Biomolecular Ligands Research Articles

Biomolecular ligands as tools to modulate the optical and chiroptical properties of gold nanoclusters

Biomolecule-stabilized gold nanoclusters (AuNCs) have become interesting functional nanomaterials because of their unique optical properties, together with excellent biocompatibility and stability in biological conditions. In this review, we explore the...

Ultrathin Pt Nanowire Network Monolayer for Oxygen Reduction Reaction

Owing to a rising energy demand with the increasing population and development of society, hydrogen fuel cells have attracted considerable attention in electrochemistry research. Compared with the hydrogen oxidation reaction at the anode, the oxygen reduction reaction (ORR) at the cathode exhibits more sluggish kinetics due to its multielectron, multistep reaction mechanism, which is the critical limiting step in hydrogen fuel cells. Therefore, developing catalysts with excellent performance for ORR is essential for developing hydrogen fuel cells. Pt nanomaterials represent the most widely explored ORR electrocatalysts for their high catalytic activities and stabilities in ORR.The morphology and surface structures of nanocatalysts are critical determinants of their catalytic performance, with both factors profoundly influenced by anisotropy. One-dimensional (1-D) and 2-D anisotropic Pt nanostructures demonstrate enhanced catalytic activity, due to structural anisotropy and larger surface area accessible for catalytic reactions. Owing to their high anisotropy and surface energy, one-dimensional (1-D) and 2-D anisotropic nanostructures are much more challenging to obtain, especially for the cubic lattice adopted by Pt with high symmetry, which leads to challenges in anisotropic growth from seed during colloidal synthesis. Anisotropic assembly of nanoscale building blocks can be an alternative strategy to synthesize anisotropic nanostructures, which may be guided by biomolecules due to their highly specific surface recognition properties.Here we report extensive macroscale 2-D platinum (Pt) nanowire network (NWN) sheets by a hierarchical self-assembly process with the assistance of biomolecular ligands. 1-D nanowires with a high density of grain boundaries are formed by the assembly of 1.9 nm 0-D Pt nanocrystals, and the interconnection of these 1-D nanowires forms the Pt NWN sheets with a centimeter-scale monolayer network structure extending into centimeter-scale size. The assembly of Pt nanocrystals occurs at the gas/liquid interfaces of the bubbles produced by sodium borohydride (NaBH4) during the synthesis process. The Pt NWN sheets are released at the gas/liquid surface after the rupture of these bubbles and merge into a centimeter-scale monolayer Pt NWN sheet. It is demonstrated that the monolayer Pt NWN sheet exhibits much-improved performance in oxygen reduction reaction (ORR), with 12.0 times and 21.2 times higher specific and mass activities than those of current state-of-the-art commercial Pt/C electrocatalysts. A 21.3 times greater mass activity than that of Pt/C is also observed in the Pt NWN sheet after 15,000 accelerated durability test cycles. The outstanding enhancement of the activity and stability in ORR can be attributed to the high electrochemical surface area and high density of defects formed during assembly.In summary, we developed an ultrathin monolayer Pt NWN sheet with abundant grain boundaries from the assembly of Pt nanocrystals with the assistance of biomolecules. The Pt NWN sheet has demonstrated outstanding catalytic activity and stability in ORR.

Trivalent Copper Ion-Mediated Dual Oxidation in the Copper-Catalyzed Fenton-Like System in the Presence of Histidine.

