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With the development of new ultra-soft materials for medical devices and biomedical applications, the comprehensive characterization of their physical and mechanical properties is both important and challenging. A modified atomic force microscopy (AFM) nanoindentation technique was applied to characterize the extremely low surface modulus of the new lehfilcon A biomimetic silicone hydrogel contact lens coated with a layer of branched polymer brush structures. This method allows precise determination of contact points without the effects of viscous extrusion when approaching branched polymers. In addition, it makes it possible to determine the mechanical characteristics of individual brush elements without the effect of poroelasticity. This is achieved by selecting an AFM probe with a design (tip size, geometry and spring rate) that is particularly suitable for measuring the properties of soft materials and biological samples. This method improves sensitivity and accuracy for accurate measurement of the very soft material lehfilcon A, which has an extremely low modulus of elasticity on the surface area (up to 2 kPa) and an extremely high elasticity in the internal (almost 100%) aqueous environment. The results of the surface study not only revealed the ultra-soft surface properties of the lehfilcon A lens, but also showed that the modulus of the branched polymer brushes was comparable to that of the silicon-hydrogen substrate. This surface characterization technique can be applied to other ultra-soft materials and medical devices.
The mechanical properties of materials designed for direct contact with living tissue are often determined by the biological environment. The perfect match of these material properties helps to achieve the desired clinical characteristics of the material without causing adverse cellular responses1,2,3. For bulk homogeneous materials, the characterization of mechanical properties is relatively easy due to the availability of standard procedures and test methods (eg, microindentation4,5,6). However, for ultra-soft materials such as gels, hydrogels, biopolymers, living cells, etc., these test methods are generally not applicable due to measurement resolution limitations and the inhomogeneity of some materials7. Over the years, traditional indentation methods have been modified and adapted to characterize a wide range of soft materials, but many methods still suffer from serious shortcomings that limit their use8,9,10,11,12,13. The lack of specialized test methods that can accurately and reliably characterize the mechanical properties of supersoft materials and surface layers severely limits their use in various applications.
In our previous work, we introduced the lehfilcon A (CL) contact lens, a soft heterogeneous material with all the ultra-soft surface properties derived from potentially biomimetic designs inspired by the surface of the cornea of ​​the eye. This biomaterial was developed by grafting a branched, cross-linked polymer layer of poly(2-methacryloyloxyethylphosphorylcholine (MPC)) (PMPC) onto a silicone hydrogel (SiHy) 15 designed for medical devices based on. This grafting process creates a layer on the surface consisting of a very soft and highly elastic branched polymeric brush structure. Our previous work has confirmed that the biomimetic structure of lehfilcon A CL provides superior surface properties such as improved wetting and fouling prevention, increased lubricity, and reduced cell and bacterial adhesion15,16. In addition, the use and development of this biomimetic material also suggests further expansion to other biomedical devices. Therefore, it is critical to characterize the surface properties of this ultra-soft material and understand its mechanical interaction with the eye in order to create a comprehensive knowledge base to support future developments and applications. Most commercially available SiHy contact lenses are composed of a homogeneous mixture of hydrophilic and hydrophobic polymers that form a uniform material structure17. Several studies have been conducted to investigate their mechanical properties using traditional compression, tensile and microindentation test methods18,19,20,21. However, the novel biomimetic design of lehfilcon A CL makes it a unique heterogeneous material in which the mechanical properties of the branched polymer brush structures differ significantly from those of the SiHy base substrate. Therefore, it is very difficult to accurately quantify these properties using conventional and indentation methods. A promising method uses the nanoindentation testing method implemented in atomic force microscopy (AFM), a method that has been used to determine the mechanical properties of soft viscoelastic materials such as biological cells and tissues, as well as soft polymers22,23,24,25. ,26,27,28,29,30. In AFM nanoindentation, the fundamentals of nanoindentation testing are combined with the latest advances in AFM technology to provide increased measurement sensitivity and testing of a wide range of inherently supersoft materials31,32,33,34,35,36. In addition, the technology offers other important advantages through the use of different geometries. indenter and probe and the possibility of testing in various liquid media.
