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Front Mater ; 82021 May.
Artigo em Inglês | MEDLINE | ID: mdl-34113623


The interfaces that biological tissues form with biomaterials are invariably defective and frequently the location where failure initiates. Characterizing the phenomena that lead to failure is confounded by several factors including heterogeneous material/tissue interfaces. To seamlessly analyze across these diverse structures presents a wealth of analytical challenges. This study aims to develop a molecular-level understanding of a peptide-functionalized adhesive/collagen hybrid biomaterial using Raman spectroscopy combined with chemometrics approach. An engineered hydroxyapatite-binding peptide (HABP) was copolymerized in dentin adhesive and dentin was demineralized to provide collagen matrices that were partially infiltrated with the peptide-functionalized adhesive. Partial infiltration led to pockets of exposed collagen-a condition that simulates defects in adhesive/dentin interfaces. The spectroscopic results indicate that co-polymerizable HABP tethered to the adhesive promoted remineralization of the defects. The spatial distribution of collagen, adhesive, and mineral as well as crystallinity of the mineral across this heterogeneous material/tissue interface was determined using micro-Raman spectroscopy combined with chemometrics approach. The success of this combined approach in the characterization of material/tissue interfaces stems from its ability to extract quality parameters that are related to the essential and relevant portions of the spectral data, after filtering out noise and non-relevant information. This ability is critical when it is not possible to separate components for analysis such as investigations focused on, in situ chemical characterization of interfaces. Extracting essential information from complex bio/material interfaces using data driven approaches will improve our understanding of heterogeneous material/tissue interfaces. This understanding will allow us to identify key parameters within the interfacial micro-environment that should be harnessed to develop durable biomaterials.

Langmuir ; 37(24): 7536-7547, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34102059


Controlling enzyme orientation and location on surfaces is a critical step for their successful deployment in diverse applications from biosensors to lab-on-a-chip devices. Functional activity of the enzymes on the surface will largely depend on the spatial arrangement and orientation. Solid binding peptides have been proven to offer versatility for immobilization of biomolecules on inorganic materials including metals, oxides, and minerals. Previously, we demonstrated the utility of a gold binding peptide genetically incorporated into the enzyme putrescine oxidase (PutOx-AuBP), enabling self-enzyme assembly on gold substrates. PutOx is an attractive biocatalyst among flavin oxidases, using molecular oxygen as an electron acceptor without requiring a dissociable coenzyme. Here, we explore the selective self-assembly of this enzyme on a range of surfaces using atomic force microscopy (AFM) along with the assessment of functional activity. This work probes the differences in surface coverage, distribution, size, shape, and activity of PutOx-AuBP in comparison to those of native putrescine oxidase (PutOx) on multiple surfaces to provide insight for material-selective enzymatic assembly. Surfaces investigated include metal (templated-stripped gold (TSG)), oxide (native SiO2 on Si(111)), minerals (mica and graphite), and self-assembled monolayers (SAMs) with a range of hydrophobicity and charge. Supported by both the coverage and the dimensions of immobilized enzymes, our results indicate that of the surfaces investigated, material-selective binding takes place with orientation control only for PutOx-AuBP onto the TSG substrate. These differences are consistent with the measurements of surface-bound enzymatic activities. Substrate-dependent differences observed indicate significant variations in enzyme-surface interactions ranging from peptide-directed self-assembly to enzyme aggregation. The implications of this study provide insight for the fabrication of enzymatic patterns directed by self-assembling peptide tags onto localized surface regions. Enabling functional enzyme-based nanoscale materials offers a fascinating path for utilization of sustainable biocatalysts integrated into multiscale devices.

Ouro , Dióxido de Silício , Enzimas Imobilizadas , Oxirredutases atuantes sobre Doadores de Grupo CH-NH , Peptídeos , Propriedades de Superfície
Langmuir ; 36(40): 11908-11917, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-32921059


Flavin oxidases are valuable biocatalysts for the oxidative synthesis of a wide range of compounds, while at the same time reduce oxygen to hydrogen peroxide. Compared to other redox enzymes, their ability to use molecular oxygen as an electron acceptor offers a relatively simple system that does not require a dissociable coenzyme. As such, they are attractive targets for adaptation as cost-effective biosensor elements. Their functional immobilization on surfaces offers unique opportunities to expand their utilization for a wide range of applications. Genetically engineered peptides have been demonstrated as enablers of the functional assembly of biomolecules at solid material interfaces. Once identified as having a high affinity for the material of interest, these peptides can provide a single step bioassembly process with orientation control, a critical parameter for functional immobilization of the enzymes. In this study, for the first time, we explored the bioassembly of a putrescine oxidase enzyme using a gold binding peptide tag. The enzyme was genetically engineered to incorporate a gold binding peptide with an expectation of an effective display of the peptide tag to interact with the gold surface. In this work, the functional activity and expression were investigated, along with the selectivity of the binding of the peptide-tagged enzyme. The fusion enzyme was characterized using multiple techniques, including protein electrophoresis, enzyme activity, and microscopy and spectroscopic methods, to verify the functional expression of the tagged protein with near-native activity. Binding studies using quartz crystal microbalance (QCM), nanoparticle binding studies, and atomic force microscopy studies were used to address the selectivity of the binding through the peptide tag. Surface binding AFM studies show that the binding was selective for gold. Quartz crystal microbalance studies show a strong increase in the affinity of the peptide-tagged protein over the native enzyme, while activity assays of protein bound to nanoparticles provide evidence that the enzyme retained catalytic activity when immobilized. In addition to showing selectivity, AFM images show significant differences in the height of the molecules when immobilized through the peptide tag compared to immobilization of the native enzyme, indicating differences in orientation of the bound enzyme when attached via the affinity tag. Controlling the orientation of surface-immobilized enzymes would further improve their enzymatic activity and impact diverse applications, including oxidative biocatalysis, biosensors, biochips, and biofuel production.

Técnicas Biossensoriais , Enzimas Imobilizadas , Ouro , Oxirredutases atuantes sobre Doadores de Grupo CH-NH , Peptídeos