Electrochemiluminescence Microscopy.

Electrochemiluminescence (ECL) is rapidly evolving from an analytical method into an optical microscopy. The orthogonality of the electrochemical trigger and the optical readout distinguishes it from classic microscopy and electrochemical techniques, owing to its near-zero background, remarkable sensitivity, and absence of photobleaching and phototoxicity. In this minireview, we summarize the recent advances in ECL imaging technology, emphasizing original configurations which enable the imaging of biological entities and the improvement of the analytical properties by increasing the complexity and multiplexing of bioassays. Additionally, mapping the (electro)chemical reactivity in space provides valuable information on nanomaterials and facilitates deciphering ECL mechanisms for improving their performances in diagnostics and (electro)catalysis. Finally, we highlight the recent achievements in imaging at the ultimate limits of single molecules, single photons or single chemical reactions, and the current challenges to translate the ECL imaging advances to other fields such as material science, catalysis and biology.

Bimodal Electrochemiluminescence Microscopy of Single Cells.

Electrochemiluminescence (ECL) microscopy is an emerging technique with new applications such as imaging of single entities and cells. Herein, we have developed a bimodal and bicolor approach to record both positive ECL (PECL: light-emitting object on dark background) and shadow label-free ECL (SECL: nonemissive object shadowing the background luminescence) images of single cells. This bimodal approach is the result of the simultaneous emissions of [Ru(bpy)3]2+ used to label the cellular membrane (PECL) and [Ir(sppy)3]3- dissolved in solution (SECL). By spectrally resolving the ECL emission wavelengths, we recorded the images of the same cells in both PECL and SECL modes using the [Ru(bpy)3]2+ (λmax = 620 nm) and [Ir(sppy)3]3- (λmax = 515 nm) luminescence, respectively. PECL shows the distribution of the [Ru(bpy)3]2+ labels attached to the cellular membrane, whereas SECL reflects the local diffusional hindrance of the ECL reagents by each cell. The high sensitivity and surface-confined features of the reported approach are demonstrated by imaging cell-cell contacts during the mitosis process. Furthermore, the comparison of PECL and SECL images demonstrates the differential diffusion of tri-n-propylamine and [Ir(sppy)3]3- through the permeabilized cell membranes. Consequently, this dual approach enables the imaging of the morphology of the cell adhering on the surface and can significantly contribute to multimodal ECL imaging and bioassays with different luminescent systems.

FERTILITY PRESERVATION IN YOUNG WOMEN WITH EARLY-STAGE BREAST CANCER.

Although breast cancer (BC) occurs more often in older women, it is the most commonly diagnosed malignancy in women of childbearing age. Owing to the overall advancement of modern medicine and the growing global trend of delaying childbirth until later age, we find ever more younger women diagnosed and treated for BC who have not yet completed their family. Therefore, fertility preservation has emerged as a very important quality of life issue for young BC survivors. This paper reviews currently available options for fertility preservation in young women with early-stage BC and highlights the importance of a multidisciplinary approach to fertility preservation as a very important quality of life issue for young BC survivors. Pregnancy after BC treatment is considered not to be associated with an increased risk of BC recurrence; therefore, it should not be discouraged for those women who want to achieve pregnancy after oncologic treatment. Currently, it is recommended to delay pregnancy for at least 2 years after BC diagnosis, when the risk of recurrence is highest. However, BC patients of reproductive age should be informed about the potential negative effects of oncologic therapy on fertility, as well as on the fertility preservation options available, and if interested in fertility preservation, they should be promptly referred to a reproductive specialist. Early referral to a reproductive specialist is an important factor that increases the likelihood of successful fertility preservation. Embryo and mature oocyte cryopreservation are currently the only established fertility preservation methods but they require ovarian stimulation (OS), which delays initiation of chemotherapy for at least 2 weeks. Controlled OS does not seem to increase the risk of BC recurrence. Other fertility preservation methods (ovarian tissue cryopreservation, cryopreservation of immature oocytes and ovarian suppression with gonadotropin-releasing hormone agonists) do not require OS but are still considered to be experimental techniques for fertility preservation.

Bacterial Pathogenesis: Assessment of Intracellular Positioning of Pathogen-Containing Vacuoles During Infection.

