the detection of target DNA in standard and spiked human serum samples. The DNA hybridization assay was developed in a microplate format without the need for sample PCR amplification, showing the potential suitability of this method in the parallel analysis of samples by low-trained personnel, such as that typically encountered in developing regions.

Optimization of the xylan degradation activity of monolithic enzymatic membranes as a function of their composition using design of experiments.

The aim of this work was the development and optimization of enzymatic monolithic membranes with high catalytic activity for the degradation of xylan into xylooligosaccharides. The chemometric tool design of experiments has been utilized here for the first time for the optimization of the enzymatic activity of the monolithic membranes based on their constituents. The effect of three process variables, including the amount of various monomer contents and the porogenic solvents ratio, has been studied on the enzymatic activity of the resulted membranes. The experimental design chosen was a central face centred with six central points in order to obtain an orthogonal model, with the precision of the results being independent of the range of values considered for each parameter. The software Modde(c) 6.0 from Umetrics(c) was used to build and analyze the results of the experimental design using partial least squares regression. The optimization of the suggested model provided the best membrane composition to achieve maximum enzymatic activity, which can be related to the amount of enzyme immobilized on the monolithic membrane. The predictive capacity of the model was evaluated performing additional experiments.

Integration of microcolumns and microfluidic fractionators on multitasking centrifugal microfluidic platforms for the analysis of biomolecules.

This work demonstrates the development of microfluidic compact discs (CDs) for protein purification and fractionation integrating a series of microfluidic features, such as microreservoirs, microchannels, and microfluidic fractionators. The CDs were fabricated with polydimethylsiloxane (PDMS), and each device contained multiple identical microfluidic patterns. Each pattern employed a microfluidic fractionation feature with operation that was based on the redirection of fluid into an isolation chamber as a result of an overflow. This feature offers the advantage of automated operation without the need for any external manipulation, which is independent of the size and the charge of the fractionated molecules. The performance of the microfluidic fractionator was evaluated by its integration into a protein purification microfluidic architecture. The microfluidic architecture employed a microchamber that accommodated a monolithic microcolumn, the fractionator, and an isolation chamber, which was also utilized for the optical detection of the purified protein. The monolithic microcolumn was polymerized "in situ" on the CD from a monolith precursor solution by microwave-initiated polymerization. This technique enabled the fast, efficient, and simultaneous polymerization of monoliths on disposable CD microfluidic platforms. The design of the CD employed allows the integration of various processes on a single microfluidic device, including protein purification, fractionation, isolation, and detection.

Fluorescence glucose detection: advances toward the ideal in vivo biosensor.

The importance of glucose monitoring for in vivo as well as for ex vivo applications has driven a vast number of scientific groups to pursue the development of an advanced glucose sensor. Such a sensor must be robust, versatile, and capable of the long-term, accurate and reproducible detection of glucose levels in various testing media. Among the different configurations and signal transduction mechanisms used, fluorescence-based glucose sensors constitute a growing class of glucose sensors represented by an increasing number of significant contributions to the field over the last few years. This manuscript reviews the progress in the development of fluorescence based glucose sensors resulting from the advances in the design of new receptor systems for glucose recognition and the utilization of new fluorescence transduction schemes.