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

Combined physical and oxidative stability of food Pickering emulsions

Many food products contain lipid droplets dispersed in an aqueous phase (e.g., milk, mayonnaise), thus are oil-in-water (O/W) emulsions. Food emulsions may be subjected to destabilization, both from a physical and a chemical perspective. Physical destabilization is generally prevented by the use of conventional emulsifiers such as surfactants and proteins. Chemical destabilization, in particular lipid oxidation, is a major concern in food products, especially when healthy polyunsaturated fatty acids are present, and this degradation is usually mitigated by the use of synthetic antioxidants, often in large amounts. The use of alternative ingredients for the formulation of food emulsions has been emerging, for example solid particles (so-called Pickering particles, that are very popular nowadays) that irreversibly adsorb to the interface and therewith provide high physical stability; or natural antioxidants such as tocopherols and rosemary extracts, which are attractive in the current clean-label trend to prevent lipid oxidation. The efficiency of these natural antioxidants is unfortunately often not optimal, which can be explained by their tendency to locate into the oil or water phase, whereas lipid oxidation is initiated at the oil-water interface, and thus is the place where antioxidants should be located to optimally exert their protective effect. The objective of this project was to develop food emulsions with a new and controlled architecture directed at yielding both excellent physical and oxidative stability. In these emulsions the oil droplets were covered by food-grade Pickering particles that exert a double role: they act as physical stabilizers, and as a reservoir for antioxidant molecules located close to the oil-water interface, therewith preventing the first lipid oxidation events, which is expected to drastically enhance antioxidant activity. The first part of this thesis focused on the preparation and characterization of a new food-grade lipid-based Pickering particles, referred to as colloidal lipid particles (CLPs). We prepared both surfactant-covered and protein-covered CLPs, and found that the type of emulsifier largely determined their morphology: protein-covered CLPs were roughly spherical, whereas surfactant-covered CLPs looked more lath-like (Chapters 3 and 6). We also showed that the lipid material alters the crystal polymorphism and subsequent CLP structure, which consequently influenced their performance as emulsion stabilizers (Chapter 3). For instance, surfactant-covered CLPs containing only high melting point lipids showed highly ordered crystalline structures, and formed jammed, cohesive interfacial layers once adsorbed onto oil droplets, whereas the ones containing a fraction of low melting point lipids showed less ordered crystalline structures and formed thin and bridged layers. Since protein-covered CLPs were particularly resilient to subsequent emulsification processes, these particles were used to study the formation of emulsion droplets in a microfluidic device and their stability to short-term coalescence (Chapter 4). We found a non-monotonic dependency of the droplet stability on the particle concentration: at low surface coverage, CLPs had a destabilizing effect as incompletely covered surfaces led to droplet-droplet bridging and subsequent coalescence, whereas at higher surface coverage, particles formed an effective barrier against droplet coalescence, resulting in physically stable emulsions over the time scales probed. As a next step, we investigated lipid oxidation in Pickering emulsions stabilized by protein-based CLPs that did not contain antioxidants (Chapter 5). We showed that these Pickering emulsions had a similar oxidative stability as conventional protein-stabilized emulsions for a similar composition of the oil droplets. Yet, when in both emulsions the same amount of solid lipids was present (either as stabilizing CLPs, or within the oil droplet core), a Pickering emulsion had a higher physicochemical stability. This shows that the location of crystallizable lipids influences lipid oxidation in O/W emulsions, and thus needs to be carefully considered in emulsion design. CLPs that did contain the lipophilic antioxidant &alpha;-tocopherol are presented in Chapter 6. The chemical stability of &alpha;-tocopherol was negatively influenced by lipid crystallization that probably promoted the localization of &alpha;-tocopherol close to the particle surface, which was further enhanced by emulsifiers that actively induce lipid crystallization. When applied as Pickering stabilizers in O/W emulsions (Chapter 7), lipid oxidation was reduced compared to control emulsions with the same composition and structure, but where the antioxidant was present in the core of the oil droplets. This confirmed that the interfacial localization of the antioxidant is crucial to prevent lipid oxidation in emulsions, and that the two main instability issues (i.e., physical and chemical instability) of emulsions can be mitigated through one single approach. After establishing the proof of concept with the CLPs, we used biobased particles (that may contain antioxidants) from various natural sources to stabilize O/W emulsions (Chapter 8). Emulsions stabilized by matcha tea powder or spinach leaf powder were both highly physically and oxidatively stable, which shows that the double functionality that we achieved using purposely built particles (CLPs) can also be achieved with naturally occurring particles. In the general discussion of the thesis (Chapter 9) we describe that the dual functionality of CLPs can also be reached using other food components, which makes this approach a generic one. We expect that the system could be further improved, for example, by increasing the residence time of antioxidants at the interface. To do so, we probably need to link the time scale at which the relevant oxidation events occur with those during which the antioxidant actually resides at the interface. Follow-up research on entrapment of antioxidants within particles is needed to reach long residence times at the interface while not compromising the ability of antioxidants to exert their chemical activity. To conclude: through our approach the highly-stable food emulsions of the future may come within reach

