Digital microfluidic platform assembled into a home-made studio for sample preparation and colorimetric sensing of S-nitrosocysteine.

Digital microfluidics (DMF) is a versatile lab-on-a-chip platform that allows integration with several types of sensors and detection techniques, including colorimetric sensors. Here, we propose, for the first time, the integration of DMF chips into a mini studio containing a 3D-printed holder with previously fixed UV-LEDs to promote sample degradation on the chip surface before a complete analytical procedure involving reagent mixture, colorimetric reaction, and detection through a webcam integrated on the equipment. As a proof-of-concept, the feasibility of the integrated system was successfully through the indirect analysis of S-nitrosocysteine (CySNO) in biological samples. For this purpose, UV-LEDs were explored to perform the photolytic cleavage of CySNO, thus generating nitrite and subproducts directly on DMF chip. Nitrite was then colorimetrically detected based on a modified Griess reaction, in which reagents were prepared through a programable movement of droplets on DMF devices. The assembling and the experimental parameters were optimized, and the proposed integration exhibited a satisfactory correlation with the results acquired using a desktop scanner. Under the optimal experimental conditions, the obtained CySNO degradation to nitrite was 96%. Considering the analytical parameters, the proposed approach revealed linear behavior in the CySNO concentration range between 12.5 and 400 µmol L-1 and a limit of detection equal to 2.8 µmol L-1. Synthetic serum and human plasma samples were successfully analyzed, and the achieved results did not statistically differ from the data recorded by spectrophotometry at the confidence level of 95%, thus indicating the huge potential of the integration between DMF and mini studio to promote complete analysis of lowmolecular weight compounds.

Recycling 3D Printed Residues for the Development of Disposable Paper-Based Electrochemical Sensors.

Here, we propose a recyclable approach using acrylonitrile-butadiene-styrene (ABS) residues from additive manufacturing in combination with low-cost and accessible graphite flakes as a novel and potential mixture for creating a conductive paste. The graphite particles were successfully incorporated in the recycled thermoplastic composite when solubilized with acetone and the mixture demonstrated greater adherence to different substrates, among which cellulose-based material made possible the construction of a paper-based electrochemical sensor (PES). The morphological, structural, and electrochemical characterizations of the recycled electrode material were demonstrated to be similar to those of the traditional carbon-based surfaces. Faradaic responses based on redox probe activity ([Fe(CN)6]3-/4-) exhibited well-defined peak currents and diffusional mass transfer as a quasi-reversible system (96 ± 5 mV) with a fast heterogeneous rate constant value of 2 × 10-3 cm s-1. To improve the electrode electrochemical properties, both the PES and the classical 3D-printed electrode surfaces were modified with a combination of multiwalled carbon nanotubes (MWCNTs), graphene oxide (GO), and copper. Both electrode surfaces demonstrated the suitable oxidation of nitrite at 0.6 and 0.5 V vs Ag, respectively. The calculated analytical sensitivities for PES and 3D-printed electrodes were 0.005 and 0.002 µA/(µmol L-1), respectively. The proposed PES was applied for the indirect amperometric analysis of S-nitroso-cysteine (CysNO) in serum samples via nitrite quantitation, demonstrating a limit of detection of 4.1 µmol L-1, with statistically similar values when compared to quantitative analysis of the same samples by spectrophotometry (paired t test, 95% confidence limit). The evaluated electroanalytical approach exhibited linear behavior for nitrite in the concentration range between 10 and 125 µmol L-1, which is suitable for realizing clinical diagnosis involving Parkinson's disease, for example. This proof of concept shows the great promise of this recyclable strategy combining ABS residues and conductive particles in the context of green chemical protocols for constructing disposable sensors.

A qnr-plasmid allows aminoglycosides to induce SOS in Escherichia coli.

