Chapter 7 - EPR studies of bionanomaterials
Introduction
Started more than two decades ago, the “nanorevolution” continues its rapid expansion today with no signs of slowdown in the near future. With billions of US dollars invested in basic academic research and corporate research and development, scientific breakthroughs and new consumer products that are based on nanotechnology are pouring out at the highest pace ever. Typically, nanotechnology is dealing with nanoscale objects, i.e., having dimensions from ca. 1 to 100 nm. At such atomic scale, small clusters of atoms and molecules are exhibiting some rather intriguing physical and chemical properties that are absent in bulk materials of the same composition.
One remarkable example of altered physical properties is provided by a three-dimensional quantum confinement for excitons in the semiconductor nanocrystals. If dimensions of the nanocrystals are smaller than the so-called Bohr exciton radius (typically a few nm), the exciton energy levels are quantized with the energy levels determined by the crystal size, thus, justifying the name of quantum dots (QD). Clearly, such quantum confinement effects would not occur in bulk macroscopic semiconductors. The fluorescent properties of QDs were found to be superior to the commonly used molecular fluorophores, and this led to far-reaching applications of QD technology in high-resolution cellular imaging at single-molecule level, in vivo observation of in-cell trafficking, tumor QD targeting, and medical diagnostics (e.g., Ref. [1]).
An example of strikingly different chemical properties at the nanoscale is given by Au nanoparticles (NPs). Gold is a noble metal that has been considered to be catalytically inactive for a long time. All that changed when chemists discovered a high catalytic activity of gold in a form of nanoparticles either unsupported or supported on oxidic carriers (e.g., aluminum oxide support) in various oxidation reactions (e.g., Refs. [2], [3]). This is just one of the examples that chemical properties of atomic clusters are drastically altered at the nanoscale.
In addition to the altered physical and chemical properties, another essential aspect of nanomaterials is the vastly increased surface-to-volume ratio. Thus, the interfacial and surface properties become the dominant factors in interactions of nanostructures with chemical and biological systems. For ultra-small particles of ca. 1–5 nm, a large fraction of atoms becomes exposed to the interface. For example, for a “magic” number Au(923) nanocluster having ca. 3.5 nm in diameter, 362 out of 923 Au atoms (i.e., 39%) are exposed to the surface assuming cuboctahedral shape [4]. With such a large number of atoms directly exposed to the interface, additional chemical modifications of the surface atoms provide for efficient tuning of the interfacial properties [5]. Furthermore, the surface ligands would not only control the interfacial properties, but also affect the electronic properties of the metallic core as demonstrated by UV/Vis absorption and EPR spectroscopy of thiol-modified Au NPs [6].
The 1–100 nm scale of nano objects provides a good match in terms of dimensions to biomolecules and their assemblies. For example, dimensions of typical water-soluble proteins are in 3–6 nm range and two-dimensional lipid bilayer membranes are about 4 nm thick. Protein oligomers are larger by a few folds. Furthermore, several oligomeric protein subunits could be assembled into larger 3D structures such as viral capsids that range from 20 to 500 nm and more in size. Thus, one can make nano objects either partially or completely from biomolecules and combine advantages of genetically programmed biological self-assembly with unique properties of manmade materials. These hybrid nano objects—termed bionanomaterials—represent a fruitful avenue of research and technology development in such areas as protein biochips and biosensors, new contrast agents, and drug and gene delivery systems, among the other rapidly expanding applications in medical and bioengineering fields.
The growth in bionanomaterials’ research creates additional demands for spectroscopic and analytical methods capable of characterizing the nanoscale objects, and especially interfacial phenomena, in a non-destructive way. While in situ capabilities of powerful SEM, TEM and AFM microscopic techniques are constantly improving (e.g., see Refs. [7], [8]), many challenges still remain. Many of the abovementioned methods are not directly capable of providing atomic scale data on structure and dynamics of molecules in the interfacial layers and the magnitude of hydrophobic effect and electrostatic interactions—the major forces that are responsible for the reactivity and biorecognition properties of bionanomaterials. Some of these gaps in characterization of bio-nano interfaces could be filled by solid-state NMR, but the low sensitivity of the method often limits its application unless the NMR signals are enhanced through dynamic nuclear polarization (DNP, e.g., Ref. [9]).
