Controlling the Graphene-Bio Interface: Dispersions in Animal Sera for Enhanced Stability and Reduced Toxicity.

Liquid phase exfoliation of graphite in six different animal sera and evaluation of its toxicity are reported here. Previously, we reported the exfoliation of graphene using proteins, and here we extend this approach to complex animal fluids. A kitchen blender with a high-turbulence flow gave high quality and maximum exfoliation efficiency in all sera tested, when compared to the values found with shear and ultrasonication methods. Raman spectra and electron microscopy confirmed the formation of three- or four-layer, submicrometer size graphene, independent of the serum used. Graphene prepared in serum was directly transferred to cell culture media without post-treatments. Contrary to many reports, a nanotoxicity study of this graphene fully dispersed to human embryonic kidney cells, human lung cancer cells, and nematodes (Caenorhabditis elegans) showed no acute toxicity for up to 7 days at various doses (50-500 µg/mL), but prolonged exposure at higher doses (300-500 µg/mL, 10-15 days) showed cytotoxicity to cells (∼95% death) and reproductive toxicity to C. elegans (5-10% reduction in brood size). The origin of toxicity was found to be due to the highly fragmented smaller graphene sheets (<200 nm), while the larger sheets were nontoxic (50-300 µg/mL dose). In contrast, graphene produced with sodium cholate as the mediator has been found to be cytotoxic to these cells at these dosages. We demonstrated the toxicity of liquid phase exfoliated graphene is attributed to highly fragmented fractions or nonbiocompatible exfoliating agents. Thus, low-toxicity graphene/serum suspensions are produced by a facile method in biological media, and this approach may accelerate the much-anticipated development of graphene for biological applications.

Tuning the activities and structures of enzymes bound to graphene oxide with a protein glue.

Graphene oxide (GO) is being investigated extensively for enzyme and protein binding, but many enzymes bound to GO denature considerably and lose most of their activities. A simple, novel, and efficient approach is described here for improving the structures and activities of enzymes bound to GO such that bound enzymes are nearly as active as those of the corresponding unbound enzymes. Our strategy is to preadsorb highly cationized bovine serum albumin (cBSA) to passivate GO, and cBSA/GO (bGO) served as an excellent platform for enzyme binding. The binding of met-hemoglobin, glucose oxidase, horseradish peroxidase, BSA, catalase, lysozyme, and cytochrome c indicated improved binding, structure retention, and activities. Nearly 100% of native-like structures of all the seven proteins/enzymes were noted at near monolayer formation of cBSA on GO (400% w/w), and all bound enzymes indicated 100% retention of their activities. A facile, benign, simple, and general method has been developed for the biofunctionalization of GO, and this approach of coating with suitable protein glues expands the utility of GO as an advanced biophilic nanomaterial for applications in catalysis, sensing, and biomedicine.

Cyclic Voltammetry Study of Noble Metals and Their Alloys for Use in Implantable Electrodes.

Innovation in the application and miniaturization of implantable electrodes has caused a spike in new electrode material research; however, few robust studies are available that compare different metal electrodes in biologically relevant media. Herein, cyclic voltammetry has been employed to compare platinum, palladium, and gold-based electrodes' potentiometric scans and their corresponding charge storage capacities (CSCs). Ten different noble metals and alloys in these families were tested under pseudophysiological conditions in phosphate-buffered saline (pH 7.4) at 37 °C. Charge storage capacity values (mC/cm2) were calculated for the oxide reduction, hydrogen adsorption, hydrogen desorption, and oxide formation peaks. Five scan rates spanning 2 orders of magnitude (10, 50, 100, 500, and 1000 mV/s) in both sparged and aerated environments were evaluated. Materials have been ranked by their charge storage capacities, reversibility, and trends discussed. Palladium-based alloys outperformed platinum-based alloys in the sparged condition and were ranked equally as high in the aerated condition. The Paliney 1100 (Pd-Re) alloy gave the highest observed calculated CSC value of 0.64 ± 0.02 mC/cm2 in the aerated condition, demonstrating 73 ± 5% reversibility. Trends between metal electrode families elicited in this study can afford valuable insight into future engineering of high performing implantable electrode materials.

One-step preparation of bioactive enzyme/inorganic materials.

