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. 2020 Oct 29;36(44):13292–13300. doi: 10.1021/acs.langmuir.0c02325

Single-Molecule Force Spectroscopy Reveals Adhesion-by-Demand in Statherin at the Protein–Hydroxyapatite Interface

Patrick Steinbauer †,‡,⊥, Andreas Rohatschek §,⊥,#, Orestis Andriotis §,⊥, Nikolaos Bouropoulos ∥,∇, Robert Liska ‡,⊥,#, Philipp J Thurner §,⊥,#,*, Stefan Baudis †,‡,⊥,#,*
PMCID: PMC7660943  PMID: 33118809

Abstract

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Achieving strong adhesion in wet environments remains a technological challenge in biomedical applications demanding biocompatibility. Attention for adhesive motifs meeting such demands has largely been focused on marine organisms. However, bioadhesion to inorganic surfaces is also present in the human body, in the hard tissues of teeth and bones, and is mediated through serines (S). The specific amino acid sequence DpSpSEEKC has been previously suggested to be responsible for the strong binding abilities of the protein statherin to hydroxyapatite, where pS denotes phosphorylated serine. Notably, similar sequences are present in the non-collagenous bone protein osteopontin (OPN) and the mussel foot protein 5 (Mefp5). OPN has previously been shown to promote fracture toughness and physiological damage formation. Here, we investigated the adhesion strength of the motif D(pS)(pS)EEKC on substrates of hydroxyapatite, TiO2, and mica using atomic force microscopy (AFM) single-molecule force spectroscopy (SMFS). Specifically, we investigated the dependence of adhesion force on phosphorylation of serines by comparing findings with the unphosphorylated variant DSSEEKC. Our results show that high adhesion forces of over 1 nN on hydroxyapatite and on TiO2 are only present for the phosphorylated variant D(pS)(pS)EEKC. This warrants further exploitation of this motif or similar residues in technological applications. Further, the dependence of adhesion force on phosphorylation suggests that biological systems potentially employ an adhesion-by-demand mechanism via expression of enzymes that up- or down-regulate phosphorylation, to increase or decrease adhesion forces, respectively.

Introduction

Understanding biological adhesion strategies ranging from permanent (e.g., barnacle cement1) to temporary (e.g., cephalopods2) to highly dynamic adhesion (e.g., gecko foot3,4) and translating them to technological applications have been the subject of a number of studies.2,5 Research in this field bears the promise of solving a remaining technological challenge: the ability of forming strong adhesion in the presence of, or during submersion in, water or physiological fluids. Based on such approaches, phosphorus- and catechol-containing organic materials for applications in the field of tissue engineering6 and dental restoratives7 have been developed. For an efficient glue, both strong adhesion to the substrate and strong cohesion of the glue itself are necessary. The latter is generally easier to achieve, e.g., via curing bulk glue with photoinitiators and UV light.8 Still, the challenge of achieving strong adhesion to a substrate in an aqueous environment remains, especially in biomedical applications, where biocompatibility is also of key importance.

As natural systems have overcome limitations of adhesion in aqueous environments, bioinspired or biomimetic adhesion underwater is of ongoing interest. Prominent examples are glue proteins of the mussel byssal thread,9,10 proteins secreted by the sandcastle worm (Phragmatopoma californica),11,12 or fibrin.2 Especially, the discoveries of the sandcastle worm and mussel foot proteins have been hailed as turning points for biomedical glues. The secreted glue of the byssal threads tethers strongly to all inorganic and organic surfaces in aqueous environments.13 The chemistry of these proteins bears some similarity; both the sandcastle worm protein Pc-311,12 and the mussel foot protein Mefp513 contain the amino acid 3,4-dihydroxy-l-phenylalanine (DOPA). DOPA has the highest share on the strong adhesion of marine mussels, and its strong adhesive properties, reaching up to 800 pN per amino acid on a Ti substrate, have been directly characterized via single-molecule force spectroscopy experiments.14 Other studies, e.g., by Krysiak et al.15 or Li et al.,16 have found varying values for DOPA adhesion forces, which are seemingly dependent on local pH at the adhesion point.

