Differential Interactions of Lipopolysaccharides with Lipid Bilayers: Applications for Pathogen Detection

This dissertation describes the development of new tailored methods for the discriminative detection of amphiphilic lipopolysaccharide (LPS) antigens, so as to improve screening methodologies for food-safety applications, and detection of amphiphiles in general. LPS is associated with the outer membrane of Gram-negative bacteria, and is a primary virulence biomarker of several pathogens. Direct detection of amphiphilic LPS in the aqueous matrices of the host/sample requires an appreciation of the complex biochemistry of the molecule, and forms the basis for this research. The unique structure of this molecule can be used for identification of both the serogroup and strain of pathogen. However, current detection methods lack sensitivity, and are also not serogroup specific. To achieve discriminative detection, we have first created a unique repertoire of associated reagents by isolating amphiphilic LPS from seven strains of Shiga toxin-producing Escherichia coli, and developing highly specific monoclonal antibodies against the O antigen regions of the same. We demonstrate the use of a targeted detection technique, called membrane insertion, which facilitates the physiological presentation of LPS by inserting the hydrophobic lipid A portion of the molecule into a lipid bilayer, leaving the O antigen exposed. This method is advantageous because it minimizes exposure of the highly conserved lipid A epitopes, and maximizes exposure of the serogroup specific O antigens. In addition, we present the first comprehensive biophysical analysis of the interaction of LPS with supported lipid bilayer architectures, and identify several novel and interesting effects of the same. Further characterization of these effects reveals the role or impact of membrane proteins and complexity on the interactions between host and pathogen biomarkers and significantly questions the design and execution of cell studies and in vitro platforms for amphiphilic targets like LPS. Cell studies clearly reveal that presentation of LPS either in buffer or in serum dramatically alters associated cytokine profiles. Our conclusions indicate that the biochemistry of amphiphilic molecules, like LPS, and their presentation, should always be considered when interfacing with physiological systems

Detection Methods for Lipopolysaccharides: Past and Present

Lipopolysaccharide (LPS) is the primary component of the outer membrane of Gram‐negativebacteria. LPS aids in protecting bacterial cells, and also defines the unique serogroups used to classify bacteria. Additionally, LPS is an endotoxin and the primary stimulator of innate immune cells in mammals, making it an ideal candidate for early detection of pathogens. However, the majority of methods for detection of LPS focus on detection of the endotoxic component of the molecule, lipid A. Since lipid A is largely conserved among bacterial species and serogroups, these detection approaches are highly nonspecific. Thus, the importance of identifying the O‐polysaccharide antigenic portion of LPS, which confers serogroup specificity, has received a great deal of attention in recent years. However, methods that are highly selective to the O‐antigens are typically less sensitive than those that target the endotoxin. Here we present a history and comparison of the sensitivity of these methods and their value for detecting bacteria in a variety of different sample types

Membrane Insertion for the Detection of Lipopolysaccharides: Exploring the Dynamics of Amphiphile-in-Lipid Assays

Shiga toxin-producing Escherichia coli is an important cause of foodborne illness, with cases attributable to beef, fresh produce and other sources. Many serotypes of the pathogen cause disease, and differentiating one serotype from another requires specific identification of the O antigen located on the lipopolysaccharide (LPS) molecule. The amphiphilic structure of LPS poses a challenge when using classical detection methods, which do not take into account its lipoglycan biochemistry. Typically, detection of LPS requires heat or chemical treatment of samples and relies on bioactivity assays for the conserved lipid A portion of the molecule. Our goal was to develop assays to facilitate the direct and discriminative detection of the entire LPS molecule and its O antigen in complex matrices using minimal sample processing. To perform serogroup identification of LPS, we used a method called membrane insertion on a waveguide biosensor, and tested three serogroups of LPS. The membrane insertion technique allows for the hydrophobic association of LPS with a lipid bilayer, where the exposed O antigen can be targeted for specific detection. Samples of beef lysate were spiked with LPS to perform O antigen specific detection of LPS from E. coli O157. To validate assay performance, we evaluated the biophysical interactions of LPS with lipid bilayers both in- and outside of a flow cell using fluorescence microscopy and fluorescently doped lipids. Our results indicate that membrane insertion allows for the qualitative and reliable identification of amphiphilic LPS in complex samples like beef homogenates. We also demonstrated that LPS-induced hole formation does not occur under the conditions of the membrane insertion assays. Together, these findings describe for the first time the serogroup-specific detection of amphiphilic LPS in complex samples using a membrane insertion assay, and highlight the importance of LPS molecular conformations in detection architectures