The plasmid-mediated quinolone resistance (PMQR) genes have been shown to promote high-level bacterial resistance to fluoroquinolone antibiotics, potentially leading to clinical treatment failures. In Escherichia coli, sub-minimum inhibitory concentrations (sub-MICs) of the widely used fluoroquinolones are known to induce the SOS response. Interestingly, the expression of several PMQR qnr genes is controlled by the SOS master regulator, LexA. During the characterization of a small qnrD-plasmid carried in E. coli, we observed that the aminoglycosides become able to induce the SOS response in this species, thus leading to the elevated transcription of qnrD. Our findings show that the induction of the SOS response is due to nitric oxide (NO) accumulation in the presence of sub-MIC of aminoglycosides. We demonstrated that the NO accumulation is driven by two plasmid genes, ORF3 and ORF4, whose products act at two levels. ORF3 encodes a putative flavin adenine dinucleotide (FAD)-binding oxidoreductase which helps NO synthesis, while ORF4 codes for a putative fumarate and nitrate reductase (FNR)-type transcription factor, related to an O2-responsive regulator of hmp expression, able to repress the Hmp-mediated NO detoxification pathway of E. coli. Thus, this discovery, that other major classes of antibiotics may induce the SOS response could have worthwhile implications for antibiotic stewardship efforts in preventing the emergence of resistance.

Evolution of nucleic acids biosensors detection limit III.

This review is an update of two previous ones focusing on the limit of detection of electrochemical nucleic acid biosensors allowing direct detection of nucleic acid target (miRNA, mRNA, DNA) after hybridization event. A classification founded on the nature of the electrochemical transduction pathway is established. It provides an overall picture of the detection limit evolution of the various sensor architectures developed during the last three decades and a critical report of recent strategies.

Functionalized Multi-Walled Carbon Nanotube-Based Aptasensors for Diclofenac Detection.

Driven by the increasing concern about the risk of diclofenac (DCF) residues as water pollutants in the aqueous environment and the growing need for its trace determination, a simple but sensitive electrochemical aptasensor for the trace detection of DCF was developed. To construct the aptasensor, the amine-terminated DCF aptamer was covalently immobilized on the surface of the carboxylic acid-functionalized multi-walled carbon nanotube (f-MWCNT)-modified glassy carbon electrode (GCE) through EDC/NHS chemistry. The f-MWCNTs provide a reliable matrix for aptamer immobilization with high grafting density, while the aptamer serves as a biorecognition probe for DCF. The obtained aptasensor was incubated with DCF solutions at different concentrations and was then investigated by electrochemical impedance spectroscopy (EIS). It displays two linear ranges of concentration for DCF detection, from 250 fM to 1pM and from 1 pM to 500 nM with an extremely low detection limit of 162 fM. Also, the developed biosensor shows great reproducibility, acceptable stability, and reliable selectivity. Therefore, it offers a simple but effective aptasensor construction strategy for trace detection of DCF and is anticipated to show great potential for environmental applications.

Speciation and quantitation of precious metals in model acidic leach liquors, theoretical and practical aspects of recycling.

Waste printed circuit boards are a major source of strategic materials such as platinum group metals since they are used for the fabrication of technological devices, such as hard drive discs, capacitors, and diodes. Because of the high cost of platinum, palladium, and gold (> 25 k€/kg), an economic and environmental challenge is their recycling from printed circuit boards that represent around 2% weight of electronic equipment. Hydrometallurgical treatments allow the recovery of these metals in solution, with a high recovery rate for a leaching liquor made of thiourea in hydrochloric acid. So as to develop an efficient recycling process from this leach liquor, one requires the speciation of these strategic metals, as well as their extraction and quantitation in the mixture. For this purpose, platinum, palladium, and gold were dissolved in model leach liquors made of hydrochloric acid and thiourea at low concentration. The identification of metal complexes was determined as a function of thiourea concentration (between 10 µmol/L and 10 mmol/L) by the combination of UV-visible spectrometry, cyclic voltammetry, and for the first time capillary electrophoresis. The electrokinetic method was then applied for the quantitation of trace metal analyses in leach samples from waste printed circuit boards reprocessing, demonstrating its applicability for industrializable recycling applications. Graphical abstract.

Label-free graphene oxide-based SPR genosensor for the quantification of microRNA21.

This work is focused on the development of a genosensor for microRNA-21 quantification using surface plasmon resonance (SPR) to transduce the hybridization event. The biosensing platform was built by self-assembling two bilayers of poly(diallyldimethylammonium chloride) (PDDA) and graphene oxide (GO) at a gold surface modified with 3-mercaptopropane sulfonate (MPS), followed by the covalent attachment of the DNA probe. GO was used in two directions, to allow the anchoring of the probe DNA and to increase the sensitivity of the biosensing event due to its field enhancer effect. The new bioanalytical platform represents an interesting alternative for the label-free biosensing of microRNA-21, with a linear range between 1.0 fM and 10 nM, a sensitivity of 5.1 ± 0.1 moM-1 and a detection limit of 0.3fM. The proposed sensing strategy was successfully used for the quantification of microRNA-21 in enriched urine samples. Graphical abstract.