In the quest for additional biocompatible adhesion motifs, able to provide adhesion to inorganic components and that would likely avoid counterproductive biological effects, we turned our focus to the human body. Bones and teeth are hard tissues containing large amounts of inorganic materials, interfaced with an organic extracellular matrix (ECM). Here, a number of proteins are involved with various functions, which may provide a basis for novel, albeit likely more specific adhesion motifs compared to DOPA. In this context, a number of proteins in the human saliva are involved in controlling crystal growth via adsorption onto dental enamel.17 A major protagonist of this process is the acidic phosphoprotein statherin (see Figure 1a), which is involved in oral calcium homeostasis. Statherin consists of 43 amino acids and shows high charge asymmetry in the primary structure. This is due to a high number of negatively charged amino acids located at the 15-amino acid N-terminus, known as SN15 (DpSpSEEKFLRRIGRFG where pS denotes phosphorylated serine), whereas the C-terminus contains uncharged residues.18,19 The secondary structure of statherin consists of an α-helical structure at the N-terminus (12 amino acids),20 whereas the six amino acid-containing sequence has no specific structure.

Figure 1.

Figure 1

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Bioinspired adhesion motifs. (a) N-terminal amino acid sequence of statherin.21,22 (b) Different non-collagenous proteins.23−25 (c) Amino acid sequence of the mussel foot protein Mefp-5.13 (d) Sandcastle worm producing a protein with many phosphorylated serines.11,26 (e) Polymers with different architectures in nature or bioinspired synthetic polymers.27,28

Long et al.21 proposed that the first six amino acids of the N-terminus of statherin (DpSpSEEK) are the responsible motif for adhesion on hydroxyapatite (HAP) surfaces due to its secondary structure and the negative charge. The motif is not exclusive to statherin; close analogues are present in bone and tooth matrix proteins, such as osteopontin (OPN) and dentin matrix protein (DMP 1). Beyond the role of determining ultrastructure, some non-collagenous proteins (NCPs), such as OPN and DMP 1 (see Figure 1b), have also been shown to directly contribute to the mechanical properties of bones.29−32 The primary structure of OPN contains 298 amino acids of which 25% are acidic (D, E) and further 19% bear negatively charged side chains at pH 7.4 due to post-translational modifications: phosphorylation or glycosylation (S, T).33 Networks of recombinant NCPs such as OPN or DMP 1 were also shown to be able to repeatedly dissipate large amounts of energy and have evidently very high cohesive strength.34−36 This dissipation was enhanced by addition of Ca2+ ions in solution, leading to ion bridges that can reversibly reform after disruption providing a self-healing character. This mechanism is even relevant for whole-bone mechanics, where the deletion of OPN in animal models led to significant reduction in material properties and nanoscale damage mechanisms.30−32 For OPN, which also controls hydroxyapatite (HAP) crystal size and shape,37 interaction is thought to be achieved via high abundance of phosphoserines (pS) and overall flexibility of the protein. Nevertheless, OPN also contains a sequence SSEEKQLY at position 10 from the N-terminus,38 which is predicted to be phosphorylated, i.e., pSpSEEK (Netphos 3.1.).37

Strong adhesion through polyelectrolyte proteins has also been described in the glue protein Pc-3 of the sandcastle worm (Figure 1d),11,12 containing 80 mol % phosphoserines (pS).26 Even the mussel foot protein Mefp5 (SSEEYKGGYYP, where Y denotes DOPA) contains a SSEE motif, albeit without phosphorylation (see Figure 1c). This suggests that the peptide sequence DpSpSEEK, specifically with the presence of pS, is a promising adhesion motif for hydroxyapatite (HAP) as it is present in many proteins with bioadhesive functions. These occurring sequences are closely observed in nature, and some research groups now focus on biomimetic polymers (see Figure 1e).

In this study, we hypothesized that the sequence DpSpSEEK is indeed an adhesion motif, foremost to calcium deficient hydroxyapatite (CDHAP) substrates but potentially also to others, and critically depends on the phosphorylation of serine. To test our hypothesis, we studied the adhesion properties of the amino acid sequences DpSpSEEK and its unphosphorylated variant DSSEEK via atomic force microscopy (AFM) in single-molecule force spectroscopy (SMFS) mode.

