# Strategies for chalcogenide thin film functionalization

Bruno Robert, Marta Martin, Raphaël Escalier, Bela Varga, Ahmad Mehdi, Caroline Vigreux, Csilla Gergely

► To cite this version:

Bruno Robert, Marta Martin, Raphaël Escalier, Bela Varga, Ahmad Mehdi, et al.. Strategies for chalcogenide thin film functionalization. Langmuir, 2020, 36 (26), pp.7691-7700. 10.1021/acs.langmuir.0c01328. hal-02859831

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<figure><img src="image_1.png" /></figure>
Strategies for chalcogenide thin film functionalization

Bruno Robert<sup>a,b</sup>, Marta Martin<sup>a</sup>, Raphaël Escalier<sup>b</sup>, Béla Varga<sup>a</sup>, Ahmad Mehdi<sup>b</sup>, Caroline Vigreux<sup>b</sup>
and Csilla Gergely<sup>a,*</sup>

<sup>a</sup>L2C, Univ Montpellier, CNRS, Montpellier, France.

<sup>b</sup>ICGM, Univ Montpellier, CNRS, ENSCM, Montpellier, France

E-mail address of authors:

bruno.robert1@umontpellier.fr, marta.martin@umontpellier.fr, raphael.escalier@umontpellier.fr,
bela.varga@umontpellier.fr, ahmad.mehdi@umontpellier.fr, caroline.vigreux@umontpellier.fr,
csilla.gergely@umontpellier.fr

*Corresponding author:

- E-mail address : csilla.gergely@umontpellier.fr

- Telephone : 33(0)4 67 14 32 48

- Postal address : Laboratoire Charles Coulomb, Université de Montpellier, Place Eugène Bataillon - CC074, F-34095 Montpellier Cedex 5, France
Abstract

We report on the functionalization of chalcogenide thin films with biotinylated 12-mer peptides SVSVGMKPSPRP and LLADTTHHRPWT exhibiting high binding affinity towards inorganic surfaces on the one hand and with (3-Aminopropyl)triethoxysilane (APTES) on the other hand. The specific biotin moieties were used to bind streptavidin proteins and demonstrate efficacy of the biofunctionalized chalcogenide thin films to capture biomolecules. Atomic force microscopy provided high resolution images of the interfaces and water contact angle measurements brought insight about the interaction mechanisms. Fourier transformed infrared spectroscopy in attenuated total reflection mode provided information on the secondary structure of the bound proteins thanks to the de-convolution of the amide I band (1700-1600 cm⁻¹). Following adsorption of the biotinylated peptides or APTES immobilization, a homogenous coverage of biotin layer exhibiting very low roughness was obtained also rendering more hydrophilic the Ge-Se-Te surfaces. Subsequent capture of streptavidin depends on the functionalization approach permitting more or less an optimal orientation of the biotin to bind streptavidin. The molecular interface layer formed on the Ge-Se-Te is crucial also for retaining the native secondary structure of the protein. All together our results demonstrate that both peptides and APTES were appropriate linkers to build a favorable interface on chalcogenide materials to capture proteins, opening hereby promising biosensing applications.

Keywords

Chalcogenide thin film, biofunctionalization, peptide, APTES, biotin, streptavidin capture
## Introduction

Chalcogenide glasses are vitreous inorganic materials consisting of one or more chalcogen elements (S, Se and Te) and more electropositive elements such as Ge, As, Ga or Sb. They are promising optical materials due to their large transparency in the middle to far infrared range, low optical losses, and high non linear properties (1). They exhibit convenient designing properties, allowing fabrication of diverse micro- and nano-structures such as optical fibers, thin films, waveguiding structures, nanoparticles or photonic crystals (2). By tuning their composition and consequent optical properties, they become propitious in infrared thermal imaging, chemical or bio-sensing, and in designing numerous components for photonic integrated circuits (2).

Among chalcogenide glasses, the Ge-Se-Te ternary system is particularly attractive (3). The tetravalent germanium offers good thermal and mechanical stability, while the selenium and tellurium proportions mostly define the transparency window. High content in selenium lowers the transparency window allowing the use of 1.3 µm and 1.55 µm telecommunication wavelengths and enhances the vitrification feasibility. The presence of tellurium insures transparency at high wavelengths, up to 20 µm. Therefore Se-rich Ge-Se-Te compositions such as Ge₁₀Se₄₀Te₅₀ (4), Ge₂₀Se₇₀Te₁₀ (5), Ge₁₀Se₆₀Te₃₀ (6) or Ge₂₅Se₆₅Te₁₀ and Ge₂₅Se₅₅Te₂₀ (7) have been identified as promising materials for their convenient optical properties. In particular, Ge₂₅Se₅₅Te₂₀ films, already selected to manufacture optical guiding structures (7) (8) (9), were chosen for this study.

