Circular Dichroism of Amino Acids: Following the Structural Formation of Phenylalanine

Circular dichroism (CD) is frequently used to assess the secondary structure of peptides and proteins, whereas less attention has been given to their building blocks, that is, single amino acids, as they do not possess a secondary structure. Here, we follow the CD signal of amino acids and reveal that several acids exhibit a unique CD pattern as a function of their concentration. Accordingly, we propose an eight‐level classification of the CD signal of the various amino acids. Special focus is given to the CD pattern of phenylalanine (Phe), for which we observe the formation of an ultra‐narrow CD peak (full width at high maximum of only 5 nm). This CD peak can be attributed to the formation of Phe‐based chiral structural features. Further support for the formation of an ordered structure is given by using NMR, and the additional self‐assembly process of Phe to tubular structures.     

A Molecular Tuning Fork in Single‐Molecule Mechanochemical Sensing

The separate arrangement of target recognition and signal transduction in conventional biosensors often compromises the real‐time response and can introduce additional noise. To address these issues, we combined analyte recognition and signal reporting by mechanochemical coupling in a single‐molecule DNA template. We incorporated a DNA hairpin as a mechanophore in the template, which, under a specific force, undergoes stochastic transitions between folded and unfolded hairpin structures (mechanoescence). Reminiscent of a tuning fork that vibrates at a fixed frequency, the device was classified as a molecular tuning fork (MTF). By monitoring the lifetime of the folded and unfolded hairpins with equal populations, we were able to differentiate between the mono‐ and bivalent binding modes during individual antibody‐antigen binding events. We anticipate these mechanospectroscopic concepts and methods will be instrumental for the development of novel bioanalyses.     

A Sensitive Electrochemical Biosensor for Detection of Histone Deacetylase Activity Using an Acetylated Peptide

A sensitive electrochemical biosensor was developed for activity detection of histone deacetylase sirtuin2 (SIRT2) using an acetylated peptide substrate. This substrate could be recognized by anti‐acetylated peptide antibody, which could be detected using secondary antibody conjugated alkaline phosphatase which provided an amplified electrochemical signal. In the presence of SIRT2, the substrate was deacetylated, resulting in a decreased electrochemical signal that was correlated to the concentration of SIRT2. Under optimized conditions, the biosensor exhibited a wide linear range from 1 nM to 500 nM with a detection limit of 0.1 nM. The proposed biosensor was also used for detection of SIRT2 inhibitor.     

Transparent Carbon Nanotube Network for Efficient Electrochemiluminescence Devices

A carbon nanotube‐based electrode that combines transparency and good conductivity was used for the first time to develop an electrochemiluminescence (ECL) device. It resulted in an excellent material for ECL applications thanks to the very favorable overpotential of amine oxidation that represents the rate‐determining step for the signal generation in both research systems and commercial instrumentation. The use of carbon nanotubes resulted in a ten times higher emission efficiency compared with commercial transparent indium tin oxide (ITO) electrodes. Moreover, application of this material for proof‐of‐principle ECL imaging was demonstrated, in which micro‐beads were used to mimic a real biological sample in order to prove the possibility of obtaining single cell visualization.     

MVF Sensor Enables Analysis of Nucleic Acids with Stable Secondary Structures

One major challenge in nucleic acids analysis by hybridization probes is a compromise between the probe's tight binding and sequence‐selective recognition of nucleic acid targets folded into stable secondary structures. We have been developing a four‐way junction (4WJ)‐based sensor that consists of a universal stem‐loop (USL) probe immobilized on an electrode surface and two adaptor strands (M and F). The sensor was shown to be highly selective towards single base mismatches at room temperature, able to detect multiple targets using the same USL probe, and have improved ability to detect folded nucleic acids. However, some nucleic acid targets, including natural RNA, are folded into very stable secondary and tertiary structures, which may represent a challenge even for the 4WJ sensors. This work describes a new sensor, named MVF since it uses three probe stands M, V and F, which further improves the performance of 4WJ sensors with folded targets. The MVF sensor interrogating a 16S rRNA NASBA amplicon with calculated folding energy of ?32.82 kcal/mol has demonstrated 2.5‐fold improvement in a signal‐to‐background ratio in comparison with a 4WJ sensor lacking strand V. The proposed design can be used as a general strategy in the analysis of folded nucleic acids including natural RNA.     

