Encyclopedia of Experiments: Biological Techniques
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Transcript
To begin the β-lactamase-based conductimetric biosensor assay, take a transducer chip with working, counter, and reference electrodes. The working electrode has a conductive polymer coating, facilitating target antigen immobilization. The reference and counter electrodes lack this polymer coating.
Add a solution containing the target antigen onto the chip. Incubate. The antigen gets immobilized on the working electrode surface via non-covalent interactions. Wash to remove unbound antigens.
Add a blocking solution. Proteins in the solution attach to unbound sites on the working electrode and block them.
Add a bifunctional chimeric protein to the chip to detect the immobilized antigen. The chimeric protein comprises a β-lactamase enzyme — a bacterial protein responsible for antibiotic resistance. The enzyme is fused to a nanobody — the N-terminal variable region of single-domain antibodies.
Upon incubation, the nanobody of the protein binds to the immobilized target antigen. Wash to remove unbound chimeric proteins.
Plug the chip into a computer-controlled digital multimeter. Add a detection solution containing the antibiotic benzylpenicillin — a substrate for β-lactamase.
Benzylpenicillin binds to the β-lactamase of the antigen-bound chimeric protein and gets hydrolyzed — causing the release of protons. The protons induce a change in the electrical conductance of the polymer.
Plot the real-time difference in conductance between the reference electrode and the working electrode with the immobilized target antigen, confirming its interaction with the chimeric protein.
