Novel in situ sensing surface forces apparatus for measuring gold versus gold, hydrophobic, and biophysical interactions

All active systems that undergo change, motion, or flow of matter (i.e., all biological systems and all mechanical systems) are subject to fundamental forces that drive and steer the way in which atoms, molecules, and ultimately macroscopic structures develop, evolve, adapt, and age. Consequently, the study of interactive forces is a shared and fundamental interest in seemingly unrelated fields such as biophysics and adhesion, or surface science and stem cell research.

Techniques such as atomic force microscopy¹ or using optical tweezers² can measure forces at molecular-scale and nanoscale, while they do not actively track distances. In contrast, the surface forces apparatus (SFA) is a device that couples the measurement of interaction forces and distances between mesoscopic surfaces. The core of the SFA is multiple beam interferometry (MBI), an optical high precision distance measuring technique based on the interference of white light passing through an optical cavity.

In the simplest case, this cavity is defined by two semitransparent (20–60 nm thick) mirrors on the backside of a transparent 2–5 $μ$m thick spacer layer.³ The spacer is necessary to generate a cavity that is in the range of the wavelength of white light. Traditionally, muscovite mica was used, as it can be cleaved into uniformly thick spacer layers with an atomically smooth surface. Based on MBI, the SFA measures distances and infers forces from distance shifts using Hooke’s law,⁴ i.e., the SFA couples force and distance measurements. In comparison, an AFM measures forces and the distance is inferred from force measurements. The latter breaks down when compressible materials, which is the usual case in biologic systems, are used.

With the simultaneous coupling of force and distance measurements in SFA, many unique contributions were made in understanding interaction force profiles between mesoscopic surfaces for a variety of scientific questions such as ligand-receptor interactions,⁵ adhesion mechanisms inspired by mussels,⁶ lipid bilayer interactions,⁷ bacteria interactions,⁸ electrochemical surface forces,⁹ or lubrication and wear.¹⁰ 

In all of these examples, the MBI was defined by two silver metal layers deposited on the back of atomically smooth muscovite mica substrates, or between a metal surface (gold, platinum, palladium, nickel, etc.) and a back-silvered mica. However, using muscovite mica for at least one of the interacting surfaces significantly limits the experimental design flexibility of an SFA experiment.

In addition, the basal plane of mica is chemically very stable, and in solution it carries a high surface charge, which makes it very challenging to chemically modify mica. In contrast to very diverse and covalently bound self-assembly routes on gold, molecular self-assembly on mica is limited to electrostatic interaction of surfactants and lipids with the negative surface charge. However, such layers are only weakly bound in an aqueous environment due to this electrostatic bonding mechanism. This limits these systems mostly to low interaction force scenarios,¹¹ and lipid and surfactant flip-over¹² as well as pluck-out⁵ render such surfaces prone to interactive change during experiments.

In recent years, several groups developed template stripped-gold surfaces as a very flexible design platform for SFA experiments, providing self-assembly¹³ and electrochemical possibilities.^10,14 The opportunity of exploring different interaction forces utilizing gold as the substrate that can be modified by thiol chemistry was already extensively explored in the last decade.¹³ Recently, we demonstrated a very stable substrate modification for cushioned supported lipid bilayers employing thiol chemistry on gold surfaces. Specifically, strong binding of the inner leaflet of a bilayer can be achieved by using the semifluid 2,3-di-O-phytanyl-sn-glycerol-1-tetraethylene glycol-D,L-$α$-lipoic acid ester (DPhyTL) as an anchoring group. This dramatically increases stability of lipid bilayers during force-distance measurements (F-D), even in highly adhesive environments.¹¹ However, also in this work, we used mica as an apposing surface.

Already in the late 1990s, alternative strategies for realizing “mica free” interferometers for MBI were suggested.¹⁵ From an experimental point of view, mica was considered an irreplaceable component in traditional SFA due to both potential roughness of multilayer interferometers, as well as limitations for analyzing interference pattern of complex (more than two metal surfaces) interferometers. To completely avoid mica as an interacting surface material a three-mirror interferometer³ must be set up, and its interference pattern must be evaluated with a proper theoretical framework. Recently, a polymer supported three-mirror interferometer was presented by Van Engers et al.¹⁶ and proof-of-principle measurements indicate that the interferometry can be analyzed using a transfer matrix method. However, with this method, the epoxy layer used as a spacer exhibits large scale thickness undulations resulting in irregular secondary fringes, which complicate the analysis and render the surface, although nanoscopic smooth, significantly undulating on a larger scale.