AFM nanoindentation can be conditionally divided into three main components: (1) equipment (sensors, detectors, probes, etc.); (2) measurement parameters (such as force, displacement, speed, ramp size, etc.); (3) Data processing (baseline correction, touch point estimation, data fitting, modeling, etc.). A significant problem with this method is that several studies in the literature using AFM nanoindentation report very different quantitative results for the same sample/cell/material type37,38,39,40,41. For example, Lekka et al. The influence of AFM probe geometry on the measured Young’s modulus of samples of mechanically homogeneous hydrogel and heterogeneous cells was studied and compared. They report that modulus values ​​are highly dependent on cantilever selection and tip shape, with the highest value for a pyramid-shaped probe and the lowest value of 42 for a spherical probe. Similarly, Selhuber-Unkel et al. It has been shown how the indenter speed, indenter size and thickness of polyacrylamide (PAAM) samples affect the Young’s modulus measured by ACM43 nanoindentation. Another complicating factor is the lack of standard extremely low modulus test materials and free test procedures. This makes it very difficult to get accurate results with confidence. However, the method is very useful for relative measurements and comparative evaluations between similar sample types, for example using AFM nanoindentation to distinguish normal cells from cancer cells 44, 45 .
When testing soft materials with AFM nanoindentation, a general rule of thumb is to use a probe with a low spring constant (k) that closely matches the sample modulus and a hemispherical/round tip so that the first probe does not pierce the sample surfaces on first contact with soft materials. It is also important that the deflection signal generated by the probe be strong enough to be detected by the laser detector system24,34,46,47. In the case of ultra-soft heterogeneous cells, tissues and gels, another challenge is to overcome the adhesive force between the probe and the sample surface to ensure reproducible and reliable measurements48,49,50. Until recently, most work on AFM nanoindentation has focused on the study of the mechanical behavior of biological cells, tissues, gels, hydrogels, and biomolecules using relatively large spherical probes, commonly referred to as colloidal probes (CPs). , 47, 51, 52, 53, 54, 55. These tips have a radius of 1 to 50 µm and are commonly made from borosilicate glass, polymethyl methacrylate (PMMA), polystyrene (PS), silicon dioxide (SiO2) and diamond-like carbon (DLC) . Although CP-AFM nanoindentation is often the first choice for soft sample characterization, it has its own problems and limitations. The use of large, micron-sized spherical tips increases the total contact area of ​​the tip with the sample and results in a significant loss of spatial resolution. For soft, inhomogeneous specimens, where the mechanical properties of local elements may differ significantly from the average over a wider area, CP indentation can hide any inhomogeneity in properties on a local scale52. Colloidal probes are typically made by attaching micron-sized colloidal spheres to tipless cantilevers using epoxy adhesives. The manufacturing process itself is fraught with many problems and can lead to inconsistencies in the probe calibration process. In addition, the size and mass of colloidal particles directly affect the main calibration parameters of the cantilever, such as resonant frequency, spring stiffness, and deflection sensitivity56,57,58. Thus, commonly used methods for conventional AFM probes, such as temperature calibration, may not provide an accurate calibration for CP, and other methods may be required to perform these corrections57, 59, 60, 61. Typical CP indentation experiments use large deviations cantilever to study the properties of soft samples, which creates another problem when calibrating the non-linear behavior of the cantilever at relatively large deviations62,63,64. Modern colloidal probe indentation methods usually take into account the geometry of the cantilever used to calibrate the probe, but ignore the influence of colloidal particles, which creates additional uncertainty in the accuracy of the method38,61. Similarly, elastic moduli calculated by contact model fitting are directly dependent on the geometry of the indentation probe, and mismatch between tip and sample surface characteristics can lead to inaccuracies27, 65, 66, 67, 68. Some recent work by Spencer et al. The factors that should be taken into account when characterizing soft polymer brushes using the CP-AFM nanoindentation method are highlighted. They reported that the retention of a viscous fluid in polymer brushes as a function of speed results in an increase in head loading and hence different measurements of speed dependent properties30,69,70,71.
