develop new strategies for signal amplification such as bifunctional linkers or
switchable linkers (Hahn et al. 2017; Lim et al. 2012). A bifunctional linker with
biotinylated specific antibacterial antibodies is developed which binds to the specific
target and leads to visible color changes due to aggregation of the target with the
probe (streptavidin–gold nanoparticles), biotin–streptavidin binding reaction (Weber
et al. 1989), and also the localized surface plasmon resonance phenomenon. This
strategy was improved by altering the amount of streptavidin–gold nanoparticles, to
result in a lower limit of detection (10 cfu/ml) of the pathogens E. coli and
Salmonella (You et al. 2018). This was tested in samples of tap water, lake water,
and milk samples, which are unaltered by matrix effects. Thus, it offers a speedy,
highly sensitive real-time pathogen detection biosensor. Apart from this, simultaneous detection of multiple analytes is one of the major requirements fulfilled using
an immunochemical method for diagnosing various infections in test samples. A
unique virulence factor, for example, pyocyanin, secreted only by P. aeruginosa
(Pastells et al. 2016), was used for its detection. By using antibodies of a specific
metabolite of the virulence factor pyocyanin, 1-hydroxyphenazine, the virulence
factor and hence the pathogen could be quantified/detected.
Antibiogram is a method of detection of early microbial growth. This method
employed the 96-well plate format which was modified by Elavarasan et al., into a
handy, polymethylmethacrylate microfluidic chip, wherein resazurin, a blue-colored
water-soluble dye, was used. The response was monitored by change of color
following the biochemical reactions specific for viable cells (Elavarasan et al.
2013; Kaur et al. 2013).
The reduction of water-soluble resazurin occurs in two steps when in contact with
viable cells (Fig. 10.7) (Brindha et al. 2018b). The first step results in an irreversible
pink colored, partially oxidized form of pink-colored resorufin. This, upon further
oxidation, yields a reversible and colorless hydroresorufin (Sarker et al. 2007). This
dye acts as an indicator to test cell viability, growth, and toxicity (Palomino et al.
2002). This immunoassay was carried out on a microfluidic chip not only for
detecting the presence of milk pathogens but also for detecting multidrug-resistant
pathogens. In this assay, as shown below in Fig. 10.7, the interpretation is based on
color developed in the presence of microbial samples. When the microorganisms are
susceptible to antibiotics, with no cell growth, the blue color is observed, while when
it is resistant, a pink/colorless solution is observed. In the case of moderate to
negligible growth of the cells, violet color is seen as a partial/complete reduction
of resazurin occurs.
10.4 Conclusion and Future Prospects
All the developments of sensors in the recent past are targeted for specificity, lower
detection time, lower detection limits, and enhanced sensitivity for detecting
analytes. Integration of multiple labeling techniques with immunoÀ/enzymatic
reagents or fluorophores in association with fiber optics or surface plasmon resonance is in practice, for the enhancement of selectivity/sensitivity of pathogen
detection. Recently, biosensor research focuses on target analyte enrichment (Zuo
284
J. Brindha et al.
switchable linkers (Hahn et al. 2017; Lim et al. 2012). A bifunctional linker with
biotinylated specific antibacterial antibodies is developed which binds to the specific
target and leads to visible color changes due to aggregation of the target with the
probe (streptavidin–gold nanoparticles), biotin–streptavidin binding reaction (Weber
et al. 1989), and also the localized surface plasmon resonance phenomenon. This
strategy was improved by altering the amount of streptavidin–gold nanoparticles, to
result in a lower limit of detection (10 cfu/ml) of the pathogens E. coli and
Salmonella (You et al. 2018). This was tested in samples of tap water, lake water,
and milk samples, which are unaltered by matrix effects. Thus, it offers a speedy,
highly sensitive real-time pathogen detection biosensor. Apart from this, simultaneous detection of multiple analytes is one of the major requirements fulfilled using
an immunochemical method for diagnosing various infections in test samples. A
unique virulence factor, for example, pyocyanin, secreted only by P. aeruginosa
(Pastells et al. 2016), was used for its detection. By using antibodies of a specific
metabolite of the virulence factor pyocyanin, 1-hydroxyphenazine, the virulence
factor and hence the pathogen could be quantified/detected.
Antibiogram is a method of detection of early microbial growth. This method
employed the 96-well plate format which was modified by Elavarasan et al., into a
handy, polymethylmethacrylate microfluidic chip, wherein resazurin, a blue-colored
water-soluble dye, was used. The response was monitored by change of color
following the biochemical reactions specific for viable cells (Elavarasan et al.
2013; Kaur et al. 2013).
The reduction of water-soluble resazurin occurs in two steps when in contact with
viable cells (Fig. 10.7) (Brindha et al. 2018b). The first step results in an irreversible
pink colored, partially oxidized form of pink-colored resorufin. This, upon further
oxidation, yields a reversible and colorless hydroresorufin (Sarker et al. 2007). This
dye acts as an indicator to test cell viability, growth, and toxicity (Palomino et al.
2002). This immunoassay was carried out on a microfluidic chip not only for
detecting the presence of milk pathogens but also for detecting multidrug-resistant
pathogens. In this assay, as shown below in Fig. 10.7, the interpretation is based on
color developed in the presence of microbial samples. When the microorganisms are
susceptible to antibiotics, with no cell growth, the blue color is observed, while when
it is resistant, a pink/colorless solution is observed. In the case of moderate to
negligible growth of the cells, violet color is seen as a partial/complete reduction
of resazurin occurs.
10.4 Conclusion and Future Prospects
All the developments of sensors in the recent past are targeted for specificity, lower
detection time, lower detection limits, and enhanced sensitivity for detecting
analytes. Integration of multiple labeling techniques with immunoÀ/enzymatic
reagents or fluorophores in association with fiber optics or surface plasmon resonance is in practice, for the enhancement of selectivity/sensitivity of pathogen
detection. Recently, biosensor research focuses on target analyte enrichment (Zuo
284
J. Brindha et al.