Monolithic centrifugal microfluidic platform for bacteria capture and concentration, lysis, nucleic-acid amplification, and real-time detection

We report the design, fabrication, and characterization of a polymer centrifugal microfluidic system for the specific detection of bacterial pathogens. This single-cartridge platform integrates bacteria capture and concentration, supernatant solution removal, lysis, and nucleic-acid sequence-based amplification (NASBA) in a single unit. The unit is fabricated using multilayer lamination and consists of five different polymer layers. Bacteria capture and concentration are accomplished by sedimentation in five minutes. Centrifugation forces also drive the subsequent steps. A wax valve is integrated in the cartridge to enable high-speed centrifugation. Oil is used to prevent evaporation during reactions requiring thermal cycling. Device functionality was demonstrated by real-time detection of E. coli from a 200-muL sample

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

Development of an on-disc isothermal in vitro amplification and detection of bacterial RNA

This document is the Accepted Manuscript version, made available under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License CC BY NC-ND 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/). The final, published version is available online at doi: https://doi.org/10.1016/j.snb.2016.08.018. Published by Elsevier B. V.We present a centrifugal microfluidic “Lab-on-a-Disc” (LoaD) system capable of implementing nucleic acid in vitro amplification using non-contact heating and fluorescence detection. The system functionality is verified by implementing a Nucleic Acid Sequence Based Amplification (NASBA) reaction, targeting the tmRNA transcript of Haemophilus influenzae. The NASBA assay incorporates fluorescent molecular beacon probes reporting target tmRNA amplification for endpoint detection. The system implements non-contact IR heating to heat the NASBA reaction to the required target temperatures during denaturation and amplification steps. The LoaD control system facilitates spin speed and chamber positioning for heating and fluorescence detection. The LoaD alignment system uses magnetic fields to locate and lock the chamber in the required position (heating or detection). The NASBA assay was implemented on the system using Haemophilus influenzae tmRNA over the range 102–104 cell equivalent (CE) units. For comparison, identical qNASBA assays were implemented on a Roche LightCycler 2.0 over this concentration range.Peer reviewe

Self-partitioning SlipChip for slip-induced droplet formation and human papillomavirus viral load quantification with digital LAMP

Human papillomavirus (HPV) is one of the most common sexually transmitted infections worldwide, and persistent HPV infection can cause warts and even cancer. Nucleic acid analysis of HPV viral DNA can be very informative for the diagnosis and monitoring of HPV. Digital nucleic acid analysis, such as digital PCR and digital isothermal amplification, can provide sensitive detection and precise quantification of target nucleic acids, and its utility has been demonstrated in many biological research and medical diagnostic applications. A variety of methods have been developed for the generation of a large number of individual reaction partitions, a key requirement for digital nucleic acid analysis. However, an easily assembled and operated device for robust droplet formation without preprocessing devices, auxiliary instrumentation or control systems is still highly desired. In this paper, we present a self-partitioning SlipChip (sp-SlipChip) microfluidic device for the slip-induced generation of droplets to perform digital loop-mediated isothermal amplification (LAMP) for the detection and quantification of HPV DNA. In contrast to traditional SlipChip methods, which require the precise alignment of microfeatures, this sp-SlipChip utilized a design of “chain-of-pearls” continuous microfluidic channel that is independent of the overlapping of microfeatures on different plates to establish the fluidic path for reagent loading. Initiated by a simple slipping step, the aqueous solution can robustly self-partition into individual droplets by capillary pressure-driven flow. This advantage makes the sp-SlipChip very appealing for the point-of-care quantitative analysis of viral load. As a proof of concept, we performed digital LAMP on an sp-SlipChip to quantify human papillomaviruses (HPVs) 16 and 18 and tested this method with fifteen anonymous clinical samples

Integrated microfluidic systems with sample preparation and nucleic acid amplification

Rapid, efficient and accurate nucleic acid molecule detection is important in the screening of diseases and pathogens, yet remains a limiting factor at point of care (POC) treatment. Microfluidic systems are characterized by fast, integrated, miniaturized features which provide an effective platform for qualitative and quantitative detection of nucleic acid molecules. The nucleic acid detection process mainly includes sample preparation and target molecule amplification. Given the advancements in theoretical research and technological innovations to date, nucleic acid extraction and amplification integrated with microfluidic systems has advanced rapidly. The primary goal of this review is to outline current approaches used for nucleic acid detection in the context of microfluidic systems. The secondary goal is to identify new approaches that will help shape future trends at the intersection of nucleic acid detection and microfluidics, particularly with regard to increasing disease and pathogen detection for improved diagnosis and treatment