In this study, we have characterized the surface modulus of the ultra-soft highly elastic material lehfilcon A CL using a modified AFM nanoindentation method. Given the properties and new structure of this material, the sensitivity range of the traditional indentation method is clearly insufficient to characterize the modulus of this extremely soft material, so it is necessary to use an AFM nanoindentation method with higher sensitivity and lower sensitivity. level. After reviewing the shortcomings and problems of existing colloidal AFM probe nanoindentation techniques, we show why we chose a smaller, custom-designed AFM probe to eliminate sensitivity, background noise, pinpoint point of contact, measure velocity modulus of soft heterogeneous materials such as fluid retention dependency. and accurate quantification. In addition, we were able to accurately measure the shape and dimensions of the indentation tip, allowing us to use the cone-sphere fit model to determine the modulus of elasticity without assessing the contact area of ​​the tip with the material. The two implicit assumptions that are quantified in this work are the fully elastic material properties and the indentation depth-independent modulus. Using this method, we first tested ultra-soft standards with a known modulus to quantify the method, and then used this method to characterize the surfaces of two different contact lens materials. This method of characterizing AFM nanoindentation surfaces with increased sensitivity is expected to be applicable to a wide range of biomimetic heterogeneous ultrasoft materials with potential use in medical devices and biomedical applications.
Lehfilcon A contact lenses (Alcon, Fort Worth, Texas, USA) and their silicone hydrogel substrates were chosen for nanoindentation experiments. A specially designed lens mount was used in the experiment. To install the lens for testing, it was carefully placed on the dome-shaped stand, making sure that no air bubbles got inside, and then fixed with the edges. A hole in the fixture at the top of the lens holder provides access to the optical center of the lens for nanoindentation experiments while holding the liquid in place. This keeps the lenses fully hydrated. 500 μl of contact lens packaging solution was used as a test solution. To verify the quantitative results, commercially available non-activated polyacrylamide (PAAM) hydrogels were prepared from a polyacrylamide-co-methylene-bisacrylamide composition (100 mm Petrisoft Petri dishes, Matrigen, Irvine, CA, USA), a known elastic modulus of 1 kPa. Use 4-5 drops (approximately 125 µl) of phosphate buffered saline (PBS from Corning Life Sciences, Tewkesbury, MA, USA) and 1 drop of OPTI-FREE Puremoist contact lens solution (Alcon, Vaud, TX, USA). ) at the AFM hydrogel-probe interface.
Samples of Lehfilcon A CL and SiHy substrates were visualized using an FEI Quanta 250 Field Emission Scanning Electron Microscope (FEG SEM) system equipped with a Scanning Transmission Electron Microscope (STEM) detector. To prepare the samples, the lenses were first washed with water and cut into pie-shaped wedges. To achieve a differential contrast between the hydrophilic and hydrophobic components of the samples, a 0.10% stabilized solution of RuO4 was used as a dye, in which the samples were immersed for 30 min. The lehfilcon A CL RuO4 staining is important not only to achieve improved differential contrast, but also helps to preserve the structure of the branched polymer brushes in their original form, which are then visible on STEM images. They were then washed and dehydrated in a series of ethanol/water mixtures with increasing ethanol concentration. The samples were then cast with EMBed 812/Araldite epoxy, which cured overnight at 70°C. Sample blocks obtained by resin polymerization were cut with an ultramicrotome, and the resulting thin sections were visualized with a STEM detector in low vacuum mode at an accelerating voltage of 30 kV. The same SEM system was used for detailed characterization of the PFQNM-LC-A-CAL AFM probe (Bruker Nano, Santa Barbara, CA, USA). SEM images of the AFM probe were obtained in a typical high vacuum mode with an accelerating voltage of 30 kV. Acquire images at different angles and magnifications to record all the details of the shape and size of the AFM probe tip. All tip dimensions of interest in the images were measured digitally.
