Metallization of Targeted Protein Assemblies in Cell‐Derived Extracellular Matrix by Antibody‐Guided Biotemplating

Abstract Biological systems are composed of hierarchical structures made of a large number of proteins. These structures are highly sophisticated and challenging to replicate using artificial synthesis methods. To exploit these structures in materials science, biotemplating is used to achieve biocomposites that accurately mimic biological structures and impart functionality of inorganic materials, including electrical conductivity. However, the biological scaffolds used in previous studies are limited to stereotypical and simple morphologies with little synthetic diversity because of a lack of control over their morphologies. This study proposes that the specific protein assemblies within the cell‐derived extracellular matrix (ECM), whose morphological features are widely tailorable, can be employed as versatile biotemplates. In a typical procedure, a fibrillar assembly of fibronectin—a constituent protein of the ECM—is metalized through an antibody‐guided biotemplating approach. Specifically, the antibody‐bearing nanogold is attached to the fibronectin through antibody–antigen interactions, and then metals are grown on the nanogold acting as a seed. The biomimetic structure can be adapted for hydrogen production and sensing after improving its electrical conductivity through thermal sintering or additional metal growth. This study demonstrates that cell‐derived ECM can be an attractive option for addressing the diversity limitation of a conventional biotemplate.

(B) Cyclic voltammograms were acquired in a non-faradaic region at different scan rates using Au@Pt-ECM (left panel).By plotting the anodic current at 0.12 V (vs.RHE) as a function of scan rate, the double-layer capacitance was calculated (right panel) to be 3.51 mF cm -2 .Figure S14.The chronopotentiometry measurement of Au@Pt-ECM to acquire 10 mA cm -2 as a function of time.S4.The information about primary and secondary antibodies and their diluted concentrations.In the case of initial concentration could not be confirmed, only the dilution ratio was noted.
Jae-Byum Chang* Movie S1.Confocal microscopy z-stack image after expansion.Movie S2. 3D visualization of the z-stack image shown in Movie S1.

Figure S1 .
Figure S1.SEM images of ECM after decellularization.Negative staining with 2% uranyl acetate was applied to highlight the dECM in an electron microscope.The bottom is a magnified image of the yellow box.

Figure S2 .
Figure S2.Confirmation of AuNP development on different metal NPs acting as seeds using high-resolution TEM images.(A) PtNPs (Sigma-Aldrich, 773875) with a diameter of about 3 nm.(B) AuNP was grown on PtNP through the catalytic reduction effect of a metal surface.

Figure S3 .
Figure S3.Fluorescence images of fibronectin structures in different cell lines and SEM images of metallic structures mimicking them through antibody-guided biotemplating.(A, C) The observed fibronectin structures were derived from monkey kidney epithelial cells (BS-C-1) and (B, D) human embryonic kidney cells (HEK-293).We performed the experiments without decellularization when each type of cell reached full confluency.

Figure S4 .
Figure S4.The size variation of grown AuNPs per the number of Au growth.(A-D) Four pairs of SEM images of metalized dECM along with fibronectin from one to four times Au growth.As shown in the inset digital images, the color of the metalized dECM darkened as the number of Au growth increased.(E-F) Diameter distribution of AuNPs after one (E) and four (F) times of Au development.

Figure S5 .
Figure S5.Compositional analysis of synthesized dECM after Au growth.(A) XRD patterns of the metalized dECM and pristine dECM.(B) High-angle annular dark-field (HAADF) image, EDS map, and corresponding spectrum of a single strand AuNPs-array that imitated the fibronectin structure.

Figure S6 .
Figure S6.Fluorescence images showing the structures of three different types of proteins made of dECM and SEM images of AuNP-assemblies templated by each protein.(A, D) Collagen type Ⅰ was found to have an intertwined fibrous mesh structure with micro-sized pores, similar to fibronectin in the fluorescence image, but AuNP-fibrous assemblies were identified with a low labeling density of AuNPs in the SEM image.(B, E) Laminin exhibited disconnected structures in both the fluorescence and SEM images.(C, F) Elastin also showed a fibrous structure in the dECM, but the fluorescence images showed a weak signal, which was corroborated by the low labeling density of AuNPs in the SEM image.