The design and manufacture of inorganic-organic interfaces have become a very active research field, providing new trends in material science (10). The functionalization of the inorganic substrate may offer biocompatibility, improved chemical specificity and selectivity, or modifications in wettability. Nevertheless, one should identify the appropriate molecular linker that will interact with both material and organic moieties, either in a covalent or non-covalent way (11). Thiolization, silanization, or peptide and protein adsorption have been used to build such interfaces. Peptides with various sequences have demonstrated specific grafting and direct adsorption on a broad set of inorganic materials. They were reported as efficient molecular linkers for subsequent biomolecule adhesion (12). Peptides bind the surface in a non-covalent way through specific affinity of an amino acid, or a short sequence of amino
acids for various materials (12) (13). Phage display technique was reported as an effective screening procedure enabling identification of unique peptide sequences revealing high affinity towards diverse inorganic surfaces (14) (15). Among them, the 12-mer peptide LLADTTHHRPWT (henceforth referred to as LLA) expresses affinity for Se-rich material such as ZnSe (16) and n-type silicon surface (17). Concerning the 12-mer peptide SVSVGMKPSPRP (henceforth referred to as SVS), it expresses affinity for a larger set of semiconductor materials (18) (19), including CdSe (14). To the best of our knowledge, no peptide functionalization has been reported on chalcogenide glass, as most of the previous studies focus on crystalline inorganic surfaces. Covalent grafting of organosilanes such as 3-aminopropyltriethoxysilane (APTES) has been extensively studied to chemically modify inorganic surfaces, including bulk chalcogenide materials. An As<sub>29</sub>Se<sub>54</sub>Te<sub>17</sub> optical fiber was reported to be functionalized with APTES thirty years ago (20) (21). More recently, a covalent Ge-O-Si bonding has been established in the functionalization mechanism of Ge-As-Se bulk glass and the silicon atom of APTES (22) (23). Disulfide group has been reported to functionalize Ge-As-Se bulk glass, under radiation inducing subsequent formation of As-S and S-Se covalent bonds (22) (24) (23). Finally, thin film deposition on chalcogenide surfaces has been conducted with polymers, such as poly(L-lysine) (25) or (Poly(p-xylylen) (26). Reporting on the difficulty of direct chalcogenide functionnalization, others proposed a preliminary gold deposition: a thin film of Au has been evaporated on the Ge<sub>28</sub>Sb<sub>12</sub>Se<sub>60</sub> surface in order to facilitate further thiol functionalization (27) (28).

Taking into account the high Se content of chalcogenide materials, we have investigated the possibility to use these peptides for their surface modification. In this paper we compare both peptides functionalization routes to the commonly used organosilane route, in order to functionalize Ge<sub>25</sub>Se<sub>55</sub>Te<sub>20</sub> thin films deposited by thermal co-evaporation. By building an organic linker, we aim to introduce the Ge-Se-Te films as promising substrates for applications in biosensing. This novel and one-step functionalization protocol for chalcogenides has been inspired from our previous work (17) in which the biotin-streptavidin couple is used to demonstrate the effectiveness of surface functionalization for bio-detection purposes. The streptavidin (STV) protein is known to exhibit very high affinity for the biotin moiety, forming one of the strongest non-covalent interactions encountered in nature. Moreover,
the capture event of the streptavidin by a biotinylated surface can be conveniently studied by atomic force microscopy (AFM) (29) or infrared spectroscopy in attenuated total reflection mode (FTIR-ATR) (30). Since streptavidin could also adsorb onto raw inorganic surfaces, the bovine serum albumin (BSA) protein is used to saturate all the non-specific adsorption sites left on the biotinylated surface (31). The functionalization process either by peptides (LLA and SVS) or organosilane (APTES) was monitored using a multitechnic characterization that combined AFM, wettability measurements and FTIR-ATR.
## Materials and Methods

### Thin film deposition

Ge<sub>25</sub>Se<sub>55</sub>Te<sub>20</sub> thin films were deposited by thermal co-evaporation of the three pure elements (Ge pieces from Goodfellow, Se pieces from ChemPur and Te pieces from Sigma-Aldrich; each one with a purity of 99,999 %) using a PLASSYS MEB500 device. Pure germanium was melted and evaporated by electron beam bombardment whereas selenium and tellurium were evaporated by current-induced heating. The average deposition rate was 67 Å.s<sup>-1</sup>, and the pressure in the chamber was below 2.10<sup>-7</sup> mbar. Sets of eight smooth glass cover slips were used as substrates for each new deposit, and heated at 70 °C during the deposition process. Three quartz crystal microbalances were used for in situ control of the three elemental deposition rates, from which the composition and thickness were deduced. A scheme of the deposition chamber is provided in SI, *figure S1*, and more details on the fabrication process can be found in a previous work (7). The film thickness was confirmed with a DEKTAK mechanical profilometer (Bruker), while the composition was checked by Energy-Dispersive X-ray Spectroscopy, using a S4500 Hitachi instrument equipped with an EDX Thermofischer.