Ruthenium(II) Complex Containing a Redox-Active Semiquinonate Ligand as a Potential Chemotherapeutic Agent: From Synthesis to In Vivo Studies.

Chemotherapy remains one of the dominant treatments to cure cancer. However, due to the many inherent drawbacks, there is a search for new chemotherapeutic drugs. Many classes of compounds have been investigated over the years to discover new targets and synergistic mechanisms of action including multicellular targets. In this work, we designed a new chemotherapeutic drug candidate against cancer, namely, [Ru(DIP)2(sq)](PF6) (Ru-sq) (DIP = 4,7-diphenyl-1,10-phenanthroline; sq = semiquinonate ligand). The aim was to combine the great potential expressed by Ru(II) polypyridyl complexes and the singular redox and biological properties associated with the catecholate moiety. Experimental evidence (e.g., X-ray crystallography, electron paramagnetic resonance, electrochemistry) demonstrates that the semiquinonate is the preferred oxidation state of the dioxo ligand in this complex. The biological activity of Ru-sq was then scrutinized in vitro and in vivo, and the results highlight the promising potential of this complex as a chemotherapeutic agent against cancer.

A Maltol-Containing Ruthenium Polypyridyl Complex as a Potential Anticancer Agent.

Cancer is one of the main causes of death worldwide. Chemotherapy, despite its severe side effects, is to date one of the leading strategies against cancer. Metal-based drugs present several potential advantages when compared to organic compounds and they have gained trust from the scientific community after the approval on the market of the drug cisplatin. Recently, we reported the ruthenium complex ([Ru(DIP)2 (sq)](PF6 ) (where DIP is 4,7-diphenyl-1,10-phenantroline and sq is semiquinonate) with a remarkable potential as chemotherapeutic agent against cancer, both in vitro and in vivo. In this work, we analyse a structurally similar compound, namely [Ru(DIP)2 (mal)](PF6 ), carrying the flavour-enhancing agent approved by the FDA, maltol (mal). To possess an FDA approved ligand is crucial for a complex, whose mechanism of action might include ligand exchange. Herein, we describe the synthesis and characterisation of [Ru(DIP)2 (mal)](PF6 ), its stability in solutions and under conditions that resemble the physiological ones, and its in-depth biological investigation. Cytotoxicity tests on different cell lines in 2D model and on HeLa MultiCellular Tumour Spheroids (MCTS) demonstrated that our compound has higher activity than cisplatin, inspiring further tests. [Ru(DIP)2 (mal)](PF6 ) was efficiently internalised by HeLa cells through a passive transport mechanism and severely affected the mitochondrial metabolism.

Increasing the Cytotoxicity of Ru(II) Polypyridyl Complexes by Tuning the Electronic Structure of Dioxo Ligands.

Due to the great potential expressed by an anticancer drug candidate previously reported by our group, namely, Ru-sq ([Ru(DIP)2(sq)](PF6) (DIP, 4,7-diphenyl-1,10-phenanthroline; sq, semiquinonate ligand), we describe in this work a structure-activity relationship (SAR) study that involves a broader range of derivatives resulting from the coordination of different catecholate-type dioxo ligands to the same Ru(DIP)2 core. In more detail, we chose catechols carrying either an electron-donating group (EDG) or an electron-withdrawing group (EWG) and investigated the physicochemical and biological properties of their complexes. Several pieces of experimental evidences demonstrated that the coordination of catechols bearing EDGs led to deep-red positively charged complexes 1-4 in which the preferred oxidation state of the dioxo ligand is the uninegatively charged semiquinonate. Complexes 5 and 6, on the other hand, are blue/violet neutral complexes, which carry an EWG-substituted dinegatively charged catecholate ligand. The biological investigation of complexes 1-6 led to the conclusion that the difference in their physicochemical properties has a strong impact on their biological activity. Thus, complexes 1-4 expressed much higher cytotoxicities than complexes 5 and 6. Complex 1 constitutes the most promising compound in the series and was selected for a more in depth biological investigation. Apart from its remarkably high cytotoxicity (IC50 = 0.07-0.7 µM in different cancerous cell lines), complex 1 was taken up by HeLa cells very efficiently by a passive transportation mechanism. Moreover, its moderate accumulation in several cellular compartments (i.e., nucleus, lysosomes, mitochondria, and cytoplasm) is extremely advantageous in the search for a potential drug with multiple modes of action. Further DNA metalation and metabolic studies pointed to the direct interaction of complex 1 with DNA and to the severe impairment of the mitochondrial function. Multiple targets, together with its outstanding cytotoxicity, make complex 1 a valuable candidate in the field of chemotherapy research. It is noteworthy that a preliminary biodistribution study on healthy mice demonstrated the suitability of complex 1 for further in vivo studies.

