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Characterizing and Patterning Polyacrylamide Substrates Functionalized with N-Hydroxysuccinimide

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Abstract

Polyacrylamide is a widely used material in cell mechanobiology research. Here, we validate and optimize a method to activate polyacrylamide substrates for protein patterning, resulting in a system with precise and independent control over the geometric and mechanical factors that cells perceive. Acrylic acid incorporated into the hydrogel provides functional sites for activation with N-hydroxysuccinimide, which in turn forms covalent bonds with proteins printed in microscale patterns. To validate and optimize substrate fabrication, we demonstrate that acrylic acid incorporates into the polymer, that is has no effect on Young’s modulus at up to 0.4 wt%, and that increasing concentrations of acrylic acid result in substrates with increasing amounts of protein bound to them. Finally, we demonstrate that cells attach and spread to substrates with protein patterned with electrohydrodynamic jet (e-jet) printing. The method represents an improvement over the most-widely used method to chemically activate polyacrylamide with sulfo-SANPAH. With further refinement, truly independent control over ligand density and stiffness is possible. These substrates are powerful platforms for exploring the interacting influence of substrate stiffness and ligand density on cell behavior.

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Abbreviations

AAm:

Acrylamide

ANOVA:

Analysis of variance

APS:

Ammonium persulfate

bis:

N,N′-methylenebisacrylamide

BSA:

Bovine serum albumin

ECM:

Extracellular matrix

EDC:

1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride

e-jet:

Electrohydrodynamic jet printing

HEPES:

4-(2-Hydroxyehtyl)-1-piperazineethanesulfonic acid

HBSS:

HEPES-buffered saline solution

MES:

2-(N-morpholino)ethane sulfonic acid

microBCA:

Micro bicinchoninic acid

NHS:

N-hydroxysuccinimide

PBS:

Phosphate-buffered saline

sulfo-SANPAH:

N-sulfosuccinimidyl-6-(4′-azino-2′-nitrophenylamino) hexanoate

TEMED:

N,N,N′,N′-tetramethylethylenediamine

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Acknowledgments

This manuscript is based on work supported by the Center for Nanoscale Chemical Electrical Mechanical Manufacturing Systems (Nano-CEMMS) at the University of Illinois, funded by the National Science Foundation under Grant DMI 0328162 and by NSF EAGER Grant CMMI 1264988. We acknowledge undergraduate researchers N. Patel, T. Perez, H. Friedman, and C. Frid for their assistance with some of the experiments.

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Authors and Affiliations

  1. Department of Bioengineering, University of Illinois at Urbana-Champaign, Digital Computer Laboratory Room 1270, 1304 W. Springfield Ave., Urbana, IL, 61801, USA

    Michael J. Poellmann

  2. Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, Mechanical Engineering Building Room 128, 1206 W. Green St., Urbana, IL, 61801, USA

    Amy J. Wagoner Johnson

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Correspondence to Amy J. Wagoner Johnson.

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Associate Editor Michael R. King oversaw the review of this article.

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Poellmann, M.J., Wagoner Johnson, A.J. Characterizing and Patterning Polyacrylamide Substrates Functionalized with N-Hydroxysuccinimide. Cel. Mol. Bioeng. 6, 299–309 (2013). https://doi.org/10.1007/s12195-013-0288-5

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