https://doi.org/10.1038/s41598-022-24701-9
“As new ultra-soft materials are being developed for medical devices and biomedical applications, the comprehensive characterization of their physical and mechanical properties is both critical and challenging. To characterize the very low surface modulus of the novel biomimetic lehfilcon A silicone hydrogel contact lens coated with a layer of a branched polymer brush structure, an improved atomic force microscopy (AFM) nanoindentation method has been applied. This technique allows for precise contact-point determination without the effects of viscous squeeze-out upon approaching the branched polymer. Additionally, it allows individual brush elements to be mechanically characterized in the absence of poroelastic effects. This was accomplished by selecting an AFM probe with a design (tip size, geometry, and spring constant) that was especially suited to measuring the properties of soft materials and biological samples. The enhanced sensitivity and accuracy of this method allows for the precise measurement of the very soft lehfilcon A material, which has an extremely low elastic modulus in the surface region (as low as 2 kPa) and extremely high elasticity (nearly 100%) in an aqueous environment. The surface-characterization results not only reveal the ultra-soft nature of the lehfilcon A lens surface but also demonstrate that the elastic modulus exhibits a 30 kPa/200 nm gradient with depth due to the disparity between the modulus of the branched polymer brushes and the SiHy substrate. This surface-characterization methodology may be applied to other ultra-soft materials and medical devices.”
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AFM nanoindentation can be broadly divided into three main components: (1) hardware (sensors, detectors, probe etc.); (2) measurement parameters (e.g., force, displacement, speed, ramp size, etc.); and (3) data processing (baseline correction, contact point estimation, data fitting, modelling etc.). One outstanding issue with the technique is that some studies in the literature that use AFM nanoindentation report very dissimilar quantitative results for the same sample/cell/material type37,38,39,40,41. For example, Lekka et al. studied and compared the effects of AFM probe geometries on the measured Young’s moduli of mechanically homogenous hydrogels and heterogenous cell samples. They reported that the modulus values were highly dependent on the choice of cantilevers and the tip shape, with pyramidal probes giving the highest values and spherical probes the lowest42. Similarly, Selhuber-Unkel et al. have shown how the indenter speed, indenter size and thickness of polyacrylamide (PAAm) samples can all influence Young’s moduli measured using AFM nanoindentation43. An additional complication is the unavailability of standard testing materials with very low elastic modulus and complimentary testing techniques. This makes it very challenging to obtain accurate results with confidence. Nevertheless, the method is extremely useful in conducting relative measurements and comparative assessments between similar sample types, for example, using AFM nanoindentation to discriminate between normal and cancerous cells44,45.
When testing a soft material with AFM nanoindentation, a general rule of thumb is to use a probe with a low spring constant (k) that closely matches the modulus of the sample and a hemispherical/rounded tip so that at the first contact with the soft sample, the probe does not pierce through the sample surface. It is also important that the probe generates a deflection signal that is high enough to be recorded by the laser detector system24,34,46,47. In the case of ultra-soft heterogenous cells, tissues, and gels, an additional challenge is overcoming the adhesive forces between the probe and the sample surface in order to assure reproducibility and reliability of the measurements48,49,50. Until very recently, most AFM nanoindentation work that focused on studying the mechanical behavior of biological cells, tissues, gels, hydrogels and biomolecules involved the use of relatively large spherical probes commonly called as colloidal probes (CP)26,43,47,51,52,53,54,55. The radius of these probes can vary from 1 to 50 µm, and they are usually made up of borosilicate glass, polymethyl methacrylate (PMMA), polystyrene (PS), silicon dioxide (SiO2) and diamond-like-carbon (DLC). Even though CP-AFM nanoindentation is often the preferred choice for the characterization of soft samples, it has its own challenges and limitations. The use of a large, micrometer-sized spherical tip increases the overall tip-sample contact area and causes significant reduction in the spatial resolution. For a soft, heterogenous sample, where the mechanical properties of the local features can be remarkably different from the average over a wider area, CP indentation can lead to obscuring of any heterogeneity in the properties on a local scale52. Colloidal probes are usually fabricated by attaching micrometer-size colloidal spheres to tipless cantilevers with epoxy-based adhesives. The fabrication process itself presents many challenges and can cause inconsistencies during calibration of the probes. In addition, the size and the mass of the colloidal particle directly impact the primary calibration parameters of the cantilever such as the resonant frequency, spring constant and the deflection sensitivity56,57,58. Therefore, the usual methods such as thermal-tune calibration used for conventional AFM probes may not provide accurate calibration for CP, and additional methods may be required to perform these corrections57,59,60,61. Typical CP indentation experiments use large cantilever deflections to examine the properties of soft samples, and this produces another challenge for calibrating the non-linear behavior of the cantilever at relatively large deflections62,63,64. Current colloidal-probe indentation methods often consider the geometry of the cantilever for probe calibration but neglect the effects of the colloidal particle, producing additional uncertainties in the accuracy of this method38,61. Similarly, calculations of the elastic modulus by the fitting of contact models are directly dependent on the geometry of the indenting probe, and a mismatch between the tip and a sample surface feature may lead to innaccuracies27,65,66,67,68. Some recent work by Spencer et al. underscores the factors to be considered when using CP-AFM nanoindentation methods to characterize soft polymer brushes. They reported that rate-dependent, viscous-fluid confinement within polymer brushes can lead to increased indenter loads and therefore give rise to apparent rate-dependent property measurements30,69,70,71.
In this study, we characterized the surface modulus of an ultra-soft, highly elastic material, lehfilcon A CL, using an improved AFM nanoindentation method. Considering the properties and the novel structure of this material, it was clear that the sensitivity range of traditional indentation methods would be inadequate to characterize the modulus of such an extremely soft material, and it would therefore be necessary to use an AFM nanoindentation technique with higher sensitivity and lower noise levels. After reviewing the inadequacies and problems of existing colloidal-probe AFM nanoindentation methods, we show why we selected a specially designed smaller AFM probe that addresses the issues of sensitivity, background noise, precise contact-point determination, rate-dependent fluid confinement and accurate quantitative modulus measurements for soft heterogenous materials. In addition, our ability to measure the shape and size of the indenting tip accurately allowed us to use a cone-sphere fitting model that enables the determination of elastic modulus without the necessity of estimating the tip-material contact area. Two implied assumptions that were made to conduct quantitative assessments in this work are fully elastic nature of the material and the modulus being independent of the indentation depth. Using this approach, we first tested an ultra-soft standard sample of known modulus to quantitatively evaluate the method, and then used the method to characterize the surface of two different contact-lens materials. This AFM nanoindentation surface-characterization methodology with enhanced sensitivity is expected to be applicable for a wide range of biomimetic heterogenous, ultra-soft materials that are of potential use in medical-device and biomedical applications.
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