Energetics and Redox Kinetics of Pure Ferrocene-Terminated N-Heterocyclic Carbene Self-Assembled Monolayers on Gold.

N-heterocyclic carbene (NHC) self-assembled monolayers (SAMs) on gold have received considerable attention, but little is known about the lateral interactions between neighboring NHC molecules, their stability when subjected to aggressive oxidizing/reducing conditions, and their interactions with solution ions, all of which are essential for their use in a wide range of applications. To address these deficiencies, we present a comprehensive investigation of two different ferrocene (Fc)-terminated NHC SAMs with different chain lengths and linking groups. Pure monolayers of Fc-terminated NHCs display only a single, symmetrical pair of redox peaks, implying the formation of a homogeneous SAM structure with uniformly distributed Fc/Fc+ redox centers. By comparison, pure Fc-alkylthiol SAMs exhibit complex and impractical redox chemistry and require surface dilution in order to achieve reproducible properties. The NHC SAMs examined in this study exhibit very fast Fc redox kinetics and comparable or even superior stability against the application of multiple potential cycles or long-time holding at constant potential compared to alkylthiol SAMs. Furthermore, ion pairing of Fc+ and hydrophobic perchlorate and other hydrophilic anions is observed with Fc-NHC SAMs, highlighting conditions favorable for future applications of these monolayers. This study should therefore shed light on the very promising characteristics of redox-active NHC SAMs as an alternative to traditional Fc-alkylthiol SAMs for multiple practical applications, including in sensors and electrocatalysis.

Developing a toll-like receptor biosensor for Gram-positive bacterial detection and its storage strategies.

The biorecognition ability of hybridized toll-like receptors (TLRs) 2 and 6 proteins on electrode surfaces has been studied. TLR biosensors have been designed to be non-specific to particular bacterial strains but rather to provide broad spectrum detection of cells and toxins containing relevant pathogen-associated molecular patterns (PAMPs). Our electrochemical TLR2/6 biosensors demonstrated selective detection towards Gram-positive bacterial whole-cells and a synthetic diacylated lipopeptide (Pam2CSK4), a PAMP. Responses towards Bacillus licheniformis (B. licheniformis) and Enterococcus hirae (E. hirae) were obtained. The biosensor was able to differentiate signals between B. licheniformis and a Gram-negative bacterial cell (control) as low as 100 CFU mL-1. One challenge in developing protein-based biosensors is to improve the shelf-life of the biosensor chips and preserve the detection activity of the protein molecules, therefore we did our first exploration into storage conditions. The activity of stored biosensors was found to be strongly dependent on storage medium, and that effective 'shelf-life' was obtained makes an important step towards creating robust sensors for real-life applications.

Synthesis and Biochemical Evaluation of Nicotinamide Derivatives as NADH Analogue Coenzymes in Ene Reductase.

Nicotinamide and pyridine-containing conjugates have attracted a lot of attention in research as they have found use in a wide range of applications including as redox flow batteries and calcium channel blockers, in biocatalysis, and in metabolism. The interesting redox character of the compounds' pyridine/dihydropyridine system allows them to possess very similar characteristics to the natural chiral redox agents NAD+ /NADH, even mimicking their functions. There has been considerable interest in designing and synthesizing NAD+ /NADH mimetics with similar redox properties. In this research, three nicotinamide conjugates were designed, synthesized, and characterized. Molecular structures obtained through X-ray crystallography were obtained for two of the conjugates, thereby providing more detail on the bonding and structure of the compounds. The compounds were then further evaluated for biochemical properties, and it was found that one of the conjugates possessed similar functions and characteristics to the natural NADH. This compound was evaluated in the active enzyme, enoate reductase; like NADH, it was shown to help reduce the C=C double bond of three substrates and even outperformed the natural coenzyme. Kinetic data are reported.

Electrochemical studies of human nAChR a7 subunit phosphorylation by kinases PKA, PKC and Src.