The Cu(II)/H2O2 system is recognized for its potential to degrade recalcitrant organic contaminants and inactivate microorganisms in wastewater. We investigated its unique dual oxidation strategy involving the selective oxidation of copper-complexing ligands and enhanced oxidation of nonchelated organic compounds. L-Histidine (His) and benzoic acid (BA) served as model compounds for basic biomolecular ligands and recalcitrant organic contaminants, respectively. In the presence of both His and BA, the Cu(II)/H2O2 system rapidly degraded His complexed with copper ions within 30 s; however, BA degraded gradually with a 2.3-fold efficiency compared with that in the absence of His. The primary oxidant responsible was the trivalent copper ion [Cu(III)], not hydroxyl radical (•OH), as evidenced by •OH scavenging, hydroxylated BA isomer comparison with UV/H2O2 (a •OH generating system), electron paramagnetic resonance, and colorimetric Cu(III) detection via periodate complexation. Cu(III) selectively oxidized His owing to its strong chelation with copper ions, even in the presence of excess tert-butyl alcohol. This selectivity extended to other copper-complexing ligands, including L-asparagine and L-aspartic acid. The presence of His facilitated H2O2-mediated Cu(II) reduction and increased Cu(III) production, thereby enhancing the degradation of BA and pharmaceuticals. Thus, the Cu(II)/H2O2 system is a promising option for dual-target oxidation in diverse applications.

Strategies for Mitigating Commercial Sensor Chip Variability with Experimental Design Controls.

Surface plasmon resonance (SPR) is a popular real-time technique for the measurement of binding affinity and kinetics, and bench-top instruments combine affordability and ease of use with other benefits of the technique. Biomolecular ligands labeled with the 6xHis tag can be immobilized onto sensing surfaces presenting the Ni2+-nitrilotriacetic acid (NTA) functional group. While Ni-NTA immobilization offers many advantages, including the ability to regenerate and reuse the sensors, its use can lead to signal variability between experimental replicates. We report here a study of factors contributing to this variability using the Nicoya OpenSPR as a model system and suggest ways to control for those factors, increasing the reproducibility and rigor of the data. Our model ligand/analyte pairs were two ovarian cancer biomarker proteins (MUC16 and HE4) and their corresponding monoclonal antibodies. We observed a broad range of non-specific binding across multiple NTA chips. Experiments run on the same chips had more consistent results in ligand immobilization and analyte binding than experiments run on different chips. Further assessment showed that different chips demonstrated different maximum immobilizations for the same concentration of injected protein. We also show a variety of relationships between ligand immobilization level and analyte response, which we attribute to steric crowding at high ligand concentrations. Using this calibration to inform experimental design, researchers can choose protein concentrations for immobilization corresponding to the linear range of analyte response. We are the first to demonstrate calibration and normalization as a strategy to increase reproducibility and data quality of these chips. Our study assesses a variety of factors affecting chip variability, addressing a gap in knowledge about commercially available sensor chips. Controlling for these factors in the process of experimental design will minimize variability in analyte signal when using these important sensing platforms.

Bubble-Mediated Large-Scale Hierarchical Assembly of Ultrathin Pt Nanowire Network Monolayer at Gas/Liquid Interfaces.

Extensive macroscale two-dimensional (2-D) platinum (Pt) nanowire network (NWN) sheets are created through a hierarchical self-assembly process with the aid of biomolecular ligands. The Pt NWN sheet is assembled from the attachment growth of 1.9 nm-sized 0-D nanocrystals into 1-D nanowires featuring a high density of grain boundaries, which then interconnect to form monolayer network structures extending into centimeter-scale size. Further investigation into the formation mechanism reveals that the initial emergence of NWN sheets occurs at the gas/liquid interfaces of the bubbles produced by sodium borohydride (NaBH4) during the synthesis process. Upon the rupture of these bubbles, an exocytosis-like process releases the Pt NWN sheets at the gas/liquid surface, which subsequently merge into a continuous monolayer Pt NWN sheet. The Pt NWN sheets exhibit outstanding oxygen reduction reaction (ORR) activities, with specific and mass activities 12.0 times and 21.2 times greater, respectively, than those of current state-of-the-art commercial Pt/C electrocatalysts.

