Exploiting Saturation Regimes and Surface Effects to Tune Composite Design: Single Platelet Nanocomposites of Peptoid Nanosheets and CaCO3

Mineral-polymer composites found in nature exhibit exceptional structural properties essential to their function, and transferring these attributes to the synthetic design of functional materials holds promise across various sectors. Biomimetic fabrication of nanocomposites introduces new pathways for advanced material design and explores biomineralization strategies. This study presents a novel approach for producing single platelet nanocomposites composed of CaCO3 and biomimetic peptoid (N-substituted glycines) polymers, akin to the bricks found in the brick-and-mortar structure of nacre, the inner layer of certain mollusc shells. The significant aspect of the proposed strategy is the use of organic peptoid nanosheets as the scaffolds for brick formation, along with their controlled mineralization in solution. Here, we employ the B28 peptoid nanosheet as a scaffold, which readily forms free-floating zwitterionic bilayers in aqueous solution. The peptoid nanosheets were mineralized under consistent initial conditions (σcalcite = 1.2, pH 9.00), with variations in mixing conditions and supersaturation profiles over time aimed at controlling the final product. Nanosheets were mineralized in both feedback control experiments, where supersaturation was continuously replenished by titrant addition and in batch experiments without a feedback loop. Complete coverage of the nanosheet surface by amorphous calcium carbonate was achieved under specific conditions with feedback control mineralization, whereas vaterite was the primary CaCO3 phase observed after batch experiments. Thermodynamic calculations suggest that time-dependent supersaturation profiles as well as the spatial distribution of supersaturation are effective controls for tuning the mineralization extent and product. We anticipate that the control strategies outlined in this work can serve as a foundation for the advanced and scalable fabrication of nanocomposites as building blocks for nacre-mimetic and functional materials.


ζ-potential of peptoid nanosheets
To measure the ζ-potential of nanosheets, we prepared nanosheets in buffers ranging from pH 6-10 (Table S1). 1 We made the nanosheets in the rocker as explained in the main text and sonicated the nanosheets for 15 min in a sonication bath to decrease the sheet size.We used deionised water to prepare stock solutions of the buffers in Table S1 with all compounds purchased from Sigma Aldrich.We calibrated the pH meter (Metroholm) using standard buffers of pH 4,7 and 9 and adjusted the pH of the buffers using HCl or NaOH.
We dialysed the nanosheets overnight in 10 mM of each of the buffers using the dialysis kit from Spectra/Por® (Float-A-Lyser G2, MWCO 100 kD, 1 mL).We added 700 μL of the dialysed nanosheets to a disposable folded capillary cell and placed the cell in a Malvern Zetasizer Nano-ZS used to conduct the measurements.We equilibrated the sample at 20 °C for 300 s and conducted the electrophoretic mobility measurements with five repetitions per sample.After the experiment, we applied the Smoluchowski approximation to the data and obtained ζ-potential of the nanosheets (Fig. S1).
Table S1: Buffers used for ζ-potential measures in the pH interval of 6-10.

PHREEQC calculations for mineralization conditions
Table S2 shows the thermodynamic calculations for solution conditions used for mineralization experiments.
We calculated σ for the different calcium carbonate polymorphs using Ksp calcite = 10 -8.48 and Ksp vaterite = 10 -7.91 , and Ksp ACC = 10 -6.40 . 3,4 he calculated ion activities are given by {Ca 2+ } and {CO3 2-}.We ran all calculations without equilibrating with CO2 in air (Keeling curve 2018, 410 ppm, log(PCO2) = -3.39),which means the listed log(PCO2) in the table show that all solutions are undersaturated with respect to CO2.To minimise the effect of this, we sealed all experiments using parafilm and filled containers used during mineralization as much as possible during experiments.Lastly, Max precip shows the maximum precipitation given in mol/L when solutions are set to change form supersaturated (σcalcite = 1.2-1.4) to calcite saturated (σcalcite = 0.0), which gives some idea to maximum material fabricated.

