Bifunctional Sensor Research Articles

DevS/DosS sensor is bifunctional and its phosphatase activity precludes aerobic DevR/DosR regulon expression in Mycobacterium tuberculosis.

Two-component systems, comprising histidine kinases and response regulators, empower bacteria to sense and adapt to diverse environmental stresses. Some histidine kinases are bifunctional; their phosphorylation (kinase) and dephosphorylation (phosphatase) activities toward their cognate response regulators permit the rapid reversal of genetic responses to an environmental stimulus. DevR-DevS/DosR-DosS is one of the best-characterized two-component systems of Mycobacterium tuberculosis. The kinase function of DevS is activated by gaseous stress signals, including hypoxia, resulting in the induction of ~ 48-genes DevR dormancy regulon. Regulon expression is tightly controlled and lack of expression in aerobic Mtb cultures is ascribed to the absence of phosphorylated DevR. Here we show that DevS is a bifunctional sensor and possesses a robust phosphatase activity toward DevR. We used site-specific mutagenesis to generate substitutions in conserved residues in the dimerization and histidine phosphotransfer domain of DevS and determined their role in kinase/phosphatase functions. In vitro and in vivo experiments, including a novel in vivo phosphatase assay, collectively establish that these conserved residues are critical for regulating kinase/phosphatase functions. Our findings establish DevS phosphatase function as an effective control mechanism to block aerobic expression of the DevR dormancy regulon. Asp-396 is essential for both kinase and phosphatase functions, whereas Gln-400 is critical for phosphatase function. The positive and negative functions perform opposing roles in DevS: the kinase function triggers regulon induction under hypoxia, whereas its phosphatase function prevents expression under aerobic conditions. A finely tuned balance in these opposing activities calibrates the dormancy regulon response output.

Wavelength-resolved simultaneous photoelectrochemical bifunctional sensor on single interface: A newly in vitro approach for multiplexed DNA monitoring in cancer cells

Currently, the photoelectrochemical (PEC) strategies can just achieve single analyte detection on a single interface with limited detection efficiency. It is highly valuable but full of challenge to develop a PEC biosensor for multiple analytes evaluation on a single interface. For this point, the wavelength-selective photoactive materials, which could generate separated photocurrents under excitation lights with certain wavelengths, were mainly important to overcome this challenge. Herein, these wavelength-selective photoactive materials were successfully synthesized and served as signal indicators to construct a novel PEC biosensor for multiple analytes evaluation on a single interface for the first time. Moreover, an enzyme-assisted target recycling amplification strategy was introduced for ultrasensitive monitoring. As a result, the proposed PEC biosensor showed excellent analytical performance for both oral cancer (ORVOA 1) gene and p53 gene down to attomolar level. In addition, the fabricated PEC biosensor was employed to evaluate ORVOA 1 gene and p53 gene in Hela cells. This assay has laid the foundation for fabrication of simple, ultrasensitive and economical PEC diagnostic devices to detect multiple analytes in cells, which paved a new avenue for early diagnosis of cancer with higher efficiency and accuracy.

Triphenylamine based lab-on-a-molecule for the highly selective and sensitive detection of Zn2+ and CN− in aqueous solution

A triphenylamine based lab-on-a-molecule TATP was designed and synthesized as a bifunctional sensor for the detection of Zn2+ and CN− through fluorescence and UV-Vis absorption channels, with detection limits of 14 nM and 0.37 μM, respectively.

An electrochemical bifunctional sensor for the detection of nitrite and hydrogen peroxide based on layer-by-layer multilayer films of cationic phthalocyanine cobalt(ii) and carbon nanotubes.

