Characterization of protein films on dental materials: Bicinchoninic acid assay (<scp>BCA</scp>) studies on loosely and firmly adsorbed protein layers

Kratz, Fabian; Müller, Christoph Michael; Körber, Nils; Umanskaya, Natalia; Hannig, Matthias; Ziegler, Christiane

doi:10.1002/pssa.201200642

Cited by 13 publications

(16 citation statements)

References 24 publications

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“…5 The pI of BSA is 4.7 to 4.9. 11 The BSA concentration of 610 lM corresponds to a clinically relevant level of albumin in human adult serum at 35-55 g/l (530-833 lM). 12 Figure 3(a) shows the relative DI D of the transistors on the first treatment wafer following deposition of PP58-NPs and 60 min exposures to KBA, increasing concentrations of SRC in KBA, and 610 lM BSA in KBA.…”

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confidence: 99%

Kinase detection with gallium nitride based high electron mobility transistors

Makowski

Bryan

Sitar

et al. 2013

Applied Physics Letters

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A label-free kinase detection system was fabricated by the adsorption of gold nanoparticles functionalized with kinase inhibitor onto AlGaN/GaN high electron mobility transistors (HEMTs). The HEMTs were operated near threshold voltage due to the greatest sensitivity in this operational region. The Au NP/HEMT biosensor system electrically detected 1 pM SRC kinase in ionic solutions. These results are pertinent to drug development applications associated with kinase sensing. V C 2013 AIP Publishing LLC. [http://dx.doi.org/10.1063/1.4812987] Quantification of cellular or tissue kinome activity is important in basic cell biology research 1 and drug development. 2 Label-free methods of kinome activity profiling are ideal due to elimination of artifacts caused by changes to kinase characteristics with labeling. 3 An important label-free method of kinome activity measurement is the kinase affinity assay. In this method, cell lysates are exposed to beads functionalized with small molecule kinase inhibitors that bind kinases in the cell lysate. The kinases are removed from the beads by elution, and the kinase profile is identified by mass spectrometry. The factors that affect kinome activity profiles are the amount of kinase present in the cell lysate and the kinase affinity to the inhibitor beads. 2 Affinity is in turn affected by kinase activation state as regulated by phosphorylation. 2,3 Basic science and clinical applications exist for the kinase affinity assay. An example basic science application was the monitoring of cell cycle kinase phosphorylation status. 1 An example clinical application was a triple negative breast cancer (TNBC) drug screening study. 2 This study monitored changes in the kinome activity profile of TNBC cells with exposure to a MEK inhibitor drug. The kinome activity data guided selection of a combination of kinase inhibitor drugs in conjunction with the MEK inhibitor that resulted in decreased drug resistance compared to using any of the drugs alone. 2 Monitoring of the entire kinome gave a better overall view of drug efficacy than by monitoring the activity of a single kinase where drug resistance often develops by compensatory upregulation of uninhibited kinases. 2 Another clinical application of the kinase affinity assay was in drug development through determining kinase inhibitor drug targets. 3 Cell lysates were treated with soluble kinase inhibitors, and the lysates were exposed to the affinity beads. The soluble drug bound its target kinases, and the remaining kinases had their ATP binding sites available to bind the beads of the affinity assay. 3 The kinases with a decreased representation based on mass spectrometry following drug treatment were identified as the drug targets. 3 In this proof-of-concept report, we have demonstrated label-free detection of a kinase by a nanoparticle (NP)/high electron mobility transistor (HEMT) biosensor system. This detection method excluded the complication of a sequential kinase affinity assay followed by mass spectrometry analysis. Gold (Au) NPs w...

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mentioning

confidence: 99%

Kinase detection with gallium nitride based high electron mobility transistors

Makowski

Bryan

Sitar

et al. 2013

Applied Physics Letters

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“…The Micro BCA™ Protein Assay Kit (Thermo Scientific, Pierce Protein Research Products, Rockfort, USA) has been successfully used before to determine the amount of protein on different dental material surfaces . In short, Cu 2+ reacts with peptide bonds and is reduced to Cu + .…”

Section: Methodsmentioning

confidence: 99%

“…The sample preparation is described in detail in Ref. . In short, a calibration curve with known albumin, respectively, lysozyme concentrations was conducted to assign the absorption values to protein concentrations.…”

Section: Methodsmentioning

confidence: 99%

Investigation of loosely and firmly bound albumin and lysozyme layers on dental materials

Kratz

Trautmann

Umanskaya

et al. 2016

Physica Status Solidi (a)

