Closure of Loop-Mediated Isothermal Amplification Chamber on Lab-On-Chip Using Thermal-Responsive Valve

Point-of-care tests for nucleic acids are important for the diagnosis and management of infectious and genetic diseases, biowarfare agents, and for drug research. Recent integration of loop-mediated isothermal amplification (LAMP) onto a lab-on-chip (LOC) platform allows sensitive and specific, detection of target DNA or RNA sequences for point-of-care (POC) applications. However, because of the high risk of contamination to LAMP products, robust and simple methods of hermetically sealing the reaction chamber are essential. In addition, having a method for isolation of nucleic acids at the level of the chip is more effective for POC applications because a laboratory setting is not required to complete the analysis. This report describes an integrated, polymer-based cassette which was designed for detection of DNA/RNA target-sequences by using LAMP and a solid-state nucleic acid purification membrane. The LAMP chamber seals using a self-actuated thermalresponsive valve made from expandable microspheres suspended in Polydimethylsiloxane (PDMS). A flow-through, Flinders Technology Associates membrane (Whatman FT A®) was installed on the cassette with the expectation of providing isolation and purification of nucleic acids. The cassette was designed, fabricated and the efficacy of the valve to seal the LAMP chamber was investigated. The valve underwent expansion and held pressures of233 +/- 5.0 kPa without signs of leakage. A portable, battery-powered, polyimide-based thin film heater, placed outside the cassette, provided thermal control. In addition, a LAMP primer set was designed for the serotonin receptor 5HTRIA promoter gene using Primer Explorer V 4 software (http:/ /primerexplorer.jp/elamp4.0.0/index.html) for LAMP detection of neurotransmitter serotonin. The integrated, portable LAMP cassette will be attractive in global health-care challenges, mainly in resource-poor locations, where the availability of laboratory equipment and/or trained personal is restricted.Digitized from a paper copy provided by the Physiological Sciences Graduate Interdisciplinary Program

User-Friendly, Low-Cost, Microfluidic Devices With Capillary Circuits For Multiplexed, Isothermal, Point-Of-Care, Nucleic Acid Amplification Tests

Rapid, sensitive, and specific detection of causative pathogens is key to personalized medicine and the prompt implementation of appropriate mitigation measures to reduce disease transmission, mortality, morbidity, and cost. Conventional molecular detection methods require trained personnel, sophisticated equipment, and specialized laboratories, which limits their use to centralized laboratories. To enable molecular diagnostics at the point of need and in resource-poor settings, inexpensive, simple devices that combine multiple unit operations and are capable of co-detecting endemic pathogens are needed. In this work, I have developed microfluidic devices with capillary circuits to automate liquid distribution, eliminating the need for expensive equipment, sophisticated laboratory facilities, and skilled personnel to enable molecular diagnostics at the point of need. Capillary valves with different sizes were developed and implemented to aliquot samples and reagents to multiple reaction chambers and to enable draining liquids from supply lines without affecting liquids in the various reaction chambers, enabling bubble-free operation. The sealing of my microfluidic devices to prevent evaporation during incubation is facilitated with phase-change materials and capillary-induced motion. When my microfluidic chip is heated to its incubation temperature, the phase-change material melts and flows to seal ports of entry and air vent. Numerical simulations were carried out to assess the viability of on-chip, in-house developed, two-stage isothermal nucleic acid amplification in the presence of diffusion and advection. An Android-based smartphone application was developed to automate real-time signal monitoring, time series image analysis, and diagnostic result interpretation. Three 3D-printed, portable, microfluidic devices with capillary circuits were designed, fabricated, and tested for single-stage and two-stage, isothermal nucleic acid amplification with either liquid reagents or pre-stored dry reagents that do not require a cold chain. All devices have proved successful for rapid, sensitive, and specific multiplexed detections of human and animal pathogens

Molecular methods for the detection of infectious diseases: bringing diagnostics to the point-of-care