In this work, we present an alternative approach to achieve a three-mirror interferometer that enables a similarly smooth gold-gold contact, with a mica spacer of constant thickness. We test this approach by measuring surface forces across gold-gold with a number of different surface modifications demonstrating stability, reproducibility, and equal applicability of this three-mirror setup, compared to traditional two-mirror setups. In addition, we show SFA force-distance measurements with our novel home-built sensing SFA (sSFA) that measures forces and distances both simultaneously and independently, in normal as well as shear direction. This new setup also includes a shear angle corrected friction configuration.

2,3-di-O-phytanyl-sn-glycerol-1-tetraethylene glycol-D,L-$α$-lipoic acid ester (DPhyTL, custom-made from Celestial Synthetics, Australia), sodium chloride and chloroform (99.9%, Sigma Aldrich), HPLC-grade ethanol, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) (DGS-NTA, Avanti Lipids) were used as supplied. Water was purified with a Milli-Q System (Merck).

High-purity gold and silver (99.99%, Goodfellow) and a titanium target (2 in., Kurt J Lesker) were used for PVD/sputtering processes. Muscovite mica layers (optical grade V1, S&J Trading Company, USA) were manually cleaved to uniform thicknesses ranging from 4 to 5 $μ$m.

Lipid bilayer preparations were adapted from Andersson et al.¹⁷ and combined with Langmuir–Blodgett trough (LBT) assembly. Therefore, gold surfaces were incubated in a 0.1 mg/ml DPhyTL ethanol solution. After 1 h, both surfaces were thoroughly rinsed with pure ethanol and gently blow-dried. The outer leaflet, consisting of DOPC mixed with NTA-DGS in a 1/49 wt%/wt%, was dissolved in chloroform and deposited on SFA disks in a LBT at a pressure of 20 or 30 mN/m, respectively. The sSFA cell was assembled in 150 mM NaCl to avoid drying out of the membranes. Measurements, if not otherwise stated, were performed in 150 mM NaCl.

All force-distance measurements were performed with a constant compression/retraction speed of 1.4 nm/s. All piezoelectric crystals and piezocontrollers are from PI. The control software is based on a home-made voltage control and output (labview interface).

F-D characteristics are constructed by matching in time the measured forces from the force sensor to the calculated separation distance, D. Forces and separation distances were recorded with 10 frames per second. Separation distance analysis from the acquired interference pattern was done using our home-built software package sfa explorer.⁴ For SAM functionalized gold surfaces, the SAM thicknesses used for the separation distance fitting were fitted to literature reported values, with DPhyTL having 5 nm,¹⁸ and a DOPC monolayer having 3 nm thickness.¹⁹ 

The sSFA was constructed solely from commercial parts that can be obtained from any supplier for optics (in this work Thorlabs). Specifically, a 2350 mW white LED source (400–700 nm), a Plan N 4$×$ Olympus objective (NA 0.1), or a Plan N 10$×$ Olympus objective (NA 0.25) was used. A Quantalux TM 2.1 MP camera was used for Newton ring imaging, and an Andor Zyla camera cooled to $−20°$C (sCMOS) was used for imaging fringes of equal chromatic order (FECO) in combination with a 500 mm Andor imaging spectrometer. A full list of parts can be obtained from the authors free of charge. Commercial parts are connected with simple home-made connectors and right-angle brackets made from drift minimized materials. Drawings can be obtained upon reasonable request. Piezoelectric OEM actuators from PI (P601 and P603) were used together with a PI closed loop controller (E610). The controller software is based on an in-house labview code.