Experimental Section

All reagents, if not otherwise mentioned, were purchased from Sigma Aldrich and were used without any further purification. Solvents were purchased and distilled prior to usage. 1H and 13C NMR spectra were recorded on a Bruker Avance 400 MHz FT-NMR spectrometer. Deuterated methanol (MeOD) was used as a solvent.

Synthesis of Dopamine-Thiol

According to Olofsson et al.,39 1 equiv. of dopamine hydrochloride (Fluka Chemical Corp.; 98%) was reacted with 1 equiv. of γ-thiobutyrolactone in water to yield 97% N-(3,4-dihydroxyphenetyl)-4-mercapto butanamide (dopamine-thiol).

1H NMR (400 MHz, MeOD) δ (ppm): 7.99 (s, 1H, −NH−), 6.70 (d, 1H, J1 = 8.0 Hz, ArH), 6.66 (d, 1H, J1 = 2.1 Hz, ArH), 6.53 (dd, 1H, J1 = 8.0 Hz, J2 = 2.1 Hz, ArH), 3.36 (t, 2H, J1 = 5.8 Hz, J2 = 2.1 Hz, −CH2–NH), 2.65 (t, 2H, J1 = 7.3 Hz, −CH2–Ar), 2.47 (t, 2H, J1 = 7.1 Hz, −CH2–SH), 2.28 (t, 2H, J1 = 7.5 Hz, −CH2–CO), 1.86 (m, 2H, −CH2–CH2–SH), 1.42 (s, 1H, −SH).

13C NMR (100 MHz, MeOD) δ (ppm): 173.9 (−C=O), 145.02 (ArC–OH), 143.61 (ArC–OH), 130.53 (ArC-CH2), 119.52 (ArC), 115.45 (ArC), 114.99 (ArC), 41.33 (−CH2–NH−), 34.51 (−CH2–CH2–NH−), 34.19 (O=C–CH2−), 29.97 (−CH2–CH2–SH), 23.20 (−CH2–SH).

AFM Tip Functionalization

The AFM tips for the force spectroscopy measurements were prepared according to functionalization protocols.40,41 Prior to functionalization of silicon nitride (Si3N4), AFM tips (Bruker MSNL-10) were spontaneously oxidized in an ambient atmosphere (atmospheric oxygen) for at least 30 min, resulting in a thin layer of silicon oxide with silanol groups (Si–OH). The AFM tips were cleaned in chloroform (3 × 5 min) and dried in a gentle nitrogen stream. Subsequently, the tips were immersed in 0.5 g mL–1 ethanolamine hydrochloride (TCI Deutschland, Eschborn, Germany) dissolved in DMSO in the presence of molecular sieves (4 Å) for 30 min, resulting in an amino-derivatized AFM tip. The aminated AFM tips were washed with DMSO (3 × 1 min) and ethanol (3 × 1 min) and dried in a gentle nitrogen stream. Maleimidopropionyl-PEG-hydroxysuccinimide esters (Mal-PEG-NHS, with an average of 27 ethylene glycol units, Polypure AS, Oslo, Norway, or Mal-PEG-NHS with an average of 162 ethylene glycol units, JenKem Technology, USA) were used as a linker and covalently coupled to the amino-functionalized tips in a Teflon reaction chamber by incubating cantilevers with a solution of 3 mg of the Mal-PEG-NHS linker dissolved in 0.5 mL of chloroform. Triethylamine (90 μL) was added to the linker solution. After 2 h of reaction time, the AFM tips were washed with chloroform (3 × 10 min) and dried in a gentle nitrogen stream. In order to attach the samples (amino acid sequences DpSpSEEKC or DSSEEKC, Epoch Life Science, Texas, USA, or dopamine-thiol) to the linker system, the sample was dissolved in 100 μL of deionized water to obtain a 10 mM solution. This solution was mixed with ethylenediamine tetraacetic acid (EDTA), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and tris(2-carboxyethyl)phosphine (TCEP; TCI Deutschland, Eschborn, Germany). Subsequently, 100 μL of this solution was pipetted onto the AFM tips, which were placed on pieces of Parafilm in Petri dishes. After 2 h reaction time, the tips were washed (3 × 5 min) and stored at 4 °C for up to two days in PBS buffer (pH 7.2–7.4; sterile filtered).