Physicochemical Characterization of Phthalocyanine-Functionalized Quantum Dots by Capillary Electrophoresis Coupled to a LED Fluorescence Detector.

Capillary zone electrophoresis (CZE) complemented with Taylor Dispersion Analysis-CE (TDA-CE) was developed to physicochemically characterize phthalocyanine-capped core/shell/shell quantum dots (QDs) at various pH and ionic strengths. An LED-induced fluorescence detector was used to specifically detect the QDs. The electropherograms and taylorgrams allowed calculating the phthalocyanine-QDs (Pc-QDs) ζ-potential and size, respectively, and determining the experimental conditions for colloidal stability. This methodology allowed evidencing either a colloidal stability or an aggregation state according to the background electrolytes nature. The calculated ζ-potential values of Pc-QDs decreased when ionic strength increased, being well correlated with the aggregation of the nanoconjugates at elevated salt concentrations. For the same reason, the hydrodynamic diameter of Pc-QDs increased with increasing background electrolyte ionic strength. The use of electrokinetic methodologies has provided insights into the colloidal stability of the photosensitizer-functionalized QDs in physiologically relevant solutions and, thereby, its usefulness for improving their design and applications for photodynamic therapy.

Integrated microfluidic device for the separation, decomposition and detection of low molecular weight S-nitrosothiols.

S-nitrosothiols (RSNOs) are very important biomolecules that play crucial roles in many physiological and physiopathological processes. They act as NO-donors and are candidates for future medicines. Their identification and quantitation are therefore important for biomedical applications. One, two or more RSNOs can then be combined to design a drug and therefore, the quantification of each is important to establish an acceptable quality control process. Till date, miniaturized devices have been used to detect RSNOs based on their total quantitation without a preceding separation step. This study reports on an original and integrated microdevice allowing for the successive electrokinetic separation of low molecular weight RSNOs, their decomposition under metal catalysis, and their quantitation by amperometric detection of the produced nitrite in the end-channel arrangement, leading to their quantitation in a single run. For this purpose, a commercial SU-8/Pyrex microfluidic system was coupled to a portable and wireless potentiostat. Different operating and running parameters were optimized to achieve the best analytical data, allowing for an LOD equal to 20 µM. The simultaneous separation of S-nitrosoglutathione and S-nitrosocysteine was successfully obtained within 75 s. The proposed methodology using SU-8/Pyrex microfluidic devices opens new possibilities to investigate future drug candidates for NO-donors.

Simultaneous Electrochemical Speciation of Oxidized and Reduced Glutathione. Redox Profiling of Oxidative Stress in Biological Fluids with a Modified Carbon Electrode.

The simultaneous electrochemical quantification of oxidized (GSSG) and reduced glutathione (GSH), biomarkers of oxidative stress, is demonstrated in biological fluids. The detection was accomplished by the development of a modified carbon electrode and was applied to the analysis of biological fluids of model organisms under oxidative stress caused by lead intoxication. Nanocomposite molecular material based on cobalt phthalocyanine (CoPc) and multiwalled carbon nanotubes functionalized with carboxyl groups (MWCNTf) was developed to modify glassy carbon electrodes (GCE) for the detection of reduced and oxidized glutathione. The morphology of the nanocomposite film was characterized by scanning electron microscopy (SEM) and profilometry. The electrochemical behavior of the modified electrode was assessed by cyclic voltammetry (CV) to determine the surface coverage (Γ) by CoPc. The electrocatalytic behavior of the modified electrode toward reduced (GSH) and oxidized (GSSG) forms of glutathione was assessed by CV studies at physiological pH. The obtained results show that the combined use of CoPc and MWCNTf results in an electrocatalytic activity for GSH oxidation and GSSG reduction, enabling the simultaneous detection of both species. Differential pulse voltammetry reveals detection limits of 100 µM for GSH and 8.3 µM for GSSG, respectively. The potential interference from ascorbic acid, cysteine, glutamic acid, and glucose was also studied, and the obtained results show limited effects from these species. Finally, the hybrid electrode was used for the determination of GSH and GSSG in rat urine and plasma samples, intoxicated or not by lead. Both glutathione forms were detected in these complex biological matrixes without any pretreatment. Our results portray the role of GSH and GSSG as markers of oxidative stress in live organisms under lead intoxication.