Figure 1(a) shows the newly constructed in situ sensing surface forces apparatus (sSFA) that measures lateral and normal forces acting across two crossed cylindrical surfaces (radius R = 1–2 cm) that form a MBI. The setup also contains an electrochemical and fluid cell, shown in panel (b). The cell is also particularly designed for simple bilayer transfers. Specifically, in Fig. 1(a), an upper surface is mounted to a high accuracy xyz-translation stage (Thorlabs). The surface mount consists of home-built right-angle bracket mounted to a linear z-piezo (100 $μ$m range, PI instruments). In contrast to traditional SFA setups, we use a highly sensitive custom designed load cell based force sensor (ME-Messysteme GmbH, detection limit $≃1μ$N or 0.1 mN/m) instead of a double cantilever spring.

This is a central advancement of this setup and allows to decouple force measurements from the interferometric distance measurement. The load sensor is very stiff (about k = 10 kN/m) and designed to deflect in normal direction with respect to the applied load with high linearity. It uses semiconductor strain gauges (SGs) for detecting forces. To minimize the drift, the SG is enclosed in a thermally isolating housing. Two of these load cells are mounted in series with $90°$ rotation for measuring normal and sliding forces in the x/y plane, making them insensitive to gravitational pull.

The second surface is mounted on an alignment tower consisting of two rotation stages, a tilt control, and a shear piezo. The advantage of a double-rotation/tilt angle-correction mechanism is to allow a compensation of the misalignment angles of $γ$ and $θ$ between the shear piezo’s direction of motion, and the two surface apex directions of the crossed cylinders, respectively [see Fig. 1(c)]. The top surface apex is pointing along the z-direction, the apex of the shearing surface is mounted along the x-direction, and the load cells are rotated for detection of shear and normal forces, respectively. Angle misalignment of $θ$ between the x-motion of the piezo (shear direction) and the cylindrical apex of the mobile (lower) substrate result in considerable cross talk in shear experiments. Such a misalignment will cause asymmetric variation of the separation distance (and hence applied load) with increasing sliding distances. This often limits lateral motion to sliding distances of a few tens of nanometers in traditional setups or to high pressures. The misalignment angle $γ$ has less influence on the shear mechanism but adds consistency to optical data quality by adjusting the apposing cylinders perfectly parallel to each other. With this mechanism, we can control a parallel alignment with lateral distance inaccuracy well below 0.1% over a few micrometers (effectively 5–10 nm misalignment per micrometer).

The fluid cell is displayed in Fig. 1(b). The optics are aligned horizontally, allowing to remove the cell without remounting of the optics. As described in our previous work, the optical path is fixed and an established contact is translated into the center of the optical beam.⁴ The fluid cell is not closed with a gas tight sealing, inert gas atmospheres can be realized by bubbling the setup and the enclosure of the SFA with inert gases such as argon or helium. The cell can be filled with a maximum of $∼20ml$ liquid volume, and the cell fits into the well of an appropriately sized LBT. For preparing bilayer surfaces, the cell is small enough that samples can be mounted under water in the LBT. Electrochemical working electrodes (WEs) can be attached to the top and/or the bottom surface, and counter electrode ring (CE) and a commercial Ag-AgCl reference electrode (RE) are mounted as indicated.

All in all, this SFA combines commercial translation and rotation stages and piezoactuators, with custom-made couplings and right-angle brackets that can be easily reproduced in any workshop. The custom-made load cells are also commercially available. The drift stability of this setup is 0.06 $μ$N/min in a standard lab environment without special precautions and remains insignificant for several hours, if the detection system is placed within a thermal isolation box that is controlled to within $±0.2°$/h. The vertical placement drift (thermal expansion/contraction of the apparatus parts, and piezo drift in open loop) is typically about 1–2 nm/min in our lab. However, in comparison to classical SFA, where forces are measured as deviation from linear approach rates, the sSFA is in principle not sensitive to the thermal drift of the system components, as it records position and force independently at any time.

In summary, this makes this sSFA an affordable yet high-performance do-it-yourself (DIY) setup that can be constructed within a few weeks of planning time. Drawings and part numbers of commercially obtained parts can be obtained from the authors upon reasonable request and free of charge.