Substrate Preparation

The preparation of the calcium-deficient hydroxyapatite (CDHAP) pellet (N. Bouropoulos, Department of Materials Science, University of Patras, Greece) can be seen in the Supporting Information, S11. The top layer of mica (Agar Scientific Ltd., Stansted, UK) was removed with adhesive tape. A silicon wafer [Si-Mat, Kaufering, Germany; crystal structure of (100)] was cleaned by a UV cleaner (Boekel Scientific, Pennsylvania, USA) for 15 min prior to usage. Afterward, it was sonicated in water, methanol, acetone, and toluene followed by coating of TiO2 nanoparticles (for detailed information, see Supporting Information, S12). The layer thickness of the TiO2-layered wafer was determined by ellipsometry (Sentech SE 500adv, Sentech Instruments, Berlin, Germany) (see Supporting Information, S12).

AFM-SMFS Measurements

All AFM experiments were performed under buffered conditions (PBS buffer) with high-quality chemicals in a temperature- and humidity-controlled room at a temperature of 20.7 ± 0.7 °C and a humidity of 47.9 ± 6.7%. The measurements were conducted on a NanoWizard ultra speed atomic force microscope (JPK Instruments, Berlin, Germany) using Bruker MSNL-10 cantilevers (<12 nm tip radius, 0.02 N m–1 spring constant) in PBS buffer (pH 7.2–7.4). The AFM was operated with an inverted optical microscope Axio Observer, AxioVert 200 (Zeiss, Germany). The NanoWizard is placed on a Halcyonics i4 vibration isolation system (Accurion, Göttingen, Germany), and the whole system is located in an acoustic enclosure from JPK, which in turn is placed on a stable base (JPK, Berlin, Germany). Prior to functionalization, AFM tips were cleaned in chloroform, and cantilever spring constants were determined under dry conditions using a thermal method (Sader method).42 Spring constants (Kc) between 7.9 and 9.9 mN m–1 were obtained. After AFM tip functionalization, the cantilever sensitivity was measured in PBS buffer prior to every SMFS measurement. In brief, 16 force curves were recorded in air (prior to functionalization) and in PBS (prior to adhesion measurements) on a stiff substrate (mica), and each curve was linear fitted. The mean of the inversed slopes was then set as the inverse optical lever sensitivity (InvOLS) of the cantilever, once for the sensitivity measurement in air and once in PBS.

Data Analysis

Data acquisition and analysis were carried out using the SPMControl and JPK data processing software. Prior to analysis, only single rupture events (adhesion events) with the specific linker stretching and ±10 nm of the calculated contour length were taken into account, ensuring over 95% probability to be sure that the adhesion event was mediated by bounds of single peptide sequences. Due to this restriction of data, some of the curves were discarded, but the others provide a narrow distribution of the pull-off forces. The effective spring constant keff (see Supporting Information, S8) is calculated using the cantilever stiffness kc and the PEG linker-sample stiffness kL, which consists of the PEG elasticity model43 and the wormlike chain model (WLC)44 describing the extension of the peptide sequence (Supporting Information, S9).

Statistical Analysis

The statistical analysis was performed with the statistic software IBM statistics SPSS. The samples were independent, and the grouping variables were the tip sample and the dwell time. To test the significance of the adhesion values of the samples measured on mica, on hydroxyapatite and on a TiO2-coated silicon wafer, all samples with a dwell time of 4 s were chosen. Before testing the significance of the mean values of adhesion, the distribution of the data was tested in normality tests. Kruskal–Wallis tests with Bonferroni post hoc tests were performed to check significance between the samples by variation of dwell times on HAP and the validation on TiO2. Due to the fact that the mean value of several not normally distributed samples has to be compared, a Mann–Whitney-U test was done (see Supporting Information, S10).

