Advances in Microfluidics and Lab-on-a-Chip Technologies

Advances in molecular biology are enabling rapid and efficient analyses for effective intervention in domains such as biology research, infectious disease management, food safety, and biodefense. The emergence of microfluidics and nanotechnologies has enabled both new capabilities and instrument sizes practical for point-of-care. It has also introduced new functionality, enhanced sensitivity, and reduced the time and cost involved in conventional molecular diagnostic techniques. This chapter reviews the application of microfluidics for molecular diagnostics methods such as nucleic acid amplification, next-generation sequencing, high resolution melting analysis, cytogenetics, protein detection and analysis, and cell sorting. We also review microfluidic sample preparation platforms applied to molecular diagnostics and targeted to sample-in, answer-out capabilities

The burden of congenital Chagas disease and implementation of molecular diagnostic tools in Latin America

It is estimated that between 8000 and 15 000 Trypanosoma cruzi infected babies are born every year to infected mothers in Chagas disease endemic countries. Currently, poor access to and performance of the current diagnostic algorithm, based on microscopy at birth and serology at 8-12 months after delivery, is one of the barriers to congenital Chagas disease (CCD) control. Detection of parasite DNA using molecular diagnostic tools could be an alternative or complement to current diagnostic methods, but its implementation in endemic regions remains limited. Prompt diagnosis and treatment of CCD cases would have a positive clinical and epidemiological impact. In this paper, we analysed the burden of CCD in Latin America, and the potential use of molecular tests to improve access to early diagnosis and treatment of T. cruzi infected newborns.Fil: Picado, Albert. Foundation for Innovative New Diagnostics; SuizaFil: Cruz, Israel. Foundation for Innovative New Diagnostics; SuizaFil: Redard Jacot, Maël. Foundation for Innovative New Diagnostics; SuizaFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Torrico, Faustino. Universidad Mayor de San Simón; Bolivia. Fundación CEADES; BoliviaFil: Sosa-Estani, Sergio Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Epidemiología y Salud Pública. Instituto de Efectividad Clínica y Sanitaria. Centro de Investigaciones en Epidemiología y Salud Pública; Argentina. Drugs for Neglected Diseases initiative; BrasilFil: Katz, Zachary. Foundation for Innovative New Diagnostics; SuizaFil: Ndung'u, Joseph Mathu. Foundation for Innovative New Diagnostics; Suiz

HCN to HCO^+ Millimeter Line Diagnostics of AGN Molecular Torus I : Radiative Transfer Modeling

We explore millimeter line diagnostics of an obscuring molecular torus modeled by a hydrodynamic simulation with three-dimensional nonLTE radiative transfer calculations. Based on the results of high-resolution hydrodynamic simulation of the molecular torus around an AGN, we calculate intensities of HCN and HCO^{+} rotational lines as two representative high density tracers. The three-dimensional radiative transfer calculations shed light on a complicated excitation state in the inhomogeneous torus, even though a spatially uniform chemical structure is assumed. Our results suggest that HCN must be much more abundant than HCO^{+} in order to obtain a high ratio ($R_{HCN/HCO+}\sim 2$) observed in some of the nearby galaxies. There is a remarkable dispersion in the relation between integrated intensity and column density, indicative of possible shortcomings of HCN(1-0) and HCO^{+}(1-0) lines as high density tracers. The internal structures of the inhomogeneous molecular torus down to subparsec scale in external galaxies will be revealed by the forthcoming Atacama Large Millimeter/submillimeter Array (ALMA). The three-dimensional radiative transfer calculations of molecular lines with high-resolution hydrodynamic simulation prove to be a powerful tool to provide a physical basis for molecular line diagnostics of the central regions of external galaxies.Comment: 29 pages, 13 figures, Accepted for publication in ApJ, For high resolution figures see http://alma.mtk.nao.ac.jp/~masako/MS72533v2.pd

