Thickness Determination and Control in Protein-Based Biomaterial Thin Films

Controlling the thickness and uniformity of biomaterial films is crucial for their application in various fields including sensing and bioelectronics. In this work, we investigated film assemblies of an engineered repeat protein—specifically, the consensus tetratricopeptide repeat (CTPR) protein —a system with unique robustness and tunability. We propose the use of microreflectance spectroscopy and apparent color inspection for the quick assessment of the thickness and uniformity of protein-based biomaterial films deposited on oxidized silicon substrates. Initially, we characterized the thickness of large, uniform, spin-coated protein films and compared the values obtained from microreflectance spectroscopy with those obtained from other typical methods, such as ellipsometry and atomic force microscopy. The excellent agreement between the results obtained from the different techniques validates the effectiveness of microreflectance as a fast, noninvasive, and affordable technique for determining the thickness of biomaterial films. Subsequently, we applied microreflectance spectroscopy to determine the thickness of drop-casted CTPR-based films prepared from small protein solution volumes, which present a smaller surface area and are less uniform compared to spin-coated samples. Additionally, we demonstrate the utility of apparent color inspection as a tool for assessing film uniformity. Finally, based on these results, we provide a calibration of film thickness as a function of the protein length and concentration for both spin-coated and drop-casted films, serving as a guide for the preparation of CTPR films with a specific thickness. Our results demonstrate the remarkable reproducibility of the CTPR film assembly, enabling the simple preparation of biomaterial films with precise thickness.


S2. Transfer-matrix method for optical contrast simulations
Following the work by Byrnes et al. [s1], we apply the transfer-matrix method to describe the propagation of light in the multilayer system constituted by the CTPR and the SiO2 films surrounded by two semi-infinite media, air, and Si, respectively (see sketch in Fig. S3).

Figure S3
. Schematics of light propagation in a multilayer system constituted by a semi-infinite air layer (layer 0), a CTPR thin film of thickness dCTPR (layer 1), an oxide layer of thickness dSiO2 (layer 2) and a semi-infinite silicon substrate (layer 3).r and t represent the reflection and transmission coefficients for the total system.
The system can be described by a matrix M relating the incident, the reflected and transmitted amplitudes: where r and t are the reflection and transmission coefficients for the whole system.
The matrix M can be written as the product of a series of matrices representing the reflection and transmission of light at each interface between layers and the propagation of light within each of the thin film layers.
The phase changes due to propagation within each layer are considered in where phase change di of light propagating in a layer of thickness di is given by di = 2pnidi/l, with l being the wavelength of light.
In our case, the transfer matrix for the system constituted by the four layers, sketched in Figure S3, is given by: Given the refractive index and thickness values for each layer, the reflection coefficient r can be calculated from eq. s1 as r = M10/M00.The reflectance R of the multilayer system is then given by R = |r| 2 .
Optical contrast simulations are performed by computing, for each wavelength value, the reflectance of the multilayer system containing the CTPR film (RCTPR) and the reflectance of the bare SiO2/Si substrate (RSi/SiO2).Optical contrast is calculated as C = (RCTPR -RSi/SiO2)/(RCTPR + RSi/SiO2).

S8. Film thickness as a function of concentration for different CTPR lengths.
Table S2.Thickness values for spin-coated CTPR films obtained from micro-reflectance measurements.Three separate samples were characterized at each concentration value.Microreflectance spectroscopy was evaluated at five different locations for each sample.The main thickness values are obtained from the average of all the measurements.Error is calculated from the standard deviation of results.

Micro-reflectance
Spin-Coating concentration (μM) CTPR 4 thickness (nm) CTPR 8 thickness (nm) CTPR 16 thickness (nm) Table S3.Thickness values for spin-coated CTPR films obtained from ellipsometry measurements.Three separate samples were characterized at each concentration value.The main thickness values are obtained from the average of all the measurements.Error is calculated from the standard deviation of results.

Ellipsometry
Spin-Coating concentration (μM) CTPR 4 thickness (nm) CTPR 8 thickness (nm) CTPR 16 thickness (nm) Table S4.Thickness values for drop-casted CTPR films obtained from micro-reflectance measurements.Three separate samples were characterized at each concentration value.Microreflectance spectroscopy was evaluated at five different locations for each sample.The main thickness values are obtained from the average of all the measurements.Error is calculated from the standard deviation of results.As a function of mass concentration, a deviation from a simple linear correlation is observed for spin-coated films of CTPR16, for which we have prepared samples at higher concentrations.Specifically, thickness data for concentrations higher than 30 mg/mL seem to follow a linear trend with higher slope than data at lower concentrations.Interestingly, the appearance of different thickness-concentration regimes has been described for spin-coated polymer films.Schubert and Dunkel [s2] attribute these distinct regimes in polystyrene films to the variations of viscosity with concentration.The results of fitting CTPR16 thickness-concentration data to two different linear regimes with different slopes are presented in Figure S10.

SDS-PAGE gel electrophoresis
Figure S1.SEM micrograph of a cross section after freeze-fracture of a drop-casted protein film (at 800 µM CTPR4) deposited on a 525 µm Si/SiO2 wafer with a ~295 nm layer of SiO2.Scale bars represent 100 μm (left image) and 5 μm (right image).

Figure S4 .
Figure S4.Optical contrast simulation for CTPR films of different thickness (tCTPR) deposited on a silicon substrate with a native oxide layer with thickness of 5 nm.
Figure S5.Optical images of spin coating CTPR4 films prepared from solutions with increasing protein concentration.Scale bar: 100 µm.

Figure S7 .
Figure S7.Refractive index values extracted from the fit to a Cauchy model of ellipsometry data for CTPR4 samples prepared from different protein concentrations.

S9.
Figure S9.Thickness of CTPR films as a function of mass concentration (mg/ml) for samples prepared by (a) spin-coating and (b) drop-casting.Solid lines represent linear fits to the data.

Figure S10 .
Figure S10.Thickness of CTPR films as a function of mass concentration (mg/ml) for samples prepared by spin-coating.Solid lines represent linear fits to the data.Data for CTPR16 has been fitted to two different linear expression for lower and higher concentration regimes, with corresponding slopes of 2.7 and 4.7 nm/mg. −1 , respectively.

Table S1 .
Summary of linear regression parameters, including slopes, intercepts, and coefficients of determination for the thickness-concentration data for CTPR films presented in Figures2 and 4in the main text.Slope and intercept errors are obtained from the 95% confidence bounds of the fits.