Structural-functional analysis of engineered protein-nanoparticle assemblies using graphene microelectrodes

Abstract

The characterization of protein-nanoparticle assemblies in solution remains a challenge. We demonstrate a technique based on a graphene microelectrode for structural-functional analysis of model systems composed of nanoparticles enclosed in open-pore and closed-pore ferritin molecules. The method readily resolves the difference in accessibility of the enclosed nanoparticle for charge transfer and offers the prospect for quantitative analysis of pore-mediated transport, while shedding light on the spatial orientation of the protein subunits on the nanoparticle surface, faster and with higher sensitivity than conventional catalysis methods.

Fig. 1. (a) Schematic of the setup for measuring spontaneous faradaic charge transfer across a pore to a graphene microelectrode in buffer solution and circuit diagram. (b) Faradaic current as a function of electrostatic potential in the buffer solution above graphene. The red line is a linear fit to the data.

Fig. 2. (a) Time trace of the charge transfer between a graphene microelectrode and mutant A. fulgidus E65R ferritin solution (AfFtn-R), and wild-type A. fulgidus ferritin solution (AfFtn). The gray-level of the data plot increases with the solution ionic strength. (b) Faradaic current for AfFtn-R (black circles) and AfFtn (red squares) based on the data in panel (a). The black curve is a fit to the data for AfFtn-R using ESI eqn (1S). (c) Faradaic current difference for AfFtn compared to AfFtn-R; the red curve is a fit based on ESI eqn (2S).

Fig. 3. (a) Difference in faradaic current for solutions of AuNP-enclosed in A. fulgidus mutant ferritin K150A/R151A (AuNP-AfFtn-AA, blue triangles) and AuNP-enclosed in wild-type ferritin (AuNP-AfFtn, red squares) compared to the baseline current set by a solution of E65R ferritin (AfFtn-R). Two data points, which almost overlap with each other, were tested at ionic strength of 340 mM for both samples. (b) Faradaic current difference for AuNP-AfFtn-AA (blue triangles) and AuNP-AfFtn (red squares) as a function of the faradaic current for AuNP. The lines are linear fits to the data. (c) Charge-transfer efficiency ξ as a function of ionic strength fitted by the formula for the model based on electrical double layer.

Fig. 4. (a) Real-time fluorescence intensity of I-BODIPY dehalogenation catalyzed by AuNP-AfFtn-AA (blue triangles) and AuNP-AfFtn (red squares) solutions. For each measurement, 10 nM AuNP-AfFtn and 1 μM I-BODIPY were mixed in 50 mM HEPES, pH 7.0 (100 μL total volume). (b) Real-time UV-visible spectroscopy of reduction of 4-nitrophenol catalyzed by AuNP-AfFtn-AA and AuNP-AfFtn solutions. For each measurement, 5 nM AuNP-AfFtn and 50 μM 4-nitrophenol were mixed in a cuvette. Freshly prepared aqueous NaBH₄ was added to a final concentration of 2.5 mM and total sample volume of 1 mL. The solid curves are fits based on first-order kinetics. The corresponding catalytic reactions are shown in panels (a and b).