Electron paramagnetic resonance (EPR), on the other hand, has sufficient sensitivity to study nanoparticle-liquid interface even at low concentrations (e.g., Refs. [10], [11], [12], [13], [14]). The method is also fully applicable to opaque materials and solutions for which the optical methods would fail. Extension of the EPR method to spin-trapping provides the most direct way to identify and quantify the free radical species (e.g., superoxide and hydroxyl radicals) that could be produced on surfaces of nanostructured metal oxides [15], [16] and related to cytotoxicity of nanomaterials [16], [17], [18]. Finally, but not lastly, site-directed spin labeling (SDSL) EPR has matured as a method to study structure and dynamics of lipid bilayers, proteins, oligonucleotides, and their complexes. SDSL EPR in a combination with time-domain and multifrequency/high magnetic field (HF) methods is also suitable to study local polarity profiles and water penetration in lipid bilayers [19], [20], [21], while pH sensitive nitroxides allow for probing local electric field potentials at interfaces [14], [22], [23], [24], [25] and effective proton activities in the nanopores [26], [27]. Thus, one can readily apply such methods to a broad range of bionanomaterials. We note that some of the applications of EPR to a broader range of nanomaterials have been reviewed back in 2007 [28] and then in 2011 [29].
The main goal of this chapter is to discuss some recent applications of EPR in the studies of bionanomaterials. We will start with reviewing briefly the use of spin-trapping EPR for characterizing unusually high surface reactivity of nanostructured materials. Such surface-mediated reactions could be beneficial for catalysis but also serve as a source of free radicals and consequent oxidative DNA damage when introduced in the cells. Then we will proceed to studies of ligand-functionalized nanoparticles and self-assembles structures by spin-labeling EPR methods. Finally, we review the use of EPR to study effects of nanoscale confinements and self-assembly with focus on nanoporous and mesoporous materials; we also discuss the use of nanopores as sample handling tools for EPR and NMR.
Section snippets
Surface-mediated production of free radicals and radical scavenging properties of nanomaterials
Rapidly evolving nanotechnology is introducing vast quantities of new engineered nanomaterials (ENMs) into the environment every day. All these materials, including nanoparticles, nanotubes/nanowires, and nano-structured substrates, are expected to interact with biological matrices and elicit biological responses in a degree greater than traditional materials for at least three primarily reasons already mentioned above: (1) the nanoscale features of some of such materials are typically smaller
Spin-labeling EPR methods for characterizing ligand-functionalized nanoparticles and hybrid nanostructures
Over the last ca. 50 years, spin labeling of biomolecules has matured into a valuable tool to study local structure and dynamics of complex macromolecules. The progress in spin labeling methodology and applications has been well documented in literature [52], [53], [54], [55]. Recent advances in EPR instrumentation and experimental methods resulted in a resurgence of interest in biophysical EPR and, especially, the applications and development of pulsed double resonance [56], [57] and high
Spin-labeling EPR methods in studies of nanoscale confinements and nanoporous and mesoporous materials
Meso- and nanoporous materials, including mesoporous molecular sieves (MMS) based on silica and nanoporous anodic aluminum oxide (AAO) membranes, have been actively researched in diverse fields including clean energy technologies, such as energy generation and storage [97], electronics and photonics, sensors and biosensors, drug delivery matrices, nanofabrication templates, as well as versatile catalysts and adsorbents for molecular separation [98]. Some of the major challenges of such
Nanoporous and mesoporous materials as tools to study biomacromolecules by EPR and NMR
As illustrated in the preceding sections of this review, EPR methods provide a wealth of data on interfacial properties nanostructured materials including nanoparticles and nanopores. It turned out that nanostructures also could be employed as tools to study biomacromolecules by EPR and NMR. Specifically, nanostructures, and particularly nanopores, could provide effective and versatile means for macroscopic alignment of biomolecules and lipid bilayers and thus, can improve the spectral
Conclusions
Overall, the field of EPR and related magnetic resonance methods in applications to bionanomaterials continues to grow while the nanotechnology itself evolves rather rapidly with no signs of slowing down. As the bionanomaterials field starts to mature, the needs for spectroscopic methods to study interfacial phenomena and local structure and dynamics should grow even further. Many of these experimental needs could be filled by EPR methods and approaches that we have attempted to review in this
Acknowledgments
The authors are thankful to the National Science Foundation grants (no. CHE-1508607 to T.I.S.) for support of work on studies lipid–NP interface and (no. CBET-1403871 to A.I.S.) for studies for NP interfacial properties. The fabrication of AAO and development of lipid nanotube technology for lipid bilayers and membrane proteins was supported by the United States Department of Energy Contract DEFG0202ER15354 to AIS. EPR instrumentation at NCSU was supported by grants from the National Institutes
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