Ultrafast Microfluidic Immunoassays Towards Real-time Intervention of Cytokine Storms

Biomarker-guided precision medicine holds great promise to provide personalized therapy with a good understanding of the molecular or cellular data of an individual patient. However, implementing this approach in critical care uniquely faces enormous challenges as it requires obtaining “real-time” data with high sensitivity, reliability, and multiplex capacity near the patient’s bedside in the quickly evolving illness. Current immunodiagnostic platforms generally compromise assay sensitivity and specificity for speed or face significantly increased complexity and cost for highly multiplexed detection with low sample volume. This thesis introduces two novel ultrafast immunoassay platforms: one is a machine learning-based digital molecular counting assay, and the other is a label-free nano-plasmonic sensor integrated with an electrokinetic mixer. Both of them incorporate microfluidic approaches to pave the way for near-real-time interventions of cytokine storms. In the first part of the thesis, we present an innovative concept and the theoretical study that enables ultrafast measurement of multiple protein biomarkers (<1 min assay incubation) with comparable sensitivity to the gold standard ELISA method. The approach, which we term “pre-equilibrium digital enzyme-linked immunosorbent assay” (PEdELISA) incorporates the single-molecular counting of proteins at the early, pre-equilibrium state to achieve the combination of high speed and sensitivity. We experimentally demonstrated the assay’s application in near-real-time monitoring of patients receiving chimeric antigen receptor (CAR) T-cell therapy and for longitudinal serum cytokine measurements in a mouse sepsis model. In the second part, we report the further development of a machine learning-based PEdELISA microarray data analysis approach with a significantly extended multiplex capacity using the spatial-spectral microfluidic encoding technique. This unique approach, together with a convolutional neural network-based image analysis algorithm, remarkably reduced errors faced by the highly multiplexed digital immunoassay at low analyte concentrations. As a result, we demonstrated the longitudinal data collection of 14 serum cytokines in human patients receiving CAR-T cell therapy at concentrations < 10pg/mL with a sample volume < 10 µL and 5-min assay incubation. In the third part, we demonstrate the clinical application of a machine learning-based digital protein microarray platform for rapid multiplex quantification of cytokines from critically ill COVID-19 patients admitted to the intensive care unit. The platform comprises two low-cost modules: (i) a semi-automated fluidic dispensing module that can be operated inside a biosafety cabinet to minimize the exposure of technician to the virus infection and (ii) a compact fluorescence optical scanner for the potential near-bedside readout. The automated system has achieved high interassay precision (~10% CV) with high sensitivity (<0.4pg/mL). Our data revealed large subject-to-subject variability in patient responses to anti-inflammatory treatment for COVID-19, reaffirming the need for a personalized strategy guided by rapid cytokine assays. Lastly, an AC electroosmosis-enhanced localized surface plasmon resonance (ACE-LSPR) biosensing device was presented for rapid analysis of cytokine IL-1β among sepsis patients. The ACE-LSPR device is constructed using both bottom-up and top-down sensor fabrication methods, allowing the seamless integration of antibody-conjugated gold nanorod (AuNR) biosensor arrays with microelectrodes on the same microfluidic platform. Applying an AC voltage to microelectrodes while scanning the scattering light intensity variation of the AuNR biosensors results in significantly enhanced biosensing performance. The technologies developed have enabled new capabilities with broad application to advance precision medicine of life-threatening acute illnesses in critical care, which potentially will allow the clinical team to make individualized treatment decisions based on a set of time-resolved biomarker signatures.PHDMechanical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/163129/1/yujing_1.pd

Polymer Micro- and Nanofluidic Systems for In Vitro Diagnostics: Analyzing Single Cells and Molecules

Polymer micro- and nanofluidic systems, with their critical dimensions, offer a potential to outperform conventional analysis techniques and diagnostic methods by enhancing speed, accuracy, sensitivity and specificity. In this work, applications of microfluidics have been demonstrated to address the existing challenges in stroke diagnosis, by mRNA expression profiling from whole blood within \u3c20 min. A brief overview of various biomarkers for stroke diagnosis is given in chapter 1 followed by design and testing of individual microfluidic modules (chapter 2 and 3) required for the development of POC diagnostic strategy for stroke. We have designed and evaluated the performance of polymer microfluidic devices for the isolation of leukocyte subsets, known for their differential gene expression in the event of stroke. Target cells (T-cells and neutrophils) were selected from with greater purities, from 50 µl whole human blood by using affinity based capture in COC devices within a 6.6 min processing time. In addition, we have also demonstrated the ability to isolate and purify total RNA by using UV activated polycarbonate solid phase extraction platform. Polymer-based nanofluidic devices were used to study the effects of surface charge on the electrodynamic transport dynamics of target molecules. In this work, we report the fabrication of mixed-scale micro- and nanofluidic networks in poly(methylmethacrylate), PMMA, using thermal nanoimprint lithography using a resin stamp and surface modification of polymer nanoslits and nanochannels for the assessment of the associated electrokinetic parameters – surface charge density, zeta potential and electroosmotic flow. This study provided information on possible routes that can be adopted to engineer proper wall chemistry of polymer nanochannels for the enhancement or reduction of solute/wall interactions in a variety of relevant single-molecule studies