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The total amount of albumin and lysozyme adsorbed on different dentally relevant materials like titanium, gold, ceramics, poly(methyl methacrylate) (PMMA) and polytetrafluoroethylene (PTFE) was measured by bicinchoninic acid assay (BCA assay) after detachment of the proteins from the samples. The total amount of protein was separated into the amount of loosely and firmly adsorbed protein. The parts of loosely and firmly bound protein differ remarkably depending on the material. For albumin, the loosely bound protein dominates the total adsorption. In contrast, for lysozyme the amount of loosely and firmly bound protein is in some cases equal and sometimes there is more firmly than loosely bound lysozyme. In general, the firmly bound protein masks the electrostatics of the surface. The amount of loosely bound protein follows no strict tendency, whereas the amount of firmly bound protein is mostly governed by entropic and hydrophobic interactions, but modulated by electrostatics and thus by the isoelectric points of the surface and of the proteins. Most protein adsorbs on the metallic surfaces, if compared to the investigated non-metallic surfaces. For the adsorption on gold, the number of cysteines in the amino acid sequence influences the amount of protein on the gold surface. Least albumin adsorbs on PMMA, least lysozyme adsorbs on PMMA and PTFE. For albumin, pH depending conformational changes play a key role for the adsorption at pH 9.0.

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“…After the rinsing process the samples were transferred into Eppendorf tubes with 50 µl RIPA buffer and then incubated for 10 min in the ultrasonic bath. It was shown elsewhere [21] that this procedure removes all proteins from the surface so that the amount of desorbed proteins is equal to the amount of formerly adsorbed proteins. After sonication 100 µl ultrapure water as well as 150 µl BCA-reagent were added to the samples in the Eppendorf tubes.…”

Section: Bca Assay Protocolmentioning

confidence: 99%

“…The rinsing step is necessary to remove all loosely bound protein from the surface. It has been shown in another work [21] that five rinsing steps are sufficient to remove all loosely bound protein from the surface so that only the initial protein film remains on the sample surface. After the rinsing process the samples were transferred into Eppendorf tubes with 50 µl RIPA buffer and then incubated for 10 min in the ultrasonic bath.…”

Section: Bca Assay Protocolmentioning

confidence: 99%

Track A

Frey

Sales

Athanasopulu

et al. 2014

Biomedical Engineering / Biomedizinische Technik

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IntroductionElectrospinning is a well investigated technique for processing ultrafine thermoplastic fibres. The published techniques are mostly based on a polymeric solution and some on melt polymers. To improve heat and chemical resistance as well as gas permeability electrospinning of silicone rubber was aimed in this project. Possible applications may be hydrophobic membranes or coatings for a wide range of applications in clinical and experimental medicine. MethodsTo be able to adapt the process on a wide spectrum of silicones, commercial room temperature vulcanizing (RTV) silicone was used for process development. The chosen two component silicone (Silpuran® 2430, Wacker AG) was modified in viscosity and conductibility. Solvents, which have been used successfully for adapting viscosity and which led to a stable electrospinning process, were acetone and petrolether. Conductibility was tried to improve with NaCl and LiBr. Furthermore, the electrospinning process needed to be adapted to the curing properties of the silicone. A heated collector and infrared-lamps were evaluated for optimal curing parameters. ResultsMembranes have been electrospun successfully out of standard RTV-silicone. The jets respectively fibres have been spun in this project with diameters around 20 µm. Membranes with a thickness of 100 µm showed little improvement concerning hydrophobic properties and permeability. In addition, electrospun membranes showed a surface enlargement in comparison with pressed test specimens. The purpose of improving conductibility of the silicone-solvent by adding NaCl and LiBr was not reached so far. ConclusionThe investigated technique showed the possibilities of an innovative processing method for silicones. With this project basics for electrospinning of silicones have been established for diverse applications, like membranes, coatings or other tissues. The process combined with silicone properties appears to be from special interest for the medical technology. Based on the presented results detailed research on the medical use of the processed silicones can be investigated in future. AbstractPolyelectrolyte multilayer (PEM) films, established using the layer-by-layer (LbL) technique, are attractive for controlled drug delivery in order to improve tissue regeneration. For instance, the LbL-deposition provides the possibility to incorporate biomolecules via electrostatic interactions for drug delivery, which might be moreover combined with a stable biofunctionalization via covalent binding of biomolecules such as cell adhesion motifs to the outer layer. Here, the influence of covalent biofunctionalization of PEM deposited on poly(L-lactide) (PLLA) on the release profiles of avidin fluorescein isothiocyanate (avidin-FITC) incorporated as model drug within the PEM is demonstrated. IntroductionTissue regeneration combines several processes that have a different temporal progress. Consequently, tissue engineering strategies afford scaffolds specifically designed to mediate tissue-scaffold interactions by sur...

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Characterization of protein films on dental materials: Bicinchoninic acid assay (BCA) studies on loosely and firmly adsorbed protein layers

Cited by 13 publications

References 24 publications

Kinase detection with gallium nitride based high electron mobility transistors

Kinase detection with gallium nitride based high electron mobility transistors

Investigation of loosely and firmly bound albumin and lysozyme layers on dental materials

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