Insulator-to-metal-like transition in thin films of a biological metal-organic framework

Temperature-induced insulator-to-metal transitions (IMTs) where the electrical resistivity can be altered by over tens of orders of magnitude are most often accompanied by structural phase transition in the system. Here, we demonstrate an insulator-to-metal-like transition (IMLT) at 333 K in thin films of a biological metal-organic framework (bio-MOF) which was generated upon an extended coordination of the cystine (dimer of amino acid cysteine) ligand with cupric ion (spin-1/2 system) – without appreciable change in the structure. Bio-MOFs are crystalline porous solids and a subclass of conventional MOFs where physiological functionalities of bio-molecular ligands along with the structural diversity can primarily be utilized for various biomedical applications. MOFs are usually electrical insulators (so as our expectation with bio-MOFs) and can be bestowed with reasonable electrical conductivity by the design. This discovery of electronically driven IMLT opens new opportunities for bio-MOFs, to emerge as strongly correlated reticular materials with thin film device functionalities.

Silicon functionalization expands the repertoire of Si-rhodamine fluorescent probes.

Fluorescent dyes such as rhodamines are widely used to assay the activity and image the location of otherwise invisible molecules. Si-rhodamines, in which the bridging oxygen of rhodamines is replaced with a dimethyl silyl group, are increasingly the dye scaffold of choice for biological applications, as fluorescence is shifted into the near-infrared while maintaining high brightness. Despite intense interest in Si-rhodamines, there has been no exploration of the scope of silicon functionalization in these dyes, a potential site of modification that does not exist in conventional rhodamines. Here we report a broad range of silyl modifications that enable brighter dyes, further red-shifting, new ways to modulate fluorescence, and the introduction of handles for dye attachment, including fluorogenic labeling agents for nuclear DNA, SNAP-tag and HaloTag labeling. Modifications to the bridging silicon are therefore of broad utility to improve and expand the applications of all Si-dyes.

Bicomponent Cellulose Fibrils and Minerals Afford Wicking Channels Stencil-Printed on Paper for Rapid and Reliable Fluidic Platforms.

Flexible and easy-to-use microfluidic systems are suitable options for point-of-care diagnostics. Here, we investigate liquid transport in fluidic channels produced by stencil printing on flexible substrates as a reproducible and scalable option for diagnostics and paper-based sensing. Optimal printability and flow profiles were obtained by combining minerals with cellulose fibrils of two different characteristic dimensions, in the nano- and microscales, forming channels with ideal wettability. Biomolecular ligands were easily added by inkjet printing on the channels, which were tested for the simultaneous detection of glucose and proteins. Accurate determination of clinically relevant concentrations was possible from linear calibration, confirming the potential of the introduced paper-based diagnostics. The results indicate the promise of simple but reliable fluidic channels for drug and chemical analyses, chromatographic separation, and quality control.

Hydrogen bond surrogate helices as minimal mimics of protein α-helices.

Examination of complexes of proteins with biomolecular ligands reveals that proteins tend to interact with partners via folded sub-domains, in which the backbone possesses secondary structure. α-Helices comprising the largest class of protein secondary structures, play fundamental roles in a multitude of highly specific protein-protein and protein-nucleic acid interactions. We have demonstrated a unique strategy for stabilization of the α-helical conformation that involves replacement of one of the main chain i and i+4 hydrogen bonds in the target α-helix with a covalent bond. We termed this synthetic strategy a hydrogen bond surrogate (HBS) approach. Two salient features of this approach are: (1) the internal placement of the crosslink allows development of helices such that none of the solvent-exposed surfaces are blocked by the constraining element, i.e., all side chains of the constrained helices remain available for molecular recognition. (2) This approach can be deployed to constrain very short peptides (<10 amino acid residues) into highly stable α-helices. This chapter presents the biophysical basis for the development of the hydrogen bond surrogate approach, as well as methods for the synthesis and conformational analysis of the artificial helices.

Quantitative Analysis of DNA-Mediated Formation of Metal Nanocrystals.