2018 Annual Research Symposium Abstract Book

2018 annual volume of abstracts for science research projects conducted by students at Trinity College

Ecosystem on a Chip: Understanding communication between plant and fungus

In jeder Symbiose ist das Verhältnis der zwei beteiligten Spezien geprägt vom Austausch und der Erkennung von Signalen. In parasitären Beziehungen, konzentriert sich die angreifende Spezies auf das Vermeiden ihrer Erkennung, während die Wirts-Spezies kontinuierlich ihre Möglichkeiten verbessert, Eindringlinge zu entdecken. Änderungen im globalen Klima ändern das Gleichgewicht, in dem sich Spezien befinden, indem sie den Wirt erheblich schwächen oder den Pathogenen ermöglichen, in neue Gebiete vorzudringen und naive Wirte zu treffen, die ihren Angriffen gegenüber anfällig sind. Um die existenzbedrohenden Ernteverluste zu reduzieren, die von Pathogenen verursacht werden, setzen Agrarwirte chemischen Pflanzenschutz ein. Dieser kann, bei exzessivem Gebrauch, den Landwirt finanziell und dem Ökosystem durch Nebenwirkungen schaden. Alternativ könnten immunoaktive Substanzen verwendet werden, um die inhärenten Verteidigungsfähigkeiten von Pflanzen zu stärken, indem man das „Defence Priming“ der Pflanzen nutzt. Hier verursacht ein vorhergehender Stress Stimulus, dass die Verteidigungsreaktion einer Pflanze schneller und stärker ausfällt, sollte ein Pathogen angreifen. So wird die Interaktion zwischen Pathogen und Wirtspflanze zu Gunsten der Pflanze manipuliert. In dieser Arbeit wurde der Wirkungsmechanismus von pilzlichen Metaboliten von Roesleria subterranea untersucht, die von unseren Projektpartnern am ibwf, Mainz als bioaktiv identifiziert wurden. Zusätzlich wollten wir Methoden etablieren, Mikrofluidische BioReaktoren (MBR) zu nutzen, um die Verteidigungsreaktion von Pflanzenzellen zu quantifizieren, die in ihnen kultiviert werden. Es sollten die Konzentrationsmessung von Reaktiven Oxygen Spezien (ROS) und eine Beobachtung des extrazellulären pHs getestet werden. Wir haben das etablierte Design des MBR geändert und getestet, um pilzliche Zellen darin zu beherbergen, um sie in möglichen Co-Kultivierungs Experimenten unter kontinuierlicher Beobachtung zu verwenden. Unter den R. subterranea Metaboliten konnten wir das AcetonAduct von Entatreventinon (AaE) als möglichen Effektor identifizieren, der in der Lage zu sein scheint, die Synthese von Phytoalexinen von dem Programmed Cell Death (PCD) einer erfolgreichen Immunreaktion der Pflanze zu trennen. Mit den MBR wurden erfolgreiche Experimente durchgeführt, in denen wir den pH kontinuierlich messen konnten und konnten signifikante Unterschiede in der H2O2 Konzentration im ausfließenden Medium des MBRs messen. Das Kultivieren von Pilz-Zellen im MBR war nur für kurze Zeiträume möglich, in denen wir jedoch ein erfolgreiches Co-Kultivierungsexperiment durchführen konnten. Zusammenfassend, konnte diese Arbeit das Potential demonstrieren, MBRs zu nutzen, um die Parameter einer pflanzlichen Immunreaktion während des Kultivierens auszulesen. Es wurden mögliche Verbesserungenmöglichkeiten im System erkannt, um die Methoden zu vereinfachen und zu beschleunigen. Zusätzlich wurde AaE als möglicher Effektor erkannt, der in der Lage zu sein scheint, die Synthese von Phytoalexinen von der Induktion des PCD zu trennen, der fester Bestandteil einer erfolgreichen Verteidigung der Wirtspflanze ist

