Stabilization of dry protein coatings with compatible solutes

Killian, Manuela S.; Taylor, Andy; Castner, David G.

doi:10.1116/1.5031189

Cited by 8 publications

(7 citation statements)

References 45 publications

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“…The samples (labeled as PCL Hy_FN) were washed with PBS and kept in PBS until further characterization. The samples were not stored in these conditions and prepared fresh just before their characterization to prevent the drying of the functionalized samples surface, which could induce protein denaturation (Killian et al, 2018).…”

Section: Methodsmentioning

confidence: 99%

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Fibronectin Functionalized Electrospun Fibers by Using Benign Solvents: Best Way to Achieve Effective Functionalization

Liverani

Killian

Boccaccını

2019

Front. Bioeng. Biotechnol.

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The aim of this study is to demonstrate the feasibility of different functionalization methods for electrospun fibers developed using benign solvents. In particular three different approaches were investigated to achieve the functionalization of poly(epsilon caprolactone) (PCL) electrospun fibers with fibronectin. Protein surface entrapment, chemical functionalization and coaxial electrospinning were performed and compared. Moreover, bilayered scaffolds, with a top patterned and functionalized layer with fibronectin and a randomly oriented not functionalized layer were fabricated, demonstrating the versatility of the use of benign solvents for electrospinning also for the fabrication of complex graded structures. Besides the characterization of the morphology of the obtained scaffolds, ATR-FTIR and ToF-SIMS were used for the surface characterization of the functionalized fibers. Cell adhesion and proliferation were also investigated by using ST-2 cells. Positive results were obtained from all functionalized scaffolds and the most promising results were obtained with bilayered scaffolds, in terms of cells infiltration inside the fibrous structure.

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Section: Methodsmentioning

confidence: 99%

“…Low energy electron flooding was used to prevent charging of the samples. The peak lists used for evaluation were derived from Killian et al (2018) and are shown in Supplementary Table 1. Signals were identified using the accurate mass as well as their isotopic pattern.…”

Section: Methodsmentioning

confidence: 99%

Fibronectin Functionalized Electrospun Fibers by Using Benign Solvents: Best Way to Achieve Effective Functionalization

Liverani

Killian

Boccaccını

2019

Front. Bioeng. Biotechnol.

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“…On potential advantage of in situ liquid SIMS to study protein films is that the protein structure is maintained in the in situ setup compared to the previous experiments done in UHV. Comparisons of the amino acid fragment patterns from a given protein analyzed in a dried state in UHV, a protected state (e.g., trehalose coated) in UHV 30,31 and in the liquid cell of laminin and vitronectin were conducted. Spectral PCA results in Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The residual protein solution was removed by washing the channel with 1 mL of pure water at a flow rate of 100 μL/min. The trehalose preserved protein film samples were prepared following the same procedure described in earlier publications 30,31 .…”

Section: Salvi Device Fabricationmentioning

confidence: 99%

In situ molecular imaging of adsorbed protein films in water indicating hydrophobicity and hydrophilicity

Zhou

Engelhard

et al. 2020

Sci Rep

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In situ molecular imaging of protein films adsorbed on a solid surface in water was realized by using a vacuum compatible microfluidic interface and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Amino acid fragments from such hydrated protein films are observed and identified in the positive ion mode and the results are in agreement with reported works on dry protein films. Moreover, water clusters from the hydrated protein films have been observed and identified in both the positive and negative ion mode for a series protein films. Thus, the detailed composition of amino acids and water molecules in the hydrated protein films can be characterized, and the protein water microstructures can be revealed by the distinct three-dimensional spatial distribution reconstructed from in situ liquid ToF-SIMS molecular imaging. Furthermore, spectral principal component analysis of amino acid fragment peaks and water cluster peaks provides unique insights into the water cluster distribution, hydrophilicity, and hydrophobicity of hydrated adsorbed protein films in water. Hydration is crucial to keep the conformation and biological activity of proteins 1,2. It is well-known that water molecules around proteins have properties quite distinct from those in the aqueous bulk phase 3,4. As a representative form of hydrated proteins, the hydrated protein films consist of a mixed layer of protein and water molecules 5,6. Thus, the revelation of the composition and structure of hydrated protein films, especially for the distribution of water molecules within them, is an effective way to understand the hydration mechanism of proteins. Adsorbed protein films on a solid surface have been widely studied because of their unique feature in cell adhesion, which has multiple applications in biology and medicine, such as cell culture 7 and implantable device manufacture 8. The composition and structure of adsorbed hydrated protein films are usually evaluated by analyzing their dry samples due to the technical and instrumental limitations to study the solid-liquid interface using surface sensitive techniques that are largely constrained because of vacuum conditions 9,10. The orientation of the adsorbed protein molecules is of great interest 9,10. However, the dry protein film is rather different from the hydrated protein film in its natural state. While dry sample surface analysis is useful to provide ensemble measurements of the chemical composition, thickness, and possibly orientation, they do not necessarily reflect the composition and structure of hydrated protein films in water. More importantly, the water microenvironment surrounding the protein and water molecules associated with the protein structure are lost in dry sample analysis. Actually, as far as we know, few experimental techniques can be used to analyze the water microenvironment surrounding the protein molecules, and most understandings in this field have been from computational efforts 1,11,12. Therefore, direct molecular evidence is of great importance to verify t...

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“…So, when using ToF-SIMS for analysis of protein conformation and orientation analysis the samples must be processed in a manner that preserves protein structure in UHV. (33,47,48) Processing all the complex information produced by ToF-SIMS analysis is challenging given the number of mass fragments present. A typical approach to address this challenge is to use multivariate analysis (MVA).…”

Section: Iiid Time-of-flight Secondary Ion Mass Spectrometry (Tof-sims)mentioning

confidence: 99%

Developments and Ongoing Challenges for Analysis of Surface-Bound Proteins

Weidner

Castner

2021

Annual Rev. Anal. Chem.

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Proteins at surfaces and interfaces play important roles in the function and performance of materials in applications ranging from diagnostic assays to biomedical devices. To improve the performance of these materials, detailed molecular structure (conformation and orientation) along with the identity and concentrations of the surface-bound proteins on those materials must be determined. This article describes radiolabeling, surface plasmon resonance, quartz crystal microbalance with dissipation, X-ray photoelectron spectroscopy, secondary ion mass spectrometry, sum frequency generation spectroscopy, and computational techniques along with the information each technique provides for characterizing protein films. A multitechnique approach using both experimental and computation methods is required for these investigations. Although it is now possible to gain much insight into the structure of surface-bound proteins, it is still not possible to obtain the same level of structural detail about proteins on surfaces as can be obtained about proteins in crystals and solutions, especially for large, complex proteins. However, recent results have shown it is possible to obtain detailed structural information (e.g., backbone and side chain orientation) about small peptides (5–20 amino sequences) on surfaces. Current studies are extending these investigations to small proteins such as protein G B1 (∼6 kDa). Approaches for furthering the capabilities for characterizing the molecular structure of surface-bound proteins are proposed. Expected final online publication date for the Annual Review of Analytical Chemistry, Volume 14 is August 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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Stabilization of dry protein coatings with compatible solutes

Cited by 8 publications

References 45 publications

Fibronectin Functionalized Electrospun Fibers by Using Benign Solvents: Best Way to Achieve Effective Functionalization

Fibronectin Functionalized Electrospun Fibers by Using Benign Solvents: Best Way to Achieve Effective Functionalization

In situ molecular imaging of adsorbed protein films in water indicating hydrophobicity and hydrophilicity

Developments and Ongoing Challenges for Analysis of Surface-Bound Proteins

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