The predictive synthesis of metal nanocrystals with desired structures relies on the precise control of the crystal formation process. Using a capping ligand is an effective method to affect the reduction of metal ions and the formation of nanocrystals. However, predictively synthesizing nanostructures has been difficult to achieve using conventional capping ligands. DNA, as a class of the promising biomolecular capping ligands, has been used to generate sequence-specific morphologies in various metal nanocrystals. However, mechanistic insight into the DNA-mediated nanocrystal formation remains elusive due to the lack of quantitative experimental evidence. Herein, we quantitatively analyzed the precise control of DNA over Ag+ reduction and the structures of resulting Au-Ag core-shell nanocrystals. We derived the equilibrium binding constants between DNA and Ag+, the kinetic rate constants of sequence-specific Ag+ reduction pathways, and the percentage of active surface sites remaining on the nanocrystals after DNA passivation. These three synergistic factors influence the nucleation and growth process both thermodynamically and kinetically, which contributed to the morphological evolution of Au-Ag nanocrystals synthesized with different DNA sequences. This study demonstrates the potential of using functional DNA sequences as a versatile and tunable capping ligand system for the predictable synthesis of metal nanostructures.

Homogeneous Oligomeric Ligands Prepared via Radical Polymerization that Recognize and Neutralize a Target Peptide

AbstractAbiotic ligands that bind to specific biomolecules have attracted attention as substitutes for biomolecular ligands, such as antibodies and aptamers. Radical polymerization enables the production of robust polymeric ligands from inexpensive functional monomers. However, little has been reported about the production of monodispersed polymeric ligands. Herein, we present homogeneous ligands prepared via radical polymerization that recognize epitope sequences on a target peptide and neutralize the toxicity of the peptide. Taking advantage of controlled radical polymerization and separation, a library of multifunctional oligomers with discrete numbers of functional groups was prepared. Affinity screening revealed that the sequence specificity of the oligomer ligands strongly depended on the number of functional groups. The process reported here will become a general step for the development of abiotic ligands that recognize specific peptide sequences.

Homogeneous Oligomeric Ligands Prepared via Radical Polymerization that Recognize and Neutralize a Target Peptide.

Abiotic ligands that bind to specific biomolecules have attracted attention as substitutes for biomolecular ligands, such as antibodies and aptamers. Radical polymerization enables the production of robust polymeric ligands from inexpensive functional monomers. However, little has been reported about the production of monodispersed polymeric ligands. Herein, we present homogeneous ligands prepared via radical polymerization that recognize epitope sequences on a target peptide and neutralize the toxicity of the peptide. Taking advantage of controlled radical polymerization and separation, a library of multifunctional oligomers with discrete numbers of functional groups was prepared. Affinity screening revealed that the sequence specificity of the oligomer ligands strongly depended on the number of functional groups. The process reported here will become a general step for the development of abiotic ligands that recognize specific peptide sequences.

Turning DNA Binding Motifs into a Material for Flow Cells.

Nanoscale assemblies of DNA strands are readily designed and can be generated in a wide range of shapes and sizes. Turning them into solids that bind biomolecules reversibly, so that they can act as active material in flow cells, is a challenge. Among the biomolecular ligands, cofactors are of particular interest because they are often the most expensive reagents of biochemical transformations, for which controlled release and recycling are desirable. We have recently described DNA triplex motifs that bind adenine‐containing cofactors, such as NAD, FAD and ATP, reversibly with low micromolar affinity. We sought ways to convert the soluble DNA motifs into a macroporous solid for cofactor binding. While assemblies of linear and branched DNA motifs produced hydrogels with undesirable properties, long DNA triplexes treated with protamine gave materials suitable for flow cells. Using exchangeable cells in a flow system, thermally controlled loading and discharge were demonstrated. Employing a flow cell loaded with ATP, bioluminescence was induced through thermal release of the cofactor. The results show that materials generated from functional DNA structures can be successfully employed in macroscopic devices.

Biocomputing with Nanostructures on Lipid Bilayers.

