Blocking Agent Optimization for Nonspecific Binding on Phage Based Magnetoelastic Biosensors

Abstract

Magnetoelastic biosensors have been studied for the past few years for use in detecting bacteria for food safety. These magnetoelastic biosensors are composed of a bio-molecular recognition element and transducer platform (magnetoelastic resonator). Phages (biomolecular recognition element) have been developed to provide very specific binding with Salmonella typhimurium bacteria. However, there is some binding (non-specific binding) of bacteria to the gold areas not covered with phage on both the measurement and control sensors. In those areas, a blocking agent is necessary to reduce the non-specific binding. The purpose of this study is to compare three of the often used blocking agents, BSA, milk and casein in different concentrations to determine the agent and the optimum concentration for blocking. To accomplish this, a set of measurement and control sensors were prepared using identical procedures except the control sensors were not immobilized with phage. The measurement and control sensors were then coated with a blocking agent of concentrations ranging from 0.1 to 5 mg/mL. SEM observations show that coating the magnetoelastic biosensors with BSA at a concentration of 1mg/mL yields the largest amount of specific binding of bacteria cells while also maintaining the largest ratio of specific to non-specific binding. This binding characteristic is a result of the physical and electrical structure of the blocking proteins. To verify that the blocking solutions made good contact with the sensor's surface, contact angle measurements were used to evaluate the wettability of the surface. Changes in the resonant frequency of the biosensors were found to agree with the results from SEM observations.

Detection of methicillin-resistant Staphylococcus aureus using novel lytic phage-based magnetoelastic biosensors

“…However, a BSA concentration of 10 mg/ml did not result in an increase in the number of MRSA bound on the measurement sensors. The higher concentration of BSA did not guarantee the maximum binding of MRSA, which was in agreement with a previously published result [22]. The reason of significantly lower binding of MRSA at 10 mg/ml of BSA concentration in both sensors is presumably associated with the shape and size of BSA.…”

“…Therefore, open areas on the sensor (between phages) must be blocked with a blocking agent to prevent non-specific binding on the sensor. A previous study [22] showed that BSA has superior blocking ability when compared to casein and fat-free milk. Therefore, BSA was selected as a blocking agent and the different concentration of BSA was used in order to determine the optimum concentration of BSA (Fig.…”

“…The reason of significantly lower binding of MRSA at 10 mg/ml of BSA concentration in both sensors is presumably associated with the shape and size of BSA. BSA is a globular protein (approximately 66,000 Da) that has a prolate ellipsoid shape with dimensions of 14 nm × 4 nm × 4 nm [22], which is much smaller than the size of lytic phage. Presumably, appropriate concentration of BSA covers non-specific binding site between adjacent phages.…”

Effects of Surface Morphologies of Fresh Produce on the Performance of Phage-Based Magnetoelastic Biosensors

“…However, the direct detection of ME biosensors without washing procedures was sometimes limited due to the non-specific bindings on the * Electrochemical Society Active Member z E-mail: parkmik@auburn.edu measurement and control sensors. Thus, Shen et al, 19 optimized the blocking condition by selecting a blocking agent and its concentration. However, fresh produce have diverse surface properties depending on surface hydrophobicity, surface constitutional characteristics, and surface morphology.…”

Novel Approach of a Phage-Based Magnetoelastic Biosensor for the Detection of Salmonella enterica serovar Typhimurium in Soil

“…The selection of the best extraction procedures solved the nonspecific binding on the sensor in a certain range; however, the extraction did not solve all possible nonspecific binding problems. The best blocking reagent needs to satisfy certain requirements, including blocking the space between the E2 phages and not interfering with the capacity of the E2 phage to interact with its target [23,24]. Since BSA, casein, and PEG have been popularly used as blocking reagents on plastic or gold surfaces [11,16,29], these reagents were selected for this study.…”