Point-of-Care Devices for Viral Detection: COVID-19 Pandemic and Beyond

The pandemic of COVID-19 and its widespread transmission have made us realize the importance of early, quick diagnostic tests for facilitating effective cure and management. The primary obstacles encountered were accurately distinguishing COVID-19 from other illnesses including the flu, common cold, etc. While the polymerase chain reaction technique is a robust technique for the determination of SARS-CoV-2 in patients of COVID-19, there arises a high demand for affordable, quick, user-friendly, and precise point-of-care (POC) diagnostic in therapeutic settings. The necessity for available tests with rapid outcomes spurred the advancement of POC tests that are characterized by speed, automation, and high precision and accuracy. Paper-based POC devices have gained increasing interest in recent years because of rapid, low-cost detection without requiring external instruments. At present, microfluidic paper-based analysis devices have garnered public attention and accelerated the development of such POCT for efficient multistep assays. In the current review, our focus will be on the fabrication of detection modules for SARS-CoV-2. Here, we have included a discussion on various strategies for the detection of viral moieties. The compilation of these strategies would offer comprehensive insight into the detection of the causative agent preparedness for future pandemics. We also provide a descriptive outline for paper-based diagnostic platforms, involving the determination mechanisms, as well as a commercial kit for COVID-19 as well as their outlook

Parallel solid-phase isothermal amplification and detection of multiple DNA targets in microliter-sized wells of a digital versatile disc

An integrated method for the parallelized detection of multiple DNA target sequences is presented by using microstructures in a digital versatile disc (DVD). Samples and reagents were managed by using both the capillary and centrifugal forces induced by disc rotation. Recombinase polymerase amplification (RPA), in a bridge solid phase format, took place in separate wells, which thereby modified their optical properties. Then the DVD drive reader recorded the modifications of the transmitted laser beam. The strategy allowed tens of genetic determinations to be made simultaneously within < 2 h, with small sample volumes (3 mu L), low manipulation and at low cost. The method was applied to high-throughput screening of relevant safety threats (allergens, GMOs and pathogenic bacteria) in food samples. Satisfactory results were obtained in terms of sensitivity (48.7 fg of DNA) and reproducibility (below 18 %). This scheme warrants cost-effective multiplex amplification and detection and is perceived to represent a viable tool for screening of nucleic acid targets.This research has been funded through projects FEDER PrometeoII/2014/040 (GVA), and CTQ/2013/45875-R (MINECO). The Spanish Ministry of Education and Science provided S.S.F. with a grant for PhD studies.Santiago Felipe, S.; Tortajada-Genaro, LA.; Puchades, R.; Maquieira Catala, Á. (2016). Parallel solid-phase isothermal amplification and detection of multiple DNA targets in microliter-sized wells of a digital versatile disc. Microchimica Acta. 183(3):1195-1202. https://doi.org/10.1007/s00604-016-1745-3S119512021833Li Y, Guo SJ, Shao N, Tu S, Xu M, Ren ZR, Ling X, Wang GQ, Lin ZX, Tao SC (2011) A universal multiplex PCR strategy for 100-plex amplification using a hydrophobically patterned microarray. Lab Chip 11:3609–3618Ng JK, Chong S (2011) Multiplexing Capabilities of Biosensors for Clinical Diagnostics. 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Nat Biotechnol 31:1126–1132McCalla SE, Tripathi A (2011) Microfluidic reactors for diagnostics applications. Annu Rev. Biomed Eng 13:321–343Westin L, Xu X, Miller C, Wang L, Edman CF, Nerenberg M (2000) Anchored multiplex amplification on a microelectronic chip array. Nat Biotechnol 18:199–204Khan Z, Poetter K, Park DJ (2008) Enhanced solid phase PCR: mechanisms to increase priming by solid support primers. Anal Biochem 375:391–393Khodakov DA, Ellis AV (2014) Recent developments in nucleic acid identification using solid-phase enzymatic assays. Microchim Acta 181:1633–1646Shin Y, Perera AP, Kim KW, Park MK (2013) Real-time, label-free isothermal solid-phase amplification/detection (ISAD) device for rapid detection of genetic alteration in cancers. Lab Chip 13:2106–2114Kersting S, Rausch V, Bier FF, von Nickisch-Rosenegk M (2014) Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis. Malaria J 13:99–107del Río JS, Yehia Adly N, Acero-Sánchez JL, Henry OY, O’Sullivan CK (2014) Electrochemical detection of Francisella tularensis genomic DNA using solid-phase recombinase polymerase amplification. Biosens Bioelectron 54: 674–678Lutz S, Weber P, Focke M, Faltin B, Hoffmann J, Müller C, Mark D, Roth G, Munday P, Armes N, Piepenburg O, Zengerle R, von Stetten F (2010) Microfluidic lab-on-a-foil for nucleic acid analysis based on isothermal recombinase polymerase amplification (RPA). Lab Chip 10:887–893Santiago-Felipe S, Tortajada-Genaro LA, Morais S, Puchades R, Maquieira A (2014) One-pot isothermal DNA amplification–Hybridisation and detection by a disc-based method. Sensor Actuat B-Chem 204:273–281Tortajada-Genaro LA, Santiago-Felipe S, Amasia M, Russom A, Maquieira A (2015) Isothermal solid-phase recombinase polymerase amplification on microfluidic digital versatile discs (DVDs). 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Advances in microfluidics and lab on a chip technologies