### Functionalization

The functionalization protocol as summarized in *scheme 1* consists in consecutive incubation steps. The peptide route protocol for chalcogenides has been inspired from our previous work (17). It involves direct incubation of biotinylated 12-mer peptides on the raw Ge-Se-Te surfaces. LLADTTHHRPWT-biotin (Mw = 1.802 kg.mol<sup>-1</sup>) and SVSVGMKPSPRP-biotin (Mw = 1.596 kg.mol<sup>-1</sup>) were synthesized by Genecust. 20 µM-biotinylated peptides diluted in Phosphate Buffered Saline-Tween (PBST/10) were drop casted on the Ge-Se-Te thin films for 1 h. Surfaces were then rinsed with 1 mL of PBST/10 and 1 mL of distilled water to remove unbounded peptides.
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<figure><img src="image_2.png" /><figcaption>**Scheme 1.** Schematic representation of the functionalization procedures for the peptide (left) and the organosilane (right) routes.</figcaption></figure>

The organosilane route first involves a well-known plasma activation of the Ge-Se-Te surface, which allows to form hydroxyl groups. The thin films were therefore treated with oxygen plasma at 0.6 mbar during 10 minutes at 120 W. Solution of (3-Aminopropyl) triethoxysilane (APTES) (99% purity, Sigma-Aldrich) were prepared in 0.5 % (v/v) anhydrous toluene. Ge-Se-Te thin films were immersed for 12 h in a hermetic container, rinsed with toluene and ethanol to remove adsorbed precursors, and finally dried 15 minutes at 110 °C. This protocol inhibits self-condensation of APTES and produces free NH₂ on the Ge-Se-Te surface. Solution of 100 µM NHS-Biotin diluted in dimethyl sulfoxide (DMSO) was then drop casted on Ge-Se-Te thin films. Surfaces were incubated for 1 h and then rinsed with 1 mL of DMSO and 1 mL of distilled water.

Both functionalization routes (via peptides and organosilane) were subsequently subjected to the same protocol. Coating of surfaces might leave unoccupied hydrophobic areas on the surface. These sites must be blocked in order to prevent nonspecific binding of subsequent reactants and the coating to not suffer from non specific adsorption. Bovine serum albumin (BSA) is one of the most commonly used protein
blockers (31), therefore we have incubated our surface with BSA to prevent subsequent nonspecific
binding of streptavidin. The biotinylated surfaces were incubated with 20 µM bovine serum albumin
(BSA, 66 kDa protein from Fluka) diluted in phosphate buffered saline solution (PBS/10). After 1 h,
surfaces were rinsed with 1 mL of PBS/10 and 1 mL of distilled water to remove unbound excess of
BSA. Finally, surfaces were incubated with 50 µM streptavidin (STV, 53 kDa protein from Sigma-
Aldrich) diluted in PBS/10. After 1 h, the surfaces were rinsed with 1 mL of PBS/10 and 1 mL of
distilled water to remove unbound STV. Note that each functionalization route was performed using
samples from the same batch. A reference sample was systematically set aside and surface modified
samples were then investigated after each incubation step.

Atomic Force Microscopy (AFM)

AFM images were recorded in air, with an Asylum Molecular Force Probe 3D head and Molecular Force
Probe 3D controller (from Asylum Research). Images were acquired in tapping mode using rectangular
OMCL-AC240TS silicon cantilevers (Olympus) with a nominal tip radius of 7 nm. Height and phase
images were recorded at 1 Hz line-scan rate and digitized in 512×216 pixels at three different scan-sizes
(10×10 µm², 2×2 µm² and 0.5×0.5 µm²). At least 3 images were recorded for each sample and the scan
size was adjusted to insure topography investigations. Typically, 0.5×0.5 µm² images exhibit clearly the
surface features after incubation steps, therefore these are discussed in the next section. Nevertheless,
2×2 µm² height images and cross section profiles are also available in *figure S2*. The height images
provide topographical data, whereas the phase images reveal the samples viscoelastic properties. Further
image corrections (removing erroneous scan lines, plane fitting and flattening) and image analysis
(roughness, cross section and particle analysis) were carried out using the implemented Asylum
software. In order to easily compare the AFM results, the height images, the cross section profiles and
phase images from the different figures are displayed at the same scale.
Attenuated Total Reflectance Infrared Spectroscopy (ATR-FTIR)

Infrared absorption spectroscopy was carried out in the Attenuated Total Reflectance mode, using a Perkin Elmer device from Frontier bearing a diamond crystal (n = 2.4) from 4000 cm⁻¹ to 650 cm⁻¹ with a 2 cm⁻¹ spectral resolution. Baseline correction, Savitzky-Golay smoothing, second derivative and spectral deconvolution were performed using Origin Pro 8.

Wettability measurements

Static water contact angle (WCA) values were measured for at least 4 different drops of 100 µL distilled water using the SCA optical contact angle device and software. Typical water drop images and explanation on the water contact angle measurement are illustrated in SI, *figure S3*.
## Results and Discussion

The elemental compositions of all the Ge-Se-Te deposited films were consistent with the targeted one, with a maximum deviation of 5 %. Their thickness was measured to be 3.0 ± 0.4 µm, as expected, and surfaces present a root mean squared roughness of around 0.35 ± 0.08 nm.

