Abstract
Here we report a protocol to investigate the electron-transfer processes of redox-active biomolecules in biological membranes by electrochemistry using biomimetic hybrid bilayer membranes (HBMs) assembled on gold electrodes. Redox-active head groups, such as the ubiquinone moiety, are embedded in HBMs that contain target molecules, e.g., nicotinamide adenine dinucleotide (NADH). By using this approach, the electron-transfer processes between redox molecules and target biomolecules are mediated by mimicking the redox cycling processes in a natural membrane. Also included is a procedure for in situ surface-enhanced Raman scattering (SERS) to confirm the electrochemically induced conformational changes of the target biomolecules in the HBMs. In addition, each step in constructing the HBMs is characterized by electrochemical impedance spectroscopy (EIS), high-resolution X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The time required for the entire protocol is ∼12 h, whereas the electrochemical measurement of electron-transfer processes takes less than 1 h to complete.
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Acknowledgements
This research was supported by the National Science Fund for Distinguished Young Scholars (21125522), the Major Research Plan of the National Natural Science Foundation of China (91027035), the Fundamental Research Funds for the Central Universities (WK1013002) and the 973 Program (2013CB733700).
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W.M. and Y.-T.L. designed and performed all the experiments and analyzed the data. W.M., Y.L.Y., L.X.Q. and H.Z. wrote the manuscript. Z.G. drew and summarized the figures. T.C.S., H.Z., D.-W.L. and H.-Y.C. finalized the preparation of the manuscript. Y.-T.L. supervised all of the work. All authors discussed the results and commented on the manuscript.
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Supplementary information
Supplementary Figure 1
Cyclic voltammetry of 0.5 mM ubiquinone 0 obtained on a gold electrode in 0.1 M PBS buffer of pH 7.0 at 25 °C. Scan rate is 100 mV s−1. Arrow indicates initial scan direction. The initial scan started from -0.6 V vs. SCE. The voltammetric data is from a multi-cycle experiment. (PDF 276 kb)
Supplementary Figure 2
Cyclic voltammograms of the ubiquinone embedded HBMs system in which NADH or NAD+ was not successfully immobilized in a lipid membrane in 0.1 M PBS buffer of pH 7.0.Q1S-embedded HBMs system (a), Q5S-embedded HBMs system (b), and Q10S-embedded HBMs system (c). Scan rate is 100 mV s−1. Arrow indicates initial scan direction. The initial scan started from -0.50 V vs. SCE for Q1S-embedded HBMs system, -0.60 V vs. SCE for Q5S-embedded HBMs system, and -0.60 V vs. SCE for Q10S-embedded HBMs system. The voltammetric data is from a multi-cycle experiment. (PDF 235 kb)
Supplementary Figure 3
Voltammetric responses of the hexanethiol-embedded HBMs system in the absence (black line) and presence (red line) NADH in lipid membrane in 0.1M PBS buffer of pH 7.0 under nitrogen atmosphere. Arrow indicates initial scan direction. The initial scan started from -0.30 V vs. SCE. The voltammetric data is from a multi-cycle experiment. (Modified with permission from ref. 5). (PDF 229 kb)
Supplementary Figure 4
Voltammetric responses of ubiquinone-embedded HBMs systems (QnS-HBMs) immersed in PBS buffer (pH 7.0) with (red line)/without (black line) NADH under nitrogen atmosphere. (A): Q1S-HBMs (B): Q5S-HBMs; (C): Q10S-HBMs. Arrow indicates initial scan direction. The initial scan started from -0.35 V vs. SCE for Q1S-HBMs system, -0.70 V vs. SCE for Q5S-HBMs system, and -0.70 V vs. SCE for Q10S-HBMs system. The voltammetric data is from a multi-cycle experiment. (Modified with permission from ref. 5). (PDF 91 kb)
Supplementary Figure 5
Nyquist plot for the Faradaic impedance before and after formation of HBMs (with and without NADH) measured on a ubiquinone-functionalized gold electrode. The spectra were recorded in 0.1 M PBS containing 1 mM [Fe(CN)6] 3−/4− as a redox probe, using a frequency range from 100 kHz to 0.1Hz with 10 mV excitation signal. The ESI was measured at formal potential of [Fe(CN)6] 3−/4− vs. SCE. The measured potentials were 0.220 V vs. SCE, 0.214 V vs. SCE, and 0.210 V vs. SCE for Q1S-SAMs, Q1S-HBMs, and Q1S-HBMs-NADH, respectively; The measured potential were 0.225 V vs. SCE, 0.234 V vs. SCE, and 0.236 V vs. SCE for Q5S-SAMs, Q5S-HBMs, and Q5S-HBMs-NADH, respectively; The measured potential were 0.220 V vs. SCE, 0.244 V vs. SCE, and 0.240 V vs. SCE for Q10S-SAMs, Q10S-HBMs, and Q10S-HBMs-NADH, respectively. (Reproduced with permission from ref.5). (PDF 346 kb)
Supplementary Figure 6
AFM images show significant changes in the microscopic features on the electrode surfaces. Shown are (A):Q5S-SAMs; (B):Q5S-HBMs; (C):Q5S-HBMs-NADH. (Reproduced with permission from ref. 5). (PDF 316 kb)
Supplementary Figure 7
High-resolution XPS spectra of S2P for QnS-SAMs, QnS-HBMs, and QnS-HBMs-NADH. Open circles represent experimental raw data, red solid lines are for the total fits, black lines are for the component-fitted peaks, and green lines are for the baselines. (Reproduced with permission from ref. 5). (PDF 306 kb)
Supplementary Figure 8
High-resolution XPS spectra of P2P for QnS-SAMs, QnS-HBMs, and QnS-HBMs-NADH. Open circles represent experimental raw data, red solid lines are for the total fits, black lines are for the component-fitted peaks, and green lines are for the baselines. (Reproduced with permission from ref. 5). (PDF 82 kb)
Supplementary Figure 9
High-resolution XPS spectra of N1s for QnS-SAMs, QnS-HBMs, and QnS-HBMs-NADH. Open circles represent experimental raw data, red solid lines are for the total fits, black lines are for the component-fitted peaks, and green lines are for the baselines. The data of N1s are fit with three components: the quaternary ammonium from lipid of egg phosphatidyl choline (N+, ∼402.5 eV), the non-conjugated nitrogen (∼400.6 eV) and the conjugated nitrogen (∼399.0 eV) from NADH. (Reproduced with permission from ref. 5). (PDF 91 kb)
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Ma, W., Ying, YL., Qin, LX. et al. Investigating electron-transfer processes using a biomimetic hybrid bilayer membrane system. Nat Protoc 8, 439–450 (2013). https://doi.org/10.1038/nprot.2013.007
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DOI: https://doi.org/10.1038/nprot.2013.007
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