Point-of-Need DNA Testing for Detection of Foodborne Pathogenic Bacteria

Foodborne pathogenic bacteria present a crucial food safety issue. Conventional diagnostic methods are time-consuming and can be only performed on previously produced food. The advancing field of point-of-need diagnostic devices integrating molecular methods, biosensors, microfluidics, and nanomaterials offers new avenues for swift, low-cost detection of pathogens with high sensitivity and specificity. These analyses and screening of food items can be performed during all phases of production. This review presents major developments achieved in recent years in point-of-need diagnostics in land-based sector and sheds light on current challenges in achieving wider acceptance of portable devices in the food industry. Particular emphasis is placed on methods for testing nucleic acids, protocols for portable nucleic acid extraction and amplification, as well as on the means for low-cost detection and read-out signal amplification

Integrated Electrochemical Biosensors for Detection of Waterborne Pathogens in Low-Resource Settings

More than 783 million people worldwide are currently without access to clean and safe water. Approximately 1 in 5 cases of mortality due to waterborne diseases involve children, and over 1.5 million cases of waterborne disease occur every year. In the developing world, this makes waterborne diseases the second highest cause of mortality. Such cases of waterborne disease are thought to be caused by poor sanitation, water infrastructure, public knowledge, and lack of suitable water monitoring systems. Conventional laboratory-based techniques are inadequate for effective on-site water quality monitoring purposes. This is due to their need for excessive equipment, operational complexity, lack of affordability, and long sample collection to data analysis times. In this review, we discuss the conventional techniques used in modern-day water quality testing. We discuss the future challenges of water quality testing in the developing world and how conventional techniques fall short of these challenges. Finally, we discuss the development of electrochemical biosensors and current research on the integration of these devices with microfluidic components to develop truly integrated, portable, simple to use and cost-effective devices for use by local environmental agencies, NGOs, and local communities in low-resource settings

Nucleic Acid-based Detection of Bacterial Pathogens Using Integrated Microfluidic Platform Systems

The advent of nucleic acid-based pathogen detection methods offers increased sensitivity and specificity over traditional microbiological techniques, driving the development of portable, integrated biosensors. The miniaturization and automation of integrated detection systems presents a significant advantage for rapid, portable field-based testing. In this review, we highlight current developments and directions in nucleic acid-based micro total analysis systems for the detection of bacterial pathogens. Recent progress in the miniaturization of microfluidic processing steps for cell capture, DNA extraction and purification, polymerase chain reaction, and product detection are detailed. Discussions include strategies and challenges for implementation of an integrated portable platform

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

Hydrogel-Based Colorimetric Assay for Multiplexed MicroRNA Detection in a Microfluidic Device

Although microRNA (miRNA) expression levels provide important information regarding disease states owing to their unique dysregulation patterns in tissues, translation of miRNA diagnostics into point-of-care (POC) settings has been limited by practical challenges. Her; we developed a hydrogel-based microfluidic platform for colorimetric profiling of miRNAs, without the use of complex external equipment for fluidics and imaging. For sensitive and reliable measurement without the risk of sequence bias, we employed a gold deposition-based signal amplification scheme and dark-field imaging, and seamlessly integrated a previously developed miRNA assay scheme into this platform. The assay demonstrated a limit of detection of 260 fM, along with multiplexing of small panels of miRNAs in healthy and cancer samples. We anticipate this versatile platform to facilitate a broad range of POC profiling of miRNAs in cancer-associated dysregulation with high-confidence by exploiting the unique features of hydrogel substrate in an on-chip format and colorimetric analysis

A Microfluidic Approach to Selection and Enrichment of Aptamers for Biomolecules and Cells

