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Approximately 14 million people die from infectious diseases annually, inflicting severe societal and economic burdens disproportionally affecting the Global South. The current standard for pathogen identification is blood culture which is too slow to guide the effective and targeted management of patients, and prevent the administration of broad-spectrum antibiotics. The analysis of microbial cell-free DNA (cfDNA), released from lysed pathogens in the infected human blood circulation, is an unbiased and sensitive way to detect infectious pathogens, including those that cannot be cultured such as viruses, fungi and protozoa. We have developed a cfDNA assay, named iSEP-SEQ, which combines a patented microfluidic system for cfDNA extraction, and real-time sequencing. The sample preparation module has been demonstrated on over 400 human samples and the iSEP-SEQ workflow is currently being piloted on clinical samples from septic patients to demonstrate its ability to identify pathogens in under six hours from blood draw.

Resistive Pulse Sensing (RPS) is a label-free and single-molecule detection approach that requires simple instrumentation to implement and as such, can be mobilized to be integrated into in vitro diagnostic assays for not only detecting but identifying key disease-associated biomarkers with high analytical sensitivity. Thus, RPS is a logical choice for coupling with liquid biopsy markers for the precision management of a variety of diseases due to the significant mass limits imposed on any assay in which a liquid biopsy marker is used. We have developed a unique measurement modality and sensor technology (dual in-plane nanopore sensor) that couples RPS to nanoscale electrophoresis, which has recently garnered attention due to unique molecular-dependent information it can provide. Not does it generate the typical RPS measurement parameters, but also the molecular-dependent electrophoretic mobility, which we call the time-of-flight (ToF). The RPS parameters coupled with the ToF and machine learning leads to high detection efficiency and classification accuracy of single molecules harvested from liquid biopsy markers, including nucleic acids and proteins. Our devices, which are made from plastics via high-scale production modalities (injection molding), consist of channels with dimensions ranging from 1 to 100 nm (effective diameter) that are 10’s of microns in length. In this talk, I will discuss the operational parameters and unique applications of our dual in-plane nanopore sensor for three compelling applications: (1) determining the fill status (empty versus full) of adeno-associated viruses (AAVs), which serve as carriers of gene therapy drugs; (2) peptide fingerprinting of single protein molecules; and (3) DNA/RNA single-molecule sequencing.

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As a person’s physiological regulation of biomarker molecules is challenged by acute illness, exposure to toxins or even surgery, the concentration these molecules can give important information about their health. For premature infants their regulation systems have yet to mature, so they can also suffer rapid changes in biomarker concentrations. Our view is that to monitor such biomarker changes effectively ideally requires moment-by-moment measurement of blood or tissue concentrations. The person acts as their own control allowing acute deterioration to be noticed quickly.

We have been developing a range of sensing and biosensing solutions for the invasive, minimally invasive, and non-invasive monitoring of people in healthcare situations. Microfluidics coupled to novel biosensors provide a valuable means of clinical sampling and robust quantification of measured biomarkers.

I will describe the key challenges in the development of such integrated microfluidic sensing devices and present our recent data from the neonatal intensive care unit amongst other projects.

In recent years, advanced in vitro pulmonary platforms have witnessed exciting developments pushing beyond traditional preclinical cell cultures at the air-liquid interface. Here, I will discuss ongoing developments that aim to deliver new generations of anatomically- and physiologically-inspired lung-on-chip platforms that coincide with the sprouting of human-relevant preclinical in vitro models. Motivated by translational endpoints. I will exemplify efforts covering inhalation-based inflammation and infection of the airways to novel strategies for improved aerosol delivery to the lungs. Lastly, we will explore the integration of induced pluripotent stem cell (iPSC) derived airway epithelial cells into lung-on-chips.

The basis of my talk is polymer microarray technology which has been developed in the Bradley group.
In my talk I will introduce polymer microarray technology and describe how this approach has been used in a number of applications – ranging from sensor optimisation/immobilisation to the provision of cellular substrates/scaffolds for a variety of screening applications. Specifically I will cover:

The use of polymer microarray technology to discover substrates with optimal binding and responsiveness of fluorescent reporters and their immobilisation onto the ends of optical fibres for measuring pH around tumours

The use of polymer microarray technology to discover polymers that bind cancer stem cells and control their differentiation) (with Tetsuya Tega, Stem Cells, 2016) with application in cancer screening.

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Getting the full picture of your rare extracellular vesicles (EVs) in cell culture or biofluid samples is a challenge – sometimes even if samples are purified. To most techniques, interference from lipoproteins, cell debris and protein aggregates can get in the way or make it hard to be confident that you're counting the right stuff. The task gets even harder when sample volumes are limited and the EV subpopulations you care about are rare. Leprechaun skips past troublesome background matrix effects by capturing EVs on its Luni consumable to analyze particle size, concentration, and phenotype for exactly the EV particles you care about. With sensitivity down to 5x10^5 particles/mL, analysis down to 35 nm, single particle phenotypic analysis and now the ability to quantify the proportion of an EV subpopulation out of the total EV concentration, Leprechaun is ready to help you paint the whole picture of your EV sample no matter how complex.