Pediatric Tuberculosis: The Impact of “Omics” on Diagnostics Development

Tuberculosis (TB) is a major public health concern for all ages. However, the disease presents a larger challenge in pediatric populations, partially owing to the lack of reliable diagnostic standards for the early identification of infection. Currently, there are no biomarkers that have been clinically validated for use in pediatric TB diagnosis. Identification and validation of biomarkers could provide critical information on prognosis of disease, and response to treatment. In this review, we discuss how the “omics” approach has influenced biomarker discovery and the advancement of a next generation rapid point-of-care diagnostic for TB, with special emphasis on pediatric disease. Limitations of current published studies and the barriers to their implementation into the field will be thoroughly reviewed within this article in hopes of highlighting future avenues and needs for combating the problem of pediatric tuberculosis

<i>In Vivo</i> Pulmonary Delivery and Magnetic-Targeting of Dry Powder Nano-in-Microparticles

This brief communication evaluates the cytotoxicity and targeting capability of a dry powder chemotherapeutic. Nano-in-microparticles (NIMs) are a dry powder drug delivery vehicle containing superparamagnetic iron oxide nanoparticles (SPIONs) and either doxorubicin (w/w solids) or fluorescent nanospheres (w/v during formulation; as a drug surrogate) in a lactose matrix. <i>In vitro</i> cytotoxicity was evaluated in A549 adenocarcinoma cells using MTS and LDH assays to assess viability and toxicity after 48 h of NIMs exposure. <i>In vivo</i> magnetic-field-dependent targeting of inhaled NIMs was evaluated in a healthy mouse model. Mice were endotracheally administered fluorescently labeled NIMs either as a dry powder or a liquid aerosol in the presence of an external magnet placed over the left lung. Quantification of fluorescence and iron showed a significant increase in both fluorescence intensity and iron content to the left magnetized lung. In comparison, we observed decreased targeting of fluorescent nanospheres to the left lung from an aerosolized liquid suspension, due to the dissociation of SPIONs and nanoparticles during pulmonary administration. We conclude that dry powder NIMs maintain the therapeutic cytotoxicity of doxorubicin and can be better targeted to specific regions of the lung in the presence of a magnetic field, compared to a liquid suspension

High-Resolution Graphene Films for Electrochemical Sensing <i>via</i> Inkjet Maskless Lithography

Solution-phase printing of nanomaterial-based graphene inks are rapidly gaining interest for fabrication of flexible electronics. However, scalable manufacturing techniques for high-resolution printed graphene circuits are still lacking. Here, we report a patterning technique [<i>i.e.</i>, inkjet maskless lithography (IML)] to form high-resolution, flexible, graphene films (line widths down to 20 μm) that significantly exceed the current inkjet printing resolution of graphene (line widths ∼60 μm). IML uses an inkjet printed polymer lacquer as a sacrificial pattern, viscous spin-coated graphene, and a subsequent graphene lift-off to pattern films without the need for prefabricated stencils, templates, or cleanroom technology (<i>e.g.</i>, photolithography). Laser annealing is employed to increase conductivity on thermally sensitive, flexible substrates [polyethylene terephthalate (PET)]. Laser annealing and subsequent platinum nanoparticle deposition substantially increases the electroactive nature of graphene as illustrated by electrochemical hydrogen peroxide (H₂O₂) sensing [rapid response (5 s), broad linear sensing range (0.1–550 μm), high sensitivity (0.21 μM/μA), and low detection limit (0.21 μM)]. Moreover, high-resolution, complex graphene circuits [<i>i.e.</i>, interdigitated electrodes (IDE) with varying finger width and spacing] were created with IML and characterized <i>via</i> potassium chloride (KCl) electrochemical impedance spectroscopy (EIS). Results indicated that sensitivity directly correlates to electrode feature size as the IDE with the smallest finger width and spacing (50 and 50 μm) displayed the largest response to changes in KCl concentration (∼21 kΩ). These results indicate that the developed IML patterning technique is well-suited for rapid, solution-phase graphene film prototyping on flexible substrates for numerous applications including electrochemical sensing

High-Resolution Graphene Films for Electrochemical Sensing <i>via</i> Inkjet Maskless Lithography

Solution-phase printing of nanomaterial-based graphene inks are rapidly gaining interest for fabrication of flexible electronics. However, scalable manufacturing techniques for high-resolution printed graphene circuits are still lacking. Here, we report a patterning technique [<i>i.e.</i>, inkjet maskless lithography (IML)] to form high-resolution, flexible, graphene films (line widths down to 20 μm) that significantly exceed the current inkjet printing resolution of graphene (line widths ∼60 μm). IML uses an inkjet printed polymer lacquer as a sacrificial pattern, viscous spin-coated graphene, and a subsequent graphene lift-off to pattern films without the need for prefabricated stencils, templates, or cleanroom technology (<i>e.g.</i>, photolithography). Laser annealing is employed to increase conductivity on thermally sensitive, flexible substrates [polyethylene terephthalate (PET)]. Laser annealing and subsequent platinum nanoparticle deposition substantially increases the electroactive nature of graphene as illustrated by electrochemical hydrogen peroxide (H₂O₂) sensing [rapid response (5 s), broad linear sensing range (0.1–550 μm), high sensitivity (0.21 μM/μA), and low detection limit (0.21 μM)]. Moreover, high-resolution, complex graphene circuits [<i>i.e.</i>, interdigitated electrodes (IDE) with varying finger width and spacing] were created with IML and characterized <i>via</i> potassium chloride (KCl) electrochemical impedance spectroscopy (EIS). Results indicated that sensitivity directly correlates to electrode feature size as the IDE with the smallest finger width and spacing (50 and 50 μm) displayed the largest response to changes in KCl concentration (∼21 kΩ). These results indicate that the developed IML patterning technique is well-suited for rapid, solution-phase graphene film prototyping on flexible substrates for numerous applications including electrochemical sensing