We also constructed a gold-gold surface setup based on a multilayer construction process using a physical vapor and magnetron sputtering deposition as shown in Fig. 2. First, standard molecularly smooth back-silvered mica substrates on cylindrical disks were prepared according to the established protocol^4,20 (steps 1–3 in Fig. 2). After the UV-glue (NOA 81) is fully cured, samples are introduced into the PVD for a second gold film evaporation on top of back-silvered mica surfaces. This process involves predeposition of an adhesion promoting layer of 3–5 nm titanium by argon sputtering (at a rate of 1 nm/min) followed by a thermal evaporation of 10 nm gold at a rate of 0.1 Å/s (steps 4 and 5) for completing the two layer mirror preparation.

This process provides a well wetting and hence well-adhering gold layer on mica; see Fig. 2 in the supplementary material²¹ for AFM imaging of the evaporated gold layer with an RMS roughness of 360 pm over $5×5μm2$⁠.

The latter compares well with the roughness of template stripped surfaces. The peak to peak roughness is slightly higher compared to template stripped surfaces, indicating statistically insignificant 5–10 peak to valley maxima of about 800 pm over $5×5μm2$⁠.

Positioning the prepared two layer mirror against another apposing template stripped-gold mirror will form a three-mirror interferometer, where two gold surfaces face each other.

As pointed out previously,¹⁶ interference pattern in a three-mirror interferometer show a pronounced overlap of primary and secondary fringes as can be seen in Fig. 2(b). This figure shows a three mirror and a standard mica versus gold experiment, prepared with similar mica thickness. Before discussing the observed pattern in detail, it is helpful to remind us of all reflecting surfaces within the interferometer again. Along the light path, the classical so called two-mirror interferometer consists of a glass-glue-Ag(40 nm)-mica(2–5 $μ$m)-gap-Au(40 nm)-glue-glass interferometer. Standing waves with high intensity are generated between the silver and gold mirror, while other interfaces contribute weak secondary standing waves. The newly designed three-mirror is setup as a glass-glue-Ag(40 nm)-mica(2–5 $μ$m)-TiAu(14 nm)-gap-Au(40 nmm)-glue-glass interferometer. Here, the main interference pattern still originates from the thick silver and gold layers, while the thin evaporated layer contributes more intense secondary FECO. The gap can be air or any liquid and the interacting surfaces are the surfaces that close the gap. Hence, in short we call these setups mica-gold and gold-gold for the interacting contact that is formed in such an interferometer, respectively.

In comparison the mica-gold and the gold-gold fringes of equal chromatic order (FECO) both show areas of alternating high and low intensity due to secondary interference pattern originating from silver-mica-TiAu (14 nm) interference corresponding to the mica thickness. Interestingly, bright spectral regions of the mica-gold setup show low intensity for the gold-gold setting. For the three-mirror interferometer the intensity is generally considerably lower and standing waves are slightly broader.

The Newton ring of the three-mirror interferometer is clear and also clearly indicates secondary FECO in the contact region, which originate from the gold-glue-glass disk interference pattern, reflecting the uneven glue layer thickness. These secondary patterns are weak and do not interfere with the experiment and analysis.

When the gap opens during a force versus distance measurement, primary fringes undergo a significant change in intensity during approach and separation of the surfaces. This can be seen in the middle line fitting in Fig. 2(b) on the right. The two fits indicate a fit in contact and at 23 nm distance. Compared to the classic two-mirror interferometer intensity drops significantly. In addition, also the wavelength shift is highly nonlinear for this interferometer. In particular, wavelength shifts in close contact are much smaller compared to a two-mirror interferometer, making the three-mirror interferometer in principle less sensitive. As we will see in the examples below, this is not significant and a typical spectrometer can still resolve the wavelength shifts with high enough accuracy to measure distances with subnanometer resolution. Also, the fit shows that the $4×4$ matrix method⁴ can very well model the obtained interference pattern, including intensity modulations.

One significant disadvantage of a direct gold-gold contacts is that it is prone to cold welding, as shown previously.¹⁶ Contacting nonfunctionalized gold surfaces very often results in rupture of the mica-supported gold thin film; see Fig. 1 in the supplementary material²¹ for a cold-welded contact and following hole of the gold layer.

