An embryo of protocells: The capsule of graphene with selective ion channels

The synthesis of artificial cell is a route for searching the origin of protocell. Here, we create a novel cell model of graphene capsules with selective ion channels, indicating that graphene might be an embryo of protocell membrane. Firstly, we found that the highly oxidized graphene and phospholipid-graphene oxide composite would curl into capsules under a strongly acidic saturated solution of heavy metallic salt solution at low temperature. Secondly, L-amino acids exhibited higher reactivity than D-amino acids on graphene oxides to form peptides, and the formed peptides in the influence of graphene would be transformed into a secondary structure, promoting the formation of left-handed proteins. Lastly, monolayer nanoporous graphene, prepared by unfocused 84Kr25+, has a high selectivity for permeation of the monovalent metal ions ( Rb+ > K+ > Cs+ > Na+ > Li+, based on permeation concentration), but does not allow Cl- go through. It is similar to K+ channels, which would cause an influx of K+ into capsule of graphene with the increase of pH in the primitive ocean, creating a suitable inner condition for the origin of life. Therefore, we built a model cell of graphene, which would provide a route for reproducing the origin of life.


External Databases Formation mechanism of CGOs
GOs contain high levels of sp2 carbons, such that the oxygen level is low. To generate a high concentration of sp3 carbon in GOs, the oxygen level must be increased. Therefore, the graphite was oxidized for different durations (from 8 to 70 h) to prepare GOs HOGOs with high oxygen levels through an improved Hummers method 1 . GO and HOGOs characterization experiments were performed through TEM, Raman spectroscopy, FTIR, Potentiometric titration curve, and XPS (Figs. S1-2). The results showed that HOGOs with more sp3 of carbons contained higher oxygen levels (Fig. S2). More wrinkles were observed on HOGOs because of the higher oxygen levels (Fig. S1). Another GO with a high level of oxygen was prepared through chemical oxidation, similar to the control group.
10 mg of HOGO (GOs-70 h) was dispersed in 2 mL of water, and ultrasonicated for 30 min. Two 5 mg samples of Pb(NO 3 ) 2 were added into the HOGO dispersion (5g/L) at 4 and 50 °C, respectively. The dispersion was then observed using TEM after a strong shock. The results showed that CGOs could only be formed in the saturated Pb(NO 3 ) 2 or Co(NO 3 ) 2 solution at 4 °C (Fig. S6). Even if CGOs were formed, they would still disappear after re-dilution. Meanwhile, the SEM and TEM analysis results showed that no cubic crystals of Pb(NO 3 ) 2 were observed in CGOs, and a large number of ~20 nm spheroids were found (Fig. S3a). These results showed that the CGO formation strongly depended on the temperature and Pb(NO 3 ) 2 concentration. In addition, Pb(NO 3 ) 2 was not detected inside CGOs, showing that CGOs could form in solution under these conditions. Therefore, Pb(NO 3 ) 2 was adsorbed on the outside surface of CGOs after entering the solution, and crystallized on the outside surface, thus fixing the shape of CGOs during the sample preparation for TEM analysis. Fig. S4c shows that CGOs were observed in HOGOs prepared through chemical oxidation, demonstrating that the CGO formation is influenced by the GO oxygen level. solutions. Researchers have reported that Pb(NO 3 ) 2 can adsorb onto the GO carboxyl groups to prepare RGO-supported palladium nanoparticles 2 . Therefore, CGO formation could be attributed to the strong Pb(NO 3 ) 2 coordination ability. CGO formation also depended on the adsorption time and the oxygen level. However, Fig.   S7 shows that the ultrasonication time could affect the shape of CGOs. We found that Pb(NO 3 ) 2 was wrapped by the GOs (Fig. S7) and evenly distributed in the inside portions of GOs at high radiation doses (Fig. S8). Previous studies have confirmed that more Pb(NO 3 ) 2 would adsorb onto the surface of GOs with higher oxygen levels.
This effect causes more Pb(NO 3 ) 2 molecules to be distributed in the inner surface of CGOs, which changes the CGO shape.
In summary, these results showed that GOs with higher oxygen levels could not uniformly disperse in aqueous solutions. As the temperature decreases, GOs would roll into capsules with lower surface energies. To observe the CGO shape through TEM is difficult, and the salting-out of Pb(NO 3 ) 2 could fix the CGO shape such that it could be observed through TEM. However, when CGOs are unstable, CGOs would open again with increasing temperature.