As vital biological mediators, the accurate detection of nitrite (NO2 -) and hydrogen peroxide (H2O2) is desirable for clinical monitoring and diagnosis. Herein, cationic 2,9,16,23-tetra[4-(N-methyl)pyridinyloxy]phthalocyanine cobalt(ii) ([TMPyPcCo]4+) and acid-treated multiwalled carbon nanotubes (aCNTs) were alternately self-assembled on the glassy carbon electrode (GCE) by means of the electrostatic interaction, leading to a 3D loose and interconnected assembly of [TMPyPcCo/aCNTs]n multilayer films. In the [TMPyPcCo/aCNTs]n films, [TMPyPcCo]4+ is anchored onto the surface of aCNTs without any inert polymer binders, which is beneficial to expose more active sites for electrocatalysis. The effective combination of [TMPyPcCo]4+ and aCNTs brings many advantages in electrochemical detection, involving the fast oriented transmission of charges, permeable channels for ion adsorption and transport, and more sensing sites, thus the [TMPyPcCo/aCNTs]n films display excellent electrochemical sensitivity towards both NO2 - and H2O2. The responses of NO2 - and H2O2 vary linearly with respect to the concentration from 5 µM to 30 mM and 10 µM to 9 mM. Furthermore, the superior cycling stability, reproducibility, and selectivity make [TMPyPcCo/aCNTs]n films suitable for the real samples.

Photochemical Green Synthesis of Nanostructured Cobalt Oxides as Hydrogen Peroxide Redox for Bifunctional Sensing Application

Photochemically synthesized cobalt oxide hydroxide (PCOH) under UV light has been successfully achieved without the addition of surfactants, templates, or organic solvents at ambient temperatures. PCOH samples can be converted to spinel cobalt oxide (PCO) after the calcination at 500°C. The in-situ growth of hierarchical nanostructures was demonstrated with a three-stage mechanism in the photochemical preparation. The development of nanostructures and elemental condensation under different irradiation time governs the bifunctional-sensing performance of the cobalt oxide products. The formation of nanostructured cobalt oxides involves a free-radical oxidation mechanism in the acidic condition. We further demonstrate that the preparation of sensor-active cobalt oxides under nature sunlight (Solar-2H) is feasible. Compared to the multi-component bifunctional electrocatalysts comprised of enzymes and noble metals, our greenly prepared electrocatalysts, as the single-species sensor, achieve bifunctional hydrogen peroxide detection effectively. The as-produced electrocatalysts utilize electro-oxidative and electro-reductive signals together as bifunctional sensor, maximizing the wide detection range of 0.005–35mM, a low detection limit of 0.7μM, selective H2O2 detection, and ∼3000-time higher sensitivity than commercial cobalt oxides.

Design of Multichannel Osmium-Based Metalloreceptor for Anions and Cations by Taking Profit from Metal-Ligand Interaction and Construction of Molecular Keypad Lock and Memory Device.

A polypyridylimidazole-based bifunctional Os(II) complex of the type [(bpy)2Os(tpy-Hbzim-dipy)](ClO4)2 (1), where tpy-Hbzim-dipy = 4'-[4-(4,5-dipyridin-2-yl-1H-imidazol-2-yl)-phenyl]-2,2';6',2″-terpyridine and bpy = 2,2'-bipyridine, has been synthesized and structurally characterized for the construction of multifunctional logic devices. After coordination of an [Os(bpy)2](2+) unit to one of the two bidentate chelating sites, the complex offers a terpyridine motif for binding with cationic guests and an imidazole moiety for interacting with selective anionic species. Consequently, the anion- and cation-binding aspects of the metallorecptor were examined in solution and in the solid state by different spectroscopic and electrochemical methods. The complex behaves as a bifunctional sensor for F(-), AcO(-), CN(-), Fe(2+), and Cu(2+) ions in acetonitrile, whereas it is a highly selective chromogenic chemosensor for only CN(-) and Fe(2+) ions in water. Based on various output signals with a particular set of anionic and cationic inputs, the complex mimics the functions of two-input INHIBIT, OR, NOR, and XNOR logic gates, as well as three-input NOR logic behavior. More importantly, the complicated functions of a keypad lock and memory device were also nicely demonstrated by the complex. Finally, density functional theory (DFT) and time-dependent density functional theory (TD-DFT) calculations also provide a rationale for properly understanding and interpreting the experimentally observed results.

A bifunctional sensor based on Au-Fe3O4 nanoparticle for the detection of Cd2+

A bifunctional sensor based on Au-Fe3O4 nanoparticles (NPs) modified by ethyl 1-(2-(3′,4′-dihydroxyphenyl)-2-oxoethyl)-1H-1,2,3-triazole-4-carboxylate (ETC) was developed. Due to the coordination interaction between cadmium (Cd2+) and ETC, Cd2+ induced the aggregation of Au-Fe3O4 NPs from dispersion, concomitant with a change in UV–vis spectra and transverse relaxation time (T2). The combined signals provided high selectively for Cd2+ ions.

