Impedance Detection of 3‐Phenoxybenzoic Acid Comparing Wholes Antibodies and Antibody Fragments for Biomolecular Recognition

Antibody fragments for detection of 3‐phenoxybenzoic acid (3‐PBA) are created by disulfide bond cleavage with tris(2‐carboxyethyl)phosphine (TCEP). These antibody fragments are employed to improve the sensitivity for 3‐PBA detection by electrochemical impedance spectroscopy (EIS). The sensitivity and detection limit are compared for biomolecular recognition by both polyclonal antibodies and antibody fragments immobilized through amide bond formation atop a self‐assembled monolayer (SAM) on an Au electrode, as well as antibody fragments directly attached to Au through Au−S covalent bond formation. The detection limit for 3‐PBA is dramatically improved (1.1×10−8 M) for direct immobilization of antibody fragments through Au−S covalent bond formation relative to immobilization of either polyclonal antibodies (2.1×10−5 M) or antibody fragments (1.7×10−5 M) through amide bond formation. This can be attributed primarily to the much lower charge transfer resistance (Rct) and higher ionic permeability obtained at biosensor interfaces that do not include an Au‐thiol SAM, allowing easier detection of further changes in Rct. In addition, the exponential dependence of the electron tunnelling rate on biomolecular film thickness increases the sensitivity of the antibody fragments modestly relative to polyclonal antibodies. This is the first quantitative comparison of the use of antibodies and antibody fragments for biomolecular recognition within impedance biosensors.