Human infectious diseases represent a leading cause of morbidity and mortality globally, caused by human-infective pathogens such as bacteria, viruses, parasites or fungi. Point-of-care (POC) diagnostics allow accessible, simple, and rapid identification of the organism causing the infection which is crucial for successful prognostic outcomes, clinical management, surveillance and isolation. The research conducted in this thesis aims to investigate novel methods for molecular-based diagnostics. This multidisciplinary project is divided into three main sections: (i) molecular methods for enhanced nucleic acid amplification, (ii) POC technologies, and (iii) sample preparation. The application, design and optimisation of loop-mediated isothermal amplification (LAMP) is investigated from a molecular perspective for the diagnostics of emerging infectious pathogens and antimicrobial resistance. LAMP assays were designed to target pathogens responsible for parasitic (malaria), bacterial and viral (COVID-19) infections, as well as antimicrobial resistance. A novel LAMP-based method for the detection of single nucleotide polymorphisms was developed and applied for diagnostics of antimicrobial resistance, emerging variants and genetic disorders. The method was validated for the detection of artemisinin-resistant malaria. Furthermore, this thesis reports the optimisation of LAMP from a biochemical perspective through the evaluation of its core reagents and the incorporation of enhancing agents to improve its specificity and sensitivity. In order to remove cold-chain storage from the diagnostic workflow, the optimised LAMP protocol was designed to be compatible with lyophilisation. Translation of LAMP to the POC demands the development of detection technologies that are compatible with the advantages offered by isothermal amplification. The use of simple, accessible and portable technologies is investigated in this thesis through the development of: (i) a novel colorimetricLAMP detection method for end-point and low cost detection, and (ii) the combination of LAMP with an electrochemical biosensing platform based on ion-sensitive field effect transistors (ISFETs) fabricated in unmodified complementary metal-oxide semiconductor (CMOS) technology for real-time detection. Lastly, current nucleic acid extraction methods are not transferable to be used outside the laboratory. Research of novel methods for low-cost and electricity-free sample preparation was carried out using cellulose matrices. A novel, rapid (under 10 min) and efficient nucleic acid extraction method from dried blood spots was developed. A sample-to-result POC test requires the implementation and integration of molecular biology, cutting-edge technology and data-driven approaches. The work presented in this thesis aims to set new benchmarks for the detection of infectious diseases at the POC by leveraging on developments in molecular biology and digital technologies.Open Acces

Improved Tools for Point-of-Care Nucleic Acid Amplification Testing

There is a critical need for improved diagnostic tools to detect infectious diseases, especially in low-resource regions. A sample-to-answer point-of-care nucleic acid amplification test (NAAT) would be incredibly valuable for many different applications (e.g. COVID-19, Chlamydia/Gonorrhoeae, Influenza, Ebola, Zika/Chikungunya/Dengue, etc.). However, sample preparation (purification of pure nucleic acids) is a challenging bottleneck. In Chapter 2, commercial NA extraction methods were studied and improved. In Chapter 3, commercial stocks of SARS-CoV-2 RNA used in FDA emergency-use authorizations were found to be inaccurate and were independently quantified using reverse transcription digital PCR. In Chapter 4, a 3D printed meter-mix device was developed for initial processing prior to the sample preparation device. In Chapter 5, a 3D printed sample-to-device interface was prototyped to facilitate loading multi-volume SlipChip devices with purified template mixed with LAMP reactants. In Chapters 6-7, advancements were made for image processing of commercial chips to study digital LAMP reactions. In Chapter 8, additional tools were developed towards sample-to-answer point-of-care NAAT including a sample preparation module, amplification module, cell-phone readout, and automated base station

Microfluidics for Molecular Measurements and Quantitative Distributable Diagnostics

A major challenge in global health care is a lack of portable and affordable quantitative diagnostic devices. This is because classic quantification of biomolecules is typically performed using kinetic assays that require strict control only found in controlled laboratory environments. By using the power of microfluidics, quantitative assays can be performed robustly in a "digital" format that is decoupled from precise kinetics through highly parallelized qualitative reactions. The benefits of performing quantitative assays in a digital format extend beyond just assay robustness to reduction of instrumental complexity, increase in quantitative precision, and an increase in the amount of information that can be gained from a single experiment. These microfluidic architectures, however, are not limited to usage in scenarios of quantification of biomolecules. These architectures can also potentially be extended to answering complex biological questions in single cells, such as determining the 3-dimensional organization of nuclear DNA and RNA

