Protein self-assembly onto nanodots leads to formation of conductive bio-based hybrids

The next generation of nanowires that could advance the integration of functional nanosystems into synthetic applications from photocatalysis to optical devices need to demonstrate increased ability to promote electron transfer at their interfaces while ensuring optimum quantum confinement. Herein we used the biological recognition and the self-assembly properties of tubulin, a protein involved in building the filaments of cellular microtubules, to create stable, free standing and conductive sulfur-doped carbon nanodots-based conductive bio-hybrids. The physical and chemical properties (e.g., composition, morphology, diameter etc.) of such user-synthesized hybrids were investigated using atomic and spectroscopic techniques, while the electron transfer rate was estimated using peak currents formed during voltammetry scanning. Our results demonstrate the ability to create individually hybrid nanowires capable to reduce energy losses; such hybrids could possibly be used in the future for the advancement and implementation into nanometer-scale functional devices.


Characterization of S-doped C-dots
The morphology of as-prepared S-doped C-dots was investigated using high-resolution transmission electron microscopy (HRTEM; Model JEM-2100). First, the samples (1 mL of purified S-doped C-dots solution was diluted into 10 mL of water), dropped onto copper wire meshes, and dried for 20 min at 55 o C. An accelerated voltage of 200 KV was used for analyses. Sample's sizes are revealed in Figure S1. Figure S1: HRTEM images of S-doped C-dots. Scale bars: a) 50 nm, b) 10 nm.

High resolution AFM image of the S-doped C-dots
In order to provide details of the hybrids structure, high-resolution AFM images were obtained. The experimental details were exactly the same as described in the manuscript. Briefly, contact mode Atomic Force Microscopy (AFM, Asylum Research, USA) with a silicon nitride tip (TR-400PB, Asylum Research, USA) in solution was used. The trigger force was kept constant at 3 nN while the spring constant of the cantilever was measured before each experiment using established method. Figure S2: High resolution AFM image of the S-doped C-dots hybridized microtubule showing S-doped C-dots incorporated into the polymerized hybrid as a bead-like geometry.
For the electrochemical impedance spectroscopy (EIS) analyses, 20 μL of the obtained "microtubule-chitosan" solution was dropped onto a cleaned electrode and incubated overnight under vacuum (same procedure as listed in the materials and methods). The EIS analyses have been performed in 50 mM potassium ferricyanide (K 3 Fe(CN) 6 ; Fisher Scientific, USA) in BRB80 buffer containing 10 μM taxol. The supporting electrolyte used in our experiments is 10 mM NaCl.
Analyses reveal no major differences between the two curves ( Figure S3).

Evaluate the change in impedance on the chitosan membrane
For the bio-hybrid synthesis, also called hybrid microtubule, first biotin-tubulin-S-doped C-dots-conjugates were formed using non-specific binding of biotin-tubulin onto S-doped C-dots scaffolds as described in the paper. Briefly, 1 μL 6 mg/mL synthesized S-doped C-dots were injected into a 600 μL eppendorf tube containing 5 μL of 4 mg/mL biotin-tubulin and the mixture was incubated for 2 h at 200 rpm in an ice bath. Subsequently, 5 μL of 4 mg/mL free biotin-tubulin was mixed with the biotin functionalized tubulin-S-doped C-dots conjugates and an additional 2.5 μL microtubule polymerization solution, and subjected to 37 o C for 30 min. When time elapsed, the hybrids were stabilized in BRB80 buffer containing 10 μM taxol. Synthesized S-doped C-dots hybridized microtubules were spun down using high-speed centrifuge (30000 rpm for 10 min at room temperature). The supernatant was removed carefully and the pellet was re-suspended in 1 mL BRB80 buffer containing 10 μM taxol.
The synthesized microtubule and hybrids (before and after the centrifugation) were immobilized onto the electrode using the methods described in the manuscript. EIS analyses are presented in Figure S4; minor changes (not statistically relevant) have been observed most likely associated with changes in the microtubule/hybrid length known to occur because of the mechanical stress imposed by centrifugation. Figure S4: EIS graph of the modified electrode. Black curve: Microtubule/Chitosan/Au. Red curve: S-doped C-dots hybridized microtubule/Chitosan/Au. Blue curve, Centrifuged S-doped C-dots hybridized microtubule/Chitosan/Au.