Microfluidic devices applied on enriching post –translational modified proteins for proteomics

In this work, microfluidic devices were developed for enriching post-translational modified proteins. Post-translational modifications (PTM) of proteins play essential roles in cellular physiology and disease. The identification of protein substrates and detection of modification site helps understand PTM-mediated regulation in essential biological pathways and functions in various diseases. However, PTM proteins are typically present only at trace levels, making them difficult to identify in mass spectrometry based proteomics. This work study is about the design, fabrication and testing of the microfluidic device for the enrichment of abundant amount of PTMs. Carbonylated protein is used as a representative PTM to illustrate the wide application of this method for any PTMs converted into a tractable tag after derivatization. The surface topography, surface functional group mapping and elemental composition changes after each modification step of the treatment process were systematically measured qualitatively and quantitatively. Quantitative study of capture efficiency and elution efficiency of the device was also studied. Furthermore, there are also ideas that this proteome enrichment device can be assembled with other lab-on-a-chip components for follow-up protein analysis. For example, coupling with mass spectrometry will allow automatic low-volume fraction deposition on mass spectrometry. As a part of the microfluidic device designing, this work also aims at optimizing the operating parameters and geometric parameters of microfluidic devices with microscale posts. The operating parameters studied are Reynolds number, Peclet number, Damköhler number, and equilibrium reaction constant. These parameters encompass the influence of velocity, diffusivity, density, viscosity, hydraulic diameter, inlet concentration of species and forward and backward reaction constants. This work theoretically analyzes the influence of the above mentioned operating parameters using finite element analysis software COMSOL Multiphysics 4.2.a. The results of this study would improve the design of microfluidic devices used for chemical reactions as well as that used for protein enrichment

Focal Spot, Winter 2009/2010

https://digitalcommons.wustl.edu/focal_spot_archives/1113/thumbnail.jp

Development towards a point-of-care system to monitor pregnancy and fertility biomarkers

The aim of this thesis was to develop a point of care (POC) device that could monitor progesterone and oestriol in saliva. These hormones have a key role to play in both female fertility and pregnancy. Understanding the concentration of progesterone in the body is key to understanding a patient’s fertility and pregnancy status. Combining progesterone and oestriol detection can give valuable insight into when labour may commence during pregnancy. This can be achieved by measuring the progesterone to oestriol ratio in saliva samples, throughout the pregnancy progesterone is the more dominant hormone but a few weeks before labour oestriol becomes the more dominant hormone. Saliva was chosen as the biological sample due to the ease of collection as well as it providing a better chemical understanding of the active hormonal concentrations compared to blood. Typical levels in saliva are 100 ng mL⁻¹ for progesterone and 0 - 5 ng mL⁻¹ for oestriol.The work presented began with the design of a chemiluminescence immunoassay which could be translated onto a microfluidic device, this approach would provide both good sensitivity and selectivity. Chemiluminescence was chosen as a detection system due to the high sensitivities that can be achieved with simple instrumentation where a CCD camera could be used to also obtain spatial information. The CL assay involved the chemical immobilisation of the antibodies onto glass slides. Silanisation with (3-aminopropyl)triethoxysilane (APTES) in combination with a N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide/N-hydroxysulfosuccinimide (EDC/sulfo-NHS) linker proved the most successful immobilisation method, with a LOD of 33 ±3 pg mL⁻¹ being achieved for progesterone in 10 mM phosphate buffered saline (PBS). This method however lacked reproducibility and did not transfer well on to polymer substrates or the microfluidic devices due to problems with the antibody immobilisation procedure. Immobilisation of anti-progesterone was then investigated on a range of electrode surfaces (Au, glassy carbon and ITO). This immobilisation procedure involved electrochemically depositing nitrobenzene onto the electrode surface followed by an electrochemical reduction of the nitro groups to the corresponding amine. To allow electrochemical detection ferrocene was tagged to the anti-progesterone antibodies to give a redox tag. The antibodies were then immobilised through an EDC/sulfo-NHS linkage. This method proved to be successful and very reproducible. By tagging ferrocene onto the antibody a rigid structure was achieved during the immobilisation procedure allowing the ideal antibody orientation, this process also allowed quantification of the concentration of antibodies on the surface (4.46 x10⁻⁷ mol m⁻²). Electrochemical based immunoassays were successfully carried with a 15 min incubation time for progesterone giving LODs of 1 pg mL⁻¹ for the gold and glassy carbon and 0.1 pg mL⁻¹ for the ITO. The ITO performed better than the other materials due to the electrode being uniformly flat enabling more efficient surface modifications. The methodology was also translated for use with artificial saliva with a LOD of 1.7 pg mL⁻¹ for progesterone.Once the electrochemical immobilisation platform had been shown to be successful this was taken forward as a potential route to carry out a CL immunoassay. This novel approach utilised the oxidised ferrocene tag on the antibody as the catalyst for the luminol CL reaction. A static system was devised in which the antibodies had been immobilised on to ITO using the electrochemical approach and the CL reagents added by pipette, LODs of 2.35 and 2.54 pg mL⁻¹ were obtained for progesterone and oestriol respectively in saliva after a 30 min incubation time. The static system could therefore be used as a POC device for these hormones meeting the aims of this thesis. The next step was then to start developing a more automated method within a flow cell. Initially the ITO electrode with the pre-prepared immobilised antibody and the oxidised ferrocene tag was incorporated into a macrofluidic device. The CL immunoassay was successfully carried out in the macrofluidic device although the LODs were an order of magnitude higher than those seen from the static system for both progesterone and oestriol in saliva due to the large volume of the flow cell. Finally the ITO electrode with pre-prepared immobilised antibody with the oxidised ferrocene tag was slotted into the microfluidic device that had been designed and measurement were made. There was however problems with the design and possible new designs are discussed