### Morphology Study

0.5×0.5 µm² AFM height images, cross section profiles and phase images of the bare and modified Ge-Se-Te surfaces are displayed in figures 1-3, corresponding to LLA (figure 1), SVS (figure 2) and APTES route functionalization (figure 3), respectively. 2×2 µm² height images are also provided in the SI, figure S2. The mean of the Root Mean Square (RMS) roughness values calculated from the 2×2 µm² height images are depicted in figure 4.

LLA-biotin incubation led to the formation of a smooth (RMS = 305 ± 20 pm, see figure 4) homogeneous layer with disappearance of features from the raw surfaces (figure 1b) and no phase contrast (figure 1b'). This can indicate a complete surface coverage that has been already reported when these 12-mer peptides were used to functionalize silicon and ZnSe surfaces (17), (32). After proteins (BSA and STV) incubation, height and phase images (figure 1, panels c and c', respectively) show the presence of some small particles that fully cover the biotinylated surface. Consequently the RMS increases to about 575 ± 25 pm (figure 4).
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<figure><img src="image_3.png" /><figcaption>**Figure 1. Functionalization via LLA-peptide.** Surface topography (0.5×0.5 µm²), phase image and cross section profile of Ge-Se-Te by AFM tapping mode. Height image of bare Ge-Se-Te (a) functionalized with LLA-biotin (b) followed by BSA and STV adsorption (c). Phase image of raw Ge-Se-Te (a') functionalized with LLA-biotin (b') followed by BSA and STV (c').</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Figure 2. Functionalization via SVS-peptide.** Surface topography (0.5×0.5 µm²), phase image and cross section profile of Ge-Se-Te by AFM tapping mode. Height image of bare Ge-Se-Te (a) functionalized with SVS-biotin (b) followed by BSA and STV adsorption (c). Phase image of raw Ge-Se-Te (a') functionalized with SVS-biotin (b') followed by BSA and STV (c').</figcaption></figure>

When the SVS peptide was used for functionalization, the SVS-biotin modified surfaces present a full coverage of small aggregates, as shown in *figure 2*, panels *b* and *b'*. The SVS peptide has been already reported to produce such kind of fine structure on inorganic surfaces [32]. Again, protein incubation led to a very good surface coverage with visible particles (*figure 2*, panels *c* and *c'*), though they seem to
adopt smaller area and bigger height compared to the LLA route modification. The roughness of the
final STV layer deposited on the SVS-route modified Ge-Se-Te surfaces is considerably higher RMS
$860 \pm 60$ pm (figure 4), compared to that obtained with the LLA peptide.

<figure><img src="image_5.png" /><figcaption>**Figure 3. Functionalization via APTES.** Surface topography (0.5×0.5 µm²), phase image and cross section profile of Ge-Se-Te by AFM tapping mode. Height image of bare Ge-Se-Te (a) functionalized with APTES and NHS-biotin (b) followed by BSA and STV adsorption (c). Phase image of raw Ge-Se-Te (a') functionalized with APTES and NHS-biotin (b') followed by BSA and STV (c').</figcaption></figure>
When the organosilane route is employed to modify the surfaces, the APTES + NHS-biotin incubation led to much rougher surfaces (RMS 600 ± 35 pm, see figure 4), compared to those obtained by the peptide-route. This probably originates in the selective anchoring of APTES via the polycondensation. Pronounced features with sizes of about 50 nm and height of few nanometers can be observed in the AFM height images (figure 3b). The subsequent BSA and STV incubations led to a layer of particles. According to the cross section in figure 3c the particles are characterized by a 4-5 nm maximum height. Moreover, according to figure 4, the formation of the layer of particles is accompanied with an increase in the RMS of 1275 ± 55 pm..

<figure><img src="image_6.png" /><figcaption>**Figure 4. RMS roughness** of the Ge-Se-Te films before and after incubation with the biotinylated precursor (LLA, SVS, APTES) followed by BSA and STV adsorption. Data represent mean ± standard deviation calculated on 2×2 µm² AFM height images. The same letter indicates that the difference between the means is statistically significant, p < 0.05, t-test.</figcaption></figure>

The AFM images and the computed roughnesses clearly indicate that though the Ge-Se-Te surfaces were successfully modified by the three functionalization routes, the obtained biotin layers exhibit different morphologies and roughness. In order to obtain more quantitative knowledge on the morphology of the formed STV layers, grain analysis of visible particles was carried out. First of all, the number of particles provides a good estimation of protein density. Accordingly, the available protein densities are 890, 1320 and 1400 / µm² for LLA, SVS and APTES route functionalization, respectively. One can notice a significantly lower protein density for the LLA route (890 protein.µm⁻²). This could be
explained by an initially lower biotin density on the surface. The particles analysis provided information on their circle equivalent area diameters (CED), center heights and circularity. The data were presented in histograms that could be fitted with a Gaussian function (examples in SI. *figures S4, S5, S6*). The average values of the Gaussian fits maxima are summarized in *figure 5*, revealing decreasing CED diameters of about 12.6, 10.5 and 9.3 nm for the LLA, SVS and APTES routes, respectively (*figure 5a*). Modification via LLA peptide led to the formation of larger features of about of 25-40 nm in diameter (*figure S4*) that were not observed on the SVS or APTES functionalized surfaces. Furthermore, as it can be noted also on the cross section profile of AFM images (*figure 1*) and roughness (*figure 4*), the particles obtained via LLA route have a significantly lower height, averaging 0.96 nm compared to those (around 1.3 nm), obtained using SVS and APTES (*figure 5 b*). Indeed, the histogram from the LLA route presents almost no particle higher than 2 nm. Finally, the Gaussian fit of the particle circularity histograms leads to mean values of 0.63, 0.68 and 0.64 for the LLA, SVS and APTES routes, respectively (*figure 4 c*), suggesting that the SVS route lead to more spherical adsorbed protein.

