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

RNA–protein binding kinetics in an automated microfluidic reactor

Microfluidic chips can automate biochemical assays on the nanoliter scale, which is of considerable utility for RNA–protein binding reactions that would otherwise require large quantities of proteins. Unfortunately, complex reactions involving multiple reactants cannot be prepared in current microfluidic mixer designs, nor is investigation of long-time scale reactions possible. Here, a microfluidic ‘Riboreactor’ has been designed and constructed to facilitate the study of kinetics of RNA–protein complex formation over long time scales. With computer automation, the reactor can prepare binding reactions from any combination of eight reagents, and is optimized to monitor long reaction times. By integrating a two-photon microscope into the microfluidic platform, 5-nl reactions can be observed for longer than 1000 s with single-molecule sensitivity and negligible photobleaching. Using the Riboreactor, RNA–protein binding reactions with a fragment of the bacterial 30S ribosome were prepared in a fully automated fashion and binding rates were consistent with rates obtained from conventional assays. The microfluidic chip successfully combines automation, low sample consumption, ultra-sensitive fluorescence detection and a high degree of reproducibility. The chip should be able to probe complex reaction networks describing the assembly of large multicomponent RNPs such as the ribosome

Development of a rapid prototyping method for hard polymer microfluidic systems tested through iterative design of a PCR chamber chip

Tese de mestrado integrado, Engenharia Biomédica e Biofísica (Engenharia Clínica e Instrumentação Médica), Universidade de Lisboa, Faculdade de Ciências, 2014One of the challenges of working with polymer microfluidics is the lack of an established prototyping method which allows for easy translation to industrial production. By combining Hot Embossing and Computer Numerically Controlled Milling a microfluidic rapid prototyping method was established for Polycarbonate and Cyclic Olefin Polymer. This method was then tested and optimized through an iterative design process of a microfluidic Polymerase-Chain Reaction chamber. The fabrication method proved to be suitable for microfluidic prototyping, allowing for rapid design changes and fabrication of good quality copies in a simple and straightforward fashion.Uma das dificuldades em trabalhar com microfluídica em polímeros é a falta da existência de um método de prototipagem que permita uma passagem simples para um ambiente de produção industrial. Neste trabalho foi desenvolvido um método de prototipagem rápida para microfluídica em Policarbonato e Cyclic Olefin Polymer utilizando uma Fresadora de Controlo Numérico Computorizado e Hot Embossing. Este método foi testado e optimizado através de um processo de design iterativo de uma câmara microfluídica de Reacção em Cadeia da Polimerase em Policarbonato. O método desenvolvido provou ser adequado para prototipagem microfluídica, permitindo alterações rápidas ao desenho e fabricação de várias cópias com boa qualidade de cada desenho

Light-actuated microfluidics

Lab-on-a-chip (LoC) technologies are nominated to revolutionize chemical and bio-chemical analysis. Although their great potential, they have not reach the consumer grade market, due to a lack of truly enabled functionality compared to current analytical methods. This works focuses on performing absorbance detection on LoC devices, by an easy to implement set-up. The present work is enclosed in a project which aims to combine, low-price absorption detection, low-cost materials and fast prototyping/fabrication method for developing an affordable and portable diagnostic tool based on enzymatic immunoassays. The obtained results are satisfactory, due to the obtained sensitivity, an order of magnitude higher than a standard microplate reader

Microfluidic-SANS: flow processing of complex fluids

Understanding and engineering the flow-response of complex and non-Newtonian fluids at a molecular level is a key challenge for their practical utilisation. Here we demonstrate the coupling of microfluidics with small angle neutron scattering (SANS). Microdevices with high neutron transmission (up to 98%), low scattering background ([Image: see text]), broad solvent compatibility and high pressure tolerance (≈3–15 bar) are rapidly prototyped via frontal photo polymerisation. Scattering from single microchannels of widths down to 60 μm, with beam footprint of 500 μm diameter, was successfully obtained in the scattering vector range 0.01–0.3 Å(−1), corresponding to real space dimensions of [Image: see text]. We demonstrate our approach by investigating the molecular re-orientation and alignment underpinning the flow response of two model complex fluids, namely cetyl trimethylammonium chloride/pentanol/D(2)O and sodium lauryl sulfate/octanol/brine lamellar systems. Finally, we assess the applicability and outlook of microfluidic-SANS for high-throughput and flow processing studies, with emphasis of soft matter

Fast prototyping microfluidics: Integrating droplet digital lamp for absolute quantification of cancer biomarkers

UID/CTM/50025/2019 UID/Multi/04378/2019 Inn-INDIGO/0002/2015 PTDC/BTM-SAL/31201/2017 SFRH/BPD/124311/2016Microfluidic (MF) advancements have been leveraged toward the development of state-of-the-art platforms for molecular diagnostics, where isothermal amplification schemes allow for further simplification of DNA detection and quantification protocols. The MF integration with loop-mediated isothermal amplification (LAMP) is today the focus of a new generation of chip-based devices for molecular detection, aiming at fast and automated nucleic acid analysis. Here, we combined MF with droplet digital LAMP (ddLAMP) on an all-in-one device that allows for droplet generation, target amplification, and absolute quantification. This multilayer 3D chip was developed in less than 30 minutes by using a low-cost and extremely adaptable production process that exploits direct laser writing technology in “Shrinky-dinks” polystyrene sheets. ddLAMP and target quantification were performed directly on-chip, showing a high correlation between target concentration and positive droplet score. We validated this integrated chip via the amplification of targets ranging from five to 500,000 copies/reaction. Furthermore, on-chip amplification was performed in a 10 µL volume, attaining a limit of detection of five copies/µL under 60 min. This technology was applied to quantify a cancer biomarker, c-MYC, but it can be further extended to any other disease biomarker.publishersversionpublishe