Ultramicroelectrode Research Articles

Enhanced signal to noise ratio of single entity electrochemistry signal of platinum nanoparticles using passive silver ultramicroelectrode

AbstractThe single‐entity electrochemistry (SEE) of electrocatalytic platinum (Pt) single nanoparticles (NPs) on a less electrocatalytic silver (Ag) ultramicroelectrode (UME) surface was investigated using the electrocatalytic amplification method. Two characteristic types of current responses—current staircases and blips (or spikes)—were observed during single NP collision experiments, depending on the applied potential at the Ag UME. Notably, at applied potentials of 0.13 and 0.17 V, the Ag UME becomes passive due to the formation of a delicate oxide layer, resulting in a highly stable background current. This leads to an enhanced signal‐to‐noise (S/N) ratio, attributed to the low background current, when using Ag UME compared to commonly used UMEs such as Au, C, Ni, and Hg for the SEE of Pt NPs. The exceptionally low background current can provide a significant advantage for detailed observation of SEE signals and further mechanistic studies based on the current response.

A study of electrochemiluminescence mechanism of Rubrene/TPrA system in an aqueous solution via stochastic collision electrochemistry in an emulsion reactor

BackgroundElectrochemiluminescence (ECL) is an electrochemically induced process in which radicals generated at the electrode surface undergo exergonic electron transfer reaction to form excited states and luminesce. ECL, with high sensitivity and superior spatiotemporal control, has been widely applied in bioanalysis and light-emitting devices. The ECL signal of rubrene (Rub) was observed in Rub/TPrA oil-in-water (o/w) emulsions, which was inconsistent with the theory of ion-transfer coupled electron-transfer in Rub emulsion droplets, and the conventional ECL mechanism in Rub/TPrA system couldn't explain this phenomenon. ResultsChoosing the toluene oil-in-water emulsion droplets as reactors, we put forward and verified a new ECL mechanism of Rub in aqueous solution on the basis of cyclic voltammograms, single-entity electrochemistry, ECL detection techniques and COMSOL finite element simulation. The new mechanism involved that Rub was reduced to Rub•- by the highly reducing species TPrA•, Rub•- was then oxidized to the excited state Rub∗ by the stable intermediate TPrA•+, and finally Rub∗ was inactivated by emitting photons. In simulation, the standard oxidation rate constant of TPrA in the emulsion droplets was deduced as 1.5 × 10−4 cm/s, and the deprotonation rate of TPrA•+ was about 200 s−1. Besides, emulsion droplets colliding on Au ultramicroelectrode (UME) were found to be able to amplify ECL signals. SignificanceThe newly proposed ECL mechanism of Rub/TPrA emulsion reactors broke the solubility problem of Rub in an aqueous, promoting the research and application of the organic luminophore Rub in bioanalysis. With the ECL signals amplification capability, the o/w emulsion reactors are promising to future design of ECL biosensors with higher sensitivity.

Ultradense Array of On-Chip Sensors for High-Throughput Electrochemical Analyses.

High-throughput sensors are valuable tools for enabling massive, fast, and accurate diagnostics. To yield this type of electrochemical device in a simple and low-cost way, high-density arrays of vertical gold thin-film microelectrode-based sensors are demonstrated, leading to the rapid and serial interrogation of dozens of samples (10 μL droplets). Based on 16 working ultramicroelectrodes (UMEs) and 3 quasi-reference electrodes (QREs), a total of 48 sensors were engineered in a 3D crossbar arrangement that devised a low number of conductive lines. By exploiting this design, a compact chip (75 × 35 mm) can enable performing 16 sequential analyses without intersensor interferences by dropping one sample per UME finger. In practice, the electrical connection to the sensors was achieved by simply switching the contact among WE adjacent fingers. Importantly, a short analysis time was ensured by interrogating the UMEs with chronoamperometry or square wave voltammetry using a low-cost and hand-held one-channel potentiostat. As a proof of concept, the detection of Staphylococcus aureus in 15 samples was performed within 14 min (20 min incubation and 225 s reading). Additionally, the implementation of peptide-tethered immunosensors in these chips allowed the screening of COVID-19 from patient serum samples with 100% accuracy. Our experiments also revealed that dispensing additional droplets on the array (in certain patterns) results in the overestimation of the faradaic current signals, a phenomenon referred to as crosstalk. To address this interference, a set of analyses was conducted to design a corrective strategy that boosted the testing capacity by allowing using all on-chip sensors to address subsequent analyses (i.e., 48 samples simultaneously dispensed on the chip). This strategy only required grounding the unused rows of QRE and can be broadly adopted to develop high-throughput UME-based sensors. In practice, we could analyze 48 droplets (with [Fe(CN)6]4-) within ∼8 min using amperometry.