Nicotinic acetylcholine receptors (nAChR) are ion channels which regulate a numerous of neurotransmitters, including acetylcholine, norepinephrine, dopamine, serotonin and glutamate. These receptors are important targets for the study of a plethora of diseases such as Alzheimer's disease, schizophrenia, Parkinson's Disease, cancer, inflammation, etc. The α7 subunits are especially interesting in that they are commonly occurring and are critical sites of regulation. Herein we report the phosphorylation of the human nAChR α7 subunits, by the kinases PKA, PKC and Src, by both biochemical and electrochemical techniques along with the kinetics of each phosphorylation reaction. Phosphorylation was investigated through changes in current density as well as impedance and X-ray photo electron spectroscopy (XPS) and the kinetics were determined electrochemically using the surface Michaelis-Menten model. Our results clearly demonstrate the phosphorylation of the nAChR α7 and the invaluable strength of surface electrochemical techniques in the investigation of protein phosphorylation.

Electron Transfer in Spacer-Free DNA Duplexes Tethered to Gold via dA10 Tags.

Electrical properties of DNA critically depend on the way DNA molecules are integrated within the electronics, particularly on DNA-electrode immobilization strategies. Here, we show that the rate of electron transport in DNA duplexes spacer-free tethered to gold via the adenosine terminal region (a dA10 tag) is enhanced compared to the hitherto reported DNA-metal electrode tethering chemistries. The rate of DNA-mediated electron transfer (ET) between the electrode and methylene blue intercalated into the dA10-tagged DNA duplex approached 361 s-1 at a ca. half-monolayer DNA surface coverage ΓDNA (with a linear regression limit of 670 s-1 at ΓDNA → 0), being 2.7-fold enhanced compared to phosphorothioated dA5* tethering (6-fold for the C6-alkanethiol linker representing an additional ET barrier). X-ray photoelectron spectroscopy evidenced dA10 binding to the Au surface via the purine N, whereas dA5* predominantly coordinated to the surface via sulfur atoms of phosphothioates. The latter apparently induces the DNA strand twist in the point of surface attachment affecting the local DNA conformation and, as a result, decreasing the ET rates through the duplex. Thus, a spacer-free DNA coupling to electrodes via dA10 tags thus allows a perspective design of DNA electronic circuits and sensors with advanced electronic properties and no implication from more expensive, synthetic linkers.

Hg(ii) interactions with T-rich regions in oligonucleotides: effects of positional variations on the electrochemical properties.

Hg(ii) binding to thymine-rich oligonucleotides (ODNs) is investigated electrochemically. The focus of this study is to probe the effects of position on the electrochemical response. For this purpose, three oligonucleotides were investigated in which the position of a hexa-thymine repeat is varied within a surface-supported oligonucleotide. The hexa repeats were placed in the top, middle, and bottom positions within the strand with respect to the gold surface. The effects were monitored by electrochemical impedance spectroscopy and scanning electrochemical microscopy. Using charge transfer resistance (RCT) and tip current (I) as a measure, it was possible to monitor the effects of Hg(ii) binding to the ds-oligonucleotide. The extent of film resistance reduces as the T-rich region moves from the bottom to top position within the film. The T-rich region closer to the gold surface probably builds less flexible and more rigid T-Hg(ii)-T basepairs compared to the other two positions and is expected to stay in the upright orientation on the surface. This in turn results in significant differences in the electrochemical readout, demonstrating that the position of T-rich sequences within an oligonucleotide strand matters.

Detection of the Lipopeptide Pam3CSK4 Using a Hybridized Toll-like Receptor Electrochemical Sensor.

Electrochemical detection of Pam3CSK4, a synthetic triacylated lipopeptide that mimics the structural moieties of its natural Gram negative bacterial pathogen-associated molecular pattern (PAMP) counterpart, has been achieved using hybridized toll-like receptors (TLR) combining TLR1 and TLR2 onto a single sensor surface. These sensors represent the first hybridized TLR sensors. The limit of detection for Pam3CSK4 attained was 7.5 µg/mL, which is within the same order of magnitude for that of the more labor-intensive and time-consuming cell-assay technique, 2.0 µg/mL. The results gathered in these electrochemical experiments show that sensors fabricated by immobilizing a mixture of cooperative TLR1 and -2 generate higher responses when exposed to the analyte in comparison to the control sensors fabricated using pure TLR1 or -2 standalone. A PAMP selectivity test was carried out in line with our inspiration from the mammalian innate immune response. TLRs1-5 as standalone biorecognition elements and the hybridized "TLR1 and 2" sensor surface were investigated, understanding the known TLR-PAMP interactions, through the exploitation of this electrochemical sensor fabrication technique. The experimental result is consistent with observations from previously published in vivo and in vitro studies, and it is the first demonstration of the simultaneous evaluation of electrochemical responses from multiple, unique fabricated TLR sensor surfaces against the same analyte.