Biophysical and Biochemical Screening Approaches for Antimicrobial Drug Discovery Targeting S. aureus ClpP

The discovery of antibacterial drugs has been among most significant achievements of mankind in saving millions of lives across the planet from infectious diseases. With rise in resistance to almost all existing chemotypes, the design of next generation novel antibiotics has become much more challenging and difficult. The early 21st century witnessed the advancement of multiple novel chemotypes during golden age of antibiotics however the pace of antibiotic drug discovery has slowed down tremendously, contributing to life threatening antimicrobial discovery void since 1980’s. Therefore the need to develop novel antibiotics with unique mechanism of action to leverage against multi drug resistance pathogens, is paramount. In this direction the Caseinolytic Protease P (ClpP) is an emerging drug discovery target with significant potential for treatment of recalcitrant biofilm forming infections from pathogens such as Methicillin-resistant Staphylococcus aureus (MRSA) This dissertation highlights the ongoing efforts to facilitate the discovery of novel non peptidic ClpP activator compounds and improvement of pharmacological profile of existing ClpP targeting Acyldepsipeptides (ADEPs) series antibiotics. The chapter one discusses the history and synopsis of conventional antibiotics drug discovery screening approaches, and transitions to modern era structure or fragment based screening approaches. The merits and challenges of such approaches of targeting a well conserved bacterial protease (ClpP) are discussed along with dissertation aims toward development of biophysical and biochemical screening approaches. Chapter two discusses optimization of thermal shift assay as primary screening assay for ClpP and its utility toward screening of fragment collections and buffer conditions. Chapter three discussed the development of a site specific Fluorescence Polarization based FP probe based on ADEP scaffold and its utility as a robust high throughput capable primary screening assay for screening of diverse collections ranging from bioactives to fragments. Chapter four discusses development of a label free Surface Plasmon Resonance (SPR) based assay geared toward screening of fragment as well as in house small and large (ADEP analogs) series compounds in addition to determining full kinetics for lead prioritization. Chapter five discusses the results of multiple screening campaigns utilizing combination of above assays to generate multiple hits with superior ligand efficiency and chemical tractability. Chapter six concludes with analysis of the best of compounds among individual series or from screening campaigns and highlights effectiveness of above screening assays toward hit exploration along with outlook on anticipated challenges and future directions

Reconfigurable multi-carrier transmitters and their application in next generation optical networks

With the advent of new series of Internet services and applications, future networks will have to go beyond basic Internet connectivity and encompass diverse services including connected sensors, smart devices, vehicles, and homes. Today’s telecommunication systems are static, with pre-provisioned links requiring an expensive and time-consuming reconfiguration process. Hence, future networks need to be flexible and programmable, allowing for resources to be directed, where the demand exists, thus improving network efficiency. A cost-effective solution is to utilise the legacy fibre infrastructure more efficiently, by reducing the size of the guard bands and allowing closer optical carrier spacing, thereby increasing the overall spectral efficiency. However, such a scheme imposes stringent transmitter requirements such as frequency stability, which would not be met with the incumbent laser-array based transmitters. An attractive alternative would be to employ an optical frequency comb (OFC), which generates multiple phase correlated carriers with precise frequency separation. The reconfigurability of such a multi-carrier transmitter would enable tuning of channel spacing, number of carriers and emission wavelengths, according to the dynamic network demands. This research thesis presents the work carried out, in the physical layer, towards realising reconfigurability of an optical multi-carrier transmitter system. The work focuses on an externally injected gain-switched laser-based OFC (EI-GSL), which is a particular type of multi-carrier source. Apart from the detailed characterisation of GSL OFCs, advances to the state of the art are achieved via comb expansion, investigating new demultiplexing methods and system implementations. Firstly, two novel broadband GS-OFC generation techniques are proposed and experimentally demonstrated. Subsequently, two flexible and compact demultiplexing solutions, based on micro-ring resonators and laser based active demultiplexers are investigated. Finally, the application of a reconfigurable multi-carrier transmitter, employed in access and data centre networks, as well as analog-radio over fibre (A-RoF) distribution systems, is experimentally demonstrated

Formal specification of requirements for analytical redundancy-based fault -tolerant flight control systems

Flight control systems are undergoing a rapid process of automation. The use of Fly-By-Wire digital flight control systems in commercial aviation (Airbus 320 and Boeing FBW-B777) is a clear sign of this trend. The increased automation goes in parallel with an increased complexity of flight control systems with obvious consequences on reliability and safety. Flight control systems must meet strict fault-tolerance requirements. The standard solution to achieving fault tolerance capability relies on multi-string architectures. On the other hand, multi-string architectures further increase the complexity of the system inducing a reduction of overall reliability.;In the past two decades a variety of techniques based on analytical redundancy have been suggested for fault diagnosis purposes. While research on analytical redundancy has obtained desirable results, a design methodology involving requirements specification and feasibility analysis of analytical redundancy based fault tolerant flight control systems is missing.;The main objective of this research work is to describe within a formal framework the implications of adopting analytical redundancy as a basis to achieve fault tolerance. The research activity involves analysis of the analytical redundancy approach, analysis of flight control system informal requirements, and re-engineering (modeling and specification) of the fault tolerance requirements. The USAF military specification MIL-F-9490D and supporting documents are adopted as source for the flight control informal requirements. The De Havilland DHC-2 general aviation aircraft equipped with standard autopilot control functions is adopted as pilot application. Relational algebra is adopted as formal framework for the specification of the requirements.;The detailed analysis and formalization of the requirements resulted in a better definition of the fault tolerance problem in the framework of analytical redundancy. Fault tolerance requirements and related certification procedures turned out to be considerably more demanding than those typically adopted in the literature. Furthermore, the research work brought up to light important issues in all fields involved in the specification process, namely flight control system requirements, analytical redundancy, and requirements engineering