Facilitating Miniaturized Bioanalytical Assays in Microfluidic Devices

This work describes several efforts in making microfluidic lab-on-a-chip technology more convenient to use for bioanalysis in limited-resource settings (Chapters 2-3), and describes a device for miniaturized multistep process execution (Chapter 4). One underlying theme of these projects is the streamlining of the 'chip-to-world' interfacing to help bring this technology from specialized labs of its developers into more widespread utilization by potential users in other disciplines. Chapter 2 outlines a portable method for achieving stable fluid pumping for sample loading and flow control in microfluidic devices. Chapter 3 details a method for digital nucleic acid test readout with unmodified smartphone cameras. Chapter 4 demonstrates a lab-on-a-chip platform capable of carrying out complex multiplexed biochemical reactions requiring multiple sequential additions of reagents by performing RNA barcoding for multiplexed cDNA library generation.</p

Optimization of Continuous Flow Polymerase Chain Reaction with Microfluidic Reactors

The polymerase chain reaction (PCR) is an enzyme catalyzed technique, used to amplify the number of copies of a specific ~gion ofDNA. This technique can be used to identify, with high-probability, disease-causing viruses and/or bacteria, the identity of a deceased person, or a criminal suspect. Even though PCR has had a tremendous impact in clinical diagnostics, medical sciences and forensics, the technique presents several drawbacks. For example, the costs associated with each reaction are high and the reaction is prone to cont,amination due its inherent efficiency and high sensitivity. By employing microfluidic' systems to perform PCR these advantages can be circumvented. This thesis addresses implementation issues that adversely affect PCR . in microdevices and aims to improve the efficiency of the reaction by introducing novel materials and methods to existing protocols. Molecule-surface-interactions and ,' temperature control/determination are the main focus within this work. Microchannels and microreactors are char:acterized by extremely high surface-tovolume ratios. This dictates that surfaces play a dominant role in defining the efficiency ofPCR (and other synthetic processes) through increased molecule-surface interactions. In a multicomponent reaction system where the concentration of several components needs to be maintained the situation is particularly complicated. For example, inhibition of PCR is commonly observed due to polymerase adsorption on channel walls. Within??????? this work a number of different surface treatments have been investigated with a view to minimizing adsorption effects on microfluidic channels. In addition, novel studies introducing the use of superhydrophobic coatings on microfluidic channels are presented. Specifically superhydrophobic surfaces exhibiting contact angles in excess of 1500 have been created by growing Copper oxide and Zinc oxide' nanoneedles and silica-sol gel micropores on microfluidic channels. Such surfaces utilize additional surface roughness to promote hydrophobicity. Aqueous solutions in contact with superhydrophobic surfaces are suspended by bridging-type wetting, and therefore the fraction of the surface in contact with the aqueous layer is significantly lower than for a flat surface. An additional difficulty associated with PCR on microscale is the detennination and control of temperature. When perfonning PCR, the ability to accurately control system temperatures is especially important since both primer annealing to singlestranded DNA and the catalytic extension of this primer to fonn the complementary strand will only proceed in an efficient manner within relatively narrow temperature ranges. It is therefore imperative to be able to accurately monitor the temperature distributions in such microfluidic channels. In this thesis, fluorescence lifetime imaging (FLIM) is used as a novel method to directly quantify temperature within microchannel environments. The approach, which includes the use of multiphoton e'xcitation to achieve optical sectioning, allows for high spatial and temporal resolution, operates over a wide temperature range and can be used to rapidly quantify local temperatures with high precision.Imperial Users onl