A Three‐Dimensional Origami Paper‐Based Device for Potentiometric Biosensing

Current paper‐based potentiometric ion‐sensing platforms are planar devices used for clinically relevant ions. These devices, however, have not been designed for the potentiometric biosensing of proteins or small molecule analytes. A three‐dimensional origami paper‐based device, in which a solid‐contact ion‐selective electrode is integrated with an all‐solid‐state reference electrode, is described for the first time. The device is made by impregnation of paper with appropriate bioreceptors and reporting reagents on different zones. By folding and unfolding the paper structures, versatile potentiometric bioassays can be performed. A USB‐controlled miniaturized electrochemical detector can be used for simple and in situ measurements. Using butyrylcholinesterase as a model enzyme, the device has been successfully applied to the detection of enzyme activities and organophosphate pesticides involved in the enzymatic system as inhibitors. The proposed 3D origami paper device allows the potentiometric biosensing of proteins and small molecules in a simple, portable, and cost‐effective way.     

Helical Propensity in an Intrinsically Disordered Protein Accelerates Ligand Binding

Many intrinsically disordered proteins fold upon binding to other macromolecules. The secondary structure present in the well‐ordered complex is often formed transiently in the unbound state. The consequence of such transient structure for the binding process is, however, not clear. The activation domain of the activator for thyroid hormone and retinoid receptors (ACTR) is intrinsically disordered and folds upon binding to the nuclear coactivator binding domain (NCBD) of the CREB binding protein. A number of mutants was designed that selectively perturbs the amount of secondary structure in unbound ACTR without interfering with the intermolecular interactions between ACTR and NCBD. Using NMR spectroscopy and fluorescence‐monitored stopped‐flow kinetic measurements we show that the secondary structure content in helix 1 of ACTR indeed influences the binding kinetics. The results thus support the notion of preformed secondary structure as an important determinant for molecular recognition in intrinsically disordered proteins.     

Real‐Time Analysis of Folding upon Binding of a Disordered Protein by Using Dissolution DNP NMR Spectroscopy

The kinase inhibitory domain of the cell cycle regulatory protein p27^Kip1 (p27) was nuclear spin hyperpolarized using dissolution dynamic nuclear polarization (D‐DNP). While intrinsically disordered in isolation, p27 adopts secondary structural motifs, including an α‐helical structure, upon binding to cyclin‐dependent kinase 2 (Cdk2)/cyclin A. The sensitivity gains obtained with hyperpolarization enable the real‐time observation of ¹³C NMR signals during p27 folding upon binding to Cdk2/cyclin A on a time scale of several seconds. Time‐dependent intensity changes are dependent on the extent of folding and binding, as manifested in differential spin relaxation. The analysis of signal decay rates suggests the existence of a partially folded p27 intermediate during the timescale of the D‐DNP NMR experiment.     

Light‐Regulated Tetracycline Binding to the Tet Repressor

Elucidation of the signal‐transmission pathways between distant sites within proteins is of great importance in medical and bioengineering sciences. The use of optical methods to redesign protein functions is emerging as a general approach for the control of biological systems with high spatiotemporal precision. Here we report the detailed thermodynamic and kinetic characterization of novel chimeric light‐regulated Tet repressor (TetR) switches in which light modulates the TetR function. Light absorbed by flavin mononucleotide (FMN) generates a signal that is transmitted to As‐LOV and YtvA‐LOV fused TetR proteins (LOV=light–oxygen–voltage), in which it alters the binding to tetracycline, the TetR ligand. The engineering of light‐sensing protein modules with TetR is a valuable tool that deepens our understanding of the mechanism of signal transmission within proteins. In addition, the light‐regulated changes of drug binding that we describe here suggest that engineered light‐sensitive proteins may be used for the development of novel therapeutic strategies.     

Localized Mixed‐Valence and Redox Activity within a Triazole‐Bridged Dinucleating Ligand upon Coordination to Palladium

The new dinucleating redox‐active ligand ( L^H4 ), bearing two redox‐active NNO‐binding pockets linked by a 1,2,3‐triazole unit, is synthetically readily accessible. Coordination to two equivalents of Pd^II resulted in the formation of paramagnetic (S= ) dinuclear Pd complexes with a κ²‐N,N′‐bridging triazole and a single bridging chlorido or azido ligand. A combined spectroscopic, spectroelectrochemical, and computational study confirmed Robin–Day Class II mixed‐valence within the redox‐active ligand, with little influence of the secondary bridging anionic ligand. Intervalence charge transfer was observed between the two ligand binding pockets. Selective one‐electron oxidation allowed for isolation of the corresponding cationic ligand‐based diradical species. SQUID (super‐conducting quantum interference device) measurements of these compounds revealed weak anti‐ferromagnetic spin coupling between the two ligand‐centered radicals and an overall singlet ground state in the solid state, which is supported by DFT calculations. The rigid and conjugated dinucleating redox‐active ligand framework thus allows for efficient electronic communication between the two binding pockets.     