In this study, we use thiol-based reagents to symmetrically modify two apposing gold surfaces to prevent cold welding. This solves cold welding; however, thin film thickness of deposited layers cannot be referenced directly against a gold-gold contact with such an approach. We measure all film thicknesses independently in classic two-mirror setups and use the measured mica thickness, and measured thin-film thicknesses and also compare to literature and use this approach for analyzing the force versus distance data with fixed thin-film thicknesses. This simplifies the experimental procedure and maintains an absolute reference frame. As an example, if the mica thickness is fitted directly from a three-mirror interferometer, the deviation compared to a fit from a 2-layer interferometer is in the range of 0.001% of the total thickness, which is insignificant.

We now demonstrate the performance of the sSFA and the three-mirror interferometer with a set of three different experiments, increasing in complexity of the surface architecture and simultaneously measured parameters.

First, we show hydrophobic interactions across two apposing DPhyTL modified gold surfaces in aerated electrolytes.

As shown in Fig. 3(a), hydrophobic force-distance (F-D) characteristic of such a system in MilliQ water and in 1 mM NaCl solution exhibits an expected long-range exponential attraction,²² as indicated by the red and blue dash line, respectively. The presence of 1 mM NaCl effectively reduces the long-range interaction from 80 nm down to 30 nm, which agrees very well with previously measured data.^9,12

However, the measured interaction is not related to a true short range hydrophobic effect (below 4–5 nm) between hydrophobic surfaces immersed in an electrolyte. In aerated fluids, two approaching hydrophobic surfaces can effectively jump into contact due to long-range electrostatic attraction or due to gas bubble formation. For experiments with lipid or surfactant monolayer, this long-range effect was explained by an alignment of patches of unstable monolayers (positively charged) on mica (negatively charged). Here, the DPhyTL layers are chemically grafted and the surface is intrinsically uncharged. Hence, a similar, long-range electrostatic effect is unlikely. However, condensation of air bubbles, nucleating from gas dissolved within the electrolyte, plays an equally important role in aerated electrolytes, as previously explained.¹² 

With the implemented top view camera,⁴ we also captured a real time image of the hydrophobic cavity formation during the attractive jump-ins as shown in the inset Newton ring photo in Fig. 3(a). It is clearly visible that gas bubbles form around the contact area during approach of the two hydrophobic surfaces. In addition, we can see the spontaneous flattening and growth of the hydrophobic contact areas (FECO flattening).

A great advantage of using sSFA in a deformable contact system is that the measured force is independent from the measured separation distance. Interestingly, and as indicated in the F-D profile (arrow), the attractive force keeps increasing after the attractive jump-in. This behavior clearly indicates that the strong hydrophobic attraction propagates in lateral direction deforming the contact. In this case, we see a continuous growth of the nucleating air cavities at the edge of the hydrophobic contact, but within the contact no significant air layer is observed. In Fig. 3(b), we also measured an exponential relation between the adhesion force to the electrolyte concentration. The decrease of adhesion with increasing salt concentration is likely related to the nucleation of air bubbles and the interfacial tension of the bubble/electrolyte interface, which results in the variations of the contact area and hence absolute adhesion. As shown in the inset figure of (b), the formed bubble remains on the surface after separation.

To further examine the property of this hydrophobic interface, we performed a set of friction experiments to analyze the coefficient of friction (COF) of hydrophobic DPhyTL-DPhyTL contacts. With our sSFA design, the shear angle misalignment can be precisely compensated down to an accuracy of $10−4°$⁠.

In addition, the force sensor pair simultaneously records the interaction force in both normal (blue) and horizontal/shear (red) direction during the shearing experiment, which provides instant online feedback and visualization for the shear profile change in response to the normal loading magnitude.

In Fig. 4(a), simultaneously recorded normal force and shear force at a constant loading are displayed. Unlike typical SFA friction measurements and comparable to the 3D-force sensing attachment for the SFA,²³ we can track the normal and the shear force simultaneously. Here, a small triangular wave observed in the normal force profile indicates a minor $±0.36mN$ magnitude crosstalk with the shear direction at a shear velocity of 1 $μ$m/s over 1 $μ$m shear distance. The absolute magnitude of the cross talk can be decreased further by limiting the travel distance.