Biocomputation is the algorithmic manipulation of biomolecules. Nanostructures, most notably DNA nanostructures and nanoparticles, become active substrates for biocomputation when modified with stimuli-responsive, programmable biomolecular ligands. This approach-biocomputing with nanostructures ("nano-bio computing")-allows autonomous control of matter and information at the nanoscale; their dynamic assemblies and beneficial properties can be directed without human intervention. Recently, lipid bilayers interfaced with nanostructures have emerged as a new biocomputing platform. This new nano-bio interface, which exploits lipid bilayers as a chemical circuit board for information processing, offers a unique reaction space for realizing nanostructure-based computation at a previously unexplored dimension. In this Concept, recent advances in nano-bio computing are briefly reviewed and the newly emerging concept of biocomputing with nanostructures on lipid bilayers is introduced.

Large-Area Biomolecule Nanopatterns on Diblock Copolymer Surfaces for Cell Adhesion Studies.

Cell membrane receptors bind to extracellular ligands, triggering intracellular signal transduction pathways that result in specific cell function. Some receptors require to be associated forming clusters for effective signaling. Increasing evidences suggest that receptor clustering is subjected to spatially controlled ligand distribution at the nanoscale. Herein we present a method to produce in an easy, straightforward process, nanopatterns of biomolecular ligands to study ligand–receptor processes involving multivalent interactions. We based our platform in self-assembled diblock copolymers composed of poly(styrene) (PS) and poly(methyl methacrylate) (PMMA) that form PMMA nanodomains in a closed-packed hexagonal arrangement. Upon PMMA selective functionalization, biomolecular nanopatterns over large areas are produced. Nanopattern size and spacing can be controlled by the composition of the block-copolymer selected. Nanopatterns of cell adhesive peptides of different size and spacing were produced, and their impact in integrin receptor clustering and the formation of cell focal adhesions was studied. Cells on ligand nanopatterns showed an increased number of focal contacts, which were, in turn, more matured than those found in cells cultured on randomly presenting ligands. These findings suggest that our methodology is a suitable, versatile tool to study and control receptor clustering signaling and downstream cell behavior through a surface-based ligand patterning technique.

Rapid one-step 18F-radiolabeling of biomolecules in aqueous media by organophosphine fluoride acceptors

Currently, only a few 18F-radiolabeling methods were conducted in aqueous media, with non-macroelement fluoride acceptors and stringent conditions required. Herein, we describe a one-step non-solvent-biased, room-temperature-driven 18F-radiolabeling methodology based on organophosphine fluoride acceptors. The high water tolerance for this isotope-exchange-based 18F-labeling method is attributed to the kinetic and thermodynamic preference of F/F over the OH/F substitution based on computational calculations and experimental validation. Compact [18/19F]di-tert-butyl-organofluorophosphine and its derivatives used as 18F-labeling synthons exhibit excellent stability in vivo. The synthons are further conjugated to several biomolecular ligands such as c(RGDyk) and human serum albumin. The one-step labeled biomolecular tracers demonstrate intrinsic target imaging ability and negligible defluorination in vivo. The current method thus offers a facile and efficient 18F-radiolabeling pathway, enabling further widespread application of 18F.

Development of protein-recognition SPR devices by combination of SI-ATRP with biomolecular imprinting using protein ligands

AbstractMolecularly imprinted polymer brush layers and gel layers with both a lectin (ConA) and an antibody-IgG as biomolecular ligands for a target protein were formed on surface plasmon resonance (SPR) sensor chips via surface-initiated atom transfer radical polymerization (SIATRP) without and with a crosslinker, respectively. While the IgG-imprinted brush layers chip had almost the same affinity constant for target IgG as the nonimprinted brush layer chip, the affinity constant of the IgG-imprinted gel layer chip was approximately twice than that of the nonimprinted gel layer chip. These indicate that chemical crosslinks are very important factor to create distinct molecular recognition sites by molecular imprinting. Thus, biomolecular imprinting that uses biomolecular ligands and crosslinkers enables us to design polymer layer chips with distinct molecular recognition sites with a strong affinity for a target biomolecule. The molecularly imprinted gel layers chips with lectin and antibody ligands are promising candidates for fabricating SPR sensor systems to monitor target biomolecules such as proteins.