A Dimension FastScan Bio Icon atomic force microscope (Bruker Nano, Santa Barbara, CA, USA) with “PeakForce QNM in Fluid” mode was used to visualize and nanoindentate lehfilcon A CL, SiHy substrate, and PAAm hydrogel samples. For imaging experiments, a PEAKFORCE-HIRS-FA probe (Bruker) with a nominal tip radius of 1 nm was used to capture high resolution images of the sample at a scan rate of 0.50 Hz. All images were taken in aqueous solution.
AFM nanoindentation experiments were carried out using a PFQNM-LC-A-CAL probe (Bruker). The AFM probe has a silicon tip on a nitride cantilever 345 nm thick, 54 µm long and 4.5 µm wide with a resonant frequency of 45 kHz. It is specifically designed to characterize and perform quantitative nanomechanical measurements on soft biological samples. The sensors are individually calibrated at the factory with pre-calibrated spring settings. The spring constants of the probes used in this study were in the range of 0.05–0.1 N/m. To accurately determine the shape and size of the tip, the probe was characterized in detail using SEM. On fig. Figure 1a shows a high resolution, low magnification scanning electron micrograph of the PFQNM-LC-A-CAL probe, providing a holistic view of the probe design. On fig. 1b shows an enlarged view of the top of the probe tip, providing information about the shape and size of the tip. At the extreme end, the needle is a hemisphere about 140 nm in diameter (Fig. 1c). Below this, the tip tapers into a conical shape, reaching a measured length of approximately 500 nm. Outside the tapering region, the tip is cylindrical and terminates in a total tip length of 1.18 µm. This is the main functional part of the probe tip. In addition, a large spherical polystyrene (PS) probe (Novascan Technologies, Inc., Boone, Iowa, USA) with a tip diameter of 45 µm and a spring constant of 2 N/m was also used for testing as a colloidal probe. with PFQNM-LC-A-CAL 140 nm probe for comparison.
It has been reported that liquid can be trapped between the AFM probe and the polymer brush structure during nanoindentation, which will exert an upward force on the AFM probe before it actually touches the surface69. This viscous extrusion effect due to fluid retention can change the apparent point of contact, thereby affecting surface modulus measurements. To study the effect of probe geometry and indentation speed on fluid retention, indentation force curves were plotted for lehfilcon A CL samples using a 140 nm diameter probe at constant displacement rates of 1 µm/s and 2 µm/s. probe diameter 45 µm, fixed force setting 6 nN achieved at 1 µm/s. Experiments with a probe 140 nm in diameter were carried out at an indentation speed of 1 µm/s and a set force of 300 pN, chosen to create a contact pressure within the physiological range (1–8 kPa) of the upper eyelid. pressure 72. Soft ready-made samples of PAA hydrogel with a pressure of 1 kPa were tested for an indentation force of 50 pN at a speed of 1 μm/s using a probe with a diameter of 140 nm.
Since the length of the conical part of the tip of the PFQNM-LC-A-CAL probe is approximately 500 nm, for any indentation depth < 500 nm it can be safely assumed that the geometry of the probe during indentation will remain true to its cone shape. In addition, it is assumed that the surface of the material under test will exhibit a reversible elastic response, which will also be confirmed in the following sections. Therefore, depending on the shape and size of the tip, we chose the cone-sphere fitting model developed by Briscoe, Sebastian and Adams, which is available in the vendor’s software, to process our AFM nanoindentation experiments (NanoScope). Separation data analysis software, Bruker) 73. The model describes the force-displacement relationship F(δ) for a cone with a spherical apex defect. On fig. Figure 2 shows the contact geometry during the interaction of a rigid cone with a spherical tip, where R is the radius of the spherical tip, a is the contact radius, b is the contact radius at the end of the spherical tip, δ is the contact radius. indentation depth, θ is the half-angle of the cone. The SEM image of this probe clearly shows that the 140 nm diameter spherical tip merges tangentially into a cone, so here b is defined only through R, i.e. b = R cos θ. The vendor-supplied software provides a cone-sphere relationship to calculate Young’s modulus (E) values ​​from force separation data assuming a > b. Relationship:
where F is the indentation force, E is Young’s modulus, ν is Poisson’s ratio. The contact radius a can be estimated using:
Scheme of the contact geometry of a rigid cone with a spherical tip pressed into the material of a Lefilcon contact lens with a surface layer of branched polymer brushes.