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>
**Figure 5. Particle analysis results.** Circle equivalent diameter (CED) (a), height (b) and circularity (c) means ± standard deviation average from at least three 0.5×0.5 µm² AFM images (* p < 0.05, t-test) recorded for the three type of functionalized surfaces: after incubation with the biotinylated precursor (LLA, SVS, APTES) followed by BSA and STV adsorption.

To explain these data, one should consider the eventual artefacts generated by the tip radius when imaging relatively small particles (33). According to the size of either BSA or STV (about 5 nm in diameter in solvated state) and the AFM tip radius (7 nm), such proteins could size up to 19 nm (tip diameter + particle size). Therefore, the observed features on the surface of STV coated Ge-Se-Te surfaces should not be protein aggregates, but rather single proteins that undergo more or less spreading through the adsorption event. The larger (20-40 nm) structures observed on the LLA modified surfaces, that are also less high then those seen on the SVS and APTES modified samples, might indicate the formation of more expanded STV layers.

The literature, over the last 30 years, provided a number of AFM studies on STV-modified surfaces (29). Some authors estimated the size of streptavidin interacting with biotin to be more elongated with dimensions of 3×10 nm² (CED of 13 nm) (34), others reported heights of 4.5 nm (35), while full coverage with STV of porous biotinylated silicon was also reported (17). These studies are in accordance with our results, indicating successful STV capture by our functionalized Ge-Se-Te substrates. According to the particles analysis, SVS and APTES routes provide higher protein density (≈1360 protein.µm⁻²) than the LLA counterpart (890 protein.µm⁻²). Furthermore, proteins adsorbed on the LLA-modified surface are more subject to conformational damage. Capturing STV through the biotin moiety is crucial to preserve the molecule structure, and to avoid direct and physical adsorption that causes structural damage (36) (37).
Hydrophilic-Hydrophobic Study

In order to evaluate the consequence of surface modifications on the hydrophilic-hydrophobic balance of the Ge-Se-Te substrates wettability measures were performed. Water contact angle is mainly governed by the droplet's wetting behavior, which is monitored by two types of forces: elastic forces within the drop, and attraction forces between the drop and the substrate. Note that the more hydrophilic is a surface, the lower the WCA value measured. The average WCA values are depicted in figure 6, while the corresponding photos of typically observed drops are gathered in figure S7.

Bare Ge-Se-Te surfaces are relatively hydrophobic according to their WCA values (0 = 84 ± 4°)
Concerning the organosilane route, oxygen plasma treatment led to the most hydrophilic surface, with
a WCA of 7 ± 5° (not shown). This is most probably due to the formation of hydroxyl groups, such as
Ge-OH, later involved in the Ge-O-Si bonding.

<figure><img src="image_10.png" /><figcaption>**Figure 6.** Water contact angles measured on the Ge-Se-Te surfaces before and after precursor incubation and after biotinylated precursor incubation followed by BSA and STV adsorption.</figcaption></figure>

Adsorption of the three non-biotinylated precursors (APTES and the two peptides) reduced the hydrophobicity and in a less accentuated manner for the SVS precursor. The literature provides several studies on the adhesion mechanism involved in peptide functionalization. Some authors reported the role of single amino acids (13) (12), such as the relevance of the proline (P) in the conformational
mechanism of peptides when adsorbed onto inorganic surfaces (38) (39). Nevertheless, most of those
works reported on modification of diverse metallic, crystallized or hydrophilic inorganic materials, but
none of hydrophobic materials. Thus, those studies might not be fully relevant for our chalcogenide
surfaces.

In order to discuss about the peptide adsorption mechanism it might be interesting to compare their
physico-chemical characteristics (figure S6). According to the instability index (II) and aliphatic index
(AI), the SVS peptide is more stable than the LLA peptide. Both peptides show more hydrophilic than
hydrophobic behavior (H<0) including a hydrophobic start chain, and a more hydrophilic sequence near
the biotin moiety (figure S8 a, b). The start chain of the LLADTTHHRPW peptide comprises two
leucine (L) and an alanine (A) amino acid, both containing CH3 groups. The SVSVGMKPSPRP peptide
has two valine (V) amino acids also conferring a hydrophobic start chain. This hydrophobic sequence
most probably interacts with the hydrophobic Ge-Se-Te surface, permitting to position the more
hydrophilic moiety on the top of the modified surface. This is consistent with the lower water contact
angle we have obtained on the peptide-modified surfaces.