Impedance spectroscopy analysis of human odorant binding proteins immobilized on nanopore arrays for biochemical detection

Human odorant-binding proteins (hOBPs) not only can bind and transport odorants in the surrounding environment for sensing smells, but also play important roles in transmitting lots of biomolecules in different organs. Utilizing the properties of hOBPs, an electrochemical biosensor with nanopore array was developed to detect specific biomolecular ligands, such as aldehydes and fatty acids. The highly ordered nanopores of anodic aluminum oxide with diameter of 20–40nm were fabricated with two-step oxidation. Through 2-carboxyethyl phosphonic acid, hOBPs were self-assembled on nanopores as the sensing membrane. With nanopore arrays, the impedance spectra showed quite different electron transfer processes in the frequency spectra, which could be characterized by the electron transfer resistance and electrical resistance of the porous membrane. Under stimulation of biomolecular ligands, series resistance of nanopores and hOBPs increased and showed a concentration-dependence feature, while the electron transfer resistance hardly changed. The nanopore based biosensor could sensitively detect biological ligands of benzaldehyde, docosahexaenoic acid, and lauric acid, which were closely related to or were potential biomarkers for cancers and other serious diseases. Equipped with hOBPs, the sensor exhibited promising potentials both in odorant and biomolecule detection for olfactory biosensing and in disease diagnosis and evaluation for biochemical detection.

Combinatorial Biomolecular Nanopatterning for High-Throughput Screening of Stem-Cell Behavior.

A novel combinatorial biomolecular nanopatterning method is reported, in which multiple biomolecular ligands can be patterned in multiple nanoscale dimensions on a single surface. The applicability of the combinatorial platform toward cell-biology applications is demonstrated by screening the adhesion behavior of a population of human dental pulp stem cell (hDPSC) on 64 combinations of nanopatterned extracellular matrix (ECM) proteins in parallel.

Thermo-responsive cell culture carriers based on poly(vinyl methyl ether)—the effect of biomolecular ligands to balance cell adhesion and stimulated detachment

Two established material systems for thermally stimulated detachment of adherent cells were combined in a cross-linked polymer blend to merge favorable properties. Through this approach poly(N-isopropylacrylamide) (PNiPAAm) with its superior switching characteristic was paired with a poly(vinyl methyl ether)-based composition that allows adjusting physico-chemical and biomolecular properties in a wide range. Beyond pure PNiPAAm, the proposed thermo-responsive coating provides thickness, stiffness and swelling behavior, as well as an apposite density of reactive sites for biomolecular functionalization, as effective tuning parameters to meet specific requirements of a particular cell type regarding initial adhesion and ease of detachment. To illustrate the strength of this approach, the novel cell culture carrier was applied to generate transplantable sheets of human corneal endothelial cells (HCEC). Sheets were grown, detached, and transferred onto planar targets. Cell morphology, viability and functionality were analyzed by immunocytochemistry and determination of transepithelial electrical resistance (TEER) before and after sheet detachment and transfer. HCEC layers showed regular morphology with appropriate TEER. Cells were positive for function-associated marker proteins ZO-1, Na+/K+-ATPase, and paxillin, and extracellular matrix proteins fibronectin, laminin and collagen type IV before and after transfer. Sheet detachment and transfer did not impair cell viability. Subsequently, a potential application in ophthalmology was demonstrated by transplantation onto de-endothelialized porcine corneas in vitro. The novel thermo-responsive cell culture carrier facilitates the generation and transfer of functional HCEC sheets. This paves the way to generate tissue engineered human corneal endothelium as an alternative transplant source for endothelial keratoplasty.