Understanding Reaction Kinetics of Iron Oxide (α- Fe2O3) Reduction with Single Entity Measurements

Emitting minimal amount of CO2 during the production of steel is one of the major challenges that can be overcome by producing iron using electrowinning method. The reaction of interest is at the cathode where the direct reduction of Iron oxide (α-Fe2O3) to iron takes place. In this study, we are investigating the electrochemical conversion of hematite (α-Fe2O3) Nanoparticles to iron in an extremely alkaline medium (50 wt% NaOH) using Single Entity Electrochemistry (SEE).Single Entity Electrochemistry is the study of single atom, molecule or particle colliding with the surface of the electrode usually by diffusion or migration. For these experiments, we are developing alkaline stable carbon-fiber (C)/Nickel (Ni) ultra-microelectrode (UME), prepared using multiple pathways such as plasma-focused ion beam.A spike in current transient during chronoamperometry (CA) experiments is observed when a hematite particle collides stochastically with the UME and gets reduced in the process. Each spike is the reduction event of one hematite nanoparticle to iron. Individual reduction events, that last 0.2-0.3 s and resulting in 0.1-0.2 nC of charge being passed were observed at Ni and C microelectrodes, this shows good agreement with the average particle radius of 100 nm. The shape of the current transient was distinct from previously reported transients for metal deposition at cathode1-3. However, A. Allanore, et.al. model was used to support a mechanism for the direct oxide reduction process at nanoscale particles4.The results presented here illustrate the reaction kinetics and the mode of reduction propagation in a single nano-sized hematite particle in alkaline conditions. The kinetics of hematite reduction in alkaline medium is considerably slow as observed from the slope of current spikes in SEE experiments. Furthermore, considering Allanore’s model, the reduction propagation occurs from the outer surface to the inner core.(1) Luo, L.; White, H. S. Electrogeneration of single nanobubbles at sub-50-nm-radius platinum nanodisk electrodes. Langmuir 2013, 29 (35), 11169-11175.(2) Xiao, X.; Fan, F.-R. F.; Zhou, J.; Bard, A. J. Current transients in single nanoparticle collision events. Journal of the American Chemical Society 2008, 130 (49), 16669-16677.(3) Bard, A. J.; Zhou, H.; Kwon, S. J. Electrochemistry of single nanoparticles via electrocatalytic amplification. Israel Journal of Chemistry 2010, 50 (3), 267-276.(4) Allanore, A.; Lavelaine, H.; Valentin, G.; Birat, J.; Delcroix, P.; Lapicque, F. Observation and modeling of the reduction of hematite particles to metal in alkaline solution by electrolysis. Electrochimica Acta 2010, 55 (12), 4007-4013.

(Invited) Electrochemistry of CdSe Quantum Dots in Organic Solvents. from Stochastic Electrochemistry to Film Behavior

We present our electrochemistry results of CdSe quantum dots in two organic solvents: neat methanol and acetonitrile. In MeOH, the quantum dots (QDs) can be detected through stochastic collisions; that is, as agglomerates of QDs collide with an electrode surface, the photocurrent changes as a result of the CdSe entities that collide with the electrode surface.1 The experiment presented here uses Pt ultramicroelectrodes (UME), that is, electrodes with a diameter of 25 μm or less. Our results show that the CdSe can accumulate on the surface of the electrode, and the technique provides a high degree of control. By controlling the time the electrode collects QDs, one can control the amount of CdSe that accumulates at the electrode surface. After collecting CdSe on the surface, we perform cyclic voltammetry experiments to confirm that the electrode surface has changed. In acetonitrile (MeCN), we can perform CVs over large potential windows to study the electrochemistry of the deposited CdSe. The electrochemical response of the deposited CdSe resembles that of a film, although the surface has sub-monolayer coverages, as will be discussed from the images of scanning electron microscopy (SEM).Figure 1 shows an example of a LSV in a region of interest where the current due to the CdSe significantly deviates from the background current. The LSV was run in the dark, so the current in the figure is a function of the density of states in the CdSe structures. It is also a function of the reversibility of the CV, i.e., mainly of the scan rate of the experiment. After the CV experiments, the Pt UME can be imaged in the SEM to correlate the electrochemical results with the CdSe deposited on the electrode surface. We will discuss our findings in terms of the results in the literature on CdSe electrochemistry (e.g., refs 2-5). References. (1) Subedi, P.; Parajuli, S.; Alpuche-Aviles, M. A., "Single Entity Behavior of CdSe Quantum Dot Aggregates During Photoelectrochemical Detection", Frontiers in Chemistry 9 (2021) DOI: 10.3389/fchem.2021.733642(2) Kohl, P. A.; Bard, A. J., "Semiconductor electrodes. 13. Characterization and behavior of n-type zinc oxide, cadmium sulfide, and gallium phosphide electrodes in acetonitrile solutions", Journal of the American Chemical Society 99, 7531-7539 (1977) DOI: 10.1021/ja00465a023(3) Hines, M. A.; Guyot-Sionnest, P., "Synthesis and characterization of strongly luminescing ZnS-capped CdSe nanocrystals", The Journal of Physical Chemistry 100, 468-471 (1996) DOI: 10.1021/jp9530562(4) Zhang, J. Z., "Interfacial Charge Carrier Dynamics of Colloidal Semiconductor Nanoparticles", The Journal of Physical Chemistry B 104, 7239-7253 (2000) DOI: 10.1021/jp000594s(5) Myung, N.; Ding, Z.; Bard, A. J., "Electrogenerated chemiluminescence of CdSe nanocrystals", Nano Letters 2, 1315-1319 (2002) Figure 1

