Ratiometric Fluorescence Probe for Monitoring Hydroxyl Radical in Live Cells Based on Gold Nanoclusters

Determination of hydroxyl radical (^•OH) with high sensitivity and accuracy in live cells is a challenge for evaluating the role that ^•OH plays in the physiological and pathological processes. In this work, a ratiometric fluorescence biosensor for ^•OH was developed, in which gold nanocluster (AuNC) protected by bovine serum albumin was employed as a reference fluorophore and the organic molecule 2-[6-(4′-hydroxy)­phenoxy-3<i>H</i>-xanthen-3-on-9-yl]­benzoic acid (HPF) acted as both the response signal and specific recognition element for ^•OH. In the absence of ^•OH, only one emission peak at 637 nm ascribed to AuNCs was observed, because HPF was almost nonfluorescent. However, fluorescence emission at 515 nm attributed to the HPF product after reaction with ^•OHdianionic fluoresceingradually increased with the continuous addition of ^•OH, while the emission at 637 nm stays constant, resulting in a ratiometric determination of ^•OH. The developed fluorescent sensor exhibited high selectivity for ^•OH over other reactive oxygen species (ROS), reactive nitrogen species (RNS), metal ions, and other biological species, as well as high accuracy and sensitivity with low detection limit to ∼0.68 μM, which fulfills the requirements for detection of ^•OH in a biological system. In addition, the AuNC-based inorganic–organic probe showed long-term stability against light illumination and pH, good cell permeability, and low cytotoxicity. As a result, the present ratiometric sensor was successfully used for bioimaging and monitoring of ^•OH changes in live cells upon oxidative stress

Wettability Switching of Electrode for Signal Amplification: Conversion of Conformational Change of Stimuli-Responsive Polymer into Enhanced Electrochemical Chiral Analysis

Signal amplification of chiral interaction is a much needed task for sensing of enantiomers due to nearly identical chemical and physical properties of the chiral isomers. In this article, we established an electrochemical chiral sensing method with high sensitivity and selectivity for monosacharrides based on the stimuli-responsive copolymer/graphene hybrid-modified screen-printed carbon electrodes. The hybrid synthesized by the “grafting from” atom transfer radical polymerization (ATRP) process not only acted as a chiral recognition element but also provided a chiral signal amplification strategy. This occurs due to high sensitivity of conformational transition of copolymer on graphene to the weak chiral interactions that greatly facilitating the diffusion of electroactive probes and monosaccharides to the electrode surface. The described method can quantify monosaccharides, even the concentration of one enantiomer is as low as 1 nM. Apart from the demonstrated chiral distinguish ability, good selectivity toward monosaccharides in comparison to potential interference molecules was also observed. The electrodes with significant analytical performance were successfully applied for discriminating glucose enantiomers in live cells and studying their different transport mechanism. Together, the results show that the coupling of amplification-by-wettability switching concept with electrochemical method offers great promises in providing a sensitive, facile, and cost-effective solution for chiral recognition of molecules in biological process

Carbon-Dot-Based Ratiometric Fluorescent Probe for Imaging and Biosensing of Superoxide Anion in Live Cells

In this article, a ratiometric fluorescent biosensor for O₂^•– was developed, by employing carbon dots (C-Dots) as the reference fluorophore and hydroethidine (HE), a specific organic molecule toward O₂^•–, playing the role as both specific recognition element and response signal. The hybrid fluorescent probe CD-HE only emitted at 525 nm is ascribed to C-Dots, while HE was almost nonfluorescent, upon excitation at 488 nm. However, after reaction with O₂^•–, a new emission peak ascribed to the reaction products of HE and O₂^•– was clearly observed at 610 nm. Meanwhile, this peak gradually increased with the increasing concentration of O₂^•– but the emission peak at 525 nm stayed constant, leading to a ratiometric detection of O₂^•–. The inorganic–organic fluorescent sensor exhibited high sensitivity, a broad dynamic linear range of ∼5 × 10^–7–1.4 × 10^–4 M, and low detection limit down to 100 nM. The present probe also showed high accuracy and excellent selectivity for O₂^•– over other reactive oxygen species (ROS), metal ions, and so on. Moreover, the C-Dot-based inorganic–organic probe demonstrated long-term stability against pH changes and continuous light illumination, good cell-permeability, and low cytotoxicity. Accordingly, the developed fluorescent biosensor was eventually applied for intracellular bioimaging and biosensing of O₂^•– changes upon oxidative stress

