Single-Molecule Protein Detection in a Biofluid Using a Quantitative Nanopore Sensor

Abstract

Protein detection in complex biological fluids has wide-ranging significance across proteomics and molecular medicine. Existing detectors cannot readily distinguish between specific and nonspecific interactions in a heterogeneous solution. Here, we show that this daunting shortcoming can be overcome by using a protein bait-containing biological nanopore in mammalian serum. The capture and release events of a protein analyte by the tethered protein bait occur outside the nanopore and are accompanied by uniform current openings. Conversely, nonspecific pore penetrations by nontarget components of serum, which take place inside the nanopore, are featured by irregular current blockades. As a result of this unique peculiarity of the readout between specific protein captures and nonspecific pore penetration events, our selective sensor can quantitatively sample proteins at single-molecule precision in a manner distinctive from those employed by prevailing methods. Because our sensor can be integrated into nanofluidic devices and coupled with high-throughput technologies, our approach will have a transformative impact in protein identification and quantification in clinical isolates for disease prognostics and diagnostics.

Keywords: FhuA; electrophysiology; ion channel; membrane protein engineering; protein dynamics; protein−protein interface; stochastic sensing.

Figure 1:. Single-molecule protein detection at single-tethered receptor resolution.

(a) Stochastic protein sensing of barstar (Bs) protein using OBn(GGS)₂t-FhuA. This protein sensor encompasses a truncated t-FhuA protein pore, a short (GGS)₂ hexapeptide tether, a barnase (Bn) protein receptor, and a dodecapeptide adapter (O). This model was generated in Pymol using the pdb files 1BY3 and 1BRS for (FhuA) and (Bn-Bs), respectively. (b) Representative single-channel electrical traces of OBn(GGS)₂t-FhuA in the presence of 12.63 nM Bs (top) and 50.5 nM Bs (bottom) at an applied transmembrane potential of −40 mV. The control experiment in the absence of Bs is shown in Supporting Information, Fig. S1. Single-channel electrical traces were further processed using a 20 Hz low-pass 8-pole Bessel filter. O_on indicates the Bs-released open substate; O_off shows the Bs-captured open substate. (c) Representative single-channel electrical traces of OBn(GGS)₂t-FhuA in the presence of 12.63 nM Bs (top) and 50.5 nM Bs (bottom) at an applied transmembrane potential of +15 mV. The control experiment in the absence of Bs is shown in Supporting Information, Fig. S6. Single-channel electrical traces were further processed using a 20 Hz low-pass 8-pole Bessel filter. O_on and O_off have the same meanings as those stated in (b). (d) Both diagrams show dependence of 1/τ_on (left) and 1/τ_off (right) on Bs concentration, [Bs]. Here, these kinetic rate constants in the form of mean ± s.e.m. are k_on = (1.59 ± 0.09) × 10⁷ M⁻¹s⁻¹ and k_off = 0.95 ± 0.02 s⁻¹. Data points in both panels represent mean ± s.d. obtained from n distinct experiments. In this case, n was 5, 3, 3, and 4, for a [Bs] of 12.63, 25.25, 50.5, and 100.01 nM, respectively. Experimental conditions in (d) were the same as those stated in (c).

Figure 2:. Single-molecule protein detection at single-tethered receptor resolution in unprocessed fetal bovine serum (FBS).

