ABSTRACT
This chapter provides an in-depth investigation about current methods for pathogen detection in water samples. Enzyme-linked assays for environmental samples are presented. We present possible sensing techniques that are already implemented for environmental applications. Finally, several most prominent high-sensitivity lab-ona-chip (LOC) platforms for detection/quantification of pathogens in water and wastewater samples are demonstrated. 6.1 IntroductionWaterborne infections can be caused by a number of bacterial, protozoan, and viral pathogens [1]. According to the report by the
World Health Organization (WHO) in 2004, about 2.5 million people die due to waterborne pathogens in a year [2]. In the U.S. alone, it is estimated that $20 billion per year of economic productivity is lost due to illnesses caused by waterborne pathogens [3]. Outbreaks of waterborne infections in the world are typically associated with treatment failures, contamination of untreated groundwater, contamination of drinking water in the distribution system, and poor sanitation of drinking water facilities. Low levels of pathogens can also be present in drinking water during normal operation of public water systems. The detection of waterborne pathogens remains a challenging and important issue for controlling water quality. The effective testing of pathogens requires methods of analysis that meet a number of challenging criteria. Higher sensitivity, rapid analysis time, and an extremely selective detection methodology is also required, because low numbers of pathogenic bacteria are often present in a complex biological environment, along with many other nonpathogenic organisms. 6.2 Bacterial and Large Entities’ Detection in
6.2.1 Recognition Receptors for Bacterial DetectionA biosensor recognizes its target analyte, and the corresponding responses triggered by target recognition are then converted into various equivalent signals by the transducer, which are in turn amplified, processed, and recorded for display, storage, and analysis. Recognition receptors play an essential role in the recognition of the target analyte and are the key to sensitivity and specificity in any biosensor technology. They are the unique component integrated within a biosensor responsible for binding/capturing the analyte of interest onto the sensor. This molecular recognition event generally refers to specific, noncovalent binding between two biological entities, typically one of which is a macromolecule or a molecular assembly and the other is the target analyte such as bacterial cells or viruses. Their interactions are mainly driven by intermolecular forces such as hydrogen bonding, electrostatic interactions, van der Waals forces, and hydrophobic interactions. Avidity in polyvalent interactions, particularly those involving bacterial and viral
analytes, further strengthens the interactions through simultaneous interactions at multiple sites. Recognition receptors can be divided into six major categories. These categories include antibodies, live cell systems, bacteriophages, proteins/peptides, oligonucleotides, and biomimetics. 6.2.1.1 AntibodiesAntibodies are common recognition receptors employed in pathogen (bacteria and viruses) biosensing applications. Antibodies offer high affinity and specificity molecular recognition and can be immobilized on a substrate such as a detector surface or a carrier. Their use in conventional and novel detection technologies continues to grow. An antibody recognizes its antigen through the unique structure formed within its variable domain, the complementarity determining regions (CDRs), which matches the three-dimensional structure of its antigen and results in a high-affinity specific interaction. The genetic mechanism of antibody production can virtually yield an antibody with limitless diversity, so theoretically it is possible to obtain an antibody that can recognize and bind to any target analyte. Antibodies can be covalently modified in many ways, leading to a wide variety of immunological methods. Covalent conjugation of a label such as biotin, fluorophore, radioactive isotope, and enzyme transforms an antibody into an ideal probe molecule. The first use of antibody-based direct detection was by Coons et al. [4], where they labeled antibodies with a fluorescent dye to identify tissue antigens. Since then, a rapid indirect method has become widely used and is still a popular method now. The enzyme-linked immunosorbent assay (ELISA) is a two-step method involving an unlabeled antibody specific for the analyte (primary antibody) and a labeled (usually with an enzyme that produces a chromogenic signal) secondary antibody that recognizes the primary antibody). Antibodies can also be immobilized on a variety of surfaces such as surface plasmon resonance (SPR) chips [5], optical fibers [6], nanowires [7], and microcantilever surfaces [8], opening new avenues for the development of novel biosensing mechanisms. 6.2.1.2 Live cell systems
Cell-based biosensors (CBBs) rely on the specific interaction between cells and the targeted pathogens and the subsequent response of
the cell to stimuli caused by the pathogens. In this system, the cells serve as the transducer, which converts the binding of pathogens into a cellular response. This cellular response is then converted into a measurable electronic signal by a second transducer with a detection mechanism depending on the type of cellular response. CBBs can have an extremely low detection limit due to significant signal amplification at the cellular and transducer levels. Because detection relies on direct measurements of physiological pathways, they have the potential to provide additional information such as functional activities of the analyte. For example, discrimination of viable and nonviable bacterial cells can be achieved with a CBB, which is a critical parameter in the determination of an outbreak. Banerjee et al. [9] demonstrated a CBB using collagen-encapsulated mammalian cells by monitoring the release of alkaline phosphatase as a measurement of cytotoxicity upon pathological bacterial infection. It was reported that nonpathogenic bacteria induced minimal cytotoxicity, which causes a significantly lower response. Carlyle et al. [10] developed a CBB assay capable of detecting toxin-producing bacterial strains. They demonstrated that these bacterial strains cause the aggregation of chromatophore cells, specific pigmented cells that undergo a vivid color response upon aggregation when exposed to specific biologically active agents.