Development of Au Disk Nanoelectrode Down to 3 nm in Radius for Detection of Dopamine Release from a Single Cell

A Au disk nanoelectrode down to 3 nm in radius was developed by a facile and reliable method and successfully applied for monitoring dopamine release from single living vesicles. A fine etched Au wire was coated with cathodic electrophoretic paint followed by polyimide, which retracted from the tip end during curing to expose the Au nanotip. By cyclic voltammetric scanning the above tip in 0.5 M KCl, the transformation of a core-shaped apex into a geometrically well-defined Au disk nanoelectrode with different dimensions can be controllably and reproducibly achieved. Scanning electron microscopy, transmission electron microscopy, and steady-state voltammetry were used to determine the size of nanoelectrodes. The results showed that the specific etching and insulation method not only avoids the use of toxic etching solution and the uncontrollable treatment to expose the tip but also makes possible the controllable and reproducible fabrication of Au disk nanoelectrode down to 3 nm in radius. The nanoelectrodes with well-demonstrated analytical performance were further applied for amperometrically monitoring dopamine release from single rat pheochromacytoma cells with high spatial resolution

Single Biosensor for Simultaneous Quantification of Glucose and pH in a Rat Brain of Diabetic Model Using Both Current and Potential Outputs

Glucose and pH are two important indicators of diabetes mellitus. However, their dynamic changes at the same time in brain are still not clear, mainly due to a lack of a single biosensor capable of simultaneous quantification of two species in a live rat brain. In this work, a selective and sensitive ratiometric electrochemical biosensor was developed for simultaneously quantifying glucose and pH using both current and potential outputs in a rat brain of diabetic model. Here, glucose oxidase was first employed as a specific recognition element for both glucose and pH because the active center (FAD) could undergo a 2H⁺/2e^– process. Moreover, an insensitive molecule toward pH and glucose was used as an inner-reference element to provide a built-in correction to improve the accuracy. The ratio between the oxidation peak current density of glucose and that of ABTS gradually increased with increasing concentration of glucose, and showed a good linearity in the range of 0.3–8.2 mM. Meanwhile, the midpotential difference between glucose oxidase and 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) positively shifted with pH decreasing, leading to accurate determination of pH in the linear range of 5.67–7.65. Thus, combined with the unique properties of carbon fiber microelectrode, including easy to insert and good biocompatibility, the developed single biosensor was successfully applied to detect pH and glucose at the same time in hippocampus, striatum, and cortex in a live rat brain of diabetic model

Biomimetic Mineralization of Gold Nanoclusters as Multifunctional Thin Films for Glass Nanopore Modification, Characterization, and Sensing

Hurdles of nanopore modification and characterization restrain the development of glass capillary-based nanopore sensing platforms. In this article, a simple but effective biomimetic mineralization method was developed to decorate glass nanopore with a thin film of bovine serum albumin-protected Au nanocluster (BSA-Au NC). The BSA-Au NC film emitted a strong red fluorescence whereby nondestructive characterization of Au film decorated at the inner surface of glass nanopore can be facilely achieved by a fluorescence microscopy. Besides, the BSA molecules played dual roles in the fabrication of functionalized Au thin film in glass nanopore: they not only directed the synthesis of fluorescent Au thin film but also provided binding sites for recognition, thus achieving synthesis-modification integration. This occurred due to the ionized carboxyl groups (-COO^–) of a BSA coating layer on Au NCs which can interacted with arginine (Arg) via guanidinium groups. The added Arg selectively led to the change in the charge and ionic current of BSA-Au NC film-decorated glass nanopore. Such ionic current responses can be used for quantifying Arg with a detection limit down to 1 fM, which was more sensitive than that of previous sensing systems. Together, the designed method exhibited great promise in providing a facile and controllable solution for glass nanopore modification, characterization, and sensing

Rational Design of a Stimuli-Responsive Polymer Electrode Interface Coupled with in Vivo Microdialysis for Measurement of Sialic Acid in Live Mouse Brain in Alzheimer’s Disease