Bifunctional sensing characteristics of chemical vapor deposition synthesized atomic-layered MoS2.

Two-dimensional (2D) molybdenum disulfide (MoS2) atomic layers have a strong potential to be adopted for 2D electronic components due to extraordinary and novel properties not available in their bulk foams. Unique properties of the MoS2, including quasi-2D crystallinity, ultrahigh surface-to-volume, and a high absorption coefficient, have enabled high-performance sensor applications. However, implementation of only a single-functional sensor presents a limitation for various advanced multifunctional sensor applications within a single device. Here, we demonstrate the charge-transfer-based sensitive (detection of 120 ppb of NO2) and selective gas-sensing capability of the chemical vapor deposition synthesized MoS2 and good photosensing characteristics, including moderate photoresponsivity (∼71 mA/W), reliable photoresponse, and rapid photoswitching (<500 ms). A bifunctional sensor within a single MoS2 device to detect photons and gas molecules in sequence is finally demonstrated, paving a way toward a versatile sensing platform for a futuristic multifunctional sensor.

A bifunctional “Turn On” fluorescent probe for trace level Hg2+ and EDTA in aqueous solution via chelator promoted cation induced deaggregation signalling

Because mercury and ethylenediaminetetreaacetic acid (EDTA) are widely used materials and are commonly found together in the environment due to anthropogenic and industrial releases, there is a great need to develop a bifunctional sensor for both mercury and EDTA. A fluorescent sensor SQ-1 based on a squaraine chromophore with dithiocarbamate (DTC) side arms has been prepared, and its aggregation behavior in aqueous solution has also been investigated. The results show that increasing water concentration in the solution causes dramatic H-aggregation of the symmetric SQ-1 and consequently provides a mercury probe with a visual color change and “turn on” fluorescence via cation induced deaggregation. EDTA, a strong chelator to mercury, accelerates the deaggregation process and enhances the fluorescent emission, which not only improves the sensitivity of SQ-1 to mercury by 10-fold, with a detection limit improvement from 1.1×10−7M to 1.3×10−8M, but also provides a way for trace level detection of EDTA, with the detection limit of 8.7×10−8M. The potential application of this technique for Hg2+ and EDTA determination of river water samples is revealed by proof-of-concept experiments.

Reciprocal regulation as a source of ultrasensitivity in two-component systems with a bifunctional sensor kinase.

Two-component signal transduction systems, where the phosphorylation state of a regulator protein is modulated by a sensor kinase, are common in bacteria and other microbes. In many of these systems, the sensor kinase is bifunctional catalyzing both, the phosphorylation and the dephosphorylation of the regulator protein in response to input signals. Previous studies have shown that systems with a bifunctional enzyme can adjust the phosphorylation level of the regulator protein independently of the total protein concentrations – a property known as concentration robustness. Here, I argue that two-component systems with a bifunctional enzyme may also exhibit ultrasensitivity if the input signal reciprocally affects multiple activities of the sensor kinase. To this end, I consider the case where an allosteric effector inhibits autophosphorylation and, concomitantly, activates the enzyme's phosphatase activity, as observed experimentally in the PhoQ/PhoP and NRII/NRI systems. A theoretical analysis reveals two operating regimes under steady state conditions depending on the effector affinity: If the affinity is low the system produces a graded response with respect to input signals and exhibits stimulus-dependent concentration robustness – consistent with previous experiments. In contrast, a high-affinity effector may generate ultrasensitivity by a similar mechanism as phosphorylation-dephosphorylation cycles with distinct converter enzymes. The occurrence of ultrasensitivity requires saturation of the sensor kinase's phosphatase activity, but is restricted to low effector concentrations, which suggests that this mode of operation might be employed for the detection and amplification of low abundant input signals. Interestingly, the same mechanism also applies to covalent modification cycles with a bifunctional converter enzyme, which suggests that reciprocal regulation, as a mechanism to generate ultrasensitivity, is not restricted to two-component systems, but may apply more generally to bifunctional enzyme systems.