Nanoimpact Electrochemistry for Unraveling the Reactivity of Diffusion Limited Enzymes

The ultimate challenge of modern electroanalytical chemistry is to reach a single-molecule level of detection. In this framework, nanoimpact electrochemistry (NIE) has been recently developed. It allows studying at high throughput the reactivity of small entities such as nanoparticles, cells, or proteins, that collide on a biased ultramicroelectrode (UME) inside a solution or suspension.1 The collisions cause disturbances in the recorded chronoamperogram such as current spikes or current steps.2 Single enzyme electrochemistry (SEE) can contribute to uncovering effects of dynamics and non-equilibrium behavior of individual enzyme molecules on catalysis, which are hidden in measurements on ensembles due to averaging of the measured parameters over the entire molecular population. However, NIE of smaller entities such as enzymes, is very challenging due to the low sensitivity of the state-of-the-art electrochemical equipment, and most single enzyme techniques are currently based on fluorescence.3 One strategy to overcome this issue is the amplification of the current produced by a single enzyme molecule by fast catalysis: the single enzyme converts several substrate molecules per second consuming several electrons leading measurable current. Some of us reported the implementation of this strategy for the first time, designing a single-entity electrochemical experiment that allowed the detection of catalytic currents from single laccase molecules.4 Soon after the initial SEE report in 2016, a similar approach was applied for investigations of catalase,5 an enzyme that breaks down H2O2 to O2 and H2O with diffusion limited kinetics. However, the unequivocal attribution of current disturbances to the reduction of O2 (produced locally by enzyme) on the electrode during enzyme-electrode collisions remains a challenge. We will address this challenge based on the analysis of SEE experimental data under various conditions, on protein film voltammetry (PFV) studies on rotating disk electrode (RDE), and on simulations. The collected data from the SEE, that is the current spike characteristics in each set of conditions, are statistically analyzed and discussed. PFV studies are used to evaluate basic thermodynamic and kinetic parameters of the system. Finally, we have built a model using Comsol Multiphysics. To the best of our knowledge, this is the first model reported so far that can satisfactorily reproduce the experimental SEE data, using experimentally evaluated parameters as input. The developed methodology is the succesfully implemented to the study of superoxide dismutase.6

An Ultramicroelectrode Electrochemistry and Surface Plasmon Resonance Coupling Method for Cell Exocytosis Study.

Exocytosis of a single cell has been extensively researched in recent years due to its close association with numerous diseases. However, current methods only investigate exocytosis at either the single-cell or multiple-cell level, and a method for simultaneously studying exocytosis at both levels has yet to be established. In this study, a combined device incorporating ultramicroelectrode (UME) electrochemistry and surface plasmon resonance (SPR) was developed, enabling the simultaneous monitoring of single-cell and multiple-cell exocytosis. PC12 cells were cultured directly on the SPR sensing Au film, with a carboxylated carbon nanopipette (c-CNP) electrode employed for electrochemical detection in the SPR reaction cell. Upon exocytosis, the released dopamine diffuses onto the inner wall of c-CNP, undergoing an electrochemical reaction to generate a current peak. Concurrently, exocytosis can also induce changes in the refractive index of the Au film surface, leading to the SPR signal. Consequently, the device enables real-time monitoring of exocytosis from both single and multiple cells with a high spatiotemporal resolution. The c-CNP electrode exhibited excellent resistance to protein contamination, high sensitivity for dopamine detection, and the capability to continuously monitor dopamine exocytosis over an extended period. Analysis of both SPR and electrochemical signals revealed a positive correlation between changes in the SPR signal and the frequency of exocytosis. This study introduces a novel method and platform for the simultaneous investigation of single-cell and multiple-cell exocytosis.