Adsorption of the three biotinylated precursors also generated a more hydrophilic behavior and in a
more accentuated manner for the SVS-biotin precursor (figure 6). The biotinylated surfaces have already
been reported to exhibit a hydrophilic behavior with a contact angle reaching about 35° in aqueous
solution (40). This could suggest that our modified surfaces have been covered by a significant fraction
of biotin. Accordingly, the SVS route modification exhibiting the highest hydrophilicity (45 ± 1°) offers
more biotin moiety on the top of the Ge-Se-Te surface.

One could have noticed the substantial hydrophobic/hydrophilic difference between the surfaces
incubated with the SVSVGMKPSPRP peptide (80 ± 5°) and its biotinylated counterpart (45 ± 1°). These
results demonstrate that in the case of the SVS-route functionalization, the hydrophilic behavior is
mainly due to the available biotin moiety anchored onto the hydrophilic part of the peptide. This
confirms that the hydrophobic start chain interacts with the hydrophobic Ge-Se-Te surface. In the case
of LLA route functionalization the difference between the contact angle measured on the peptide and
the biotinylated peptide itself is non significant.
Finally, after BSA and STV proteins incubation, one can observe a slight decrease in the hydrophilic behavior, whatever the functionalization route, suggesting successful surface modification.
Chemical Study

Monitoring molecules adsorption on chalcogenides can be conveniently done by FTIR spectroscopy in ATR mode, as the spectrum of the bare Ge-Se-Te surface presents a wide transparency window in the IR wavelengths (figure S9). When the surfaces were incubated with the biotinylated precursors (LLA-biotin, SVS-biotin or APTES + NHS-biotin), they did not exhibit compelling changes compared to the raw surface spectrum. Since the penetration depth of the interacting light is about few microns, this is not surprising, and it suggests that the biotinylated layer on the top of Ge-Se-Te surfaces is very thin.

<figure><img src="image_11.png" /><figcaption>**Figure 7.** FTIR-ATR spectra of the functionalized Ge-Se-Te surfaces after proteins incubation: BSA adsorption (dotted line) and STV adsorption (straight line).</figcaption></figure>

The spectra recorded after proteins incubation (BSA then STV), are presented in *figure 7* for each functionalization route. The presence of the NH stretching, CH stretching, amide I and amide II characteristic bands at 3450-3400 cm⁻¹, 2900 cm⁻¹, 1650 cm⁻¹ and 1550 cm⁻¹, respectively, confirmed the presence of proteins on the Ge-Se-Te surface regardless of the functionalization route, as suggested by AFM height images. Note that the high intensities of CHₓ bands obtained for the APTES route modification corroborate the coverage of the Ge-Se-Te surface by APTES molecules along with polycondensation. Comparison of the spectra from BSA modified surfaces and STV modified surfaces
indicates an augmentation of amide I content. Indeed, the area under the curve from 1700 cm⁻¹ to 1600 cm⁻¹ extends by 18%, 43% and 21% for the LLA, SVS and APTES route, respectively, as it can be seen in figure 8. It demonstrates that streptavidin effectively binds the surface. Since BSA is supposed to have already blocked all the non-specific sites, STV attachment must have occurred through the remaining specific binding-sites of biotin. Accordingly, the SVS route seems to have led to more STV capture events, and therefore to be the best functionalization route compared to LLA and APTES.

### Protein secondary structure study

Proteins often undergo conformational damage while being adsorbed onto surfaces. ATR-FTIR is a valuable tool to investigate protein structure during absorption events (41) (42) (43). It has already been used to study STV binding onto biotinylated surfaces (34) (30) (44), or to monitor conformational change of proteins such as BSA while adsorption (36) (37) (45). Proteins can exhibit various secondary structures and local conformations can adopt a non-ordered arrangement (random coil or extended chain), or a periodically structured arrangement, such as α-helix, β-sheet, or β-turn. As depicted in figure S10, bovine serum albumin and streptavidin adopt a specific secondary structure, in which BSA is mostly composed of α-helices and STV mostly of β-sheets.