Bifunctional polymeric membrane ion selective electrodes using phenylboronic acid as a precursor of anionic sites and fluoride as an effector: A potentiometric sensor for sodium ion and an impedimetric sensor for fluoride ion

A new concept in ion sensing mimicking allosteric membranes in biological systems was proposed and demonstrated using fluoride-effector Na+ selective electrodes (Na-ISEs). DOS-plasticized PVC membranes containing p-tert-butylcalix[4]arene tetraethyl ester (NaX) and 4-[(4-tert-butyl-2-methylphenoxy)methyl]phenyl boronic acid (50mol% compared to NaX) were found to give the best characteristic of Na-ISEs upon conditioning the membranes in 10−2M NaF: a response slope of 57.48mV per decade and detection limit of 7.42×10−7M. The selectivity coefficients (log KNa,jpot) for the optimal electrode were −2.29, −2.84, −3.16, −3.42, and −3.01 for K+, Li+, Cs+, Mg2+ and Ca2+, respectively. The optimal electrodes also gave good reversibility and could be used in a wide range of pH (pH 3.6–9.7). The fabricated Na-ISEs were found to have membrane resistances (Rm) varied with concentrations of fluoride ions used in membrane conditioning steps. Electrochemical impedance spectra showed a linear relationship between Rm and the logarithm of the concentration range of fluoride ions from 1×10−1 to 1×10−8M. Conditioning optimal membranes with various concentrations of Bu4NF gave a non-linear relationship. The proposed membrane electrode, thus, showed permselectivity toward Na+ using F− as an effector. Therefore, the proposed new concept led to the fabrication of a bifunctional sensor: a potentiometric sensor for sodium ion and an impedimetric sensor for fluoride ion.

Bifunctional sensor of pentachlorophenol and copper ions based on nanostructured hybrid films of humic acid and exfoliated layered double hydroxide via a facile layer-by-layer assembly

A new, highly sensitive bifunctional electrochemical sensor for the simultaneous determination of pentachlorophenol (PCP) and copper ions (Cu2+) has been developed, where organic–inorganic hybrid ultrathin films were fabricated by alternate assembly of humic acid (HA) and exfoliated Mg–Al-layered double hydroxide (LDH) nanosheets onto ITO substrates via a layer-by-layer (LBL) approach. The multilayer films were then characterized by means of UV–vis spectrometry, scanning electron microscopy (SEM), and atomic force microscope (AFM). These films were found to have a relatively smooth surface with almost equal amounts of HA incorporated in each cycle. Its electrochemical performance was systematically investigated. Our results demonstrate that such a newly designed (LDH/HA)n multilayer films, combining the individual properties of HA (dual recognition ability for organic herbicides and metal ions) together with LDH nanosheets (a rigid inorganic matrix), can be applied to the simultaneous analysis of PCP and Cu(II) without interference from each other. The LBL assembled nanoarchitectures were further investigated by X-ray photoelectron spectroscopy (XPS) and infrared spectroscopy (IR), which provides insight for bifunctional sensing behavior. Under the optimized conditions, the detection limit was found to be as low as 0.4nM PCP, well below the guideline value of PCP in drinking water (3.7nM) set by the United States Environmental Protection Agency (U.S. EPA), and 2.0nM Cu2+, much below the guideline value (2.0mgL−1, ~31.2nM) from the World Health Organization (WHO), respectively. Toward the goal for practical applications, this simple and cost-effective probe was further evaluated by monitoring PCP and Cu(II) in water samples.

Electrochemical determination of nicotinamide adenine dinucleotide and hydrogen peroxide based on poly(xanthurenic acid), flavin adenine dinucleotide and functionalized multi-walled carbon nanotubes

A bifunctional sensor for determination of reduced β-nicotinamide adenine dinucleotide (NADH) and hydrogen peroxide (H2O2) has been successfully fabricated by xanthurenic acid (Xa), flavin adenine dinucleotide (FAD), and functionalized carbon nanotubes (CNTs). The hybrid composites can be easily prepared by the electrocodeposition of polyXa/FAD and the adsorption of functionalized CNTs. These composites can be constructed of “polyXa/FAD/CNTs” and “CNTs/polyXa/FAD” according to the immobilization order of polyXa/FAD “before” and “after” the adsorption of CNTs. They are surface-confined and stable in various scan rates and different pH conditions. Particularly, the polyXa/FAD/MWCNTs show more efficient activity to target species due to the compact structure and more active surface area. It provides linear concentration range of 5×10−6–1.7×10−4M and 1×10−4–2.9×10−3M, with sensitivity of 155μAmM−1cm−2 and 60μAmM−1cm−2, and detection limit of 10−6M and 10−4M (S/N=3) for NADH and H2O2, respectively. No interference except of the case for NADH determination in the presence of AA. It can be a good electrocatalyst to determine NADH and H2O2 and the linear sweep voltammetry (LSV) technique can help to analyze NADH from the AA interference.