This thesis presents microfluidic devices for selection and amplification of nucleic acids (aptamers) that bind to specific targets. Aptamers are very attractive molecules in many biological applications due to their interesting properties including high target binding affinities and stability. Using conventional platforms for aptamer generation (SELEX, systematic evolution of ligands by exponential enrichment) is labor-intensive and time consuming. Microfluidic devices have been developed to improve the aptamer enrichment efficiency. However, aptamer generation using these devices is still inefficient because they require complicated flow control components for sample and reagent handling and additional off-chip processes. We developed microfluidic SELEX platforms for rapid isolation of aptamers that possess greatly simplified designs which enable easy chip fabrication and operation. The simplicity of the devices is achieved by utilizing a combination of bead-based selection and amplification of target binding nucleic acids, and gel-based electrokinetic transfer of nucleic acids. In the devices, nucleic acids that bind to targets are isolated on target-functionalized microbeads or target cells in a microchamber and electrokinetically transported to another chamber through a gel-filled microchannel by an electric field. The strands are then hybridized onto reverse primers immobilized on microbeads and amplified via polymerase chain reaction (PCR) using on-chip temperature control. The amplified strands are separated from the beads and electrophoretically transferred back into the selection chamber for subsequent SELEX rounds. Using the devices, we demonstrated enrichment of target-binding nucleic acids against human immunoglobulin E (IgE), the glucose-boronic acid complex, and MCF-7 cancer cells. With the physical and functional integration allowed by the monolithic design realized in our devices, the total process time for selection of aptamers was drastically reduced compared with that required by conventional aptamer selection platforms. Moreover, the binding affinities of the selected strands using our devices are comparable to those of aptamers obtained using the conventional platforms

Advanced Evanescent-Wave Optical Biosensors for the Detection of Nucleic Acids : An Analytic Perspective

Evanescent-wave optical biosensors have become an attractive alternative for the screening of nucleic acids in the clinical context. They possess highly sensitive transducers able to perform detection of a wide range of nucleic acid-based biomarkers without the need of any label or marker. These optical biosensor platforms are very versatile, allowing the incorporation of an almost limitless range of biorecognition probes precisely and robustly adhered to the sensor surface by covalent surface chemistry approaches. In addition, their application can be further enhanced by their combination with different processes, thanks to their integration with complex and automated microfluidic systems, facilitating the development of multiplexed and user-friendly platforms. The objective of this work is to provide a comprehensive synopsis of cutting-edge analytical strategies based on these label-free optical biosensors able to deal with the drawbacks related to DNA and RNA detection, from single point mutations assays and epigenetic alterations, to bacterial infections. Several plasmonic and silicon photonic-based biosensors are described together with their most recent applications in this area. We also identify and analyse the main challenges faced when attempting to harness this technology and how several innovative approaches introduced in the last years manage those issues, including the use of new biorecognition probes, surface functionalization approaches, signal amplification and enhancement strategies, as well as, sophisticated microfluidic solutions

Low-cost genotyping method based on allele-specific recombinase polymerase amplification and colorimetric microarray detection