Photo-stimulation of persistent luminescence nanoparticles enhances cancer cells death.

Persistent luminescence nanoparticles made of ZnGa1.995Cr0.005O4 (ZGO-NPs) are innovative nanomaterials that emit photons during long periods of time after the end of the excitation, allowing their use as diagnosis probes for in vivo optical imaging. During the excitation process, a part of the energy is stored in traps to further emit photons over long time. However, we observed in this study that some of the energy reduces molecular oxygen to produce reactive oxygen species (ROS). Following this observation, theoxidative stress induction and cytotoxic effects of these NPs were investigated on human breast cancer cells. The results indicate that ROS production was stimulated by exposition of the hydroxylated ZGO-NPs to UV or visible light, and the oxidative stress induced in cells after internalization can be directly correlated to their dose-dependent inhibition of cell viability. On the contrary, PEGylated ZGONPs were not uptaken by cells and have no effect on the production of ROS. Thus, the cell viability was not altered by these nanoparticles. This study reveals the importance of considering light irradiation and surface coating of luminescent nanoparticles toxicity which open new perspectives for their use in photodynamic therapy.

Analysis of the evolution of the detection limits of electrochemical nucleic acid biosensors II.

This critical review of electrochemical biosensors allowing direct detection of nucleic acid targets reports on different transduction pathways and their latest breakthroughs. A classification of the various strategies based on the nature of the electrochemical transduction is established to emphasize the efficiency of each of them. It provides an overall picture of the detection limit of the various approaches developed during the last two decades. Graphical Abstract Detection limits evolutions of electrochemical DNA biosensors.

Aptamer entrapment in microfluidic channel using one-step sol-gel process, in view of the integration of a new selective extraction phase for lab-on-a-chip.

There is a great demand for integrating sample treatment into µTASs. In this context, we developed a new sol-gel phase for extraction of trace compounds in complex matrices. For this purpose, the incorporation of aptamers in silica-based gel within PDMS/glass microfluidic channels was performed for the first time by a one-step sol-gel process. The effective gel attachment onto microchannel walls and aptamer incorporation in the polymerized gel were evaluated using fluorescence microscopy. A good gel stability and aptamer incorporation inside the microchannel was demonstrated upon rinsing and over storage time. The ability of gel-encapsulated aptamers to interact with its specific target (either sulforhodamine B as model fluorescent target, or diclofenac, a pain killer drug) was assessed too. The binding capacity of entrapped aptamers was quantified (in the micromolar range) and the selectivity of the interaction was evidenced. Preservation of aptamers binding affinity to target molecules was therefore demonstrated. Dissociation constant of the aptamer-target complex and interaction selectivity were evaluated similar to those in bulk solution. This opens the way to new selective on-chip SPE techniques for sample pretreatment.

Rhenium Complexes Based on 2-Pyridyl-1,2,3-triazole Ligands: A New Class of CO2 Reduction Catalysts.

A series of [Re(N^N)(CO)3(X)] (N^N = diimine and X = halide) complexes based on 4-(2-pyridyl)-1,2,3-triazole (pyta) and 1-(2-pyridyl)-1,2,3-triazole (tapy) diimine ligands have been prepared and electrochemically characterized. The first ligand-based reduction process is shown to be highly sensitive to the nature of the isomer as well as to the substituents on the pyridyl ring, with the peak potential changing by up to 700 mV. The abilities of this class of complexes to catalyze the electroreduction and photoreduction of CO2 were assessed for the first time. It is found that only Re pyta complexes that have a first reduction wave with a peak potential at ca. -1.7 V vs SCE are active, producing CO as the major product, together with small amounts of H2 and formic acid. The catalytic wave that is observed in the CVs is enhanced by the addition of water or trifluoroethanol as a proton source. Long-term controlled potential electrolysis experiments gave total Faradaic yield close to 100%. In particular, functionalization of the triazolyl ring with a 2,4,6-tri-tert-butylphenyl group provided the catalyst with a remarkable stability.