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Technological developments in microfluidics have greatly contributed to the field of liquid biopsy especially in oncology. The presentation will first cover early development of droplet-based digital PCR for the detection of rare mutations and methylation markers. It will then present pertinent clinical applications of ddPCR for cancer patients follow-up.

Cancer-related deaths are mainly caused by metastatic spread of tumor cells from the primary lesion to distant sites via the blood circulation. Understanding the mechanisms of blood-borne tumor cell dissemination by the detection and molecular characterization of circulating tumor cells (CTCs) in the blood of patients with cancer has opened a new era in cancer research. However, blood is known to be a hostile environment for CTCs. Although the primary tumor presumably sheds thousands of cells into the bloodstream every day, only a very small percentage of these cells survive in the bloodstream and become detectable as CTCs in a blood sample. Within the immunological synapse, a multitude of inhibitory receptors have been identified. Programmed cell death protein-1 (PD-1) and its ligand, PD-L1, have been one of the most prominent examples to antagonize immune escape mechanisms employed by tumor cells.

In my talk, I will define the new concept of Liquid Biopsy as well as discuss about (i) CTCPD-L1(+) plus extracellular vesicles expressing PD-L1 as important biomarkers in liquid biopsy in breast and non-small cell lung cancers as well as (ii)metastasis-competent CTCs from colon and breast cancers to discover new targetable immune checkpoint inhibitors. Indeed, these more aggressive and selected clones of CTCs have the capacity to initiate secondary tumors in distant organs; interestingly, they are not expressing PD-L1 but survived the constant immune attacks.

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Sensitive and accurate detection of microRNA panels is critical to enable their use as disease biomarkers. I will introduce the Digiplex technology, a microRNA profiling method that combines the multiplexing power of a DNA-grafted suspension array with the accuracy of a digital readout and the simplicity flow cytometry. In this approach, fluorescently encoded DNA-grafted particle populations are used to capture the microRNAs, following a Poisson distribution. These particles are then isolated in microfluidic droplets, where single captured miRNA molecules trigger an isothermal exponential amplification, activating a fluorescent probe on the particle surface. Flow cytometry analysis yields the ratio of positive and negative particles for each target, enabling the rapid reconstruction of multidimensional concentration profiles. We applied Digiplex to total RNA extracts, successfully developing a 10-plex assay with femtomolar sensitivity.

Cell-free DNA (cfDNA) in human blood plasma is a valuable biomarker for various physiological and pathological conditions. Specific genetic and epigenetic alterations associated with diseases can be detected in the blood, offering a non-invasive method for monitoring at-risk patients and assessing therapy efficacy. While high-sensitivity PCR-based technologies have demonstrated proof-of-principle, the low concentration of cfDNA complicates analytical routines, especially when rapid results are needed. Additionally, these technologies often require knowledge of the disease's genetic background, increasing the risk of false-negative diagnostics. We have developed the µLAS technology for high-sensitivity DNA sizing in just 25 minutes. µLAS utilizes electrohydrodynamic separation and concentration of DNA in funnel-like microfluidic channels. Unlike competing assays, µLAS operates effectively in plasma with high salt concentrations, enabling the collection of cfDNA biomarkers with excellent selectivity, independent of prior genetic knowledge. In this talk, we will present the µLAS technology and discuss our findings from clinical applications.

Circulating cell-free nucleic acids in bodily fluids (such as blood or interstitial skin fluid) hold great promise as clinically useful molecular biomarkers for a broad range of pathologies including most cancer types and adverse pregnancy outcomes. Their diagnostic or predictive value make them particularly attractive for the development of new, minimally invasive tests that could be easily implemented into primary care to improve patient triage and quality of care. However, the development of such tests has been hampered by technical challenges, mostly associated with the way these biomarkers are sampled and then detected. The lack of standardized protocols (for both sampling and detection) is indeed a major source of error and variation between studies and is responsible for many discrepancies in the literature. The low natural abundance of these biomarkers in liquid biopsies and their high sequence homology make them particularly challenging to detect with suitable levels of sensitivity and specificity. Most detection methods require heaving sample pre-processing (an often cumbersome and multistep process) and target or signal amplification. These processes are prone to contamination and error and generally low throughput, which is a major issue for the discovery and clinically validation of these biomarker in large clinical studies. The technologies involved are also not easily amenable to miniaturisation, as would be required for future implementation in real-life settings. Here, we report on the development of innovative and minimally invasive screening tests for the early diagnosis of skin cancer and the early stratification of women at risk of preterm birth. These tests have been designed so that can be easily implemented into clinical practice, through the NHS, to significantly improve patient stratification.

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