Optimization of Nanoporus Anodic Aluminum Sensor-Based Lspr for Methicillin-Resistant Staphylococcus Aureus detection

Methicillin-resistant Staphylococcus aureus (MRSA) causes foodborne illnesses in approximately 241,000 persons in the United States annually, despite of greatly underreported (about 25 fold) and underdiagnosed (about 29 fold) ratio. MRSA has considered as one of the major foodborne pathogens, and it is considered as one of the most significant threats to public health. Recently, MRSA has gained substantial attention due to its increasing resistance against a broad range of antibiotics. The timely detection and quantification of MRSA are of key importance for the prevention of MRSA outbreaks. Considerable efforts have been directed towards the development of rapid and practical biosensor methods for MRSA detection. To keep up this trend, SPR method has been combined with a nanoporous anodic aluminum (NAA) sensor in this study, which is called as NAA sensor -based localized surface plasmon resonance (LSPR) system (Figure 1). This method will overcome the limitations of SPR method due to larger surface-to-volume ratio of NAA sensor.

This study aims to optimize the pore size of NAA prior to employment of the NAA-based LSPR system for MRSA detection. For the fabrication of three different sizes of NAA sensor, a two-step anodizing method was employed using various anodization voltage and time, and electrolyte solution. The topology of NAA sensors were observed by employing FE-SEM for measuring the pore diameter, inter-pore distance, and pore length. The reflectance spectrum and fringe pattern of each NAA sensor were measured by using LSPR for the selection of optimum NAA sensor for MRSA detection. The NAA sensor was modified with 11-mercaptoundecanoic acid and immobilized with anti-S. aureus polyclonal antibody. The NAA immunosensors were then exposed to serially diluted MRSA culture starting from 10¹ CFU/mL to 10⁸ CFU/mL for the measurement of wavelength shift. The attachment of MRSA on the surface of NAA immunosensor was confirmed by SEM observation.

Three different pore sizes of sensors were successfully fabricated in the range of 50 to 200 nm by the modification of fabrication conditions. The inter-pore distance and pore length of each sensor with different pore size were 99.7 ± 4.13 nm and 1.02 ± 0.01 µm for 50 nm size sensor, 201.5 ± 2.12 nm and 1.01 ± 0.01 µm for 100 nm size sensor, and 260 ± 1.40 nm and 1.00 ± 0.01 µm for 200 nm size sensor, respectively. The reflectance spectrum and fringe pattern of 200 nm size of sensor showed clearer and greater reflectance spectrum than those  of 50 nm and 100 nm pore size of sensor. In addition, the sensitivity of 50, 100 and 200 nm pore size of NAA sensors were determined to be 747 nm/RIU, 1000 nm/RIU, and 1534 nm/RIU, respectively. The wavelength shift of MRSA on the NAA immunosensor was increased as the concentration of MRSA increased. The confirmed numbers of MRSA captured on the NAA immunosensor were increased in a concentration-dependent manner. Detection limit of NAA-based LSPR method was determined to be 10² ± 0.79 CFU/mL. Taking the excellent advantage in cost-effectiveness and simple fabrication of NAA sensor, an optimized NAA sensor-based LSPR method could be applied to detect MRSA in food with successful practicability.