If a ≤ b, the relation reduces to the equation for a conventional spherical indenter;
We believe that the interaction of the indenting probe with the branched structure of the PMPC polymer brush will cause the contact radius a to be greater than the spherical contact radius b. Therefore, for all quantitative measurements of the elastic modulus performed in this study, we used the dependence obtained for the case a > b.
The ultrasoft biomimetic materials studied in this study were comprehensively imaged using scanning transmission electron microscopy (STEM) of the sample cross section and atomic force microscopy (AFM) of the surface. This detailed surface characterization was performed as an extension of our previously published work, in which we determined that the dynamically branched polymeric brush structure of the PMPC-modified lehfilcon A CL surface exhibited similar mechanical properties to native corneal tissue 14 . For this reason, we refer to contact lens surfaces as biomimetic materials14. On fig. 3a,b show cross sections of branched PMPC polymer brush structures on the surface of a lehfilcon A CL substrate and an untreated SiHy substrate, respectively. The surfaces of both samples were further analyzed using high-resolution AFM images, which further confirmed the results of the STEM analysis (Fig. 3c, d). Taken together, these images give an approximate length of the PMPC branched polymer brush structure at 300–400 nm, which is critical for interpreting AFM nanoindentation measurements. Another key observation derived from the images is that the overall surface structure of the CL biomimetic material is morphologically different from that of the SiHy substrate material. This difference in their surface morphology can become apparent during their mechanical interaction with the indenting AFM probe and subsequently in the measured modulus values.
Cross-sectional STEM images of (a) lehfilcon A CL and (b) SiHy substrate. Scale bar, 500 nm. AFM images of the surface of the lehfilcon A CL substrate (c) and the base SiHy substrate (d) (3 µm × 3 µm).
Bioinspired polymers and polymer brush structures are inherently soft and have been widely studied and used in various biomedical applications74,75,76,77. Therefore, it is important to use the AFM nanoindentation method, which can accurately and reliably measure their mechanical properties. But at the same time, the unique properties of these ultra-soft materials, such as extremely low elastic modulus, high liquid content and high elasticity, often make it difficult to choose the right material, shape and shape of the indenting probe. size. This is important so that the indenter does not pierce the soft surface of the sample, which would lead to errors in determining the point of contact with the surface and the area of ​​contact.
For this, a comprehensive understanding of the morphology of ultra-soft biomimetic materials (lehfilcon A CL) is essential. Information about the size and structure of the branched polymer brushes obtained using the imaging method provides the basis for the mechanical characterization of the surface using AFM nanoindentation techniques. Instead of micron-sized spherical colloidal probes, we chose the PFQNM-LC-A-CAL silicon nitride probe (Bruker) with a tip diameter of 140 nm, specially designed for quantitative mapping of the mechanical properties of biological samples 78, 79, 80, 81, 82, 83, 84 The rationale for using relatively sharp probes compared to conventional colloidal probes can be explained by the structural features of the material. Comparing the probe tip size (~140 nm) with the branched polymer brushes on the surface of CL lehfilcon A, shown in Fig. 3a, it can be concluded that the tip is large enough to come into direct contact with these brush structures, which reduces the chance of the tip piercing through them. To illustrate this point, in Fig. 4 is a STEM image of the lehfilcon A CL and the indenting tip of the AFM probe (drawn to scale).