Reference study of CaCO3 precipitation in standard feedback control setup
No nanosheets or seeds were added to this experiment.Supersaturated solutions at σcalcite = 1.2 were prepared and samples were taken after 15 min (before any titrant addition), after 3 and 5 hours by placing a sample droplet (approx.3 uL) on a Si wafer and letting it dry.At the end of the experiment, the solution was vacuum filtered (filter pore size 200 nm), and no particles could be detected on the filter.For the 15 min sample, calcite crystals of approx.6 μm precipitated (Fig. S6).Since no calcite crystals were found on the 200 nm filter at the end of the experiment, these crystals must be an effect of sample drying.
No calcite was seen for samples taken at 3 h and 5 h.Morphology is instead spherical here with smaller size scale.The polymorph was not investigated, yet, observed precipitates are believed to be drying artifacts due to lack of precipitation observed when solution was filtered.
Theoretical calculations of the amount of ACC that can form during drying was made by assuming evaporation of a 3 µL sampled solution at σcalcite = 1.2 to 1 µL.Supersaturation with respect to ACC upon evaporation was calculated to be =0.499and the theoretical amount of ACC that can precipitate was calculated to be 4.61E-04 mol/L.

Reference study of CaCO3 precipitation to investigate drying artifacts
In order to verify the source of precipitation observed on nanosheets and on wafer, further investigations were carried out by drop-casting small aliquots of supersaturated mineralization solution (σcalcite = 1.2 and pH 9.0) with and without nanosheets on silicon wafers and let dry.In the absence of sheets, SEM images showed precipitation of CaCO3 particles with typical vaterite and calcite morphology (Fig. S5).Drying of the supersaturated mineralization solution that contained 1 mM of nanosheets on wafer similarly resulted in observations of precipitation, yet, the nanosheets were not heavily decorated as observed for crystallization experiments conducted with the standard feedback-controlled setup (Fig. S6).Both large crystals with typical vaterite and calcite morphology, and small ACC-like particles were observed on the sheets.

Figure S1 :
Figure S1: The isoelectric point (IP) of the nanosheets is below pH 6.5, which means that the nanosheets at mineralisation conditions (pH 9) are negatively charged.As reference, the IP of single B28 polymers is also plotted in the figure.2

Figure S2 :
Figure S2: Illustration of the standard feedback control setup.The setup consists of a cone-shaped reaction vessel and the volume of the reaction solution is 112.5 mL.A mechanical stirrer is used to ensure solution mixing.A pH probe with 0.01-unit resolution is inserted in the reaction vessel to measure and record solution pH.Two titrant tubings are introduced into the reaction vessel from the same inlet for addition of titrant solutions of CaCl2 and Na2CO3 via a peristaltic pump, which is controlled by the detected pH changes due to calcium carbonate precipitation.

Figure S4 :
Figure S4: SEM images of samples collected from a standard feedback control experiment after 15 min, 3h and 5h.

Figure S6 :
Figure S6: (a-f) SEM images of samples formed on wafer by drying aliquots of supersaturated mineralization solution (σcalcite = 1.2 and pH 9.0) with 1 mM of nanosheets.(a,b,d) Large crystals with typical vaterite and calcite morphology were observed on the sheets as shown by red arrows.Nanosheets were not heavily mineralized upon drying.e) A region marked with the red circle shows ACC-like particles sparsely localized on the nanosheet surface, f) Image shows the marked region in (e) with a higher magnification.

Figure
Figure S9: a) EDXS of mineralized nanosheet in standard feedback control setup.The sheet is rich in C, O, and Ca with traces of Na and we also measure Si from the substrate.b) The cross in the image shows where the EDXS spectrum is measured.

Figure S10 :
Figure S10: SEM images of a) sample withdrawn after aging of nanosheets mineralized in standard feedback control setup showing decorated nanosheets and b) a higher magnification image focusing on a single sheet with vaterite mineralization.

Figure S12 :
Figure S12: SEM images of samples collected from modified feedback control experiments under poor mixing conditions and 200 mM titrant concentration without peptoid nanosheets at varying time points, a-c) 20 min, d) 35 min, e) 45 min, f) 65 min.

Table S2 :
PHREEQC of the final mixed solutions.