Lipopolysaccharide (LPS) is an amphiphilic lipoglycan that is the primary component of the outer membrane of Gramnegative bacteria. Classified as a pathogen associated molecular pattern (PAMPs), LPS is an essential biomarker for identifying pathogen serogroups. Structurally, LPS is comprised of a hydrophobic lipophilic domain that partitions into the outer membrane of Gram-negative bacteria. Previous work by our team explored biophysical interactions of LPS in supported lipid bilayer assemblies (sLBAs), and demonstrated LPS-induced hole formation in DOPC lipid bilayers. Here, we have incorporated cholesterol and sphingomyelin into sLBAs to evaluate the interaction of LPS in a more physiologically relevant system. The goal of this work was to determine whether increasing membrane complexity of sLBAs, and changing physiological parameters such as temperature, affects LPS-induced hole formation. Integrating cholesterol and sphingomyelin into sLBAs decreased LPS-induced hole formation at lower concentrations of LPS, and bacterial serotype contributed to differences in hole formation as a response to changes in temperature. We also investigated the possibility of LPS-induced hole formation in cellular systems using the cytokine response in both TLR4 (+)/(-) murine macrophages. LPS was presented to each cell line in murine serum, delipidated serum, and buffer (i.e. no serum), and the resulting cytokine levels were measured. Results indicate that the method of LPS presentation directly affects cellular cytokine expression. The two model systems presented in this study provide preliminary insight into the interactions of LPS in the host, and suggest the significance of amphiphile-carrier interactions in regulating host-pathogen biology during infection.
Direct ultra-sensitive detection of pathogen biomarkers in blood could provide a universal strategy for diagnosis of bacterial infections, which remain a leading cause of morbidity and mortality in many areas of the world. Many factors complicate diagnosis, including the presence of multiple co-infections in a given patient, and lack of infrastructure in rural settings. In some pediatric patients, such as those in areas with poor resources, an additional challenge exists with low sample volumes due to age and other health factors such as anemia and dehydration. Our team is working on developing novel diagnostic assays, with a waveguide-based biosensor platform, to rapidly and specifically identify pathogen biomarkers from small samples of serum or plasma, allowing for the timely and sensitive diagnosis of infection at the point of care. In addition to the platform, we have developed novel membrane insertion and lipoprotein capture assay methods, to capture lipidated pathogen biomarkers in aqueous blood, by virtue of their interactions with host lipoprotein carriers. Herein, we demonstrate our efforts to adapt the lipoprotein capture assay for the detection of small concentrations of pathogen-secreted lipopolysaccharides in aqueous blood, with the ultimate aim of diagnosing Gram-negative infections effectively.
Shiga toxin-producing <i>Escherichia coli</i> (STEC) poses a serious threat to human health through the consumption of
contaminated food products, particularly beef and produce. Early detection in the food chain, and discrimination from
other non-pathogenic <i>Escherichia coli</i> (E. coli), is critical to preventing human outbreaks, and meeting current
agricultural screening standards. These pathogens often present in low concentrations in contaminated samples, making
discriminatory detection difficult without the use of costly, time-consuming methods (e.g. culture). Using multiple signal
transduction schemes (including novel optical methods designed for amphiphiles), specific recognition antibodies, and a
waveguide-based optical biosensor developed at Los Alamos National Laboratory, we have developed ultrasensitive
detection methods for lipopolysaccharides (LPS), and protein biomarkers (Shiga toxin) of STEC in complex samples
(e.g. beef lysates). Waveguides functionalized with phospholipid bilayers were used to pull down amphiphilic LPS,
using methods (membrane insertion) developed by our team. The assay format exploits the amphiphilic biochemistry of
lipoglycans, and allows for rapid, sensitive detection with a single fluorescent reporter. We have used a combination of
biophysical methods (atomic force and fluorescence microscopy) to characterize the interaction of amphiphiles with lipid
bilayers, to efficiently design these assays. Sandwich immunoassays were used for detection of protein toxins.