Combined ultra‐microelectrode: Exploring new potentials for in vivo/in situ ascorbic acid electroanalysis

AbstractMiniaturized and portable analytical tools show promise for sophisticated analysis, particularly in biological systems such as fruits, and they are suitable for advanced agriculture and related food industries. In this study, we developed combined ultra‐microelectrodes (UME) by modifying a microscale carbon fiber electrode (33 μm) coated with an Au nano‐film in a micropipette‐tip system. The proposed UME@Au exhibited a linear response to AC concentrations ranging from 30 to 1400 μM, with a 16 μM limit of detection. It demonstrated the ability to perform in vivo‐in vitro AC analysis in micro‐zones and volumes, such as different points of fruit tissue (Such as lemon) and within the body of a living plant (Such as Cactus arms and trunk), serving as a tiny implanted probe.In the first part of our study, we analyzed AC levels in lemon tissue directly. Our measurements revealed that AC levels are distributed heterogeneously in a single fruit. Additionally, stored AC levels depend on the color of the lemon (yellow ones have higher levels than the green ones). Furthermore, the UME was applied to control AC levels in different storage conditions, including opened containers, airtight containers, with and without exposing daylight, etc.In the second part, the UME@Au was utilized as an implanted sensor for in vivo analysis of AC in different parts of the cactus, recognized as a source of AC. No sample preparation is needed with minimum damage. The implanted microsensor could perform electroanalysis inside the live plant and stored parenchyma cells, etc. Notably, our results showed that AC levels are higher in the younger arms compared to the older ones, and so on.Based on our findings, the miniaturized, small, cheap, user‐friendly electrode demonstrated many capabilities, such as being implantable, having satisfactory stability, and not requiring sample preparations for analysis. It can open up a new window for micro‐electroanalysis in food and analytical plant sciences. We predict that this microscale platform can be modified and used for bioanalysis of other (bio)targets, such as vitamins, ions, and even the detection of plant pathogens in plants and crops directly. This involvement in the smart and modern farming industry is anticipated in the near future.

Enhancing low-concentration cell detection in single entity electrochemical systems through forced convection

This study advances the detection of bacteria at low concentrations in single-entity electrochemistry (SEE) systems by integrating forced convection. Our results show that forced convection significantly improves the mass transfer rate of electrolyte, with the mass transfer coefficient demonstrating a proportional relationship to the flow rate to the power of 1.37. Notably, while the collision frequency of E. coli initially increases with the flow rate, a subsequent decrease is observed at higher rates. This pattern is attributed to the mechanics of cell collision under forced convection. Specifically, while forced convection propels cells towards the ultra-microelectrode (UME), it does not aid in their penetration through the boundary layer, leading to cells being driven away from the UME at higher flow rates. This hypothesis is supported by the statistical analysis of collision data, including signal heights and rise times. By optimizing the flow rate to 2 mL/min, we achieved enhanced detection of E. coli in concentrations ranging from 0.9 × 107 to 5.0 × 107 cells/mL. This approach significantly increased collision frequency by elevating the mass transfer of cells, with the mass transfer coefficient rising from 0.1 × 10−5 m/s to 0.9 × 10−5 m/s. It provides a viable solution to the challenges of detecting bacteria at low concentrations in SEE systems.

Investigation of the Electrocatalytic Reduction of Peroxydisulfate Using Scanning Electrochemical Microscopy.