Establishing a Mouse Model of Glioblastoma (GBM) And Developing Strategies to Eliminate Cancer Cells.

As the tenth leading cause of cancer death in the United States with a 6.8% five-year survival rate, glioblastoma multiforme (GBM) is a very deadly disease with a poor prognosis. Therefore, a curative treatment is in high demand. Among the widely used anti-metabolites (Chemotherapeutic agents, CAs), each compound approved for human use by the Food and Drug Administration (FDA) has side effects. Three main phenomena contribute to the side effects and the reappearance of cancer: (a) the cancer cells become resistant to chemo- and radiation therapy; (b) this chemo-resistance prompts the use of higher doses of the chemotherapeutic agents; and (c) the higher doses of the CAs also kills the proliferating immune cells, thereby causing immune-suppression and associated infections that often kill the patients. Amid such mixed success, a latent question has remained, “Could we invent an effective but simple strategy of turning at least some of the chemotherapeutic agents into side effect-free anti-metabolites that could bring about long-term cancer remission?” Our earlier publications targeted curcumin (CC) to antibodies, an approach that killed melanoma and glioblastoma brain cancer cells. In the current study, we developed methods for brain cancer cell implantation, immunohistochemistry of the tumor tissue, and tumor-cell dispersion for flow cytometry to help study curcumin-mediated repolarization of TAMs and recruitment of NK cells to eliminate GBM. Additionally, we attempted to develop CA-CC adducts to achieve repolarization of TAMs and eliminate chemoresistance in the GBM cells

ClpX interactions with ClpP, SspB, protein substrate and nucleotide

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, February 2006.Includes bibliographical references.ClpXP and related ATP-dependent proteases are implements of cytosolic protein destruction. They couple chemical energy, derived from ATP hydrolysis, to the selection, unfolding, and degradation of protein substrates with the appropriate degradation signals. The ClpX component of ClpXP is a hexameric enzyme that recognizes protein substrates and unfolds them in an ATP-dependent reaction. Following unfolding, ClpX translocates the unfolded substrate into the ClpP peptidase for degradation. The best characterized degradation signal is the ssrA-degradation tag, which contains a binding site for ClpX and an adjacent binding site for the SspB adaptor protein. I show that the close proximity of these binding elements causes SspB binding to mask signals needed for ssrA-tag recognition by ClpX. The SspB dimer overcomes this signal masking by tethering itself and bound substrate to ClpX, via docking sites located in the dimeric N-terminal domain of ClpX. Because this N-domain dimer binds only a single SspB subunit, the ClpX hexamer can accommodate just one SspB dimer per hexamer. Other adaptor proteins that use these same tethering sites must compete with SspB for access to ClpXP. Substrates bearing ssrA tags with increased spacing between the SspB and ClpX binding elements are degraded more efficiently at low concentrations by ClpXP.(cont.) This mechanism in which the adaptor first obstructs and then stimulates substrate recognition may have evolved to permit an additional level of regulation of substrate choice. SspB binding to ssrA-tagged substrate is a highly dynamic process, allowing rapid transfer of substrates from SspB to ClpX. Although the ClpX hexamer is composed of six identical polypeptides, individual subunits assume at least three distinct conformations. Using a hexamer that was engineered to prevent nucleotide hydrolysis, I show that some nucleotide-binding sites in ClpX release ATP rapidly, others release ATP slowly, and at least two sites remain nucleotide free. Occupancy of both the slow sites by ATP and the fast sites by either ATP or ADP is required to bind the degradation tags of protein substrates. The ability of ClpX to retain binding of substrate with ATP or ADP in the fast sites suggests that nucleotide hydrolysis in the fast sites, but not in the slow sites, will allow repeated unfolding attempts without substrate release over multiple ATPase cycles. My results rule out ATPase models including ClpX6eATP6 or ADP6 and also suggest that the enzyme hydrolyzes only a fraction of bound ATP in a single turnover event. Short peptide motifs of ClpX, known as IGF loops, interact with ClpP and change conformation as a response to nucleotide binding by ClpX.(cont.) As ClpX varies its nucleotide content during the ATP hydrolysis cycle, it also varies its affinity for ClpP. Processing of substrates is coupled to the ATP-hydrolysis cycle of ClpX and appears to modulate ClpX's affinity for ClpP by changing how long each ClpX subunit spends in each nucleotide state.by Greg Louis Hersch.Ph.D