Diagnostics of the Molecular Component of PDRs with Mechanical Heating

Context. Multitransition CO observations of galaxy centers have revealed that significant fractions of the dense circumnuclear gas have high kinetic temperatures, which are hard to explain by pure photon excitation, but may be caused by dissipation of turbulent energy. Aims. We aim to determine to what extent mechanical heating should be taken into account while modelling PDRs. To this end, the effect of dissipated turbulence on the thermal and chemical properties of PDRs is explored. Methods. Clouds are modelled as 1D semi-infinite slabs whose thermal and chemical equilibrium is solved for using the Leiden PDR-XDR code. Results. In a steady-state treatment, mechanical heating seems to play an important role in determining the kinetic temperature of the gas in molecular clouds. Particularly in high-energy environments such as starburst galaxies and galaxy centers, model gas temperatures are underestimated by at least a factor of two if mechanical heating is ignored. The models also show that CO, HCN and H2 O column densities increase as a function of mechanical heating. The HNC/HCN integrated column density ratio shows a decrease by a factor of at least two in high density regions with n \sim 105 cm-3, whereas that of HCN/HCO+ shows a strong dependence on mechanical heating for this same density range, with boosts of up to three orders of magnitude. Conclusions. The effects of mechanical heating cannot be ignored in studies of the molecular gas excitation whenever the ratio of the star formation rate to the gas density is close to, or exceeds, 7 \times 10-6 M yr-1 cm4.5 . If mechanical heating is not included, predicted column densities are underestimated, sometimes even by a few orders of magnitude. As a lower bound to its importance, we determined that it has non-negligible effects already when mechanical heating is as little as 1% of the UV heating in a PDR.Comment: 26 pages, 14 figures in the text and 13 figures as supplementary material. Accepted for publication in A&

Non-coding RNAs in saliva: emerging biomarkers for molecular diagnostics.

Saliva is a complex body fluid that comprises secretions from the major and minor salivary glands, which are extensively supplied by blood. Therefore, molecules such as proteins, DNA, RNA, etc., present in plasma could be also present in saliva. Many studies have reported that saliva body fluid can be useful for discriminating several oral diseases, but also systemic diseases including cancer. Most of these studies revealed messenger RNA (mRNA) and proteomic biomarker signatures rather than specific non-coding RNA (ncRNA) profiles. NcRNAs are emerging as new regulators of diverse biological functions, playing an important role in oncogenesis and tumor progression. Indeed, the small size of these molecules makes them very stable in different body fluids and not as susceptible as mRNAs to degradation by ribonucleases (RNases). Therefore, the development of a non-invasive salivary test, based on ncRNAs profiles, could have a significant applicability to clinical practice, not only by reducing the cost of the health system, but also by benefitting the patient. Here, we summarize the current status and clinical implications of the ncRNAs present in human saliva as a source of biological information

Sample preparation for point of care molecular diagnostics of STIs

This paper was presented at the 4th Micro and Nano Flows Conference (MNF2014), which was held at University College, London, UK. The conference was organised by Brunel University and supported by the Italian Union of Thermofluiddynamics, IPEM, the Process Intensification Network, the Institution of Mechanical Engineers, the Heat Transfer Society, HEXAG - the Heat Exchange Action Group, and the Energy Institute, ASME Press, LCN London Centre for Nanotechnology, UCL University College London, UCL Engineering, the International NanoScience Community, www.nanopaprika.eu.Brunel DoCLab is part of the esti2 consortium developing electronic self-testing instruments for sexually transmitted infections using nucleic acid amplification testing (NAAT). A proprietary sample collection device has been designed to integrate directly with a microfluidic cartridge. Cell lysis was conducted using a chemical method and nucleic acid purification was done on an activated cellulose membrane. The microfluidic device incorporates passive mixing of the lysis-binding buffers and sample. Preliminary results have shown extraction efficiencies for this new membrane of 69% and 57% compared to the commercial Qiagen extraction method of 85% and 59.4% for 0.1ng/μL and 100ng/μL salmon sperm DNA spiked in phosphate buffered solution. Preliminary extraction experiments in the passive mixer cartridges with lysis and nucleic acid purification showed extraction efficiency around 80% of the commercial Qiagen kit. Isothermal amplification was conducted using thermophillic helicase dependant amplification. A low cost benchtop real-time isothermal amplification platform has been developed capable of running six amplifications simultaneously. Work to integrate sample collection, nucleic acid extraction and isothermal amplification is currently underway

Diagnostic applications of next generation sequencing: working towards quality standards

Over the past 6 years, next generation sequencing (NGS) has been established as a valuable high-throughput method for research in molecular genetics and has successfully been employed in the identification of rare and common genetic variations. All major NGS technology companies providing commercially available instruments (Roche 454, Illumina, Life Technologies) have recently marketed bench top sequencing instruments with lower throughput and shorter run times, thereby broadening the applications of NGS and opening the technology to the potential use for clinical diagnostics. Although the high expectations regarding the discovery of new diagnostic targets and an overall reduction of cost have been achieved, technological challenges in instrument handling, robustness of the chemistry and data analysis need to be overcome. To facilitate the implementation of NGS as a routine method in molecular diagnostics, consistent quality standards need to be developed. Here the authors give an overview of the current standards in protocols and workflows and discuss possible approaches to define quality criteria for NGS in molecular genetic diagnostics