Characterization of phthalocyanine functionalized quantum dots by dynamic light scattering, laser Doppler, and capillary electrophoresis.

In this work, we characterized different phtalocyanine-capped core/shell/shell quantum dots (QDs) in terms of stability, ζ-potential, and size at various pH and ionic strengths, by means of capillary electrophoresis (CE), and compared these results to the ones obtained by laser Doppler electrophoresis (LDE) and dynamic light scattering (DLS). The effect of the phthalocyanine metallic center (Zn, Al, or In), the number (one or four), and nature of substituents (carboxyphenoxy- or sulfonated-) of functionalization on the phthalocyanine physicochemical properties were evaluated. Whereas QDs capped with zinc mono-carboxyphenoxy-phtalocyanine (ZnMCPPc-QDs) remained aggregated in the whole analyzed pH range, even at low ionic strength, QDs capped with zinc tetracarboxyphenoxy phtalocyanine (ZnTPPc-QDs) were easily dispersed in buffers at pH equal to or higher than 7.4. QDs capped with aluminum tetrasulfonated phthalocyanine (AlTSPPc-QDs) and indium tetracarboxyphenoxy phthalocyanines (InTCPPc-QDs) were stable in aqueous suspension only at pH higher than 9.0 due to the presence of functional groups bound to the metallic center of the phthalocyanine. The ζ-potential values determined by CE for all the samples decreased when ionic strength increased, being well correlated with the aggregation of the nanoconjugates at elevated salt concentrations. The use of electrokinetic methodologies has provided insights into the colloidal stability of the photosensitizer-functionalized QDs in physiological relevant solutions and thereby, its usefulness for improving their design and applications for photodynamic therapy. Graphical Abstract Schematic illustration of the phthalocyanine capped QDs nanoconjugates and the capillary electrophoresis methods applied for size and ζ-potential characterization.

Colorimetric analysis of the decomposition of S-nitrosothiols on paper-based microfluidic devices.

A disposable microfluidic paper-based analytical device (µPAD) was developed to easily analyse different S-nitrosothiols (RSNOs) through colorimetric measurements. RSNOs are carriers of nitric oxide (NO) that play several physiological and physiopathological roles. The quantification of RSNOs relies on their decomposition using several protocols and the colorimetric detection of the final product, NO or nitrite. µPADs were fabricated by wax printing technology in a geometry containing one central zone for the sample inlet and eight circular detection zones interconnected by microfluidic channels for decomposition and posterior detection of decayed products. Different decomposition protocols including mercuric ions and light (UV, visible, and infrared) were tested on µPADs. For this purpose, a 3D printed holder was coupled with µPADs to easily design a simultaneous decomposition procedure using different light sources. The Griess reagent was added to detect NO and nitrite produced by the different decomposition methods. µPADs were then scanned using a flat board scanner and calibration curves based on color intensity were plotted. The limit of detection (LOD) values achieved for nitrite (used as a reference compound) and S-nitrosoglutathione (GSNO) using mercuric decomposition were 3 and 4 µM, respectively. The LOD reported herein for nitrite is considered among the lowest LODs already reported for this compound using µPADs. The results also show that low-molecular-weight RSNO, namely S-nitrosocysteine, decomposes more easily than high-molecular-weight RSNOs with light. As a proof of concept, RSNOs in human plasma were successfully detected on µPADs. For this purpose, a preliminary treatment step was optimized and the presence of high-molecular-weight (HMW) RSNOs was evidenced in the available plasma samples. The concentrations of HMW-RSNOs and nitrite in the various samples ranged from 5 to 16 µM and from 37 to 58 µM, respectively.

Electrochemical Detection of Nitric Oxide in Plant Cell Suspensions.

Nitric oxide is a hydrophobic radical acting as a physiological mediator in plants. Because of its unique properties, the detection of NO in plant tissues and cell suspensions remains a challenge. For this purpose, several techniques are used, each having certain advantages and limitations such as interferences with other species, questionable sensitivity, and/or selectivity or ex situ measurement. Here we describe a very attractive approach for tracking NO in plant cell suspensions using a NO-sensitive homemade platinum/iridium-based electrochemical microsensor. This method constitutes the absolute real-time proof of the production of free NO in physiological conditions.