Collagen Assembly As Examined By Atomic Force Microscopy

Author : Hanna Cho
Publisher :
ISBN 13 :
Total Pages : pages
Book Rating : 4.:/5 (125 download)

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Download or read book Quantitative Characterization Of Piezoelectricity In Collagen Type I Fibrils Via Piezoresponse Force Microscopy written by Hanna Cho and published by . This book was released on 2017 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: DISCLOSURES: Jinha Kwon (N), Do-Gyoon Kim (N), Hanna Cho (N) INTRODUCTION: As a main component of bone, type I collagen has piezoelectricity, which is one of the candidates to transduce mechanical inputs to physiological signals in bone1. Although piezoelectricity of collagen was experimentally confirmed for a long time1,2, it is still challenging to quantitatively measure the piezoelectric property of a single collagen fibril due to its small piezoresponse. Piezoresponse Force Microscopy (PFM) based on Atomic Force Microscopy (AFM) has been successfully applied to measure shear piezoelectricity in a single collagen fibril,3,4 but previous studies used high voltage inputs up to 30V to induce a piezoelectric stain large enough to be measured by an AFM tip. The issue for these studies is that biological samples like collagen is vulnerable to a high electrical input. Moreover, previous works did not carefully examine the effect of substrateu2019s conductivity and the contribution of parasitic electrostatic forces between the tip and sample, which should be critical to precisely determine the quantitative piezoelectric properties of collagen. In this study, we utilized the contact resonance of an AFM cantilever to amplify piezoresponse signal of collagen with a small electrical input up to 5V. We also carefully examined the effect of substrateu2019s conductivity by measuring collagen fibrils on bare and gold-coated glass slides. Moreover, the contribution of electrostatic forces to the PFM results were investigated while they are varied by applying different DC offsets. As a results, the piezoelectric properties of a single collagen fibril was precisely obtained in both vertical and shear directions and its heterogeneous nature within a fibril was revealed. METHODS: Type I collagen fibrils from bovine achilles tendon (SIGMA-ALDTICHu00ae) powder was used to prepare the sample. The powder was dissolved in 0.01M sulfidic acid at 4 u2103 overnight. After then, the collagen was shredded by using a commercial blender (Type 4185, BRAUNu00ae) and phosphate buffered saline was added to get final concentration of 4 u00b5g/ml. Upon completion, glass slides, cleaned by sonicating for 30 seconds, were immerged in the collagen solution for 1 hour at room temperature, to adhere collagen fibrils on the surface. To study the effect of substrateu2019s conductivity on the applied electric field, we prepared the sample on bare and gold-coated glass slides. Finally, the slides were gently rinsed with DI water to eliminate mineral composite on the top of collagen fibrils. Subsequently, PFM was performed in a commercial AFM system (MFP-3D infinity). To avoid damage to the sample during scanning and increase sensitivity of measurements, a soft conductive AFM cantilever was used (2.5 N/m stiffness, 3XC-GG, OPUSu00ae). Each probe was carefully calibrated in both vertical and lateral directions by measuring the force curves on a flat sapphire surface as a reference. After then, the collagen sample was located and aligned to the tip perpendicularly and an AC voltage was applied to the fibril through the conductive AFM tip. By sweeping the frequency of the applied voltage from 50 kHz to 300 kHz, the vertical and lateral resonance frequencies (70 kHz and 260 kHz, respectively) was identified and used for PFM measurements. In addition, DC voltage was applied and varied simultaneously to compensate the electrostatic force contribution. Finally, the piezoelectric property of the collagen was calculated by fitting the measured piezoresponse vs. applied voltage graph. RESULTS SECTION: Figure 1 a) shows the piezoresponse of a single type I collagen fibril depending on the substrate types, gold and slide glass. Totally, five collagen fibrils were measured in each substrate and 110 data points of each fibril depending on input voltage was measured (n=550). The piezoelectric coefficient in the case of the collagen coated on gold and glass in lateral direction shows 0.56 pm/V and 0.14 pm/V, respectively. Figures 1 b) and c) show the PFM maps combining structural and piezoresponse information, in which the structure of the map represents topography of the collagen fibril and color map represents its piezoresponse amplitude, to reveal the heterogeneous nature of collagen piezoelectricity. In addition, the effect of electrostatic force on piezoresponse result was investigated depending on the measurement direction and substrates as seen in figure 2. For the results in the vertical direction, the electrostatic force was compensated at a certain DC voltage offset (e.g., 500 mV on gold and 800 mV on glass), while the electrostatic force does not alter the PFM amplitude in the lateral direction. DISCUSSION: The piezoresponse of a single Type I collagen fibril was qualitatively characterized through a resonance-enhanced PFM technique. The piezoresponse of the collagen coated on a gold substrate is similar to reported value4 and almost four times larger than the case of the collagen coated on a glass slide. Because the glass substrate cannot provide a good electrical ground, which reduce the actual electrical field induced across the collagen fibril. Thatu2019s why it is important to use a conductive substrate to quantify collagenu2019s piezoelectricity. Moreover, it is shown that the electrostatic force only alter the PFM amplitude in the vertical measurement direction. After compensating the electrostatic force contribution in the vertical direction, the piezoresponse amplitude was reduced below the noise level even with the resonance amplification, which means there is no significant piezoresponse of collagen in the vertical direction. One the other hand, the piezoresponse in the lateral direction was not affected by a DC voltage offset, meaning the PFM result represents piezoresponse of the collagen fibril only without the electrostatic contribution. This study suggests methodology to use the PFM technique without signal distortion to qualitatively characterize piezoelectricity of biological samples such as the collagen.SIGNIFICANCE/CLINICAL RELEVANCE: The resonance-enhanced PFM method to precisely quantify piezoelectricity of collagen fibrils, which can be applied to investigate abnormal collagen in disease. REFERENCES: [1] Ahn, A.C., et al. Medical Engineering & Physics 31, 2009;733u2013741. [2] Fukada, E., et al. Jpn. J. Appl. Phys. 3, 1964;117. [3] Denning, D., et al. ACS Biomater. Sci. Eng. 3, 2017;929u2013935. [4] Minary MJ and Yu M-F, ACS Nano. 2019:1859-1863. [5] Kim, S et al., Sci Rep 7, 2017.