(a) Schematic representation of stochastic protein sensing of Bs using OBn(GGS)₂t-FhuA in the presence of FBS. (b) Representative single-channel electrical traces of OBn(GGS)₂t-FhuA in 5% (v/v) FBS and in the presence of 12.63 nM Bs (top) and 50.5 nM Bs (bottom). Experiments were conducted at an applied transmembrane potential of +15 mV. Single-channel electrical traces were further processed using a 20 Hz low-pass 8-pole Bessel filter. Bs was added to the cis side of the chamber. O_on and O_off stand for the same meanings as in Fig. 1a. Single-molecule protein captures are indicated by upwards current transitions (to O_off) from basal current level (O_on) of OBn(GGS)₂t-FhuA. ΔI_FBS represents spectrum of FBS-induced current transitions either departed from either O_on or O_off substate. (c) Representative standard histograms of the release (τ_on; left) and capture (τ_off; right) durations at 12.63 nM Bs. The τ_on and τ_off values obtained from the fits (mean ± s.e.m.) were 4,188 ± 429 ms (n = 96) and 1,220 ± 90 ms (n = 97), respectively. (d) Representative standard histograms of the release (τ_on; left) and capture (τ_off; right) durations at 50.5 nM Bs. The τ_on and τ_off values obtained from the fits were 1,150 ± 57 ms (n = 239) and 1,223 ± 25 ms (n = 243), respectively. Experimental conditions in (c) and (d) were the same as in (b). (e) Dependence of 1/τ_on (left) and 1/τ_off (right) on [Bs] when measurements were conducted in 5% FBS. Here, k_on = (1.67 ± 0.09) × 10⁷ M⁻¹s⁻¹ and k_off = 0.86 ± 0.03 s⁻¹. Data points in both panels represent mean ± s.d. obtained from n = 3 distinct experiments. (f) Event dwell time histograms of FBS-induced current blockades in 5% (v/v) FBS as well as in the presence of 12.63 nM Bs (left) and 50.5 nM Bs (right). The numbers of FBS-induced events were 3,040 and 5,007, respectively. The best-fit model was a multiexponential function with four terms (Supporting Information, Tables S4–S5). (g) Event probability (left) and dwell time (right) values of FBS-induced current blockades (FBS_{event x}) are independent of [Bs]. Data points in both panels represent mean ± s.d. obtained from n = 3 distinct experiments. Experimental conditions in panels (c) - (g) were the same as those stated in (b).

Figure 3:. Single-molecule protein detection in HI-FBS.

(a) Single-channel electrical traces of OBn(GGS)₂t-FhuA in either 5% (v/v) (top) or 10% (bottom) HI-FBS and in presence of 12.63 nM Bs. The applied transmembrane potential was +15 mV. Single-channel electrical traces were further processed using a 20 Hz low-pass 8-pole Bessel filter. Bs was added to the cis side of the chamber. O_on and O_off have the same meanings as in Fig. 1a. ΔI_HI-FBS represents spectrum of HI-FBS-induced current transitions. (b) Dependence of 1/τ_on (left) and 1/τ_off (right) on the HI-FBS concentration. Linear fits of both plots provide evidence that the average of 1/τ_on and 1/τ_off of the Bs captures at 12.63 nM Bs are independent of the tested HI-FBS concentration. Here, 1/τ_on = 0.227 ± 0.014 s⁻¹ and 1/τ_off =0.934 ± 0.030 s⁻¹. Data points represent mean ± s.d. obtained from n = 3 distinct experiments. Experimental conditions in panel (b) were the same as those stated in (a). (c) Dwell time histograms of HI-FBS-induced current blockades in either 5% (left) or 10% HI-FBS (right), and in the presence of 12.63 nM Bs. The number of HI-FBS-induced events were 42 and 515, respectively. For the top graph, the best fit model was a double-exponential distribution function with corresponding dwell time and probability values (mean ± s.e.m.), as follows: τ_off-1 = 0.33 ± 0.23 ms, P₁ = 0.88 ± 0.14, τ_off-2 = 16.57 ± 2.09 ms; P₂ = 0.12 ± 0.13. For the bottom graph, the best fit model was a four-exponential distribution function with corresponding dwell time and probability values (mean ± s.e.m.), as follows: τ_off-1 = 1.06 ± 0.33 ms, P₁ = 0.63 ± 0.39, τ_off-2 = 2.93 ± 1.74 ms, P₂ = 0.19 ± 0.31, τ_off-3 = 13.67 ± 1.14 ms, P₃ = 0.13 ± 0.13, τ_off-4 = 131.28 ± 1.19 ms, P₄ = 0.06 ± 0.05.