An innate immune system-mimicking, real-time biosensing of infectious bacteria

Abstract

An animal cell-based biosensor was investigated to monitor bacterial contamination in an unattended manner by mimicking the innate immune response. The cells (RAW 264.7 cell line) were first attached onto the solid surfaces of a 96-well microtiter plate and co-incubated in the culture medium with a sample that might contain bacterial contaminants. As Toll-like receptors were present on the cell membrane surfaces, they acted as a sentinel by binding to pathogen-associated molecular patterns (PAMPs) of any contaminant. Such biological recognition initiates signal transmission along various pathways to produce different proinflammatory mediators, one of which, tumor necrosis factor-α (TNF-α) was measured using an immunosensor. To demonstrate automated bacterium monitoring, a capture antibody specific for TNF-α was immobilized on an optical fiber sensor tip and then used to measure complex formation in a label-free sensor system (e.g., Octet Red). The sensor response time depended significantly on the degree of agitation of the culture medium, controlling the biological recognition and further autocrine/paracrine signaling by cytokines. The response, particularly under non-agitated conditions, was also influenced by the medium volume, revealing a local gradient change of the cytokine concentration and also acidity, caused by bacterial growth near the bottom surfaces. A biosensor system retaining 50 μL medium and not employing agitation could be used for the early detection of bacterial contamination. This novel biosensing model was applied to the real-time monitoring of different bacteria, Shigella sonnei, Staphylococcus aureus, and Listeria monocytogenes. They (<100 CFU mL(-1)) could be detected automatically within the working time. Such analysis was carried out without any manual handling regardless of the bacterial species, suggesting the concept of non-targeted bacterial real-time monitoring. This technique was further applied to real sample testing (e.g., with milk) to exemplify, for example, the food quality control process without using any additional sample pretreatment such as magnetic concentration.

Bacterial Sensing and Biofilm Monitoring for Infection Diagnostics

“…These type I transmembrane glycoproteins play important functions in the innate immune system, as they recognize the highly conserved biomarkers of bacterial and viral origin known as a pathogen-associated molecular pattern (PAMP). [107,108] When binding to their targets, activated TLRs form dimers and initiate immune cell signalling that facilitates the appropriate immune response. [109] Each of the different TLRs (TLR I-XIII) specifically interact with PAMPs, such as nucleic acid, LPS, cell wall components, and flagellins.…”

“…[ 109 ] Each of the different TLRs (TLR I‐XIII) specifically interact with PAMPs, such as nucleic acid, LPS, cell wall components, and flagellins. [ 107 ] The strong affinity interactions make TLRs powerful natural biorecognition elements for early detection of pathogenic invasions.…”

Real-time Monitoring of Biomarkers in Serum for Early Diagnosis of Target Disease

“…As an alternative biosensing surrogate, proinflammatory mediators such as tumor necrosis factor- (TNF-) can be measured via the antigen-antibody reaction. For demonstration, an antibody specific to TNF- was used as the capture binder and complex formation was then measured in a label-free sensor system 27 . Such analytical concepts may be useful for non-targeted, bacterial real-time monitoring.…”

Antimicrobial Peptides: Powerful Biorecognition Elements to Detect Bacteria in Biosensing Technologies

“…This is the case of human cells. Mammalian cells express a wide range of proteins and receptors to perceive their environment, which make them appealing bioreceptors to sense bacteria [ 21 , 26 , 27 ]. Bacteriophages may fall within this category as well.…”