Uni-molecular detection and quantification of selected β-lactam antibiotics with a hybrid α-hemolysin protein pore

Single-nanopores have recently been used to electrically detect a wide range of analytes. Similarly, using electrophysiology, we demonstrate how a system comprised of an ion channel formed by α-hemolysin (α-HL) and single-cyclic γ-cyclodextrin (γ-CD) molecule permits the detection of, and differentiation between three different antibiotics from the β-lactam family. Specifically, histograms of the time between the successive binding events, and the residence time distributions of the antibiotic in the γ-CD molecular adapter vary with the antibiotic type. The results show that the association times of amoxicillin, azlocillin, and ampicillin are τ(on) = 2.1 ± 0.2, 2.2 ± 0.3, and 3.1 ± 0.4 ms, respectively. Interestingly, we found that the residence time of the bulkier and negatively charged azlocillin (τ(off) = 0.008 ± 0.0005 ms) is much less than that of ampicillin (τ(off) = 0.07 ± 0.005 ms) and amoxicillin (τ(off) = 0.1 ± 0.02 ms), even though the γ-CD-α-HL complex is anionic selective. The data were also used to estimate the standard free energy of binding between ampicillin to γ-CDs binding (-12 kJ mol(-1) [corrected]). The difference in association times might be due to γ-CDs-imposed steric hindrance or an energetically more expensive desolvation step for the antibiotics to gain access to the binding site in the CD. We suggest that this technique may be used to detect other analytes used in pharmaceutical applications.