Biomarkers were spiked into homogenated ground beef samples to determine performance and limit of detection. Future
work will focus on the development of discriminatory antibodies for STEC serotypes, and using quantum dots as the
fluorescence reporter to enable multiplex screening of biomarkers.
Laser-Induced Breakdown Spectroscopy (LIBS) and Raman Spectroscopy have rich histories in the analysis of a wide variety of samples in both in situ and remote configurations. Our team is working on building a deployable, integrated Raman and LIBS spectrometer (RLS) for the parallel elucidation of elemental and molecular signatures under Earth and Martian surface conditions. Herein, results from remote LIBS and Raman analysis of biological samples such as amino acids, small peptides, mono- and disaccharides, and nucleic acids acquired under terrestrial and Mars conditions are reported, giving rise to some interesting differences. A library of spectra and peaks of interest were compiled, and will be used to inform the analysis of more complex systems, such as large peptides, dried bacterial spores, and biofilms. These results will be presented and future applications will be discussed, including the assembly of a combined RLS spectroscopic system and stand-off detection in a variety of environments.
Although bio-detection strategies have significantly evolved in the past decade, they still suffer from many
disadvantages. For one, current approaches still require confirmation of pathogen viability by culture, which is the ‘gold-standard’
method, and can take several days to result. Second, current methods typically target protein and nucleic acid
signatures and cannot be applied to other biochemical categories of biomarkers (e.g.; lipidated sugars). Lipidated sugars
(e.g.; lipopolysaccharide, lipoarabinomannan) are bacterial virulence factors that are significant to pathogenicity. Herein,
we present two different optical strategies for biodetection to address these two limitations. We have exploited bacterial
iron sequestration mechanisms to develop a simple, specific assay for the selective detection of viable bacteria, without
the need for culture. We are currently working on the use of this technology for the differential detection of two different
bacteria, using siderophores. Second, we have developed a novel strategy termed ‘membrane insertion’ for the detection
of amphiphilic biomarkers (e.g. lipidated glycans) that cannot be detected by conventional approaches. We have
extended this technology to the detection of small molecule amphiphilic virulence factors, such as phenolic glycolipid-1
from leprosy, which could not be directly detected before. Together, these strategies address two critical limitations in
current biodetection approaches. We are currently working on the optimization of these methods, and their extension to
real-world clinical samples.
We have developed a waveguide-based optical biosensor for the sensitive and specific detection of biomarkers associated with disease. Our technology combines the superior optical properties of single-mode planar waveguides, the robust nature of functionalized self-assembled monolayer sensing films and the specificity of fluorescence sandwich immunoassays to detect biomarkers in complex biological samples such as serum, urine and sputum. We have previously reported the adaptation of our technology to the detection of biomarkers associated with breast cancer and anthrax. However, these approaches primarily used phospholipid bilayers as the functional film and organic dyes (ex: AlexaFluors) as the fluorescence reporter. Organic dyes are easily photodegraded and are not amenable to multiplexing because of their narrow Stokes' shift. Here we have developed strategies for conjugation of the detector antibodies with quantum dots for use in a multiplex detection platform. We have previously evaluated dihydroxylipoic acid quantum dots for the detection of a breast cancer biomarker. In this manuscript, we investigate the detection of the Bacillus anthracis protective antigen using antibodies conjugated with polymer-coated quantum dots. Kinetics of binding on the waveguide-based biosensor is reported. We compare the sensitivity of quantum dot labeled antibodies to those labeled with AlexaFluor and demonstrate the photostability of the former in our assay platform. In addition, we compare sulfydryl labeling of the antibody in the hinge region to that of nonspecific amine labeling. This is but the first step in developing a multiplex assay for such biomarkers on our waveguide platform.
Our team has developed polyethylene glycol (PEG)-modified, self-assembled monolayers (SAMs) for biological
detection on either planar or spherical substrates, which resist non-specific binding while facilitating specific ligand
attachment. The preparation and characterization of these thin films, their validation against B. anthracis protective
antigen (PA) in a sandwich assay format, and the application of these thin films for quantitative analysis of several
medically interesting targets (breast cancer, tuberculosis, and influenza) will be shown.