Results and Discussion

Single-Molecule Adhesion Force of DpSpSEEK

To characterize specific adhesion motifs in a wet environment on different substrates, AFM tips were first functionalized with a flexible linker and second with a specific amino acid sequence to measure the pull-off force on different substrates. The adhesion forces of these amino acid sequences were investigated on mica, on calcium-deficient hydroxyapatite (CDHAP) to replicate the mineral phase of the bone and tooth, and on a TiO2-coated silicon wafer to mimic implants in a physiological environment. To simulate such conditions, adhesions motifs were measured in PBS buffer, keeping in mind that the surface of the substrates could potentially be saturated with phosphate ions. The functionalization of silicon nitride (Si3N4) AFM tips with nominal spring constants of 0.02 N m–1 followed an established protocol for antigen–antibody interaction measurements.40,45 After amino-functionalization with ethanolamine, a PEG-linker with an N-hydroxysuccinimide (NHS) group, with a length of about 72 nm, was attached to the tip (see Figure 2a). The other end of the linker contained a maleimide group, where site-specific coupling of amino acid sequences with a cysteine at the C-terminal end was performed via Michael-type maleimide-click chemistry. The N-terminal end of the sequences was acetyl (Ac) protected to avoid interactions of the amine with the substrates.

Figure 2.

Figure 2

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(a) AFM chip functionalization with a maleimide-PEG-NHS linker and following adhesion motif attachment. (b) Selected force–distance curves of SMFS measurement with DpSpSEEKC on CDHAP with different dwell times and a constant retraction velocity of 1000 nm s–1. (c) Mean adhesion values of different dwell times (error bars indicate standard deviations). For statistical analysis, Kolmogorov–Smirnov tests of every sample for normal distribution (rejected) were performed followed by a Mann–Whitney-U test to test the significance between two independent samples.

In a typical pull-off experiment, a functionalized AFM tip, submerged in PBS (pH 7.2), was extended with a constant displacement rate toward the target surface (CDHAP, TiO2, or mica) until contact with the surface reaches a predefined (set point) force. Then, the contact was held for different time periods (0, 2, 4, and 8 s). These dwell times at the extended position, i.e., at contact with the substrate, allow the adhesion motif to orientate and adhere to the surface. Finally, the functionalized tip with the attached adhesion motif was retracted from the surface with different velocities (100 to 2000 nm s–1) while recording force vs. extension data. For the data presented in the figures below, we used a maleimide-PEG-NHS linker system with an average of 162 ethylene glycol units (with an estimated length of 72 nm) for a clear separation between unspecific and specific adhesion events. For the chosen linker system, specific adhesion events were recorded at tip-sample separation distances of 60 to 150 nm, well-distanced from short-range, unspecific adhesion events. We note here that also, a shorter linker (27 ethylene glycol units) produced similar results, but no clear separation of specific adhesion events was observed (see Supporting Information, S1 and S2). For further validation, also the adhesion properties of tips during the different functionalization steps were checked showing that “true adhesion events”, as per the definition above, are only present when an adhesion motif is indeed attached to the substrate (see Supporting Information, S3). We also estimated the maximum density of molecules (molecules/μm2) that can be bound on the apex of the AFM tip, using data from a quantification of coupling sites by a marker enzyme assay done by Ebner et al.46 and information of the AFM tip geometry (see Supporting Information, Figure S4a). This shows that effectively, only one molecule and one single adhesion motif interact with the substrate. Multiple interactions and dissociation events were present in about 10% of the data as is usually the case for this SMFS technique47 and show very similar adhesion forces (see Supporting Information, Figure S4b).

The dependence of adhesion of the motif DpSpSEEKC on CDHAP on the surface dwell time was investigated by varying the dwell time from 0 to 8 s and using a constant retraction velocity of 1000 nm s–1 (Figure 2b). The adhesion force increased significantly with increasing dwell times from 0 to 4 s, whereas dwell times above 4 s did exhibit similar adhesion force values (Figure 2c). Each bar in the chart presented in Figure 2c consists of 500 measurements from four cantilevers. These results clearly show that sufficient dwell time is important for adhesion motifs to orient and arrange themselves properly on the surface. Notably, the adhesion force at dwell times of 4 s and above was 1.054 ± 0.026 nN, which is slightly higher compared to the single-molecule adhesion force of DOPA on a Ti substrate as reported by Lee et al.14 However, these pull-off forces at a dwell time of 4 s are in perfect agreement to measurements of Krysiak et al.15 The mechanism of the dwell time phenomenon remains unclear; we assume that a longer dwell time is caused by the replacement of the occupying phosphonate ions from the PBS buffer through phosphonates on the adhesion motif on the surface. Furthermore, the adhesion motifs need some time to achieve the optimal conformation for interface formation.48 There are indications that a “standing up/lying down” mechanism49 or a “rolling” into minima of the free energy50 plays an important role in catechols. In addition to dwell time, the dependence of adhesion force on the loading rate was investigated. This showed the expected rise of adhesion force with increasing loading rates, as shown in Figures 3c and 4c, similar to reported adhesion measurements on DOPA.14 From these data, the bond lengths and bond dissociation energies were calculated (see Supporting Information, S7) according to the Bell–Evans model51 as described by Friedsam et al.52

Figure 3.

