Competitive Reporter Monitored Amplification (CMA) - Quantification of Molecular Targets by Real Time Monitoring of Competitive Reporter Hybridization

Background: State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests. Methodology and Principal Findings: The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique. Conclusions and Significance: The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution fo

Microfluidic analysis techniques for safety assessment of pharmaceutical nano- and microsystems

This chapter reviews the evolution of microfabrication methods and materials, applicable to manufacturing of micro total analysis systems (or lab‐on‐a‐chip), from a general perspective. It discusses the possibilities and limitations associated with microfluidic cell culturing, or so called organ‐on‐a‐chip technology, together with selected examples of their exploitation to characterization of pharmaceutical nano‐ and microsystems. Materials selection plays a pivotal role in terms of ensuring the cell adhesion and viability as well as defining the prevailing culture conditions inside the microfluidic channels. The chapter focuses on the hepatic safety assessment of nanoparticles and gives an overview of the development of microfluidic immobilized enzyme reactors that could facilitate examination of the hepatic effects of nanomedicines under physiologically relevant conditions. It also provides an overview of the future prospects regarding system‐level integration possibilities facilitated by microfabrication of miniaturized separation and sample preparation systems as integral parts of microfluidic in vitro models.Non peer reviewe

A microfluidic device with fluorimetric detection for intracellular components analysis

An integrated microfluidic system that coupled lysis of two cell lines: L929 fibroblasts and A549 epithelial cells, with fluorescence-based enzyme assay was developed to determine β-glucocerebrosidase activity. The microdevice fabricated in poly(dimethylsiloxane) consists of three main parts: a chemical cell lysis zone based on the sheath flow geometry, a micromeander and an optical fibers detection zone. Unlike many methods described in literature that are designed to analyse intracellular components, the presented system enables to perform enzyme assays just after cell lysis process. It reduces the effect of proteases released in lysis process on determined enzymes. Glucocerebrosidase activity, the diagnostic marker for Gaucher’s disease, is the most commonly measured in leukocytes and fibroblasts using 4-methylumbelliferyl-β-D-glucopyranoside as synthetic β-glucoside. The enzyme cleavage releases the fluorescent product, i.e. 4-methylumbelliferone, and its fluorescence is measured as a function of time. The method of enzyme activity determination described in this paper was adapted for flow measurements in the microdevice. The curve of the enzymatic reaction advancement was prepared for three reaction times obtained from application of different flow rates of solutions introduced to the microsystem. Afterwards, determined β-glucocerebrosidase activity was recalculated with regard to 105 cells present in samples used for the tests. The obtained results were compared with a cuvette-based measurements. The lysosomal β-glucosidase activities determined in the microsystem were in good correlation with the values determined during macro-scale measurements

School Smoking Policy Characteristics and Individual Perceptions of the School Tobacco Context: Are They Linked to Students’ Smoking Status?

The purpose of this study was to explore individual- and school-level policy characteristics on student smoking behavior using an ecological perspective. Participants were 24,213 (51% female) Grade 10–11 students from 81 schools in five Canadian provinces. Data were collected using student self-report surveys, written policies collected from schools, interviews with school administrators, and school property observations to assess multiple dimensions of the school tobacco policy. The multi-level modeling results revealed that the school a student attended was associated with his/her smoking behavior. Individual-level variables that were associated with student smoking included lower school connectedness, a greater number of family and friends who smoked, higher perceptions of student smoking prevalence, lower perceptions of student smoking frequency, and stronger perceptions of the school tobacco context. School-level variables associated with student smoking included weaker policy intention indicating prohibition and assistance to overcome tobacco addiction, weaker policy implementation involving strategies for enforcement, and a higher number of students smoking on school property. These findings suggest that the school environment is important to tobacco control strategies, and that various policy dimensions have unique relationships to student smoking. School tobacco policies should be part of a comprehensive approach to adolescent tobacco use

Development of a microfluidic confocal fluorescence detection system for the hyphenation of nano-LC to on-line biochemical assays

One way to profile complex mixtures for receptor affinity is to couple liquid chromatography (LC) on-line to biochemical detection (BCD). A drawback of this hyphenated screening approach is the relatively high consumption of sample, receptor protein and (fluorescently labeled) tracer ligand. Here, we worked toward minimization of sample and reagent consumption, by coupling nano-LC on-line to a light-emitting diode (LED) based capillary confocal fluorescence detection system capable of on-line BCD with low-flow rates. In this fluorescence detection system, a capillary with an extended light path (bubble cell) was used as a detection cell in order to enhance sensitivity. The technology was applied to a fluorescent enhancement bioassay for the acetylcholine binding protein, a structural analog of the extracellular ligand-binding domain of neuronal nicotinic acetylcholine receptors. In the miniaturized setup, the sensitive and low void volume LED-induced confocal fluorescence detection system operated in flow injection analysis mode allowing the measurement of IC(50) values, which were comparable with those measured by a conventional plate reader bioassay. The current setup uses 50 nL as injection volume with a carrier flow rate of 400 nL/min. Finally, coupling of the detection system to gradient reversed-phase nano-LC allowed analysis of mixtures in order to identify the bioactive compounds present by injecting 10 nL of each mixture