In any case, with this setup, such systematic errors can be corrected mathematically using the force projection of the shear motion on the two force sensors, respectively, as shown in the inset figure in Fig. 4(b). With these corrections, we measured two COF for the hydrophobic DPhyTL-DPhyTL contact. Under low normal force, the measured COF $μ1$ was 0.57, while the value increased to 0.73 at normal force loadings higher than 2 mN. Again, the increase in the COF indicates the presence of bubbles.²⁴ Apparently, increased loading removes a thin cushion (bubble) layer and, therefore, results in an increase of the COF.

In summary, the new sSFA design and the three-mirror interferometer allow a new experimental insight into hydrophobic effects under very stable conditions, which will be subject to further in depth studies. Of particular interest will be completely degassed electrolytes.

After examining the gas bubble mediated hydrophobic-hydrophobic interaction, we can now look into the transition from pure hydrophobic SAM toward the symmetric interaction of bilayer systems, which are constructed from the same DPhyTL base with adding DOPC as the outer layer.

Adding of a second lipid layer of DOPC mixed with positive charge carrying NTA-DGS to the DPhyTL monolayer via a deposition in a LBT in 150 mM NaCl solution results in a very stable, symmetric lipid bilayer system. In Fig. 5(a), we show a F-D profile comparison of two lipid bilayer systems with different lateral pressures (⁠$Π$⁠) during LBT deposition: a system with lower deposition pressure of 20 mN/m (⁠$Π20$⁠) shown in red and higher pressure of 30 mN/m (⁠$Π30$⁠) shown in blue.

As expected, we observed a sharp contrast of the bilayer hemifusion mechanism between the systems. Both systems start to show interaction forces at a larger distance (I), suggesting the same bilayer thickness after LBT deposition for both lateral pressures (about 10 nm thickness for each bilayer).

However, $Π20$ exhibits a strong long-range attraction below distances of 20 nm (I) ending up in an attractive minimum at distance (II $Π20$⁠) before going into full hemifusion at distance (III). System $Π30$ also shows a long-range interaction starting at (I), which transitions into a strong repulsion until distance (II $Π30$⁠), where hemifusion to distance (III) starts. After hemifusion, both systems reach the same hardwall thickness of ca. 10 nm, indicating the pushing out of the outer DOPC layer and simultaneous formation of a DPhyTL-DPhyTL hydrophobic hardwall contact similar to the one observed in Fig. 3(a) This is also confirmed by the flattening of the FECO [inset in Fig. 5(a)].

The data are interesting and directly reflect the different pressures of the outer layers. First, a deposition of the outer DOPC layer at 20 mN/m reflects a layer in a disordered liquid form. It is hence not completely screening the hydrophobic interaction between the DPhyTL layers. This leads to hydrophobic interaction overpowering the steric repulsion at a distance of about 20 nm (I), resulting in an attractive jump-in to 12–13 nm (II $Π20$⁠). This also compares well with the DPhyTL-DPhyTL contact as seen for the purely hydrophobic experiments in Fig. 3, showing similar jump-in distances (⁠$Djump−in=25nm$⁠); however, adhesion forces (⁠$Fadh=71mN/m$⁠) reduced in magnitude due to the presence of an outer DOPC layer. Second, the bilayer system $Π30$ [Fig. 5(a), blue] constitutes a more dense and ordered state of the outer layer. Since the outer layer is well structured, electric double layer (EDL) and steric forces dominate the compression starting at about 20 nm (I) until the increasing normal load is causing the DOPC monolayers to squeeze out at 14–15 nm (II $Π30$⁠). At this distance, the normal pressure already resulted in a significant compressive expelling and hence increased expulsion of DOPC and hence exposure of the hydrophobic area of the DPythL supporting layer. This increased the exposure of hydrophobic area results in an increasing contribution of the hydrophobic attraction.