Intracellular bacterial pathogens implement a diverse array of strategies to target host cells and establish infection. For vacuolar pathogens, the process of pathogen-containing vacuole movement within host cells, termed intracellular trafficking, is central to both pathogen survival and infection progression. Typically a process mediated by secreted virulence factors that manipulate the host cytoskeletal machinery, internalized pathogen-containing vacuoles traffic to the site of replication to establish a unique replicative niche, and if applicable, traffic back toward the host cell periphery for cell-to-cell spread. As such, the intracellular positioning of pathogen-containing vacuoles represents a fundamental measure of infection progression. Here, we describe a fluorescence microscopy-based method to quantitatively assess bacterial intracellular positioning, using Salmonella enterica serovar Typhimurium infection of epithelial cells as a model. This experimental approach can be modified to study infection in diverse host cell types, and with a broad array of pathogens. The system can also be adapted to examine the kinetics of infection, identify secreted virulence factors that mediate host trafficking, investigate host factors that are targeted by the pathogen for trafficking, and assess functional domains within a virulence factor responsible for mediating the phenotype. Collectively, these tools can provide fundamental insight into the pathogenesis of a diverse array of intracellular bacterial pathogens, and new host factors that are hijacked to mediate infection. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culture and preparation of host cells Alternate Protocol: Culture and preparation of host cells to assess host factor contribution to bacterial positioning Basic Protocol 2: Infection of epithelial cells with S. Typhimurium Basic Protocol 3: Fluorescence staining for analysis of bacterial positioning Basic Protocol 4: Fluorescence microscopy analysis of bacterial positioning.

Redox-mediated electrochemiluminescence enhancement for bead-based immunoassay.

Electrochemiluminescence (ECL) is a highly sensitive mode of detection utilised in commercialised bead-based immunoassays. Recently, the introduction of a freely diffusing water-soluble Ir(iii) complex was demonstrated to enhance the ECL emission of [Ru(bpy)3]2+ labels anchored to microbeads, but a comprehensive investigation of the proposed 'redox-mediated' mechanism was not carried out. In this work, we select three different water-soluble Ir(iii) complexes by virtue of their photophysical and electrochemical properties in comparison with those of the [Ru(bpy)3]2+ luminophore and the TPrA co-reactant. A systematic investigation of the influence of each Ir(iii) complex on the emission of the Ru(ii) labels on single beads by ECL microscopy revealed that the heterogeneous ECL can be finely tuned and either enhanced up to 107% or lowered by 75%. The variation of the [Ru(bpy)3]2+ ECL emission was correlated to the properties of each Ir(iii)-based mediator, which enabled us to decipher the mechanism of interaction and define guidelines for the future design of novel Ir(iii) complexes to further enhance the ECL emission of bead-based immunoassays. Ultimately, we showcase the potential of this technology for practical sample analysis in commercial instruments by assessing the enhancement of the collective ECL intensity from a bead-based system.

The environmentally friendly approaches based on the heterojunction interface of the LaFeO3/Fe2O3@g-C3N4 composite for the disposable and laboratory sensing of triclosan.

Triclosan (TCS) is a polychlorinated phenoxy phenol (PCPPs) used as a disinfectant and a broad-spectrum antibacterial and antifungal agent in personal hygiene products. TCS easily forms diphenyl ethers and dioxins, which are persistent organic pollutants. This work used a double approach for the TSC sensing: a) screen-printed (SPE) electrochemical platform for on-site application, modified with lanthanum iron oxide and graphitic carbon nitride composite (LaFeO3/Fe2O3@g-C3N4/SPE); and b) carbon paste electrode (CPE), modified with the same material and used in laboratory conditions. Linear range from 0.1 µM to 10 µM, the limit of detection (LOD) of 29 nM and the limit of quantification (LOQ) of 91 nM were obtained for CP electrode in BRBS pH 8. SPE showed the best analytical parameters in BRBS at pH 3, with a linear range from 0.3 µM to 7 µM, LOD of 0.09 µM and LOQ of 0.28 µM. Furthermore, the influence of potential interferents was investigated and proven to be negligible. Determination of TSC was performed to estimate the environmental impact of this compound as well as the practical usefulness of the proposed sensor in the real sample analysis, confirmed with a HPLC analysis.

Direct glyphosate soil monitoring at the triazine-based covalent organic framework with the theoretical study of sensing principle.

Covalent organic frameworks (COFs) are emerging as promising sensing materials due to their controllable structure and function properties, as well as excellent physicochemical characteristics. Here, specific interactions between a triazine-based COF and a mass-used herbicide - glyphosate (GLY) have been utilized to design a disposable sensing platform for GLY detection. This herbicide has been extensively used for decades, however, its harmful environmental impact and toxicity to humans have been recently proven, conditioning the necessity for the strict control and monitoring of its use and its presence in soil, water, and food. Glyphosate is an organophosphorus compound, and its detection in complex matrices usually requires laborious pretreatment. Here, we developed a direct, miniaturized, robust, and green approach for disposable electrochemical sensing of glyphosate, utilizing COF's ability to selectively capture and concentrate negatively charged glyphosate molecules inside its nanopores. This process generates the concentration gradient of GLY, accelerating its diffusion towards the electrode surface. Simultaneously, specific COF-glyphosate binding catalyses the oxidative cleavage of the C-P bond and, together with pore nanoconfinement, enables sensitive glyphosate detection. Detailed sensing principles and selectiveness were scrutinized using DFT-based modelling. The proposed electrochemical method has a linear working range from 0.1 µM to 10 µM, a low limit of detection of 96 nM, and a limit of quantification of 320 nM. The elaborated sensing approach is viable for use in real sample matrices and tested for GLY determination in soil and water samples, without pretreatment, preparation, or purification. The results showed the practical usefulness of the sensor in the real sample analysis and suggested its suitability for possible out-of-laboratory sensing.