Figure 3

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SMFS measurement of DSSEEKC and DpSpSEEKC on CDHAP with 4 s dwell time and a retraction velocity of 1000 nm s–1. (a) Selected FD curves of DSSEEKC and DpSpSEEKC. (b) Histograms (n = 500) of adhesion force values of DSSEEKC (242 ± 26 pN) and DpSpSEEKC (1054 ± 30 pN) (p < 0.001) measured with four AFM tips. For statistical analysis, Kolmogorov–Smirnov tests of every sample for normal distribution (rejected) were performed followed by a Mann–Whitney-U test to show significance between two independent samples. (c) Force plotted against the corresponding loading rates (LR) on a logarithmic abscissa with fit. The loading rate is the product of pulling velocity and effective spring constant (keff). (d) Schematic illustration of statherin where its phosphorylated serines on the N-terminus contribute to adhesion on CDHAP via H-bonds, ionic interactions, and calcium coordination bonds.

Figure 4.

Figure 4

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SMFS measurement of DSSEEKC and DpSpSEEKC on TiO2 with 4 s dwell time and a retraction velocity of 1000 nm s–1. (a) Representative FD curves of DSSEEKC and DpSpSEEKC. (b) Histograms (n = 500) of DSSEEKC (223 ± 20 pN) and DpSpSEEKC (775 ± 23 pN) showing a significant difference of pull-off forces (p < 0.001). For statistical analysis, Kolmogorov–Smirnov tests of every sample for normal distribution (rejected) were performed followed by a Mann–Whitney-U test to show significance between two independent samples. (c) Force plotted against the corresponding loading rates (LR) on a logarithmic abscissa with fit. The loading rate is the product of pulling velocity and effective spring constant (keff). (d) Schematic illustration of statherin where its phosphorylated serines on the N-terminus contribute to adhesion on a TiO2-coated silicon wafer via Ti binding.

Adhesion Force Is Dependent on Serine Phosphorylation

To investigate how phosphorylation influences adhesion behavior, we tested the two variants of the motif: one with (DpSpSEEKC) and one without (DSSEEKC) phosphorylation of the serines. Figure 4a shows the comparison of selected retraction FD curves on a CDHAP substrate for each of these two peptide sequences, with 4 s dwell time and 1000 nm s–1 pull-off velocity. More than 500 FD curves per pulling velocity and dwell time were recorded on three spots on the substrate. Plotting the detected adhesion forces from curves showing a distinct dissociation event as histograms (Figure 3b) shows that adhesion forces for both motifs are not normally distributed (Kolmogorov–Smirnov normality test). Furthermore, significantly higher adhesion forces could be identified for the phosphorylated peptide sequence. This difference can be explained with the deprotonated hydroxyl groups presented by the phosphorylated serines of the negatively charged phosphorylated adhesion motif at pH 7.2 in PBS, as sketched in Figure 3d. In this neutral milieu, the point of zero charge (pzc) at the HAP surface is 7.4.53 The hydroxyl groups are expected to interact with the CDHAP surface through similar mechanisms as described by Waite,13 that is, H-bonds, ionic interactions with the surface, and coordination bonds to calcium.

To gain further insights into the free-energy landscape of the phosphorylated and unphosphorylated amino acid sequences, SMFS measurements were performed by varying the loading rate, which is the product of pulling velocity and effective spring constant (see Figures 3c and 4c). The calculations for DpSpSEEKC on CDHAP revealed a bond length (xb) of 1.85 ± 0.12 Å and a bond dissociation energy (ΔEb) of 23.81 ± 1.22 kcal mol–1, whereas DSSEEKC shows an xb of 2.35 ± 0.16 Å and a ΔEb of 4.15 ± 0.07 kcal mol–1.