Simultaneous exfoliation of crystalline α-zirconium phosphate (α-ZrP) nanosheets and enzyme binding, induced by shearing, without the addition of any toxic additives is reported here for the first time. These materials were thoroughly characterized and used for applications. The bulk α-ZrP material (20 mg mL-1) was exfoliated with low concentrations of a protein such as bovine serum albumin (BSA, 3 mg mL-1) in a shear reactor at 10k rpm for <80 minutes. Exfoliation was monitored by powder X-ray diffraction with samples displaying a gradual but complete loss of the 7.6 Å (002) peak, which is characteristic of bulk α-ZrP. The fully exfoliated sample loaded with the protein was characterized by transmission and scanning electron microscopy in addition to other biophysical methods. Lysozyme, glucose oxidase, met-hemoglobin, and ovalbumin also induced exfoliation and directly produced enzyme/ZrP biocatalysts. Thus, exfoliation, biophilization and enzyme binding are accomplished in a single step. Several factors contributed to the exfoliation kinetics, and the rate increased with α-ZrP and BSA concentrations and decreased with pH. However, the exfoliation efficiency inversely depended on the isoelectric point of the protein with ovalbumin (pI = 4.5) being the best and lysozyme (pI = 11.1) being the worst. A strong correlation between the protein size and exfoliation efficiency was noted, and the latter suggests the role of hydrodynamic factors in the process. Exfoliation was also achieved by simple stirring using a magnetic stirrer, under low volumes, and model enzymes, indicating 60-90% retention of bound enzymatic activities. The addition of BSA to enzymes as the diluent and stabilizing agent also prevents enzymes from the denaturing effect caused by stirring. This new method requires no pre-treatment of α-ZrP with toxic exfoliating agents such as tetrabutyl ammonium hydroxide and provides bioactive enzyme/inorganic materials in a single step. These protein-loaded biocompatible nanosheets may be useful for biocatalysis and biomedical applications.

Engineering functional inorganic nanobiomaterials: controlling interactions between 2D-nanosheets and enzymes.

A better understanding of the enzyme-nanosheet interface is imperative for the design of functional, robust inorganic nanobiomaterials and biodevices, now more than ever, for use in a broad spectrum of applications. This feature article discusses recent advances in controlling the enzyme-nanosheet interface with regards to α-zirconium(iv) phosphate (α-ZrP), graphene oxide (GO), graphene, and MoS2 nanosheets. Specific focus will be placed on understanding the mechanisms with which these materials interact with enzymes and elaborate on particular ways to engineer and control these interactions. Our main discoveries include: (1) upon adsorption to the nanosheet surface, a decrease in the entropy of the enzyme's denatured state enhances stability; (2) proteins are used to create biophilic landing pads for increased enzyme stability on many different types of nanosheets; (3) proteins and enzymes are used as exfoliants by shear force to produce biofunctionalized nanosheet suspensions; and (4) bionfunctionalized nanosheets exhibit no acute toxicity. Recognizing proper methods to engineer the interface between enzymes and 2D-nanosheets, therefore, is an important step towards making green, sustainable, and environmentally conscious inorganic bionanomaterials for sensing, catalysis and drug delivery applications, as well as towards the successful manipulation of enzymes for advanced applications.

Exfoliated and water dispersible biocarbon nanotubes for enzymology applications.

In this chapter, we report a simple and facile method to armor enzymes with carbon nanotubes (CNTs) which are exfoliated, and debundled using bovine serum albumin (BSA). The fabricated CNT/BSA dispersions are biofriendly, biocompatible, defect-free, and highly stable solutions. BSA gives maximum exfoliation efficiency, exceeding the 4mg/mL of CNT concentration compared to any previous reports. Further, the produced bioCNT dispersions were characterized by UV-visible, Raman, circular dichroism spectroscopy, and scanning electron microscopy (SEM). Exfoliation and debundling of the bioCNT dispersions is possible due to the π-π interaction, hydrogen bonding, hydrophobic interaction, and electrostatic attractive forces driving the adsorption of BSA on CNTs surface. Protein adsorption then makes a highly stable suspension in water that can be stored for a prolonged period. CNT dispersions are stable over a wide range of pH from 3 to 10 and at 4°C or 25°C for more than 2 months. Here, we also report the facile, inexpensive and green-chemistry method to fabricate a buckypaper (CNT paper), composed of the high packing density, self-assembled and randomly oriented bioCNTs, and these assemblies could be used in many emerging applications like air and water purification, nanocomposites, energy storage, and biosensing. Moreover, the CNT dispersions stabilized by BSA were successfully used in enzyme binding and kinetic studies and bound enzyme retained substantial catalytic activity. The current approach may facilitate bulk production of water dispersed CNTs in both academic and industrial laboratories. This is done by a simple method of stirring, which provides new opportunities for a wider range of CNT applications.