Tropolone as a High‐Performance Robust Anchoring Group for Dye‐Sensitized Solar Cells

A tropolone group has been employed for the first time as an anchoring group for dye‐sensitized solar cells (DSSCs). The DSSC based on a porphyrin, YD2‐o‐C8T, with a tropolone moiety exhibited a power‐conversion efficiency of 7.7 %, which is only slightly lower than that observed for a reference porphyrin, YD2‐o‐C8 , with a conventional carboxylic group. More importantly, YD2‐o‐C8T was found to be superior to YD2‐o‐C8 with respect to DSSC durability and binding ability to TiO₂. These results unambiguously demonstrate that tropolone is a highly promising dye‐anchoring group for DSSCs in terms of device durability as well as photovoltaic performance.     

Intermolecular Communication on a Liposomal Membrane: Enzymatic Amplification of a Photonic Signal with a Gemini Peptide Lipid as a Membrane‐Bound Artificial Receptor

A supramolecular system that can activate an enzyme through photo‐isomerization was constructed by using a liposomal membrane scaffold. The design of the system was inspired by natural signal transduction systems, in which enzymes amplify external signals to control signal transduction pathways. The liposomal membrane, which provided a scaffold for the system, was prepared by self‐assembly of a photoresponsive receptor and a cationic synthetic lipid. NADH‐dependent L ‐lactate dehydrogenase, the signal amplifier, was immobilized on the liposomal surface by electrostatic interactions. Recognition of photonic signals by the membrane‐bound receptor induced photo‐isomerization, which significantly altered the receptor’s metal‐binding affinity. The response to the photonic signal was transmitted to the enzyme by Cu²⁺ ions. The enzyme amplified the chemical information through a catalytic reaction to generate the intended output signal.     

Macrocyclic Protease Inhibitors with Reduced Peptide Character

There is a real need for simple structures that define a β‐strand conformation, a secondary structure that is central to peptide–protein interactions. For example, protease substrates and inhibitors almost universally adopt this geometry on active site binding. A planar pyrrole is used to replace two amino acids of a peptide backbone to generate a simple macrocycle that retains the required geometry for active site binding. The resulting β‐strand templates have reduced peptide character and provide potent protease inhibitors with the attachment of an appropriate amino aldehyde to the C‐terminus. Picomolar inhibitors of cathepsin L and S are reported and the mode of binding of one example to the model protease chymotrypsin is defined by X‐ray crystallography.     

Molecular ME‐ToF‐SIMS yield as a function of DHB matrix layer thicknesses obtained from brain sections coated by sublimation/deposition techniques

Time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) can be used to image biological samples with nanometer‐scale resolution, albeit with the drawback that it often cannot detect large molecular signals. One way to increase secondary ion molecular yield is to chemically modify the surface in the so‐called matrix‐enhanced SIMS (ME‐SIMS) approach, which is based on embedding analyte molecules in low‐weight organic matrices. In this study, a solvent‐free sample preparation technique was employed using sublimation/deposition for coating a mouse brain section with a thin layer of a 2,5‐dihydroxybenzoic acid (DHB) matrix. Using this preparation technique, signal enhancements of up to a factor of 18 could be detected. It was found that the matrix layer thickness plays an important role in the efficiency of yield enhancement. Also, a complex influence of the matrix layer on various signals was observed. Copyright © 2015 John Wiley & Sons, Ltd.     

Effective length calibration method for processing the fluorescence signal detected by charge‐coupled device in capillary electrophoresis

A novel method named effective length calibration method has been developed to process the fluorescence signal detected by charge‐coupled device during capillary electrophoresis. The new method treated each pixel as an individual point detector, and effectively binned a large number of pixels into a final electropherogram without losing the narrow detection window defined by a single pixel. Capillary electrophoresis separations of DNA were carried out and detected by charge‐coupled device and conventional detector (photomultiplier tube). Detection properties including signal‐to‐noise ratio, peak width, detection frequency, and tilt of detector were investigated. It was found that the new method achieved much higher signal‐to‐noise ratio and smaller peak width than the conventional detector did. A Detection width of 0.5 μm was easily achieved.     

A Direct Competitive Homogeneous Immunoassay for Progesterone – the Redox Quenching Immunoassay

A direct competitive amperometric immunoassay format for the detection of haptens and proteins was developed. The method is based on the quenching of electroactivity of ferrocenium, which is coupled to the antigen and used as the primary reporter, upon binding to a monoclonal anti‐ferrocenium antibody, which is coupled to the detection antibody and used as a secondary reporter. A separation‐free progesterone immunoassay with a lower detection limit of 1 ng mL^?1 (3.18 nmol L^?1) in 1 : 2 diluted blood serum was realised by combining two bifunctional conjugates, a ferrocenium‐PEG‐progesterone tracer and a bioconjugate of one anti‐progesterone and one anti‐ferrocenium antibody. The immune complex is formed within 30 s upon addition of progesterone, resulting in a total analysis time of 1.5 min.     