In order to resolve such an effect in more depth, we performed experiments with the $Π30$ bilayer at varying compressions. The positive axis of Fig. 5(b) presents a semilog plot of a series of consecutive F-D curves under different compression loadings: light compression at 0.2 mN/m (green), compression with subsequent fusion at 2 mN/m (black) and hard compression $>10mN/m$ (blue) with a clear fusion during approach. Here, we observe that the F-D characteristic of light compression (green) shows the same force profile during compression and only a weak adhesive hysteresis during separation. This confirms the assumption that the well-ordered external lipid layer is screening/overpowering the hydrophobic interaction. Increasing the compression, the external lipid layer squeezes out (II $Π30$⁠), weakening the screening effect and resulting in an adhesive jump into contact [Fig. 5(b), black]. Interestingly, the adhesive contact does not reach the same interaction compared to a pressure induced full hemifusion. In contrast, during separation the surfaces continue to approach into a closer contact, yet they approach to a distance about 1–2 nm further out compared to the F-D profile recorded at highest applied normal load (blue curve). Also, the adhesive force, with 28 mN/m, is well below adhesion forces measured for the highest compressive load. Specifically, only at normal loads above 3–4 mN/m (blue), the squeezing process continues into a direct hydrophobic contact (III) with 43 mN/m adhesive force recorded during separation. Interestingly, during separation, both surfaces indicate an extension before the surfaces separate fully. This separation is consistent with an extension of the DPhyTL layer due to polymeric relaxation effects of the poly-ethylenglycol (PEG) chain within the DPhyTL layer.¹¹ In Fig. 5(b), we also observe an initial recovery of the bilayer during decreasing of the force $δ=1.8±0.2$ nm, followed by separation at constant load. In our previous work¹¹ with asymmetric systems, we found that for a DPhyTL based bilayer facing a mica surface, an about half as pronounced extension of $δ∗=1.3±0.2$ nm was observed, fitting well to our observed results for a symmetric system with two DPhyTL layers.

In this work, we present the successful implementation of symmetric surface modification in a three-mirror interferometer in SFA. Our sSFA setup opens possibilities to simultaneously measure interaction forces in normal and shear configuration while independently measuring distances between symmetrically or asymmetrically modified gold surfaces.

The measured data compare well with previous observations with simpler two-mirror interferometers. Also, contact geometries and bubble formations can be readily resolved, providing detailed insight into an adhesive contact during formation and separation. The slightly increased peak-to-peak roughness of 800 pm compared to template stripped-gold surfaces is not problematic, in particular, when surfaces are functionalized with few nanometer thick fluid-like SAM architectures. We show that stable, consistent interference pattern and force measurements can be conducted with the developed three-mirror interferometer on hydrophobic surfaces as well as on tethered lipid bilayer systems. In particular, with the possibility for functionalizing gold with stable PEG-cushioned lipid bilayers, we were able to look closer into hemifusion and adhesion behavior of semifluid membranes that resemble biologic membranes more closely compared to supported LB on mica. We also found interesting forces evolving in time, for a direct hydrophobic contact, which we currently interpret as continuous bubble coalescence in the aerated electrolyte. Such forces would not appear in classic SFA measurements, where distance and force measurements are entangled.

Hence, using the three-mirror interferometer in an sSFA may allow for new insight into the kinetic behavior during active surface interactions in well defined, stable systems where substrate modification can be achieved by thiol-gold chemistry. With this setup, it should be possible to measure pure hydrophobic interactions in an SFA setup, as well as symmetric and asymmetric bilayer interactions including strong ligand-receptor interactions.

The authors thank the European Research Council for the support given through the ERC Grant No. 677663 (CSI.interface).

The data that support the findings of this study are available from the corresponding author upon reasonable request.

 Markus Valtiner is a professor of applied interface physics at the Vienna University of Technology (TU Wien). For his Ph.D. work at the Max-Planck-Institut für Eisenforschung(MPIE) in Düsseldorf, Germany (2005–2008), he received the Otto-Hahn medal of the Max-Planck-Society. From 2009 to 2012, he worked as a postdoctoral researcher in the Department of Chemical Engineering, University of California, Santa Barbara. In 2012, he again joined the MPIE as a group leader, and from 2016 to 2017, he was a professor of physical chemistry at the Freiberg University of Technology. In 2017, he received the Peter Mark Award of the American Vacuum society. His scientific interests are focused on solid/liquid interfaces, single-molecule interactions, and adhesion as well as corrosion in confined spaces using force probe experiments and, in particular, the surfaces forces apparatus. Since 2020, he has also been an enthusiastic expert for online teaching.