Similarly to CDHAP, adhesion to TiO2 was investigated. TiO2 is a material commonly used in orthopedic implants; hence, strong adhesion to TiO2 substrates is also desirable. Figure 4a shows the comparison of FD curves of DSSEEKC and DpSpSEEKC on a TiO2-coated silicon wafer with 4 s dwell time and 1000 nm s–1 pull-off velocity. The noticeable event around 10 nm tip-sample separation is a non-specific tip-surface interaction. After data post-processing (i.e., excluding curves not showing a clear adhesion event; example curves (shown in Supporting Information, S1)) and statistical analysis, similar to experiments on CDHAP, the histograms in Figure 4b show the same dependence of adhesion force on TiO2 from phosphorylation of serines; the phosphorylated peptide sequence shows significantly higher adhesion on TiO2 compared to the unphosphorylated sequence. Similar to adhesion on CDHAP, the difference can be explained with the presence or absence of the acidic hydroxyl groups, which interact with the TiO2 surface. At pH 7.2 in PBS, the surface is charged more positively because the pzc of TiO2 (rutile and anatase) is at 5.5–7.5.54 The interactions likely include H-bonds, ionic interactions of deprotonated hydroxyl groups, and coordination bonds to Ti as sketched in Figure 4d. In addition, adhesion forces exhibited the expected rise with the loading rate (Figure 4c) and enable the determination of xb and ΔEb. The calculations for DpSpSEEKC on TiO2 revealed a bond length (xb) of 1.98 ± 0.06 Å and a bond dissociation energy (ΔEb) of 17.76 ± 0.4 kcal mol–1, whereas DSSEEKC shows an xb of 2.57 ± 0.22 Å and a ΔEb of 4.44 ± 0.1 kcal mol–1 (see Supporting Information, S7).

Our SMFS measurements on CDHAP and on TiO2 clearly proved the hypothesis that the phosphorylation of serines plays a decisive role in specifying adhesion of the motif both to CDHAP and TiO2 substrates (see Figures 3d and 4d). Bond dissociation energy and bond length determinations are in perfect agreement to density functional theory calculations for phosphorylated amino acids exhibit ΔEb values of 25–30 kcal mol–1 and bond lengths of 1.3–3 Å.55 Phosphorylation and related interactions with the surface lead to higher dissociation energies and shorter bond length to the surfaces.

Additionally, the short sequence DpSpSEEKC leads to less sterical hindrance and better mobility, which likely promotes the adhesion of the phosphorylated serines.56 These conditions are also present in the longer sequence of 15 amino acids (SN15 at the N-terminus), where adhesion is improved through the α-helical structure.20 As expected, additional measurements on mica (muscovite) substrates, which at physiological pH are expected to present a negative surface charge and no coordination bond due to lack of calcium ions, unveil no beneficial effect of phosphorylation of serines; nevertheless, mean values and distribution of pull-off forces of the two peptide sequences (DpSpSEEKC, 278 ± 30 pN; DSSEEKC, 250 ± 25 pN) were significantly different (see Supporting Information, S6).

Comparison to DOPA Adhesion

To directly compare adhesion properties of adhesion motif DpSpSEEKC with previously reported properties of DOPA,14 we produced probes, where a thiol-modified dopamine was attached to the Mal-PEG-NHS-functionalized AFM tip. Adhesion force was measured on the same TiO2 substrates as above and compared to published values.14 The mean adhesion force value was 859 ± 157 pN (0 s dwell time) and further increased with higher dwell time (see Figure 5). This means that the adhesion motif DpSpSEEKC investigated here produces adhesion comparable to DOPA for the investigated substrates CDHAP and TiO2. Some research groups stated possible rupture forces of different bonds related to the linker system or sample attachment via thiol-maleimide coupling.57,58 During our adhesion measurements, no bond ruptures due to excessive adhesion of the motifs on the surface were observed. Otherwise, the following pull-off attempts would have revealed non-reproducible and very low adhesion values.

Figure 5.

