Selective Detection of Protein Homologues in Serum Using an OmpG Nanopore

Abstract

Outer membrane protein G is a monomeric β-barrel porin that has seven flexible loops on its extracellular side. Conformational changes of these labile loops induce gating spikes in current recordings that we exploited as the prime sensing element for protein detection. The gating characteristics, open probability, frequency, and current decrease, provide rich information for analyte identification. Here, we show that two antibiotin antibodies each induced a distinct gating pattern, which allowed them to be readily detected and simultaneously discriminated by a single OmpG nanopore in the presence of fetal bovine serum. Our results demonstrate the feasibility of directly profiling proteins in real-world samples with minimal or no sample pretreatment.

Figure 1

The open (2IWV) and closed (2IWW) structures of OmpG with the loop 6 highlighted in red. The ionic current trace was obtained in 10mM sodium phosphate pH 6, 300mM KCl buffer.

Figure 2

Detection of SB58C by OmpG-biotin nanopore. (a,b) The electrophysiology traces and all events histograms of unbound (UB) and SB-bound states of OmpG-biotin at −50 mV and +50 mV. (c) Zoomed-in electrophysiology traces and all-events histograms of the unbound state and (d) the three independent SB-binding states types A, B and C. Buffer used was 10 mM sodium phosphate pH 6, 300 mM KCl. 1nM SB antibody was added to the recording chamber. SB binding was recorded with a 2 kHz Bessel filter at a sampling rate of 100 μs.

Figure 3

The effect of serum on the gating behavior of OmpG-biotin. 100 μl of FBS was added to the loop-containing chamber to a final concentration of 10% (v/v). The buffer was 10 mM sodium phosphate pH 6.0, 300 mM KCl. OmpG was recorded with a 2 kHz Bessel filter at a sampling rate of 100 μs.

Figure 4

Discrimination of two antibodies in the presence of serum. (a) Binding of SB (blue) and BT (red) to OmpG-biotin in the absence of serum. (b) Electrophysiology traces and (c) histograms of the unbound state in comparison with the BT and SB bound states. Buffer used was 10 mM sodium phosphate pH 6.0, 300 mM KCl and recorded at −50 mV. SB (1 nM) and BT (5 nM) were added to the recording chamber. (d) BT and SB binding in the presence of serum. (e) Electrophysiology traces and (f) histograms of BT and SB binding in the presence of serum. In addition to SB and BT, serum (10% v/v) was added to the loop-containing chamber. SB and BT binding were recorded with a 2 kHz Bessel filter at a sampling rate of 100 μs.

Figure 5

The effect of serum on the fingerprint pattern of BT and SB. The gating events of different analyte protein binding states were characterized by five parameters, i.e. open probability, gating frequency, inter-event duration, event duration and the conductance of the open pore state. Changes of these parameters relative to the no binding state generate the fingerprint unique for each antibody. For SB, the three types of gating pattern were analyzed separately. The error bars indicate standard deviations from at least three independent pores.