A Bioorganometallic Approach to Study Histidine Kinase Autophosphorylations.

Auto-phosphorylation of bacterial histidine kinases PhoR, PhoQ, and EnvZ has been investigated using adenosine-5'-[γ-ferrocene] triphosphate (Fc-ATP) as a cosubstrate for the first time. The study has been carried out in solution and on surface. Results from biochemical multiplex assay and surface electrochemical/optical methods are consistent, which successfully demonstrates that Fc-ATP is an efficient cosubstrate for histidine kinase auto-phosphorylations. The study also has discovered that the concentration of Fc-ATP influences the autophosphorylation efficiency. This developed methodology will provide a powerful tool in studying such biological processes towards further understanding of the involved mechanism.

Interactions of Hg(ii) with oligonucleotides having thymine-thymine mispairs. Optimization of an impedimetric Hg(ii) sensor.

The present work describes the effect of the number of thymine-thymine mispairs in single strand DNA probes on Hg(ii) interactions and further to develop a highly sensitive DNA based impedimetric sensor for Hg(ii) detection. To achieve this goal, the influence of the number of T-T mispairs on the signal response prompted by DNA-Hg(ii) binding interactions was examined on three designed DNA probes: 5'-OH-(CH2)6-S-S-(CH2)6-AGTCCACACGTTCCTTACGC-3', 5'-OH-(CH2)6-S-S-(CH2)6-AGTCCACATTTTCCTTTTGC-3', 5'-OH-(CH2)6-S-S-(CH2)6-AGTCCATTTTTTCCTTTTTT-3' having 2T-T, 4T-T and 6T-T mispairs with identical length, respectively. This study revealed that the number of T-T mispairs plays a critical role in maximizing the signal intensity of DNA-Hg(ii) binding interactions. Based on these results, DNA comprising maximum number of T-T mispairs was further utilized for construction of the Hg(ii) sensor, which exhibited a linear correlation between the change in charge transfer resistance (ΔRCT) and the concentration of Hg(ii) over the range of 1.0 × 10-5 M to 1.0 × 10-10 M with a lower detection limit of 3.2 × 10-11 M. The selectivity was tested against 12 different metal ions including Hg(ii). The ΔRCT response from Hg(ii) is 3 times higher than the nearest competitor Pb(ii) and approximately 10 times than other ions. The potential application of such a robust and label-free DNA sensor was demonstrated by analyzing environmental samples collected from Lake Ontario.

DNA Films Containing the Artificial Nucleobase Imidazole Mediate Charge Transfer in a Silver(I)-Responsive Way.

The first sequence-dependent study of DNA films containing metal-mediated base pairs was performed to investigate the charge transfer resistance (RCT ) of metal-modified DNA. The imidazole (Im) deoxyribonucleoside was chosen as a highly AgI -specific ligandoside for the formation of Im-AgI -Im complexes within the duplexes. This new class of site-specifically metal-modified DNA films was characterized by UV, circular dichroism (CD), and X-ray photoelectron spectroscopy (XPS). The electrochemical properties of these systems were investigated by means of electron impedance spectroscopy and scanning electrochemical microscopy. Taken together, these experiments indicated that the incorporation of AgI ions into the DNA films leads to reduced electron transfer through the DNA films. A simple device was proposed that can be switched reversibly between two distinct states with different charge transfer resistance.

Effects of bipyramidal gold nanoparticles and gold nanorods on the detection of immunoglobulins.