α-Fe2O3 nanorod arrays for bioanalytical applications: nitrite and hydrogen peroxide detection

A novel biosensor was fabricated by the anodic electrochemical deposition of α-Fe2O3 nanorod (NR) arrays onto an iron substrate in an aqueous (NH4)2Fe(SO4)2 solution. The NR array electrode exhibits superior electroactivity for the oxidation of nitrite and the reduction of hydrogen peroxide. A wide linear response range (2 × 10−7–5 × 10−3 M), high sensitivity (135.36 μA mM−1 cm−2) and low detection limit (1 × 10−7 M) were obtained for the oxidation of nitrite. This α-Fe2O3 NR array electrode was further utilized in the detection of H2O2 with a detection limit of 2 × 10−7 M, linearity of 5 × 10−7–3 × 10−3 M and a sensitivity of 77.30 μA mM−1 cm−2. The stability and selectivity of the biosensor were also evaluated. The results indicate that the α-Fe2O3 NR arrays are efficient bifunctional sensors, with potential applications in biological and medical systems.

Bifunctional potentiometric sensor based on MoO3 nanorods

A solid-phase film electrode with an ion-sensitive membrane based on MoO3 nanorods was developed. Depending on the pH of test solution, the proposed electrode can be used as a sensor for measuring the concentration of hydrogen or alkali metal ions. In the range of 1 ≤ pH ≤ 5, the electrode reacts to changes in the concentration of hydrogen ions with an electrode response slope of 54 ± 2 mV/pH. The electrode is selec- tive to alkali metal cations in the concentration ranges of \(0 \leqslant pc_{M^ + } \leqslant 4\) for M = Na, K, and Li and \(1 \leqslant pc_{M^ + } \leqslant 5\) for M = Rb and Cs and the solution acidity 5 ≤ pH ≤ 13.5 with a nearly theoretical slope. The selectivity coefficients of the electrode to the ions of Na(I), Rb(I), Cs(I), Mg(II), Ca(II), Sr(II), and Ba(II) were determined.

Design and characterization of a novel sensor combined imaging and zinc ion sensing

A novel fluorescent sensor for Zn(II) detection and imaging is developed by incorporating 1-(2-pyridylazo)-2-naphthol (PAN) and Rhodamine B (RB) in a sol–gel film on an end face of a gradient index (GRIN) lens rod which is tightly connected with an imaging fiber. This technique has high-quality imaging capabilities for observing remote sample while measuring Zn(II). The RB and PAN employed in combination as the indicator for zinc-sensing is much cheaper than that of the other zinc-sensitive fluorescent dyes. The sol is prepared by base catalyzed hydrolysis of tetraethylorthosilicate (TEOS) with the addition of 3-glycidyloxypropyltrimethoxysilane (GLYMO) and 3-aminopropyltriethoxysilane (APTES), which is more suitable for Zn(II) detection. The sensor has the linear range of Zn(II) from 0.1 to 1mmol/L. Simultaneously, it has the capabilities of real-time imaging with the whole system’s resolution of 18.73 line pairs/mm. This bi-functional sensor has potential applications in in situ biosensing and other remote measurements.