TGA curve of GO-arginine and GO-proline in nitrogen flow
There was an obvious weight loss process in 150-200 o C, which is caused by the pyrolysis of oxygen containing functional groups 3,4 . However, the weight loss process caused by nitrogen-containing groups is not obvious 5 . So, the weight process in 150-200 o C could be used to represent the content of oxygen groups, and the higher degree of weight loss in 150-200 o C shows more oxygen groups and fewer nitrogen groups. Thus, we could use the slope of curves (K) to represent the degree of weight loss. Compared with GO-amino acids, K of GOs is the biggest (K A1 =K B1 =0.170). The K of GO-D-amino acids (K A2 =K B2 =0.060) is higher than that of GO-L-amino acids (K A2 =0.053, K B3 =0.050), which could be due to the higher oxygen content of GO-D-amino acids. The weight loss curve is odd for GO-L-proline. The brief increase of weight could be observed in 450-550 o C, which might be due to the adsorption of N 2 or density changes caused by the N 2 flow on GOs. Moreover, the weight loss process of 550-700 o C is due to the desorption of L-proline and 4 sp 2 -hybridzied carbon atoms 6 .

Preparation of permeation equipment
The filter equipment was successfully designed and prepared according to experimental requirements (Fig. S10). The two MNGM-PET sides have different physical and chemical properties. The PET side is unstable under basic conditions, and the PET membrane may affect the metal ion permeation, such that the metal ions with high pH would be poured into the sink close to the PET side. This sink was referred to as the permeation sink. HCl was used as the driving liquid, and was poured into the sink close to the graphene side.

Selective permeation of differential valence metal ions
KCl, CaCl 2 , and FeCl 3 , and NaCl, MgCl 2 , and FeCl 3 (0.1 mol/L per ion, pH 2.5) were, respectively, divided into two groups. The filtration experiments of the two groups were performed using the above methods, and all ions were ranked in a filter sequence. The filter sequence of the metal ions with different valence is K + > Fe 3+ > Na + > Ca 2+ , Mg 2+ (Fig. S11). This sequence could be attributed to the properties of each ion. Fe 3+ has a higher charge, and is a large hydrated ion. Mg 2+ and Ca 2+ are lower down the sequence relative to Na + , and this result could also be attributed to the lower mobility and larger hydrated ionic radius (Tables S1 and S2).

Effect of pH gradient on permeation
Considering that the filtering capability of K + is the strongest among all metal ions, this ion was used to study the effect of the pH gradient on permeation. The results showed that the permeation strongly depended on the pH gradient (Fig. 14), indicating that high H + concentrations could enhance metal ion permeation, which is ascribed to ion exchange. The poor performance of the MNGM-PET and PET membranes resulted from the shorter filtration time.

Simulation of the permeation process using the law of Poisewille before 30 s
What pattern could be used to describe the pattern of rapid filtration before 30 s?
Supposing that permeation is similar to liquid flow across tubes, and the flow state is coupled with the law of Poisewille, then K + transport across the conical hole could be measured through Poisewille formula 7 for liquid laminar flow. 5 Based on the Poisewille formula given as: = π 4 8 , where r is the radius of tube; Δp is difference between the pressures; η is solution viscosity; and L is tube length. As the tube has conical holes, the Q of the flow could be represented as:

Permeation of ions through PET membrane
Several works have reported that the nuclear pore membrane could be used to selectively filter some ions [8][9][10] . Fig. 6 shows that K + and Cs + could be limitedly separated through a single PET membrane; however, Fig. S13 shows that ion selectivity was not observed for the metal ions with different valence. Fig. 5c shows that the PET membrane has nanopores with larger scales (small-scale is ~15 nm, large scale is ~50 nm) and an extremely long tube (~20 ì m). The PET tube membrane has a large number of carboxyl groups 11,12 , which induce strong interactions with the inner surface of the tube. In fact, this process is utilized in chromatographic columns for separating metal ions, and causes a certain degree of ion selectivity for K + and Cs + . As the adsorption of metal ions onto carboxyl groups increases, the adsorption rapidly reaches equilibrium. Therefore, a certain degree of ion selectivity for K + and Cs + before 30 s was achieved. However, this selectivity would change after 30 s. As the PET membrane nanopores are large (Fig. 1b), they could not be used as the "filter-tip"        The view highlights the network of aromatic ammo acids surrounding the selectivity filter. Tyroslne-78 from the selectivity filter (Y78) interacts through hydrogen bonding and van der Waals contacts with two Trp (W67,W68) from the pore helix.