Single-Cell Genomics of Uncultivated Marine Bacteria

Historically, the difficulty of obtaining pure cultures of abundant marine microbial plankton has an obstacle to reconstructing the underlying mechanisms of biogeochemistry in the ocean. While a number of dominant marine species from the ocean surface have been cultured, the dominant microbial plankton of the dark ocean proved far more difficult to tame. Genomic analyses of single cells emerged as a powerful means to expand knowledge of the diverse biochemical potential of these communities. Chapter 1 reviews the timeline of events in this field and summarizes current research with single-cell genomics and metagenomics within the framework of marine microbial ecology. The defining step in single-cell genomics approaches to environmental studies is the physical isolation of wild-type cells from heterogeneous microbial populations. In Chapter two I detail the construction and application of new instrumentation for optical trapping in conjunction with microfluidic devices (optofluidics) that allows for the selection of individual cells for genome amplification and sequencing. This approach has unique advantages for analyses of rare community members, cells with irregular morphologies, small quantity samples, and studies that employ advanced optical microscopy approaches to cell visualization.Fluorescence-activated cell sorting (FACS) approaches to single-cell genomics have reached full development and have been applied effectively to explore microbial diversity in the deep. In Chapter 3 I explore single amplified genomes obtained with FACS approaches, from several single-amplified genomes (SAGs) of the SAR202 clade, which has been shown to be ubiquitously abundant in the meso- and bathypelagic waters of the open ocean. Prior to this study the metabolism and geochemical role of the SAR202 clade was unknown, but their high abundance suggested they played an important role in nutrient cycling in the dark ocean. Due to their distinctive vertical profile, early accounts of the SAR202 clade speculated that they might be major mediators of recalcitrant organic carbon sequestration and turnover in the deep ocean, contributing to the "microbial carbon pump" through the conversion of labile carbon forms to more heterogeneous and refractory forms that could remain in the deep sea for thousands of years. I discovered that SAR202 encodes several families of oxidative enzymes and hypothesize that they are involved in the cycling of a major class of refractory deep-water marine dissolved organic matter (DOM), known as carboxyl-rich alicyclic matter, or CRAM. In Chapter 4 I revisit the optofluidic approach and describe its use to isolate single-amplified genomes (SAGS) from the marine environment. Several of these SAGs were shown to be representatives of groups of microbial plankton that are abundant in the ocean but not represented by genome sequences. In this chapter we evaluate the performance of this technique for single-cell genomics and outline the encoded metabolic features of three relatively-unstudied groups of marine microbes isolated using this technique. In Chapter 5, I outline potential areas of improvement for the optofluidic technology described in this thesis and discuss where the future of single-cell genomics technology

Evaluation of new diagnostic technologies for rapid detection of urinary pathogens and their antibiotic resistances

Background: Most urinary tract infections (UTIs) are trivial; but complicated UTIs are a growing reason for hospitalisation in the UK, and are among the commonest sources of sepsis. Increasing resistance among uropathogens complicates treatment and drives wider empirical use of previously-reserved antibiotics. Rapid precise detection of pathogens and resistances, without culture, might better guide early therapy in deteriorating UTI patients. Methods: Two approaches were evaluated: i) MALDI-TOF mass spectrometry for direct identification of pathogens from urine together with multiplex, tandem PCR (MT-PCR) for resistance gene profiling. MALDI-TOF was also explored for rapid detection of β-lactamase activity in bacteria harvested from urine; ii) MinION sequencing for bacterial and resistance gene identification, again directly from urine. As background, an epidemiological surveillance of uropathogens from the Norfolk and Norwich University Hospital in July and November 2014 was performed. Results: Direct MALDI-TOF on urines could achieve rapid bacterial identification within 1.5 h and also allowed direct detection of extended-spectrum β-lactamase (ESBL) activity. MT-PCR showed satisfactory results in detecting the commonest resistance genes in Enterobacteriaceae directly from urines and cultivated isolates within 3 h. Weaker association was found between streptomycin resistance and aadA1/A2/A3 genes. Fluoroquinolone-susceptible and -resistant Escherichia coli were distinguished by the melting temperatures of their gyrA product. MinION sequencing correctly identified uropathogens and their resistances in all urine samples within <5 h, without culture. Acquired resistance genes agreed with resistance phenotypes and closely matched Illumina sequencing, albeit with poor discrimination within some β-lactamase families (e.g. blaTEM). Epidemiological surveillance showed E. coli predominant in all age groups and location types, with high resistance rates to amoxicillin and trimethoprim. Conclusion: Either a MALDI-TOF plus PCR or a sequencing approach could significantly shorten the time required for microbiological investigation of urosepsis, allowing clinicians to adjust therapy before the second dose of a typical (i.e. q8h) antibiotic