Negative control in two-component signal transduction by transmitter phosphatase activity

Bifunctional sensor transmitter modules of two-component systems exert both positive and negative control on the receiver domain of the cognate response regulator. In negative control, the transmitter module accelerates the rate of phospho-receiver dephosphorylation. This transmitter phosphatase reaction serves the important physiological functions of resetting response regulator phosphorylation level and suppressing cross-talk. Although the biochemical reactions underlying positive control are reasonably well understood, the mechanism for transmitter phosphatase activity has been unknown. A recent hypothesis is that the transmitter phosphatase reaction is catalysed by a conserved Gln, Asn or Thr residue, via a hydrogen bond between the amide or hydroxyl group and the nucleophilic water molecule in acyl-phosphate hydrolysis. This hypothetical mechanism closely resembles the established mechanisms of auxiliary phosphatases such as CheZ and CheX, and may be widely conserved in two-component signal transduction. In addition to the proposed catalytic residues, transmitter phosphatase activity also requires the correct transmitter conformation and appropriate interactions with the receiver. Evidence suggests that the phosphatase-competent and autokinase-competent states are mutually exclusive, and the corresponding negative and positive activities are likely to be reciprocally regulated through dynamic control of transmitter conformations.

The cell envelope stress response mediated by the LiaFSR Lm three-component system of Listeria monocytogenes is controlled via the phosphatase activity of the bifunctional histidine kinase LiaS Lm

Most members of the phylum Firmicutes harbour a two-component system (TCS), LiaSR, which is involved in the response to cell envelope stress elicited most notably by inhibitors of the lipid II cycle. In all LiaSR systems studied in detail, LiaSR-mediated signal transduction has been shown to be negatively controlled by a membrane protein, LiaF, encoded upstream of liaSR. In this study we have analysed the LiaSR orthologue of Listeria monocytogenes (LiaSR(Lm)). Whole-genome transcriptional profiling indicated that activation of LiaSR(Lm) results in a remodelling of the cell envelope via the massive upregulation of membrane-associated and extracytoplasmic proteins in the presence of inducing stimuli. As shown for other LiaSR TCSs, LiaSR(Lm) is activated by cell wall-active antibiotics. We demonstrate that the level of phosphorylated LiaR(Lm), which is required for the induction of the LiaSR(Lm) regulon, is controlled by the interplay between the histidine kinase and phosphatase activities of the bifunctional sensor protein LiaS(Lm). Our data suggest that the phosphatase activity of LiaS(Lm) is stimulated by LiaF(Lm) in the absence of cell envelope stress.

A Multilayer Film for Bifunctional Electrocatalyst and Electrochemical Sensor Based on Polyoxometalate and Tris(1,10-Phenanthroline)Ruthenium

The multilayer films containing tris(1,10-phenanthroline) ruthenium(II) Ru(phen)3Cl2 (abbr. Ru(phen)3, phen = 1,10-phenanthroline) and polyoxometalate [P2W18O62]6− (abbr. P2W18) through a layer-by-layer (LBL) process were fabricated. UV–vis absorption spectra showed that the subsequent growth of the multilayer film was regular and highly reproducible from layer to layer. X-ray photoelectron spectra (XPS) confirmed the incorporation of Ru(phen)3 and P2W18 into the film. The result of atomic force microscopy (AFM) revealed a relatively uniform surface morphology of the multilayer film. The multilayer film exhibited remarkable electrocatalytic activities toward the reduction of $$ {\text{IO}}_{3}^{ - } $$ and the oxidation to l-cysteine. The value of I pc was as a function with the concentration of iodate and l-cysteine, which indicated that the film can be a candidate for bifunctional electrocatalyst and electrochemical sensor.

Identification of structural and functional determinants of Ca2+‐dependent kinetics of voltage‐gated calcium channel regulation by CaM

Calmodulin (CaM), a widely expressed and highly conserved Ca2+‐sensing regulatory protein with at least 300 known targets, has been recently implicated in the Ca2+‐dependent inactivation and facilitation of voltage‐gated calcium channels. Due to its bi‐lobed structure, CaM is able to serve as a bi‐functional sensor and regulator of channel activity. The particular contribution of each of the two lobes and the associated forms of regulation are not universally understood or generalized for each channel type, and even less so are the particular channel regions responsible for the association and regulation by CaM. In this study we examined unique regions outside the canonical channel CaM‐binding domains for their ability to dictate the electrophysiological profile of any channel type. Channel chimaeras were constructed and patch‐clamp recordings were performed to determine the Ca2+‐dependent kinetics profiles. Furthermore, the peptides corresponding to putative CaM‐binding regions of the channel were synthesized and the binding properties were analyzed. The implications for the channel regulatory model and some unifying principles are discussed. This work received NSERC funding.