Stirred Not Shaken: Facile Production of High-Quality, High-Concentration Graphene Aqueous Suspensions Assisted by a Protein.

A simple method to produce record concentrations (up to 10 mg mL-1) of high-quality aqueous graphene suspensions by using an ordinary benchtop magnetic stirrer is reported. The shear rates employed here are almost 10 times less than those in previous reports, and graphene is efficiently separated from unexfoliated graphite during the synthesis. Systematic optimization of synthesis parameters, such as pH, protein concentration, temperature, stirrer speed, and volume of solution, afforded efficient conversion (100%) of graphite to graphene-aqueous suspensions. The synthesis is readily scaled-up with a continuous flow reactor where the graphene is produced and separated 24/7, with little or no human intervention. Raman spectroscopy confirmed little to no sp3 or oxidative defects, and that the graphene nanosheets consisted of three to five layers. The graphene suspensions were coated on aluminum and tested for thermal conductivity applications. The thermal conductivity of our graphene sample was calculated to be 684 W m-1 K-1, a value greater than that of a commercial sample. The activation energy measured for shear exfoliation by stirring was found to be over 45 billion times smaller than the corresponding thermal activation energy, affording physical insight into the process. We hypothesize that stirring selectively populates translational states that are necessary for exfoliation and thus requires far less energy than conventional exfoliation methods, where the energy is uniformly distributed among all available modes. Therefore, an efficient, convenient, and inexpensive method for graphene production in limited-resource settings is reported here.

Interlocking Enzymes in Graphene-Coated Cellulose Paper for Increased Enzymatic Efficiency.

A simple method for interlocking glucose oxidase and horseradish peroxidase in a network of cellulose fibers coated with bovine serum albumin (BSA)-exfoliated graphene (biographene) is reported here. The resulting paper reactor is inexpensive and stable. Biographene is expected to function as an electron shuttle, making the reaction between the enzyme and the substrate more efficient, and this hypothesis is examined here. The BSA used to separate the sheets of graphene provides extra carboxylic acid groups and primary amines to help interlock the enzymes and the graphene in between the fibers. The decrease in entropy associated with interlocking the enzymes on a solid support is likely responsible for the increase in enzymatic stability/activity observed. Each cellulose disk contained 5.2mg of enzyme per gram of paper and 93% of the enzyme is retained after washing for 0.5-2h. This simple methodology provides a low cost, effective approach for achieving high enzymatic activity and good loadings on a benign, versatile support.

A Simple Flow Reactor for Continuous Synthesis of Biographene for Enzymology Studies.

The unique properties of graphene make it an intriguing platform for the attachment and enhancement of biological molecules, but it has yet to achieve its full potential in terms of biological applications. Single-layer graphene is expensive, making alternatives to this material highly desired for applications that require high-quality graphene in large quantities. In this context, we report a simple, environmentally friendly, nonlabor-intensive method for the synthesis of colloidal graphene suspensions of 3-5 layers, stabilized by bovine serum albumin, in water. The method involves a flow reactor designed to continually yield high-quality graphene colloids, synthesized, purified, and optimized all in one setup. The flow reactor is able to produce colloidal graphene sheets on a multigram scale, and these colloids were characterized by Raman spectroscopy, electron microscopy, and zeta potential studies. The average size of the sheets is 0.16µm2, each consisting of 3-5 layers of graphene with little or no sp3 defects. These graphene colloids stabilized by the protein were successfully used in protein kinetic studies as well as in surface plasmon resonance protein binding studies. The ease of synthesis of these high-quality graphene colloidal suspensions in water provides an exciting opportunity for biographene to be used on an industrial scale for electronic, thermal, and enzymology applications.