Biomimetic nanosensors for measuring pathogenic bacteria in complex food matrices (Conference Presentation)

Listeria monocytogenes and Salmonella spp. are among the most common cause of foodborne illnesses that negatively affect consumers’ health and food producers’ finances and credibility. Techniques used to detect pathogens (e.g., total viable counts, polymerase chain reaction, and enzyme-linked immunosorbent assays) are time consuming and costly as they require laboratory conditions with trained personnel. To meet this demand without compromising public health concerns, highly sensitive and rapid sensors are needed in food processing facilities for pathogen detection to reduce cost and holding time for food products. Ideally, these sensors should be small, label-free, low cost, portable, and highly sensitive/selective. This study describes some recent approaches for creating biomimetic sensors by optimizing the bacteria capture efficiency without the need for pre-concentration and pre-labeling steps. Two in-field biosensors were developed for measuring pathogenic bacteria in food matrices. The first example consists of pH-responsive polymer nanobrushes embedded with platinum nanoparticles platform with enhanced limit of detection and sensitivity for quantification of Listeria monocytogenes in fresh vegetables. A new approach using a one-step metal and polymer simultaneous deposition was tested using two pH-sensitive polymers and a thiol-terminated DNA aptamer selective to surface protein internalin A of Listeria monocytogenes. The second example demonstrates development of pathogenic biosensors for chicken broth using antibodies and DNA aptamers selective to Salmonella Typhimurium adsorbed to aerosolized graphene interdigitated electrodes (IDEs). Devices were printed in polyimide tape and aerosolized graphene was thermally annealed. The integrity of the substrate was analyzed and the nano-biosensors were characterized for topography, pH-actuation, graphene content, and electroactivity using electron microscopy, cyclic voltammetry, and multiple spectroscopy techniques (Raman, Fourier-transform infrared, and electrochemical impedance). Electrochemical impedance spectroscopy was used to evaluate the signal and determine the limit of detection by evaluating the change in charge transfer resistance. The nano-biosensors have a detection limit of approximately 5 CFU.mL-1, and a response time of approximately 17 minutes (15 minutes incubation period). The pH-sensitive nanobrushes and graphene-based biosensors have a selectivity for the target pathogen of approximately 95% in vegetable and chicken broth, respectively. The designed biosensor platform showed great potential to replace current standard methods used by the food industry for rapid foodborne pathogenic bacteria detection

Presentation matters: Impact of association of amphiphilic LPS with serum carrier proteins on innate immune signaling

<div><p>Recognition of Pathogen-associated Molecular Patterns (PAMPs) by Toll-like receptors is central to innate immunity. Many bacterial PAMPs such as lipopolysaccharide (LPS) and lipoteichoic acid have amphiphilic properties. The hydrophobicity of amphiphilic PAMPs contributes to increasing entropy and causes these molecules to self-aggregate or bind host carrier proteins in aqueous physiological environments. The goal of this work was to determine how innate immune signaling is impacted by physical presentation and association of amphiphilic PAMPs with serum carrier proteins, using LPS as an example molecule. Specifically, we measured LPS-induced cytokine profiles in murine macrophages when the antigen was presented associated with the various serum carrier proteins in serum versus a serum-depleted system. Our study demonstrates that the observed cytokine profiles are dramatically different when LPS is presented in buffer, versus in serum when it is associated with proteins, specifically with respect to inhibition of pro-inflammatory cytokines in the latter. These studies suggest that LPS-mediated cytokine expression is dependent on its presentation in physiological systems. The amphiphilicity of bacterial PAMPs and consequent association with lipoproteins is a feature, which should be taken into account in the design of <i>in vitro</i> experiments. Further studies of the interdependencies of different serum carriers can identify pathways for drug delivery and diagnostics.</p></div

Heat map distribution of cytokines in each condition.

<p>Displays intensity profile of cytokine expression in TLR4(+) cells in conditions 1, 2, and 3, both with (+) and without (-) LPS stimulation. LPS condition is labeled on the upper axis, while serum condition is labeled on the lower axis. Scale bar indicates that 1 is the lowest (white color) and 4 is the highest (dark teal) intensity. Values were plotted as the mean value of the base 10 logarithm of median fluorescence intensity, n = 3.</p