Label-free Electrochemiluminescent Biosensor For Staphylococcus Aureus And Pseudomonas Aeruginosa Detection

Electrochemiluminescent(ECL) bioassay not only retains many advantages of chemiluminescent(CL) analysis, but also posses lots of other merits including strong controllability, good reproducibility, high selectivity and capability for in-situ detection. Usually the probe labelling format is adopted by most of the reported ECL bioassays, however, the labelling techniques is considered to be labor-intensive and time-consuming. Additionally, the conjugation chemistry involved in the labelling process increases the risk of damaging the activities of the biomolecules such as antibody and enzyme, affecting their analytical performance. The label-free format seems to show greater promise in ECL biosensor due to avoiding the probe labeling process and increasing the analytical performance. Herein, two novel label-free ECL biosensors were proposed for the detection of Staphylococcus aureus(S. aureus) and Pseudomonas aeruginosa detection(PA1):Part 1 Detection of Staphylococcus aureus utilizing the binding between immunoglobulin G and Staphylococcus aureus protein AA facile label-free ECL biosensor was developed for detection of S. aureus based on the specific binding between Fc region of immunoglobulin G(IgG) and S. aureus protein A(SPA) in the cell wall. Carboxyl graphene, with large surface and excellent electron transfer ability, was used as the carrier of IgG for fabrication of ECL biosensor. This biosensor was constructed by depositing carboxyl graphene/porcin IgG composite on the surface of a glassy carbon electrode. The specific reaction between SPA and IgG resulted in a decrease of ECL signal because the bound S. aureus interrupted the interfacial electron transfer and hindered the diffusion of the ECL active substances. The ECL intensity decreased linearly with S. aureus concentrations in the range of 1.0 × 103-1.0 × 109 colony-forming units(CFU) mL-1, with a detection limit of 3.1 × 102 CFU mL-1. The whole assay could be accomplished within 70 min when a ready-for-use biosensor was applied. The recovery test for food, environmental and biological samples showed recoveries between 75.0% and 116.7%. This developed biosensor displayed ideal specificity, facile manipulation, simple fabrication and short assay time, thus provided a new pathway for pathogenic bacteria rapid screening.Part 2 Detection of Pseudomonas aeruginosa based on Pseudomonas aeruginosa phage specifically binding with Pseudomonas aeruginosaA novel label-free ECL biosensor was developed for detection of PA1 based on the specific binding between Pseudomonas aeruginosa phage(PaP1) and PA1. Carboxyl graphene, with large surface and excellent electron transfer ability, was used as the carrier of PaP1 for fabrication of ECL biosensor. This biosensor was constructed by depositing carboxyl graphene/PaP1 composite on the surface of a glassy carbon electrode. The specific reaction between PA1 and PaP1 resulted in a decrease of ECL signal because the bound PA1 interrupted the interfacial electron transfer and hindered the diffusion of the ECL active substances. The ECL intensity decreased linearly with PA1 concentrations in the range of 1.4 × 102-1.4 × 106 CFU mL-1, with a detection limit of 56 CFU mL-1. The whole assay could be accomplished within 30 min when a ready-for-use biosensor was applied. The recovery test for food, drug and biological samples showed recoveries between 78.6% and 114.3%. This proposed biosensor shows simple manipulation, low-cost, rapid detection, high sensitivity and specificity, thus opens up a new pathway for pathogen screening.