Nanozyme-Mediated Dual Immunoassay Integrated with Smartphone for Use in Simultaneous Detection of Pathogens

Abstract

Nanozymes are an excellent class of optical reporters for the development of sensitive biosensors for widespread applications. In this study, mesoporous core-shell palladium@platinum (Pd@Pt) nanoparticles were synthesized and then applied as signal amplifier in a dual lateral flow immunoassay (LFIA) and integrated with a smartphone-based device for use in simultaneous detection of Salmonella Enteritidis and Escherichia coli O157:H7. After optimization, the limit of detections were calculated to be ∼20 cfu/mL for S. Enteritidis and ∼34 cfu/mL for E. coli O157:H7, respectively. The greatly improved sensitivity was contributed by the peroxidase-like catalytic activity of the Pd@Pt nanoparticles for signal enhancement and the parallel design of dual detection for eliminating the cross-interference. The estimated recoveries of the dual LFIA range from 91.44 to 117.00%, which indicated that the developed method is capable of detecting live bacteria in food samples. This approach provides an attractive platform for S. Enteritidis and E. coli O157:H7 detection using a smartphone-based device as the sole piece of equipment, indicating great promise for foodborne pathogen analysis or in-field food safety tracking.

Keywords: E. coli O157:H7; Pt−Pd nanoparticles; Salmonella Enteritidis; lateral flow immunoassay; milk; smartphone.

Figure 1.

Synthesis scheme and TEM imaging. (A) Schematic illustration of Pd@Pt nanoparticles formation. (B, C) TEM images of Pd@Pt nanoparticles, with a mean diameter of 35 nm. (D) TEM image of E. coli O157:H7 captured by antibody-modified Pd@Pt nanoparticle conjugations. (E) TEM image of S. Enteritidis captured by antibody-modified Pd@Pt nanoparticle conjugations. (F, G) TEM images of details for the recognition function of antibodies-modified Pd@Pt nanoparticle conjugations.

Figure 2.

Assay optimization with respect to the peak area of test lines with or without TMB and H₂O₂. (A) Effect of different amounts of antibody-modified Pd@Pt nanoparticle conjugations: 0.5, 1, 1.5, 2, 2.5, and 3 μL; concentration of target bacteria: 10⁸ cfu/mL. (B) Effect of reaction time after adding TMB solution: from 1 to 16 min; concentration of target bacteria: 10⁸ cfu/mL. (C) Effect of the amount of BSA in running buffer: 0, 1, 2, 3, 4, and 5%; concentration of target bacteria: 10⁷ cfu/mL. (D) Effect of amount of Tween-20 in running buffer: 0, 0.0675, 0.125, 0.25, 0.5, and 1%; concentration of bacteria: 10⁷ cfu/mL. Error bars indicate standard deviations of the three measurements.

Figure 3.

Sensitivity of dual LFIA for simultaneous detection of S. Enteritidis and E. coli O157:H7. Left: dual LFIAs without TMB solution. Right: dual LFIAs with TMB solution. (A, D) Typical photo image of dual LFIAs with different bacterial concentrations ranging from 0 to 10⁸ cfu/mL. (B, E) Quantitative detections were performed by plotting the peak of two test lines (S and E). (C, F) Calibration curves of peak areas of test lines vs target concentrations. Error bars indicate standard deviations of the three measurements.

Scheme 1.

Scheme of Smartphone-Based Dual Lateral Flow Immunoassays for Simultaneous Detection of S. Enteritidis and E. coli O157:H7 Using Pd@Pt Nanoparticles as Signal Amplification^{a a}(A) Dual lateral flow immunoassays; (B) Pd@Pt nanoparticles for signal amplification; and (C) smartphone-based device.