Proceedings of Abstracts, School of Physics, Engineering and Computer Science Research Conference 2022

© 2022 The Author(s). This is an open-access work distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. For further details please see https://creativecommons.org/licenses/by/4.0/. Plenary by Prof. Timothy Foat, ‘Indoor dispersion at Dstl and its recent application to COVID-19 transmission’ is © Crown copyright (2022), Dstl. This material is licensed under the terms of the Open Government Licence except where otherwise stated. To view this licence, visit http://www.nationalarchives.gov.uk/doc/open-government-licence/version/3 or write to the Information Policy Team, The National Archives, Kew, London TW9 4DU, or email: [email protected] present proceedings record the abstracts submitted and accepted for presentation at SPECS 2022, the second edition of the School of Physics, Engineering and Computer Science Research Conference that took place online, the 12th April 2022

Root-TRAPR: a modular plant growth device to visualize root development and monitor growth parameters, as applied to an elicitor response of Cannabis sativa

Background Plant growth devices, for example, rhizoponics, rhizoboxes, and ecosystem fabrication (EcoFAB), have been developed to facilitate studies of plant root morphology and plant-microbe interactions in controlled laboratory settings. However, several of these designs are suitable only for studying small model plants such as Arabidopsis thaliana and Brachypodium distachyon and therefore require modification to be extended to larger plant species like crop plants. In addition, specific tools and technical skills needed for fabricating these devices may not be available to researchers. Hence, this study aimed to establish an alternative protocol to generate a larger, modular and reusable plant growth device based on different available resources. Results Root-TRAPR (Root-Transparent, Reusable, Affordable three-dimensional Printed Rhizo-hydroponic) system was successfully developed. It consists of two main parts, an internal root growth chamber and an external structural frame. The internal root growth chamber comprises a polydimethylsiloxane (PDMS) gasket, microscope slide and acrylic sheet, while the external frame is printed from a three-dimensional (3D) printer and secured with nylon screws. To test the efficiency and applicability of the system, industrial hemp (Cannabis sativa) was grown with or without exposure to chitosan, a well-known plant elicitor used for stimulating plant defense. Plant root morphology was detected in the system, and plant tissues were easily collected and processed to examine plant biological responses. Upon chitosan treatment, chitinase and peroxidase activities increased in root tissues (1.7- and 2.3-fold, respectively) and exudates (7.2- and 21.6-fold, respectively). In addition, root to shoot ratio of phytohormone contents were increased in response to chitosan. Within 2 weeks of observation, hemp plants exhibited dwarf growth in the Root-TRAPR system, easing plant handling and allowing increased replication under limited growing space. Conclusion The Root-TRAPR system facilitates the exploration of root morphology and root exudate of C. sativa under controlled conditions and at a smaller scale. The device is easy to fabricate and applicable for investigating plant responses toward elicitor challenge. In addition, this fabrication protocol is adaptable to study other plants and can be applied to investigate plant physiology in different biological contexts, such as plant responses against biotic and abiotic stresses