Label Free QCM Immunobiosensor for AFB1 Detection Using Monoclonal IgA Antibody as Recognition Element

Abstract

This study introduces the use of an IgA isotype aflatoxin (AF) specific monoclonal antibody for the development of a highly sensitive Quartz Crystal Microbalance (QCM) immunobiosensor for the detection of AF in inhibitory immunoassay format. The higher molecular weight of IgA antibodies proved an advantage over commonly used IgG antibodies in label free immunobiosensor measurements. IgA and IgG antibodies with similar affinity for AF were used in the comparative studies. Sensor surface was prepared by covalent immobilization of AFB1, using self assembled monolayer (SAM) formed on gold coated Quartz Crystal, with 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxy succinimide (EDC/NHS) method using a diamine linker. Nonspecific binding to the surface was decreased by minimizing the duration of EDC/NHS activation. Sensor surface was chemically blocked after AF immobilization without any need for protein blocking. This protein free sensor chip endured harsh solutions with strong ionic detergent at high pH, which is required for the regeneration of the high affinity antibody-antigen interaction. According to the obtained results, the detection range with IgA antibodies was higher than IgG antibodies in QCM immunosensor developed for AFB1.

Quartz Crystal Microbalance With Dissipation Monitoring: A Powerful Method to Predict the in vivo Behavior of Bioengineered Surfaces

“…In the past 15 years, QCM-D has been increasingly used as a tool to characterize the biological interactions of engineered surfaces. To date, QCM-D crystals have been mainly applied to monitor the binding efficacy of probing surface of biosensors (Chen et al, 2010 ; Xu et al, 2011 ; Ertekin et al, 2016 ) as well as to verify the biofunctionalization of nanoengineered surfaces through self-assembled monolayers (SAMs; Minsky et al, 2016 ) or layer-by-layer (LbL) technologies (Chen et al, 2010 ). In addition, proteins adsorption competitive studies on biomedical device surfaces have been performed thanks to the possibility to measure at the same time the mass and the viscoelastic properties of absorbed biomolecules, which can be related to protein conformation.…”

Label-Free QCM Immunosensor for the Detection of Ochratoxin A

“…When the surface is functionalized for specific detection of an analyte, the binding of the recognition element can be observed in real-time with an appropriate frequency reader [ 24 ]. QCM is widely used in the detection of various analytes in low concentrations, such as bacteria [ 27 ], mycotoxins [ 28 , 29 ], disease markers [ 30 ], viruses [ 31 ], and many other analytes due to its simplicity, low cost, and sensitivity [ 32 ]. Another advantage of QCM over other endpoint measuring tools is its ability to make real-time measurements.…”

“…In these studies, protein conjugates of OTA were used for sensor surface preparation [ 42 , 43 , 44 ]. However, the use of protein on the sensor surface may be challenging, especially when the used antibody requires denaturing conditions for regeneration, which is a case observed for hydrophobic analytes like mycotoxins [ 29 ]. Direct covalent immobilization of OTA to the sensor surface without the use of proteins confers the sensor surface stability and durability when exposed to harsh regeneration solutions [ 29 ].…”

A New Method for Immobilization of His-Tagged Proteins with the Application of Low-Frequency AC Electric Field

“…A number of different methods have been reported for immobilizing proteins on a variety of surfaces [ 3 , 4 , 5 , 6 ]. Proteins may be immobilized by physical absorption (e.g., dot blot [ 7 , 8 ] and polyacrylamide gel [ 9 , 10 ] methods), through covalent coupling or cross-linking [ 3 , 11 , 12 , 13 , 14 , 15 ], as a self-assembled monolayer (SAM) [ 16 , 17 , 18 , 19 ], or through affinity interactions (e.g., avidin/biotin [ 3 , 20 , 21 ], nickel-nitrilotriacetic acid (Ni-NTA)/His-tagged protein [ 3 , 22 , 23 , 24 ], and intein methods [ 1 , 25 , 26 ].…”