Electrochemiluminescence Amplification in Bead-Based Assays Induced by a Freely Diffusing Iridium(III) Complex.

Heterogeneous electrochemiluminescence (ECL) assays employing tri-n-propylamine as a co-reactant and a tris(2,2'-bipyridine)ruthenium(II) ([Ru(bpy)3]2+) derivative as an emissive label are integral to the majority of academic and commercial applications of ECL sensing. This model system is an active research area and constitutes the basis of successfully commercialized bead-based ECL immunoassays. Herein, we propose a novel approach to the enhancement of such conventional ECL assays via the incorporation of a second metal coordination complex, [Ir(sppy)3]3- (where sppy = 5'-sulfo-2-phenylpyridinato-C2,N), to the experimental system. By employing ECL microscopy, we are able to map the spatial distribution of ECL emission at the surface of the bead, from [Ru(bpy)3]2+ labels, and solution-phase emission, from [Ir(sppy)3]3-. The developed [Ir(sppy)3]3--mediated enhancement approach elicited a significant improvement (70.9-fold at 0.9 V and 2.9-fold at 1.2 V vs Ag/AgCl) of the ECL signal from [Ru(bpy)3]2+ labels immobilized on the surface of a polystyrene bead. This dramatic enhancement in ECL signal, particularly at low oxidation potentials, has important implications for the improvement of existing heterogeneous ECL assays and ECL-based microscopy, by amplifying the signal, opening new bioanalytical detection schemes, and reducing both electrode surface passivation and deleterious side reactions.

La(OH)3 Multi-Walled Carbon Nanotube/Carbon Paste-Based Sensing Approach for the Detection of Uric Acid-A Product of Environmentally Stressed Cells.

This paper aims to develop an amperometric, non-enzymatic sensor for detecting and quantifying UA as an alert signal induced by allergens with protease activity in human cell lines (HEK293 and HeLa). Uric acid (UA) has been classified as a damage-associated molecular pattern (DAMP) molecule that serves a physiological purpose inside the cell, while outside the cell it can be an indicator of cell damage. Cell damage or stress can be caused by different health problems or by environmental irritants, such as allergens. We can act and prevent the events that generate stress by determining the extent to which cells are under stress. Amperometric calibration measurements were performed with a carbon paste electrode modified with La(OH)3@MWCNT, at the potential of 0.3 V. The calibration curve was constructed in a linear operating range from 0.67 µM to 121 µM UA. The proposed sensor displayed good reproducibility with an RSD of 3.65% calculated for five subsequent measurements, and a low detection limit of 64.28 nM, determined using the 3 S/m method. Interference studies and the real sample analysis of allergen-treated cell lines proved that the proposed sensing platform possesses excellent sensitivity, reproducibility, and stability. Therefore, it can potentially be used to evaluate stress factors in medical research and clinical practice.

Facile Synthesis of Low-Cost Copper-Silver and Cobalt-Silver Alloy Nanoparticles on Reduced Graphene Oxide as Efficient Electrocatalysts for Oxygen Reduction Reaction in Alkaline Media.

Copper-silver and cobalt-silver alloy nanoparticles deposited on reduced graphene oxide (CuAg/rGO and CoAg/rGO) were synthesized and examined as electrocatalysts for oxygen reduction reaction (ORR) and hydrogen peroxide reduction reaction (HPRR) in alkaline media. Characterization of the prepared samples was done by transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), Raman spectroscopy, X-ray diffraction analysis (XRD), and scanning electron microscopy with integrated energy-dispersive X-ray spectroscopy (SEM-EDS). CuAg/rGO and CoAg/rGO nanoparticles diameter ranged from 0.4 to 9.2 nm. The Ag loading was ca. 40 wt.% for both electrocatalysts, with that for Cu and Co being 35 and 17 wt.%, respectively. CoAg/rGO electrocatalyst showed a Tafel slope of 109 mV dec-1, significantly lower than that for CuAg/rGO (184 mV dec-1), suggesting faster ORR kinetics. Additionally, a higher diffusion current density was obtained for CoAg/rGO (-2.63 mA cm-2) than for CuAg/rGO (-1.74 mA cm-2). The average value of the number of electrons transferred during ORR was 2.8 for CuAg/rGO and 3.3 for CoAg/rGO electrocatalyst, further confirming the higher ORR activity of the latter. On the other hand, CuAg/rGO showed higher peak current densities (-3.96 mA cm-2) for HPRR compared to those recorded for CoAg/rGO electrocatalyst (-1.96 mA cm-2).