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.
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 Escherichia coli (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 Escherichia coli (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.
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