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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Engineering

Force-Clamp Rheometry for Characterizing Protein-based Hydrogels

Published: August 21st, 2018

DOI:

10.3791/58280

1Department of Physics, University of Wisconsin-Milwaukee

A new force-clamp rheometry technique is used to investigate the mechanical properties of low-volume protein-based hydrogel samples tethered between a voice-coil motor and a force sensor. An analog proportional-integral-derivative (PID) system allows for the 'clamping' of the force experienced to the desired protocol.

Here, we describe a force-clamp rheometry method to characterize the biomechanical properties of protein-based hydrogels. This method uses an analog proportional-integral-derivative (PID) system to apply controlled-force protocols on cylindrical protein-based hydrogel samples, which are tethered between a linear voice-coil motor and a force transducer. During operation, the PID system adjusts the extension of the hydrogel sample to follow a predefined force protocol by minimizing the difference between the measured and set-point forces. This unique approach to protein-based hydrogels enables the tethering of extremely low-volume hydrogel samples (< 5 µL) with different protein concentrations. Under force-ramp protocols, where the applied stress increases and decreases linearly with time, the system enables the study of the elasticity and hysteresis behaviors associated with the (un)folding of proteins and the measurement of standard elastic and viscoelastic parameters. Under constant-force, where the force pulse has a step-like shape, the elastic response, due to the change in force, is decoupled from the viscoelastic response, which comes from protein domain unfolding and refolding. Due to its low-volume sample and versatility in applying various mechanical perturbations, force-clamp rheometry is optimized to investigate the mechanical response of proteins under force using a bulk approach.

Apart from having unique physical properties, protein-based hydrogels hold the promise of revolutionizing force spectroscopy by enabling the measurement of several billion molecules in one 'pull', thus enabling the study of proteins in crowded environments, similar to those encountered in skin and other tissues. Protein domains remain folded inside hydrogels, allowing the study of their biomechanical response to force, binding partners, and chemical conditions. Additionally, the biomechanical response of protein domains inside hydrogels resembles the response seen with single-molecule force spectroscopy techniques. For example, chemical denaturants and oxidizi....

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1. Reagents Solution Preparation

  1. Prepare a starting protein solution by dissolving/diluting the protein of interest to the desired concentration, using a Tris buffer [20 mM tris(hydroxymethyl)aminomethane and 150 mM NaCl, pH 7.4].
    NOTE: The smallest protein concentration for which cross-linking leads to hydrogels depends on the protein used and is typically > 1 mM.
  2. Prepare stocks of ammonium persulfate (APS) (1 M) and tris(bipyridine)ruthenium(II) chloride ([Ru(bpy)3]2+

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Figure 1A shows the scheme of the photoactive reaction used to synthesize the L-EGP/L8 hydrogel. Figure 1B shows the hydrogel mixture in the PTFE tube before and after the photoactivation. Figure 1C presents the extruded L-eGFP-L8 hydrogel inside a Tris solution. The hydrogel sample has no structural defects such as notches. Hydrogels with clearly visible damage should be discar.......

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Herein, we describe a force-clamp rheometry technique to investigate the biomechanical response of low-volume protein-based hydrogels. Additionally, a protocol is provided to synthesize a uniform cylindrical low-volume protein hydrogel sample. A protocol is also presented which describes how to tie different types of protein-based hydrogels with various elasticities without causing any mechanical deformation or damage to the protein-based hydrogel samples or slippage of the gel on the hooks. The analog PID system, togeth.......

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We acknowledge financial support from Research Growth Initiative (Award No. 101X340), National Science Foundation, Major Research Instrumentation Program (Grant No. PHY-1626450), Greater Milwaukee Foundation (Shaw Award) and University of Wisconsin System (Applied Research Grant).

....

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Name Company Catalog Number Comments
SI-KG4A force transducer World Precision Instruments (WPI) SI-KG4A
Linear Voice Coil Motor Equipement Solutions LFA2010
Bovine serum albumin Rocky Mountain Biologicals (RMBIO) BSA-AAF-1XG / 100 G
Trizma Sigma-Aldrich T1503-1KG
Sodium chloride Sigma-Aldrich S7653-1KG
Ammonium persulfate Sigma-Aldrich 248614-100G
Tris(bipyridine)ruthenium(II) chloride Sigma-Aldrich 544981-1G
EXPRESS MEDICAL SUPPLIES 6-0 NYLON SUTURE 12/PK Fisher Scientific NC0395626
1mL Syringe Only, Luer-Lok Tip BD 309628
Silane, Sigmacote Sigma-Aldrich SL2-25ML
Microbore PTFE Tubing, 0.022"ID x 0.042"OD, 100 ft/roll Cole-Parmer EW-06417-21
Hypodermic Needle, 23 Gauge Healthcare Supply Pros 305194
Jensen Global JG24-1.5X Red IT Dispensing Tips - 24 gauge KIMCO JG24-1.5X
USH-103D USHIO 100W Short Arc Mercury Lamp ALB USH-103D USHIO
Medical Tweezers
Medical scissors
Olympus
The computer code and CAD design of the custom parts can be made available on request to the corresponding author.

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