<table><caption>**Table 1.** Secondary structure analysis of Bovine Serum Albumin based on the deconvolution of Amide I region into Gaussian component bands. All frequencies values are in cm⁻¹.</caption><thead><tr><th></th><th>Assignment</th><th>β-turn</th><th>α-helix</th><th>β-sheet (antiparallel)</th><th>β-sheet intermol.</th><th>Other</th></tr></thead><tbody><tr><td rowspan="3">Literature study</td><td>Adsorbed on TiO<sub>2</sub> (37)</td><td>1680 (10%)</td><td>1653 (66%)</td><td>1632 (22%)</td><td>1614 (2%)</td><td>-</td></tr><tr><td>In H<sub>2</sub>O (37)</td><td>1680 (9%)</td><td>1655 (67%)</td><td>1632 (23%)</td><td>1614 (1%)</td><td>-</td></tr><tr><td>In H<sub>2</sub>O (46)</td><td>1684 (4%)</td><td>1654 (53%)</td><td>1625 &amp; 1632 (14%)</td><td>-</td><td>-</td></tr><tr><td rowspan="3">Our study</td><td>LLA</td><td>1683 (10%)</td><td>1656 (39%)</td><td>1625 (51%)</td><td>-</td><td>1590</td></tr><tr><td>SVS</td><td>1682 (11%)</td><td>1653 (44%)</td><td>1626 (45%)</td><td>-</td><td>1606</td></tr><tr><td>APTES</td><td>1685 (11%)</td><td>1654 (44%)</td><td>1624 (44%)</td><td>-</td><td>1590</td></tr></tbody></table>
<table><caption>**Table 2.** Secondary structure analysis of adsorbed Streptavidin based on the deconvolution of Amide I region into Gaussian component bands. In our study, one should remember that BSA was previously adsorbed on surfaces. All frequencies values are in cm⁻¹.</caption><thead><tr><th></th><th>Assignment</th><th>β-turn or β-sheet (parallel)</th><th>α-helix or unordered</th><th>β-sheet (antiparallel)</th><th>Other</th></tr></thead><tbody><tr><td rowspan="3">Literature study</td><td>In D<sub>2</sub>O (47)</td><td>1673</td><td>-</td><td>1637</td><td>-</td></tr><tr><td>On biotin-surface (30)</td><td>~1695 ~1680</td><td>-</td><td>1640</td><td>-</td></tr><tr><td>On biotin-surface (44)</td><td>1689 ~1668</td><td>-</td><td>1632</td><td>-</td></tr><tr><td rowspan="3">Our study</td><td>LLA</td><td>1682 (12%)</td><td>1654 (47%)</td><td>1625 (41%)</td><td>1598</td></tr><tr><td>SVS</td><td>1686 (9%)</td><td>1656 (52%)</td><td>1626 (40%)</td><td>1601</td></tr><tr><td>APTES</td><td>1677 (19%)</td><td>1655 (28%)</td><td>1629 (53%)</td><td>1590</td></tr></tbody></table>

In the infrared spectrum, hydrogen bonding and therefore the secondary structure impacts the amide I absorption frequency. The amide I region mainly stands for the C=O stretching vibration centered around 1650 cm⁻¹. It comprises the random coil structure with a featureless and large peak around 1650 cm⁻¹, α-helix vibrations at 1655 cm⁻¹, β-sheet vibrations at 1630 cm⁻¹, including a smaller contribution at 1685 cm⁻¹ and β-turn vibrations around 1670 cm⁻¹ (41) (42). Furthermore, some authors noticed that during protein adsorption, the β-sheet contribution was shifted to lower wavenumbers, corresponding to intermolecular β-sheets (37) (48) (49). Thus, infrared spectroscopy can aide investigating BSA and STV interactions with the biotinylated Ge-Se-Te films. Comparing the secondary structure of the native proteins in liquid with those obtained after adsorption can provide valuable information in this regard (46) (47). Tables 1 and 2 reference the absorption frequencies of the different conformations encountered in BSA and STV, according to literature. The preponderant α-helix structure of BSA (table 1) arises at 1653-1655 cm⁻¹ and represents 48-67 % of the whole amide I area. The majority β-sheet structure of STV (table 2) arises at 1632-1640 cm⁻¹ with a smaller contribution around 1680 cm⁻¹ and weights for two thirds of the whole amide I area.
<figure><img src="image_12.png" /><figcaption>**Figure 8.** Deconvolution of the amide I mode, from 1725 to 1575 cm⁻¹ after BSA adsorption (upper row) and the subsequent STV adsorption (lower row)</figcaption></figure>

To obtain the secondary structure of the adsorbed proteins on our functionalized Ge-Se-Te surfaces, the amide I region of the IR spectra was subjected to a second derivative and a curve-fitting procedure. It allowed to determine the absorption frequencies and to assign a weight to each single peak. The deconvolutions of the six spectra, in the domain of 1725 cm⁻¹ to 1575 cm⁻¹, of the modified Ge-Se-Te surfaces are displayed in *figure 8*. The absorption frequencies for both BSA and STV were identified and summarized in *tables 1 and 2*, along with the reference frequencies. Wavenumbers are in accordance with the already reported works, although slightly lower frequencies from the main β-sheet adsorption at 1624-1629 cm⁻¹ are observed. Variation in the absorption frequency indicates structural modification.

In our case, the lower frequency of the main β-sheet absorption at 1625-1629 cm⁻¹ may indicate a significant intermolecular contribution due to protein aggregation as suggested by AFM images.

Nevertheless, there is a small difference in the structure of STV depending on whether it is adsorbed via the peptides (LLA or SVS) or APTES routes. The β-sheet contribution arises at 1625-1626 cm⁻¹ and at 1629 cm⁻¹ respectively, suggesting less conformational damage of STV through the APTES route.
Tables 1 and 2 also indicate the relative area of the main contributions of the amide I band after each incubation step. After BSA incubation, the percentage of the β-turn contribution matches with percentages found in literature, although the α-helix contain is lower (and concurrently the β-sheet contribution is higher). This confirms that the BSA adsorption on the non-specific sites occurred with a substantial conformational change through the formation of antiparallel β-sheets (36).

