Optical Immunosensor for the Detection of Listeria monocytogenes in Food Matrixes

Abstract

In this paper, simple imine-based organic fluorophore 4-amino-3-(anthracene-9 yl methyleneamino) phenyl (phenyl) methanone (APM) has been synthesized via a greener approach and the same was used to construct a fluorescent immunoassay for the detection of Listeria monocytogenes (LM). A monoclonal antibody of LM was tagged with APM via the conjugation of the amine group in APM and the acid group of anti-LM through EDC/NHS coupling. The designed immunoassay was optimized for the specific detection of LM in the presence of other interfering pathogens based on the aggregation-induced emission mechanism and the formation of aggregates and their morphology was confirmed with the help of scanning electron microscopy. Density functional theory studies were done to further support the sensing mechanism-based changes in the energy level distribution. All photophysical parameters were measured by using fluorescence spectroscopy techniques. Specific and competitive recognition of LM was done in the presence of other relevant pathogens. The immunoassay shows a linear appreciable range from 1.6 × 10⁶-2.7024 × 10⁸ cfu/mL using the standard plate count method. The LOD has been calculated from the linear equation and the value is found as 3.2 cfu/mL, and this is the lowest LOD value reported for the detection of LM so far. The practical applications of the immunoassay were demonstrated in various food samples, and their accuracy obtained was highly comparable with the standard existing ELISA method.

Scheme 1. Scheme Illustrating the Greener Synthesis of APM

Figure 1

Specific fluorescence response of APM/anti-LM toward LM in the presence of other pathogens (A) and the corresponding bar diagram (B) (Concentration of APM is 0.001 M; bulk concentration of anti-LM is 4.1 mg/1000 μL and APM/anti-LM is 0.0082 mg/2000 μL; LM and other pathogens are 10^–1 cfu/g in PBS buffer pH = 7.4; incubation time is 5 min). [(A) E. coli, (B) B. cereus, (C) S. paratyphi, (D) S. aureus, and (E) K. pneumoniae].

Figure 2

Emission response of APM/anti-LM while varying the concentrations of LM bacteria (A), and corresponding linear regression plot (B) (concentration of APM is 0.001 M; bulk concentration of anti-LM is 4.1 mg/1000 μL and APM/anti-LM is 0.0082 mg/2000 μL; LM is 1.6×10⁸ to 2.710⁶ in PBS buffer pH = 7.4).

Figure 3

Competitive study of the developed immunoassay, APM/anti-LM toward LM in the presence of relevant pathogenic bacteria (A), and corresponding bar diagram (B) [(A) E. coli, (B) E. coli + B. cereus, (C) E. coli + B. cereus + S. paratyphi, (D) E. coli + B. cereus + S. paratyphi + S.aureus, and (E) E. coli + B.cereus + S. paratyphi + S. aureus + K. pneumoniae] (Concentration of APM is 0.001 M; bulk concentration of anti-LM is 4.1 mg/1000 μL and APM/anti-LM is 0.0082 mg/2000 μL; LM and other pathogens are 10^–1 cfu/g in PBS buffer pH = 7.4; incubation time is 5 min.)

Scheme 2. Scheme Illustrating the Tentative Mechanism of Detection of LM Using APM/Anti-LM

Figure 4

round-state optimized geometry of the APM molecule.

Figure 5

alculated frontier molecular orbital with energy levels at the B3LYP/6-311G(d,p) level of theory. The isodensity surface contour value is set to be 0.02 au.