Schematic showing STEM image of lehfilcon A CL and an ACM indentation probe (drawn to scale).
In addition, the tip size of 140 nm is small enough to avoid the risk of any of the sticky extrusion effects previously reported for polymer brushes produced by the CP-AFM nanoindentation method69,71. We assume that due to the special cone-spherical shape and relatively small size of this AFM tip (Fig. 1), the nature of the force curve generated by lehfilcon A CL nanoindentation will not depend on the indentation speed or the loading/unloading speed. Therefore, it is not affected by poroelastic effects. To test this hypothesis, lehfilcon A CL samples were indented at a fixed maximum force using a PFQNM-LC-A-CAL probe, but at two different velocities, and the resulting tensile and retract force curves were used to plot the force (nN) in separation (µm) is shown in Figure 5a. It is clear that the force curves during loading and unloading completely overlap, and there is no clear evidence that the force shear at zero indentation depth increases with indentation speed in the figure, suggesting that the individual brush elements were characterized without a poroelastic effect. In contrast, fluid retention effects (viscous extrusion and poroelasticity effects) are evident for the 45 µm diameter AFM probe at the same indentation speed and are highlighted by the hysteresis between the stretch and retract curves, as shown in Figure 5b. These results support the hypothesis and suggest that 140 nm diameter probes are a good choice for characterizing such soft surfaces.
lehfilcon A CL indentation force curves using ACM; (a) using a probe with a diameter of 140 nm at two loading rates, demonstrating the absence of a poroelastic effect during surface indentation; (b) using probes with a diameter of 45 µm and 140 nm. s show the effects of viscous extrusion and poroelasticity for large probes compared to smaller probes.
To characterize ultrasoft surfaces, AFM nanoindentation methods must have the best probe to study the properties of the material under study. In addition to the tip shape and size, the sensitivity of the AFM detector system, sensitivity to tip deflection in the test environment, and cantilever stiffness play an important role in determining the accuracy and reliability of nanoindentation. measurements. For our AFM system, the Position Sensitive Detector (PSD) limit of detection is approximately 0.5 mV and is based on the pre-calibrated spring rate and the calculated fluid deflection sensitivity of the PFQNM-LC-A-CAL probe, which corresponds to the theoretical load sensitivity. is less than 0.1 pN. Therefore, this method allows the measurement of a minimum indentation force ≤ 0.1 pN without any peripheral noise component. However, it is nearly impossible for an AFM system to reduce peripheral noise to this level due to factors such as mechanical vibration and fluid dynamics. These factors limit the overall sensitivity of the AFM nanoindentation method and also result in a background noise signal of approximately ≤ 10 pN. For surface characterization, lehfilcon A CL and SiHy substrate samples were indented under fully hydrated conditions using a 140 nm probe for SEM characterization, and the resulting force curves were superimposed between force (pN) and pressure. The separation plot (µm) is shown in Figure 6a. Compared to the SiHy base substrate, the lehfilcon A CL force curve clearly shows a transitional phase starting at the point of contact with the forked polymer brush and ending with a sharp change in slope marking contact of the tip with the underlying material. This transitional part of the force curve highlights the truly elastic behavior of the branched polymer brush on the surface, as evidenced by the compression curve closely following the tension curve and the contrast in mechanical properties between the brush structure and bulky SiHy material. When comparing lefilcon. Separation of the average length of a branched polymer brush in the STEM image of the PCS (Fig. 3a) and its force curve along the abscissa in Fig. 3a. 6a shows that the method is able to detect the tip and the branched polymer reaching the very top of the surface. Contact between brush structures. In addition, close overlap of the force curves indicates no liquid retention effect. In this case, there is absolutely no adhesion between the needle and the surface of the sample. The uppermost sections of the force curves for the two samples overlap, reflecting the similarity of the mechanical properties of the substrate materials.