Figure 5

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Validation of SMFS measurement by comparing the pull-off forces of DpSpSEEKC and dopamine-functionalized tips on TiO2 at 4 s dwell time and a retraction velocity of 1000 nm s–1. (a) Representative FD curves. (b) Bars showing a significant difference of pull-off forces for DpSpSEEKC (775 ± 23 pN) and DOPA (1232 ± 43 pN) (p < 0.001). For statistical analysis, Kolmogorov–Smirnov tests of every sample for normal distribution (rejected) were performed followed by a Mann–Whitney-U test to test the significance between two independent samples.

In the context of bioinspired glues, especially for orthopedic applications, the adhesion motif presented here may be an interesting candidate for further exploitation. If not in its natural state, an alternative may be the use of phosphorylated groups also in synthetic polymers to achieve similar adhesion in a wet environment. From a chemical perspective, the fact that adhesion of the motif DpSpSEEKC depends so strongly on phosphorylation can be well explained as the binding through hydroxyl groups is similar to DOPA. From a biomechanical perspective, the phosphorylation dependence shines important light on how adhesion can be steered in biological systems, i.e., through enzymatic phosphorylation and dephosphorylation. In this context, our results corroborate recently reported correlation of phosphorylation levels of NCPs in bones and especially of OPN with crack propagation toughness, i.e., the slope of fracture resistance curves.59 Seemingly, an older bone has lower NCP phosphorylation likely leading to lower adhesion and cohesion34 of the interfaces that are the first ones to fail during bone damage and fracture.60 Therefore, phosphorylation is not only an interesting feature when designing bioinspired adhesion motifs for application in biomedical science, i.e., as components of bone or dental adhesives, but also for considering novel translational approaches to improve the material properties of aged and elderly bones.

Conclusion

Strong adhesion in wet environments still remains a challenge for bonding technology in biomedical applications. While several adhesion strategies in the animal kingdom were evaluated in the past, we have focused on an adhesion motif that is present in the human body in the saliva protein statherin DpSpSEEKC as well as in non-collagenous proteins in bones. This motif showed significant adhesion on hydroxyapatite and on a TiO2-coated silicon wafer, which was critically dependent on phosphorylation. The unphosphorylated variant DSSEEKC exhibited significantly lower adhesion. Adhesion properties were significantly lower to previously reported ones for DOPA. Bond dissociation energy values on CDHAP and TiO2 clearly corroborate the adhesion dependency through serine phosphorylation. This means that the motif DpSpSEEKC can indeed be considered as a strong and fully biocompatible adhesion motif for future biomedical glues. Additionally, our results suggest that in the human body, adhesion of this motif can be adjusted by phosphorylation or dephosphorylation, which is in agreement with reduced phosphorylation levels and impaired mechanical properties of bones with old age.

Acknowledgments

The authors would like to thank Thorsten Hugel for helpful discussions regarding SMFS. P.J.T. would like to gratefully acknowledge discussions with and inspiration for this work by J. Herbert Waite. Funding by the Christian Doppler Research Association (Christian Doppler Laboratory for Advanced Polymers for Biomaterials and 3D Printing), the Austrian Federal Ministry for Digital and Economic Affairs, the National Foundation for Research, Technology and Development, and the TU Wien Biointerface doctorate school are gratefully acknowledged. A.R. is a recipient of a DOC Fellowship of the Austrian Academy of Sciences at the Institute of Lightweight Design and Structural Biomechanics at TU Wien.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.langmuir.0c02325.

  • Classification of force distance curves, investigation of the influence on length of the linker system, investigation of adhesion forces of every single functionalization step, determination of the number of anchored molecules on the apex of the AFM tip, SMFS measurement on mica, determination of bond length and bond dissociation energy, calculation of PEG-peptide extension via a WLC model, statistical analysis, and a materials and methods section (PDF)

Author Contributions

○ P.J.T. and S.B. contributed equally to this manuscript. P.S. performed the laboratory and SMFS experiments, interpreted the data, prepared the figures, and wrote the manuscript. A.R. assisted with the experiments and SMFS measurement and revised and edited the manuscript. N.B. produced and analyzed the CDHAP substrates. O.A., P.J.T., R.L., and S.B. supervised experimental concept and data interpretation and revised and edited the manuscript. All the authors critically evaluated the manuscript prior to submission.

The authors declare no competing financial interest.

Supplementary Material

la0c02325_si_001.pdf (1.7MB, pdf)

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Supplementary Materials

la0c02325_si_001.pdf (1.7MB, pdf)

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