Sensitive and selective monitoring of sialic acid (SA) in cerebral nervous system is of great importance for studying the role that SA plays in the pathological process of Alzheimer’s disease (AD). In this work, we first reported an electrochemical biosensor based on a novel stimuli-responsive copolymer for selective and sensitive detection of SA in mouse brain. Notably, through synergetic hydrogen-bonding interactions, the copolymer could translate the recognition of SA into their conformational transition and wettability switch, which facilitated the access and enrichment of redox labels and targets to the electrode surface, thus significantly improving the detection sensitivity with the detection limit down to 0.4 pM. Besides amplified sensing signals, the proposed method exhibited good selectivity toward SA in comparison to potential interference molecules coexisting in the complex brain system due to the combination of high affinity between phenylboronic acid (PBA) and SA and the directional hydrogen-bonding interactions in the copolymer. The electrochemical biosensor with remarkable analytical performance was successfully applied to evaluate the dynamic change of SA level in live mouse brain with AD combined with in vivo midrodialysis. The accurate concentration of SA in different brain regions of live mouse with AD has been reported for the first time, which is beneficial for progressing our understanding of the role that SA plays in physiological and pathological events in the brain

Two-Photon Ratiometric Fluorescent Sensor Based on Specific Biomolecular Recognition for Selective and Sensitive Detection of Copper Ions in Live Cells

In this work, we develop a ratiometric two-photon fluorescent probe, ATD@QD-E₂Zn₂SOD (ATD = amino triphenylamine dendron, QD = CdSe/ZnSe quantum dot, E₂Zn₂SOD = Cu-free derivative of bovine liver copper–zinc superoxide dismutase), for imaging and sensing the changes of intracellular Cu²⁺ level with clear red-to-yellow color change based on specific biomolecular recognition of E₂Zn₂SOD for Cu²⁺ ion. The inorganic–organic nanohybrided fluorescent probe features two independent emission peaks located at 515 nm for ATD and 650 nm for QDs, respectively, under two-photon excitation at 800 nm. Upon addition of Cu²⁺ ions, the red fluorescence of QDs drastically quenches, while the green emission from ATD stays constant and serves as a reference signal, thus resulting in the ratiometric detection of Cu²⁺ with high accuracy by two-photon microscopy (TPM). The present probe shows high sensivity, broad linear range (10^–7–10^–3 M), low detection limit down to ∼10 nM, and excellent selectivity over other metal ions, amino acids, and other biological species. Meanwhile, a QD-based inorganic-organic probe demonstrates long-term photostability, good cell-permeability, and low cytotoxicity. As a result, the present probe can visualize Cu²⁺ changes in live cells by TPM. To the best of our knowledge, this is the first report for the development of a QD-based two-photon ratiometric fluorescence probe suitable for detection of Cu²⁺ in live cells

Hierarchically Porous CuO Hollow Spheres Fabricated via a One-Pot Template-Free Method for High-Performance Gas Sensors

CuO hollow spheres with hierarchical pores, that is, quasi-micropores (1.0–2.2 nm), mesopores (5–30 nm), and macropores (hollow cores, 2–4 μm), have been synthesized via a simple one-pot template-free method. The CuO hollow spheres also show a hierarchical architecture, namely, the primary CuO nanograins, the quasi-single-crystal nanosheets assembled by nanograins, and the spheres composed of the nanosheets. A mechanism involving an “oriented attachment” growth step followed by an “Ostwald ripening” process has been proposed for the hierarchical structure and pore formation of the typical CuO hollow spheres. With such unique hierarchical pores and architecture, the CuO hollow spheres display excellent sensing performance toward H₂S as gas sensing material, such as low detection limit of 2 ppb, high sensitivity at parts per billion level concentration, broad linear range, short response time of 3 s, and recovery time of 9 s. The excellent performance is ascribed to a synergetic effect of the hierarchical structure of the unique CuO spheres: the quasi-micropores offer active sites for effectively sensing, the mesopores facilitate the molecular diffusion kinetics, and the macropores serve as gas reservoirs and minimize diffusion length, while good conductivity of the quasi-single-crystal nanosheets favors fast charge transportation, which contribute to the high sensitivity, quick response, and recovery of the H₂S sensor, respectively