(a) AFM nanoindentation force curves for lehfilcon A CL substrates and SiHy substrates, (b) force curves showing contact point estimation using the background noise threshold method.
In order to study the finer details of the force curve, the tension curve of the lehfilcon A CL sample is re-plotted in Fig. 6b with a maximum force of 50 pN along the y-axis. This graph provides important information about the original background noise. The noise is in the range of ±10 pN, which is used to accurately determine the contact point and calculate the indentation depth. As reported in the literature, the identification of contact points is critical to accurately assess material properties such as modulus85. An approach involving automatic processing of force curve data has shown an improved fit between data fitting and quantitative measurements for soft materials86. In this work, our choice of points of contact is relatively simple and objective, but it has its limitations. Our conservative approach to determining the point of contact may result in slightly overestimated modulus values ​​for smaller indentation depths (< 100 nm). The use of algorithm-based touchpoint detection and automated data processing could be a continuation of this work in the future to further improve our method. Thus, for intrinsic background noise on the order of ±10 pN, we define the contact point as the first data point on the x-axis in Figure 6b with a value of ≥10 pN. Then, in accordance with the noise threshold of 10 pN, a vertical line at the level of ~0.27 µm marks the point of contact with the surface, after which the stretching curve continues until the substrate meets the indentation depth of ~270 nm. Interestingly, based on the size of the branched polymer brush features (300–400 nm) measured using the imaging method, the indentation depth of the CL lehfilcon A sample observed using the background noise threshold method is about 270 nm, which is very close to the measurement size with STEM. These results further confirm the compatibility and applicability of the shape and size of the AFM probe tip for indentation of this very soft and highly elastic branched polymer brush structure. This data also provides strong evidence to support our method of using background noise as a threshold for pinpointing contact points. Thus, any quantitative results obtained from mathematical modeling and force curve fitting should be relatively accurate.
Quantitative measurements by AFM nanoindentation methods are completely dependent on the mathematical models used for data selection and subsequent analysis. Therefore, it is important to consider all factors related to the choice of indenter, material properties and the mechanics of their interaction before choosing a particular model. In this case, the tip geometry was carefully characterized using SEM micrographs (Fig. 1), and based on the results, the 140 nm diameter AFM nanoindenting probe with a hard cone and spherical tip geometry is a good choice for characterizing lehfilcon A CL79 samples. Another important factor that needs to be carefully evaluated is the elasticity of the polymer material being tested. Although the initial data of nanoindentation (Figs. 5a and 6a) clearly outline the features of the overlapping of the tension and compression curves, i.e., the complete elastic recovery of the material, it is extremely important to confirm the purely elastic nature of the contacts. To this end, two successive indentations were performed at the same location on the surface of the lehfilcon A CL sample at an indentation rate of 1 µm/s under full hydration conditions. The resulting force curve data is shown in fig. 7 and, as expected, the expansion and compression curves of the two prints are almost identical, highlighting the high elasticity of the branched polymer brush structure.
Two indentation force curves at the same location on the surface of lehfilcon A CL indicate the ideal elasticity of the lens surface.
Based on information obtained from SEM and STEM images of the probe tip and lehfilcon A CL surface, respectively, the cone-sphere model is a reasonable mathematical representation of the interaction between the AFM probe tip and the soft polymer material being tested. In addition, for this cone-sphere model, the fundamental assumptions about the elastic properties of the imprinted material hold true for this new biomimetic material and are used to quantify the elastic modulus.