As already noticed, STV capture yielded to higher protein contribution in the spectra, demonstrating protein deposition. Table 2 summarizes the results of amide I band deconvolution corresponding to STV adsorption. We note the apparition of unordered structures for the two peptide routes and at a lesser extent for the APTES route. Moreover, the APTES route allowed to preserve a higher β-sheet content (+ 11 %). These trends suggest that in the case of the peptide (either LLA or SVS) routes, STV adsorption occurred with more conformational modification. On the contrary, the APTES route allowed better preservation of the streptavidin secondary structure. This could be explained by the better availability of free biotin moiety on the top of APTES modified Ge-Se-Te surfaces.
Conclusions

This study provides an insight to the molecular layers constituting the interface between biomolecules and chalcogenide thin films. Two strategies to functionalize Ge-Se-Te thin films were proved to be viable to accept biotin molecules and capture streptavidin : the use of small adhesion peptides, either LLADTTHHRPW or SVSVGMKPSPRP, and the use of APTES.

Atomic force microscopy, wettability and infrared spectroscopy studies demonstrated that Ge-Se-Te surfaces were undergoing topographical, wettability and chemical changes after adsorption of molecules. Incubation with biotinylated peptide led to the formation of a thin, high quality film on the top of the Ge-Se-Te surface, of very low roughness(less then 0.5 nm). The peptide route functionalization was possible due to the high affinity of the peptides for the chalcogenide surface governed by hydrophobic interactions. Through this mechanism, the peptides hydrophilic moiety along with the biotin are presented on the top of the surface, allowing the successfull capture of streptavidin.

The wettability study also suggested that the SVS route modification offered more biotin moiety on the top of the Ge-Se-Te surface. According to our atomic force microscopy results, organosilane grafting was shown to undergo concurrently with polycondensation since the deposited layer exhibits aggregations of a few nanometers in height. The particles analysis supported higher protein density for the SVS and APTES routes (≈1360 protein.µm⁻²). The lower protein density for the LLA route went along with more spread and damaged structure. Furthermore, infrared spectroscopy results indicated that bovine serum albumin adsorbs on non-specific sites along with significant conformational modification. Streptavidin incubation led to increase of the protein content, suggesting binding through the specific site via free biotin moieties. Through the organosilane route, streptavidin amply conserved its secondary structure, with predominance of β-sheet arrangement, demonstrating effectiveness of the streptavidin binding through biotin moiety.

The functionalization strategies presented in this work revealed the possibility to modify the hydrophobic Ge-Se-Te thin films for molecular detection. Together with the large opportunities offered by chalcogenide based optical devices, it opens promising avenues for their future use as biosensors.
## Supporting Information

* Further details on the chalcogenide deposition and WCA measurement

* Further insight on peptide and protein properties and structures

* Supplementary and raw data of the AFM, infrared, and WCA study

## Acknowledgements

This work was publicly funded through ANR (the French National Research Agency) under the Investissements d'avenir program with the reference ANR-16-IDEX-0006. The authors are grateful to Sikémia company (Montpellier, France) for having kindly allowed us to use their plasma device.

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Figure and Table

**Figure 1.** Functionalization via LLA-peptide

**Figure 2.** Functionalization via SVS-peptide
**Figure 3.** Functionalization via APTES

**Figure 4.** RMS roughness

**Figure 5.** Particle analysis results

**Figure 6.** Water contact angles

**Figure 7.** FTIR-ATR spectra of the functionalized Ge-Se-Te surfaces

**Table 1.** Secondary structure analysis of Bovine Serum Albumin

**Table 2.** Secondary structure analysis of adsorbed Streptavidin

**Figure 8.** Deconvolution of the amide I mode

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<figure><img src="image_13.png" /></figure>
<table><tr><td>APTES</td><td>(3-Aminopropyl)triethoxysilane</td></tr><tr><td>LLA</td><td>LLADTTHHRPWT peptide</td></tr><tr><td>SVS</td><td>SVSVGMKPSPRP peptide</td></tr><tr><td>BSA</td><td>Bovine serum albumin</td></tr><tr><td>AFM</td><td>Atomic force microscopy</td></tr><tr><td>FTIR-ATR</td><td>Infrared spectroscopy in attenuated total reflection mode</td></tr><tr><td>EDX</td><td>Energy dispersive X-ray spectrometry</td></tr><tr><td>PBST</td><td>Phosphate buffered saline-tween</td></tr><tr><td>NHS</td><td>N-hydroxysuccinimide</td></tr><tr><td>DMSO</td><td>Dimethyl sulfoxide</td></tr><tr><td>PBS</td><td>Phosphate buffered saline solution</td></tr><tr><td>STV</td><td>Streptavidin</td></tr><tr><td>WCA</td><td>Water contact angle</td></tr><tr><td>RMS</td><td>Root mean square</td></tr><tr><td>CED</td><td>Circle equivalent area diameters</td></tr></table>