Two types of gold nanoparticles are compared for their use in a sensor for immunoglobulin detection - bipyramidal gold nanoparticles (GBPs) and gold nanorods (GNRs). Using surface plasmon resonance spectroscopy and square wave voltammetry allowed us to evaluate the utilities of these two types of gold nanoparticles in a label-free detection of the analyte IgG. However both systems showed a significant enhancement in sensitivity over a nanoparticle free system, showing a 64-fold enhancement for the GBP-containing sensor and a 16-fold enhancement for the GNR-containing sensor systems.

Investigation of the utility of complementary electrochemical detection techniques to examine the in vitro affinity of bacterial flagellins for a toll-like receptor 5 biosensor.

An initial investigation of the fabrication of a novel biosensor utilizing toll-like receptor 5 (TLR5) has been conducted. The detection assay using this sensor platform has been carried out using two complementary electrochemical techniques. The electrochemical properties of the modified bare gold surface following TLR5 immobilization were characterized. The electrochemical response to changes in the sensor film resistance and electron charge-transfer permittivity triggered by independent exposures to flagellins from Salmonella typhimurium (S. typhimurium) and Bacillus subtilis (B. subtilis) were examined and observed. The quantified film resistance data gathered using electrochemical impedance spectroscopy (EIS) over a macroscopic scale are in significant agreement with the corresponding electron charge-transfer permittivity measured locally by scanning electrochemical microscopy (SECM). Unlike other sensors that exploit pathogen recognition elements, TLR5 biosensors have the potential to carry out broad-spectrum detection of flagellated bacterial pathogens in near real time. This broad-spectrum detection platform is a significant step toward the development of fast, inexpensive clinical tools for early warning diagnoses and immediate on-site treatment.

Clickable 5'-γ-ferrocenyl adenosine triphosphate bioconjugates in kinase-catalyzed phosphorylations.

Clickable co-substrate: A tri-functional 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) derivative containing a clickable site was synthesized. This compound is an effective co-substrate in kinase-catalyzed phosphorylation reactions, which can be detected by both electrochemical and immunoassay detection methods. The clickable reaction site makes direct modification possible, which greatly expands its application.

Empowerment of Water-Evaporation-Induced Electric Generators via the Use of Metal Electrodes.

As water rises in the pores of a partially immersed porous film due to capillary action, it carries along ions that are dissociated from the pore walls, generating a streaming current and potential. The water and current flows are sustained due to water evaporation from the unsubmerged surfaces. Traditionally, inert graphite (C) electrodes are used to construct water-evaporation-induced generators (WEIGs) that harness this electricity. WEIGs are environmentally friendly but have weak power outputs. Herein, we report on C/metal WEIGs that feature C top electrodes and metal bottom electrodes, as well as metal/metal WEIGs. Operating in a NaCl solution that facilitates the Galvanic corrosion of the metal (Cu, steel, and Al) electrodes, these Galvanic WEIGs outperform a C/C WEIG by thousands of times in power output. Equally interestingly, the asymmetric environments and potential differences between the two electrodes of a WEIG facilitate metal corrosion and fabrication of compact Galvanic WEIGs. This study clearly shows that one should choose electrodes with caution for the construction of true WEIGs.

Investigating electrochemical deposition of gold on commercial off-the-shelf 3-D printing materials towards developing sensing applications.

The COVID-19 pandemic highlighted the inaccessibility of quick and affordable clinical diagnostics. This led to increased interest in creating low-cost portable electrochemical (EC) devices for environmental monitoring and clinical diagnostics. One important perspective is to develop new fabrication methods for functional and low-cost electrode chips. Techniques, such as electron beam and photolithography, allow precise and high-resolution electrode fabrication; however, they are costly and can be time-consuming. More recently, fused deposition modeling three-dimensional (3-D) printing is being used as an alternative fabrication technique due to the low-cost of the printer and rapid prototyping capability. In this study, we explore enhancing the conductivity of 3-D printed working electrodes with EC gold deposition. Two commercial conductive filament brands were used and investigated to fabricate electrode chips. Furthermore, strategies to apply epoxy glue and conductive silver paint were investigated to control the electrode surface area and ensure good electrical connection. This device enables detection at drinking water concentration thresholds. The practical application of the fabricated electrodes is demonstrated by detecting Cu2+ using anodic stripping voltammetry.