After a comprehensive evaluation of the AFM nanoindentation method and its components, including indentation probe properties (shape, size, and spring stiffness), sensitivity (background noise and contact point estimation), and data fitting models (quantitative modulus measurements), the method was used. characterize commercially available ultra-soft samples to verify quantitative results. A commercial polyacrylamide (PAAM) hydrogel with an elastic modulus of 1 kPa was tested under hydrated conditions using a 140 nm probe. Details of module testing and calculations are provided in the Supplementary Information. The results showed that the average modulus measured was 0.92 kPa, and the %RSD and percentage (%) deviation from the known modulus were less than 10%. These results confirm the accuracy and reproducibility of the AFM nanoindentation method used in this work to measure the moduli of ultrasoft materials. The surfaces of the lehfilcon A CL samples and the SiHy base substrate were further characterized using the same AFM nanoindentation method to study the apparent contact modulus of the ultrasoft surface as a function of indentation depth. Indentation force separation curves were generated for three specimens of each type (n = 3; one indentation per specimen) at a force of 300 pN, a speed of 1 µm/s, and full hydration. The indentation force sharing curve was approximated using a cone-sphere model. To obtain modulus dependent on indentation depth, a 40 nm wide portion of the force curve was set at each increment of 20 nm starting from the point of contact, and measured values ​​of the modulus at each step of the force curve. Spin Cy et al. A similar approach has been used to characterize the modulus gradient of poly(lauryl methacrylate) (P12MA) polymer brushes using colloidal AFM probe nanoindentation, and they are consistent with data using the Hertz contact model. This approach provides a plot of apparent contact modulus (kPa) versus indentation depth (nm), as shown in Figure 8, which illustrates the apparent contact modulus/depth gradient. The calculated elastic modulus of the CL lehfilcon A sample is in the range of 2–3 kPa within the upper 100 nm of the sample, beyond which it begins to increase with depth. On the other hand, when testing the SiHy base substrate without a brush-like film on the surface, the maximum indentation depth achieved at a force of 300 pN is less than 50 nm, and the modulus value obtained from the data is about 400 kPa, which is comparable to the values ​​of Young’s modulus for bulk materials.
Apparent contact modulus (kPa) vs. indentation depth (nm) for lehfilcon A CL and SiHy substrates using AFM nanoindentation method with cone-sphere geometry to measure modulus.
The uppermost surface of the novel biomimetic branched polymer brush structure exhibits an extremely low modulus of elasticity (2–3 kPa). This will match the free hanging end of the forked polymer brush as shown in the STEM image. While there is some evidence of a modulus gradient at the outer edge of the CL, the main high modulus substrate is more influential. However, the top 100 nm of the surface is within 20% of the total length of the branched polymer brush, so it is reasonable to assume that the measured values ​​of the modulus in this indentation depth range are relatively accurate and do not strongly depend on the effect of the bottom object.
Due to the unique biomimetic design of lehfilcon A contact lenses, consisting of branched PMPC polymer brush structures grafted onto the surface of SiHy substrates, it is very difficult to reliably characterize the mechanical properties of their surface structures using traditional measurement methods. Here we present an advanced AFM nanoindentation method for accurately characterizing ultra-soft materials such as lefilcon A with high water content and extremely high elasticity. This method is based on the use of an AFM probe whose tip size and geometry are carefully chosen to match the structural dimensions of the ultra-soft surface features to be imprinted. This combination of dimensions between probe and structure provides increased sensitivity, allowing us to measure the low modulus and inherent elastic properties of branched polymer brush elements, regardless of poroelastic effects. The results showed that the unique branched PMPC polymer brushes characteristic of the lens surface had an extremely low elastic modulus (up to 2 kPa) and very high elasticity (nearly 100%) when tested in an aqueous environment. The results of AFM nanoindentation also allowed us to characterize the apparent contact modulus/depth gradient (30 kPa/200 nm) of the biomimetic lens surface. This gradient may be due to the modulus difference between the branched polymer brushes and the SiHy substrate, or the branched structure/density of the polymer brushes, or a combination thereof. However, further in-depth studies are needed to fully understand the relationship between structure and properties, especially the effect of brush branching on mechanical properties. Similar measurements can help characterize the mechanical properties of the surface of other ultra-soft materials and medical devices.
Datasets generated and/or analyzed during the current study are available from the respective authors upon reasonable request.
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