[EN] The costs of current genotyping methods limit their application to personalized therapy. The authors describe an alternative approach for the detection of single-point-polymorphisms using recombinant polymerase amplification as an allele-specific technique. The use of short and chemically modified primers and locked nucleic acids allowed for a selective isothermal amplification of wild-type or mutant variants at 37 °C within 40 min. An amplification chip platform containing 100 wells was manufactured with a 3D printer and using thermoplastic polylactic acid. The platform reduces reagent consumption and allows parallelization. As a proof of concept, the method was applied to the genotyping of four SNPs that are related to the treatment of tobacco addiction. The target polymorphisms included rs4680 (COMT gene), rs1799971 (OPRM1 gene), rs1800497 (ANKK1 gene), and rs16969968 (CHRNA5 gene). The genotype populations can be well discriminated.The authors acknowledge the financial support received from the Generalitat Valenciana (GVA-PROMETEOII/2014/040 project and GRISOLIA/2014/024 PhD grant) and the Spanish Ministry of Economy and Competitiveness (MINECO CTQ2013-45875-R project).Yamanaka, E.; Tortajada-Genaro, LA.; Maquieira, A. (2017). Low-cost genotyping method based on allele-specific recombinase polymerase amplification and colorimetric microarray detection. Microchimica Acta. 184(5):1453-1462. https://doi.org/10.1007/s00604-017-2144-0S145314621845Manolio TA, Chisholm RL, Ozenberger B, Roden DM, Williams MS, Wilson R et al (2013) Implementing genomic medicine in the clinic: the future is here. Genitourin Med 15:258–267Scott SA (2013) Clinical pharmacogenomics: opportunities and challenges at point-of-care. Clin Pharmacol Ther 93:33Limaye N (2013) Pharmacogenomics, Theranostics and personalized medicine-the complexities of clinical trials: challenges in the developing world. Appl Transl Genomics 2:17–21Abul-Husn NS, Owusu Obeng A, Sanderson SC, Gottesman O, Scott SA (2014) Implementation and utilization of genetic testing in personalized medicine. Pharmacogenomics Pers Med 7:227–240Knez K, Spasic D, Janssen KP, Lammertyn J (2014) Emerging technologies for hybridization based single nucleotide polymorphism detection. Analyst 139:353–370Shen W, Tian Y, Ran T, Gao Z (2015) Genotyping and quantification techniques for single-nucleotide polymorphisms. TrAC Trends Anal Chem 69:1–13Milbury CA, Li J, Makrigiorgos GM (2009) PCR-based methods for the enrichment of minority alleles and mutations. Clin Chem 55:632–640Asari M, Watanabe S, Matsubara K, Shiono H, Shimizu K (2009) Single nucleotide polymorphism genotyping by mini-primer allele-specific amplification with universal reporter primers for identification of degraded DNA. Anal Biochem 386:85–90Taira C, Matsuda K, Yamaguchi A, Sueki A, Koeda H, Takagi F, Kobayashi Y, Sugano M, Honda T (2013) Novel high-speed droplet-allele specific-polymerase chain reaction: application in the rapid genotyping of single nucleotide polymorphisms. Clin Chim Acta 424:39–46Tortajada-Genaro LA, Mena S, Niñoles R, Puigmule M, Viladevall L, Maquieira A (2016) Genotyping of single nucleotide polymorphisms related to attention-deficit hyperactivity disorder. Anal Bioanal Chem 408:2339–2345Woolley CF, Hayes MA (2014) Emerging technologies for biomedical analysis. Analyst 139:2277–2288Craw P, Balachandran W (2012) Isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review. Lab Chip 12:2469–2486Zhang L, Zhang Y, Wang C, Feng Q, Fan F, Zhang G, Kang X, Qin X, Sun J, Li Y, Jiang X (2014) Integrated microcapillary for sample-to-answer nucleic acid pretreatment, amplification, and detection. Anal Chem 86:10461–10466Chen F, Zhao Y, Fan C, Zhao Y (2015) Mismatch extension of DNA polymerases and high-accuracy single nucleotide polymorphism diagnostics by gold nanoparticle-improved isothermal amplification. Anal Chem 87:8718–8723Li J, Macdonald J (2015) Advances in isothermal amplification: novel strategies inspired by biological processes. Biosens Bioelectron 64:196–211Santiago-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. Sens Actuator B-Chem 204:273–281Santiago-Felipe S, Tortajada-Genaro LA, Puchades R, Maquieira Á (2016) Parallel solid-phase isothermal amplification and detection of multiple DNA targets in microliter-sized wells of a digital versatile disc. Microchim Acta 183:1195–1202Tortajada-Genaro LA, Santiago-Felipe S, Amasia M, Russom A, Maquieira A (2015) Isothermal solid-phase recombinase polymerase amplification on microfluidic digital versatile discs (DVDs). RSCAdv 5:29987–29995Li Z, Liu Y, Wei Q, Liu Y, Liu W, Zhang X, Yu Y (2016) Picoliter well Array Chip-based digital recombinase polymerase amplification for absolute quantification of nucleic acids. PLoS One 11:e0153359Daher RK, Stewart G, Boissinot M, Boudreau DK, Bergeron MG (2015) Influence of sequence mismatches on the specificity of recombinase polymerase amplification technology. Mol Cell Probes 29:116–121Shin 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 13:2106–2114NgePN RCI, Woolley AT (2013) Advances in microfluidic materials, functions, integration, and applications. Chem Rev 113:2550–2583Bhattacharjee N, Urrios A, Kang S, Folch A (2016) The upcoming 3D-printing revolution in microfluidics. Lab Chip 16:1720–1742Waheed S, Cabot JM, Macdonald NP, Lewis T, Guijt RM, Paull B, Breadmore MC (2016) 3D printed microfluidic devices: enablers and barriers. Lab Chip 16:1993–2013Bierut LJ, Madden PA, Breslau N, Johnson EO, Hatsukami D, Pomerleau OF, Swan GE, Rutter J, Bertelsen S, Fox L, Fugman D, Goate AM, Hinrichs AL, Konvicka K, Martin NG, Montgomery GW, Saccone NL, Saccone SF, Wang JC, Chase GA, Rice JP, Ballinger DG (2007) Novel genes identified in a high-density genome wide association study for nicotine dependence. Hum MolGen 16:24–35Carpenter MJ, Jardin BF, Burris JL, Mathew AR, Schnoll RA, Rigotti NA, Cummings KM (2013) Clinical strategies to enhance the efficacy of nicotine replacement therapy for smoking cessation: a review of the literature. Drugs 73:407–426Moody C, Newell H, Viljoen H (2016) FA mathematical model of recombinase polymerase amplification under continuously stirred conditions. Biochem Eng J 112:193–201Dimitrov RA, Zuker M (2004) Prediction of hybridization and melting for double-stranded nucleic acids. Biophys J 87:215–226Zhang C, Xing D (2007) Miniaturized PCR chips for nucleic acid amplification and analysis: latest advances and future trends. Nucleic Acids Res 35:4223–4237Liu B, Huang PJJ, Zhang X, Wang F, Pautler R, IpACF LJ (2013) Parts-per-million of polyethylene glycol as a non-interfering blocking agent for homogeneous biosensor development. Anal Chem 85:10045–10050Wu J, Kodzius R, Cao W, Wen W (2014) Extraction, amplification and detection of DNA in microfluidic chip-based assays. Microchim Acta 181:1611–1631Li J, Wang L, Mamon H, Kulke MH, Berbeco R, Makrigiorgos GM (2008) Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing. Nat Med 14:579–584Shen R, Fan JB, Campbell D, Chang W, Chen J, Doucet D, Yeakley J, Bibikova M, Garcia EW, McBride C, Steemers F, Garcia F, Kermani BG, Gunderson K, Oliphant A (2005) High-throughput SNP genotyping on universal bead arrays. Mut Res Fund Mol M 573:70–82David SP, Strong DR, Leventhal AM, Lancaster MA, McGeary JE, Munafò MR, Bergen AW, Swan GE, Benowitz NL, Tyndale RF, Conti DV, Brown RA, Lerman C, Niaura R (2013) Influence of a dopamine pathway additive genetic efficacy score on smoking cessation: results from two randomized clinical trials of bupropion. Addiction 108:2202–221