Transcranial direct current stimulation over the right dorsolateral prefrontal cortex has distinct effects on choices involving risk and ambiguity.

Human's uncertain decision-making involves choices of risk (with known probabilities) and ambiguity (with unknown probabilities). For risky and ambiguous decision-making processes, neural differences are rarely exhibited. To clarify the causal role of the right dorsolateral prefrontal cortex (DLPFC) in uncertain situations, we used transcranial direct current stimulation (tDCS) to demonstrate the involvement of the right DLPFC in decisions involving risk and ambiguity. Participants received either anodal or cathodal tDCS at 1.5 mA or sham stimulation over the right DLPFC and subsequently undertook tasks of risk and ambiguity. The results revealed that a preference for ambiguity could be measurably increased in individuals through anodal stimulation, but no significant differences were observed in the preferences for risky choices among groups. These findings suggest that different neural mechanisms underlie risky and ambiguous decisions because the right DLPFC primarily affects ambiguous behavior.

Realizing new designs of multiplexed electrode chips by 3-D printed masks.

Creating small and portable analytical methods is a fast-growing field of research. Devices capable of performing bio-analytical detection are especially desirable with the onset of the global pandemic. Lab-on-a-chip (LOC) technologies, including rapid point-of-care (POC) devices such as glucose sensors, are attractive for applications in resource-poor settings. There are many challenges in creating such devices, from sensitive molecular designs to stable conditions for storing the sensor chips. In this study we have explored using three-dimensional (3D) printing to create shadow masks as a low-cost method to produce multiplexed electrodes by physical vapour deposition. Although the dimensional resolution of the electrodes produced by using 3D printed masks is inferior to those made through photolithography-based techniques, their dimensions can be readily tailored ranging from 1 mm to 3 mm. Multiple mask materials were tested, such as polylactic acid and polyethylene terephthalate glycol, with acrylonitrile butadiene styrene shown to be the best. Simple strategies in making chip holders by 3D printing and controlling working electrode surface area with epoxy glue were also investigated. The prepared chips were tested by performing surface chemistry with thiol-containing molecules and monitoring the signals electrochemically.

N-Heterocyclic carbenes meet toll-like receptors.

Combining the stability of the N-heterocyclic carbenes (NHCs) and broad-spectrum recognition of toll-like receptor (TLR) proteins, we report new electrochemical biosensors for bacteria detection. Instead of traditional thiol-gold chemistry, newly synthesized NHCs are employed as the linker molecules to immobilize TLR bio-recognition elements on gold electrodes. Our proof-of-concept methodology includes testing the fidelity of TLR-based electrochemical sensors with NHC linkers. The performance of the biosensors is demonstrated using whole-cell bacterial cultures.

Causal Role of the Right Dorsolateral Prefrontal Cortex in Organizational Fairness Perception: Evidence From a Transcranial Direct Current Stimulation Study.

The right dorsolateral prefrontal cortex (rDLPFC) plays an essential role in social decision-making. Although several neural imaging studies have provided evidence that the rDLPFC is correlated with fairness perception, little research has investigated the causal effect of this encephalic region on individuals' consciousness, particularly perceptions of organizational fairness. The present study explores the causal relationship between the rDLPFC and organizational fairness perception by using brain modulation techniques. Healthy participants received transcranial direct current stimulation (tDCS) and fulfilled the modified ultimatum game (UG) in the sham-controlled experiment. Our results showed that only cathodal stimulation of the rDLPFC resulted in increasing rejection offers compared with the sham stimulation in conditions of disadvantageous inequity. No differences were found between the anodal and sham stimulation in any inequity condition. This study strengthens the main functional effects of the rDLPFC in negative emotional control in relation to organizational fairness perceptions.

N-Heterocyclic carbene and thiol micropatterns enable the selective deposition and transfer of copper films.

Microcontact printed patterns of N-heterocyclic carbenes (NHCs) and thiols were prepared on gold substrates and utilized as templates for the creation of metallic Cu structures using electroplating. The presence of the NHC in the pattern is essential to enable the transfer of the resulting copper microstructures to a second substrate.