pre-printAdvances in molecular biology are enabling rapid and efficient analyses for effective intervention in domains like biology research, infectious disease management, food safety and bio-defense. The emergence of microfluidics and nanotechnologies has enabled both new capabilities and instrument sizes practical for point-of-care (POC). They have also introduced new functionality, enhanced the sensitivity, and reduced the time and cost involved in conventional molecular diagnostic techniques. This chapter reviews the application of microfluidics for molecular diagnostics methods like 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

Challenges and opportunities of centrifugal microfluidics for extreme point-of-care testing

The advantages offered by centrifugal microfluidic systems have encouraged its rapid adaptation in the fields of in vitro diagnostics, clinical chemistry, immunoassays, and nucleic acid tests. Centrifugal microfluidic devices are currently used in both clinical and point-of-care settings. Recent studies have shown that this new diagnostic platform could be potentially used in extreme point-of-care settings like remote villages in the Indian subcontinent and in Africa. Several technological inventions have decentralized diagnostics in developing countries; however, very few microfluidic technologies have been successful in meeting the demand. By identifying the finest difference between the point-of-care testing and extreme point-of-care infrastructure, this review captures the evolving diagnostic needs of developing countries paired with infrastructural challenges with technological hurdles to healthcare delivery in extreme point-of-care settings. In particular, the requirements for making centrifugal diagnostic devices viable in developing countries are discussed based on a detailed analysis of the demands in different clinical settings including the distinctive needs of extreme point-of-care settings.ope

Advances in Microfluidics Technology for Diagnostics and Detection

Microfluidics and lab-on-a-chip have, in recent years, come to the forefront in diagnostics and detection. At point-of-care, in the emergency room, and at the hospital bed or GP clinic, lab-on-a-chip offers the potential to rapidly detect time-critical and life-threatening diseases such as sepsis and bacterial meningitis. Furthermore, portable and user-friendly diagnostic platforms can enable disease diagnostics and detection in resource-poor settings where centralised laboratory facilities may not be available. At point-of-use, microfluidics and lab-on-chip can be applied in the field to rapidly identify plant pathogens, thus reducing the need for damaging broad spectrum pesticides while also reducing food losses. Microfluidics can also be applied to the continuous monitoring of water quality and can support policy-makers and protection agencies in protecting the environment. Perhaps most excitingly, microfluidics also offers the potential to enable entirely new diagnostic tests that cannot be implemented using conventional laboratory tools. Examples of microfluidics at the frontier of new medical diagnostic tests include early detection of cancers through circulating tumour cells (CTCs) and highly sensitive genetic tests using droplet-based digital PCR.This Special Issue on “Advances in Microfluidics Technology for Diagnostics and Detection” aims to gather outstanding research and to carry out comprehensive coverage of all aspects related to microfluidics in diagnostics and detection