Theoretical Assessment of Binding and Mass‐Transport Effects in Electrochemical Affinity Biosensors That Utilize Nanoparticle Labels for Signal Amplification

This paper presents a theoretical study of electrochemical affinity biosensors for the detection of DNA/protein that utilize nanoparticle labels for signal amplification. This study analyzes the effects of binding and mass transport of the analytes on biosensor performance by using numerical simulations. Four cases were considered: 1) nanoparticles used to increase the loading of an electroactive species, or used as catalysts under pseudo‐first‐order conditions; 2) nanoparticles used as ultramicroelectrode arrays for the electrolysis of large concentrations of substrate; 3) nanoparticles used as seeds to deposit electrochemically detectable species; and 4) nanoparticles used to mediate the deposition of electrocatalysts. By using nanoparticle labels, high sensitivity is possible under all conditions considered. However, theoretical findings suggested that nonspecific adsorption could be more problematic in cases 2–4 due to the mismatch between the chemistry of surface binding and the principle of signal amplification that originates from the effect of mass transport. Under these conditions, any given signal would plateau at a much lower analyte concentration, well before the analyte binding had actually reached a plateau. Views on possible solutions to the above limitations are also presented.     

Polymer‐Templated Perylene‐Probe Noncovalent Self‐Assembly: A New Strategy for Label‐Free Ultrasensitive Fluorescence Turn‐On Biosensing

A new label‐free fluorescence turn‐on strategy for highly sensitive biosensing has been developed. A negatively charged perylene probe was synthesized. Polycations could induce aggregation of the perylene probe through noncovalent interactions and the fluorescence of the probe’s monomer was efficiently quenched. Upon addition of a single‐stranded nucleic acid, competitive binding of the negatively charged nucleic acid (a polyanion) to the cationic polymer resulted in the release of a monomer and thus a turn‐on fluorescence signal was detected. Without the use of any amplification techniques, a detection limit of 2 pM DNA was obtained. Based on these results, an assay strategy for the highly sensitive detection of alkaline phosphatase (ALP) activity has been demonstrated. λ Exonuclease (λ exo) could degrade 5′‐phosphorylated single‐stranded DNA. However, when the DNA sample was treated with ALP, the phosphate functional group was removed by ALP and it could no longer be degraded by λ exo. Binding of the DNA to the perylene probe–polycation complex resulted in a turn‐on fluorescence signal, which could be used for sensing of ALP. The method is highly sensitive, a limit of detection as low as 0.02 mU mL^?1 ALP was obtained. Our method is simple, convenient, highly sensitive, and inexpensive.     

Micro‐Scale Device—An Alternative Route for Studying the Intrinsic Properties of Solid‐State Materials: The Case of Semiconducting TaGeIr

An efficient application of a material is only possible if we know its physical and chemical properties, which is frequently obstructed by the presence of micro‐ or macroscopic inclusions of secondary phases. While sometimes a sophisticated synthesis route can address this issue, often obtaining pure material is not possible. One example is TaGeIr, which has highly sample‐dependent properties resulting from the presence of several impurity phases, which influence electronic transport in the material. The effect of these minority phases was avoided by manufacturing, with the help of focused‐ion‐beam, a μm‐scale device containing only one phase—TaGeIr. This work provides evidence for intrinsic semiconducting behavior of TaGeIr and serves as an example of selective single‐domain device manufacturing. This approach gives a unique access to the properties of compounds that cannot be synthesized in single‐phase form, sparing costly and time‐consuming synthesis efforts.     

Structural Elucidation of the Bispecificity of A Domains as a Basis for Activating Non‐natural Amino Acids

Many biologically active peptide secondary metabolites of bacteria are produced by modular enzyme complexes, the non‐ribosomal peptide synthetases. Substrate selection occurs through an adenylation (A) domain, which activates the cognate amino acid with high fidelity. The recently discovered A domain of an Anabaenopeptin synthetase from Planktothrix agardhii (ApnA A₁) is capable of activating two chemically distinct amino acids (Arg and Tyr). Crystal structures of the A domain reveal how both substrates fit into to binding pocket of the enzyme. Analysis of the binding pocket led to the identification of three residues that are critical for substrate recognition. Systematic mutagenesis of these residues created A domains that were monospecific, or changed the substrate specificity to tryptophan. The non‐natural amino acid 4‐azidophenylalanine is also efficiently activated by a mutant A domain, thus enabling the production of diversified non‐ribosomal peptides for bioorthogonal labeling.