Doctor of Philosophy

dissertationThis project will produce an automated microfluidic system capable of extracting and purifying nucleic acids from raw samples for detection and analysis. The first step will be the development and characterization of microfluidic components and fabrication methods that will be implemented into the final device. The gas permeability properties of PDMS will be utilized to demonstrate integrated components for pumping, gas bubble trapping and removal, and enhanced mixing. Next, a three-layer PDMS with silicone membrane microfluidic platform will be developed to control fluid flow for nucleic acid purification processes. This microfluidic chip will be capable of taking a raw biological sample through the steps of cell lysis and solid phase nucleic acid extraction to deliver purified DNA or RNA for testing and analysis. The microfluidic chip will be mounted on a portable, desktop control system to allow automated device operation in clinics, laboratories, or the field. Finally, a disposable oscillatory flow PCR chip will be made from polycarbonate to amplify low concentrations of nucleic acid. The PCR module will also be controlled by the same instrument used for nucleic acid extraction. Temperature control will be provided by external heating blocks, and internal chip fluid temperature will be determined by numerical simulations. This device will be a step towards having a universal nucleic acid purification device to fill the much-needed niche in sample preparation for lab-on-a-chip applications

Point of Care Diagnostics in the Age of COVID-19.

The recent outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated serious respiratory disease, coronavirus disease 2019 (COVID-19), poses a major threat to global public health. Owing to the lack of vaccine and effective treatments, many countries have been overwhelmed with an exponential spread of the virus and surge in the number of confirmed COVID-19 cases. Current standard diagnostic methods are inadequate for widespread testing as they suffer from prolonged turn-around times (>12 h) and mostly rely on high-biosafety-level laboratories and well-trained technicians. Point-of-care (POC) tests have the potential to vastly improve healthcare in several ways, ranging from enabling earlier detection and easier monitoring of disease to reaching remote populations. In recent years, the field of POC diagnostics has improved markedly with the advent of micro- and nanotechnologies. Due to the COVID-19 pandemic, POC technologies have been rapidly innovated to address key limitations faced in existing standard diagnostic methods. This review summarizes and compares the latest available POC immunoassay, nucleic acid-based and clustered regularly interspaced short palindromic repeats- (CRISPR)-mediated tests for SARS-CoV-2 detection that we anticipate aiding healthcare facilities to control virus infection and prevent subsequent spread