Figure S7 .
Figure S7.Comparison of the labeling density according to the size of AuNPs conjugated with the secondary antibody.(A) Illustration of an experiment using a secondary antibody-bearing 10 nm colloidal AuNPs.(B) In the fluorescence image, the fibronectin structure was clearly visible in the use of the secondary antibody-bearing 10 nm colloidal AuNPs.(C, D) Nanoscale measurements using STEM verified that the labeling density of the 10 nm colloidal AuNPs was insufficient due to the low staining efficiency of the secondary antibody containing the 10 nm colloidal AuNPs.(E, F) In contrast, a high labeling density of AuNPs was ensured using secondary antibody-bearing nanogold.To accurately compare with (C, D), slight Au growth was applied to the nanogold, growing their size to about 15 nm, and measurements were taken at the same magnification as in (C, D).

Figure S8 .
Figure S8.(A, B) Low and high magnification SEM images of Au-ECM sintered at 250 ℃. (C, D) Low and high magnification SEM images of Au-ECM sintered at 450 ℃.

Figure S9 .
Figure S9.Electrical conductivity of dECM treated with thermal sintering at 350 ℃. (A) Bright-field images of the interface region between the Au electrodes (left panel), and an enlargement of the red box (right panel).(B) The current-voltage curve of thermally sintered dECM.

Figure S10 .
Figure S10.Measurement of the thickness of Au-ECM sintered at 350 ℃ using a surface profiler.The fluctuating graph originated from the fibrous structure of metalized dECM.

Figure S11 .
Figure S11.Zeta potential measurement of AuNPs and UV-Vis spectrum of H2PtCl6•6H2O in D.I. (A) The zeta potential of AuNPs was measured as a function of solution pH, revealing that the AuNPs had a positive charge in the synthesis condition.The AuNPs used to measure the zeta potential were obtained through electroless plating of Au on a 1.4 nm nanogold not conjugated with antibodies (Nanoprobes, #2025).(B) The generation of PtCl6 2-was verifiedby the appearance of peaks at wavelength of 200 and 260 nm.[1]

Figure S12 .
Figure S12.XRD pattern of Au@Pt-ECM.The weak peaks marked by red stars indicated that a Pt shell was formed.

Figure S16 .
Figure S16.As observed by SEM and EDS maps, the formation of Au@PdNP could not be achieved when [PdCl(OH2)3] + was used as a Pd precursor due to the electrostatic repulsion between the positively charged AuNP surface and positively charged Pd complex ions.

Figure S17 .
Figure S17.Measurement of the thickness of Pd-ECM and PdxPt1-x-ECM using a surface profiler.

Figure S18 .
Figure S18.The dissociated PdNPs from Pd-ECM and their hydrogen gas sensing performance.(A) SEM images of the dissociated PdNPs.(B) Response results of the drop-casted PdNPs on sensing substrate toward different concentrations of hydrogen.

Figure S21 .
Figure S21.Fluorescence images of several ECM protein assemblies in different mouse organ slices.As illustrated in the fluorescence images, a variety of morphologies, including (A) vascular, (B) porous, and (C) fibrous structures, existed in mouse organ-derived ECM.

Figure S22 .
Figure S22.Super-resolution volumetric imaging of mouse brain slices via expansion microscopy.(A) 3D view of an expanded mouse brain slice (blue: DAPI, red: TH, green: MAP2).The size of the image was 82 μm by 82 μm by 80 μm before expansion.(B-D) Singleplane confocal microscopy image of the focal planes of the specimen shown in (A).(E) Magnified view of the 3D visualization in (A).The length scales are presented in pre-expansion dimensions.DAPI: 4′,6-Diamidino-2-Phenylindole; TH: Tyrosine Hydroxylase.

Table S1 .
Summary of the electrical conductivity of metalized dECM in this study.Data are presented as mean ± SD, n = 3 (each independent experiment).

Table S2 .
Performance comparison of the existing Pt-based catalysts for hydrogen evolution reaction.

Table S3 .
Performance comparison of the existing Pd-based hydrogen sensing materials.