The elementary steps of the electrocatalytic reduction of S2O82- using the Ru(NH3)63+/2+ redox couple were investigated using scanning electrochemical microscopy (SECM) and steady-state voltammetry (SSV). SECM investigations were carried out in a 0.1 M KCl solution using a 3.5 μm radius carbon ultramicroelectrode (UME) as the SECM tip and a 25 μm radius platinum UME as the substrate electrode. Approach curves were recorded in the positive feedback mode of SECM by reducing Ru(NH3)63+ at the tip electrode and oxidizing Ru(NH3)62+ at the substrate electrode, as a function of the tip-substrate separation and S2O82- concentration. The one-electron reaction between electrogenerated Ru(NH3)62+ and S2O82- yields the unstable S2O83•-, which rapidly dissociates to produce highly oxidizing SO4•-. Because SO4•- is such a strongly oxidizing species, it can be further reduced at both the tip and the substrate, or it can react with Ru(NH3)62+ to regenerate Ru(NH3)63+. SECM approach curves display a complex dependence on the tip-substrate distance, d, due to redox mediation reactions at both the tip and the substrate. Finite element method (FEM) simulations of both SECM approach curves and SSV confirm a previously proposed mechanism for the mediated reduction of S2O82- using the Ru(NH3)63+/2+ redox couple. Our results provide a lower limit for dissociation rate constant of S2O83•- (∼1 × 106 s-1), as well as the rate constants for electron transfer between SO4•- and Ru(NH3)62+ (∼1 × 109 M-1 s-1) and between S2O82- and Ru(NH3)62+ (∼7 × 105 M-1 s-1).

Artificial Intelligence-Assisted Multiparameter Size Discrimination of Silver Nanoparticles through Electrochemical Collision.

Single particle collision is an important tool for size analysis at the individual particle level; however, due to complex dynamic behaviors of nanoparticles on the surface of an electrode, the accuracy of size discrimination is limited. A silver (Ag) nanoparticle (NP) was chosen as the research target, and the dynamic behavior of Ag NPs was simplified by enhancing adsorption between Ag NP and Au ultramicroelectrode (UME) in alkaline media. Immediately after, accurate dynamic and thermodynamic information on single Ag NP was accurately extracted from collision events, including current intensity, transferred charge, and duration time. On the basis that there were differences between parameters of different-sized Ag NPs, multiparameter size discrimination was proposed, which improved the accuracy compared to single-parameter discrimination. More intriguingly, multiparameter analysis was combined with artificial intelligence, a tool adept at processing multidimensional data, for the first time. Finally, artificial intelligence-assisted multiparameter size discrimination was successfully used to intelligently distinguish mixed Ag NPs, with an optimal accuracy of more than 95%. To sum up, the artificial intelligence-assisted multiparameter method showed an excellent ability to quickly achieve the most accurate size discrimination of nanoparticles at the level of individual particle and provide an effective guidance for the application of nanoparticles.

In-situ fabrication of tungsten oxide film pH micro-sensor and its application on the pH monitoring of Fe-Cu galvanic corrosion

Localized and interface pH values are important parameters for studying the corrosion progress and complex interface reactions. Although commercial glass pH sensors have been widely used to measure solution pH value due to their advantages of long stability and high sensitivity, they were unable to determine the interface and microregion pH value for their difficulty in miniaturization. In this work, an all-solid-state pH micro-sensor with strong adhesion strength was in-situ fabricated on diameter 15 μm tungsten ultramicroelectrode (UME) using constant current anodizing. The pH micro-sensor showed good linear response (R2 = 0.999), excellent sensitivity (with a slope of −53.8 mV/pH), quick response (<0.1 s), long lifetime (>44 days) and great anti-interference ability to most of the ions in marine (selectivity coefficients pKij > 7), such as SO42−, Cl−, NO3−, K+, Mg2+ and Fe2+. Then, the pH micro-sensor, combined with the potentiometric mode of SECM, was further employed to explore the localized pH value distribution during the galvanic corrosion of Fe-Cu couple in 0.1 M NaCl solution. To further verify the anti-interference ability of pH micro-sensor, potentiometric SECM experiment was also conducted in artificial seawater. Results showed that both acidic sites and alkalizing sites appeared on the Fe surface, while homogeneous alkalization took place overall Cu surface. This was consistent with the SVET results that anodic and cathodic areas existed simultaneously on Fe surface. Thus, it could be concluded that local corrosion occurred on Fe surface and only part of the Fe surface acted as the anodic even under galvanic effect. However, Cu acted as cathode with the homogeneously distributing cathodic current on the whole surface during the galvanic corrosion.

Probing Local pH Change during Electrode Oxidation of TEMPO Derivative: Implication of Redox-Induced Acidity Alternation by Imidazolium-Linker Functional Groups.

The chemical degradation of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-based aqueous energy storage and catalytic systems is pH sensitive. Herein, we voltammetrically monitor the local pH (pHlocal) at a Pt ultramicroelectrode (UME) upon electro-oxidation of imidazolium-linker functionalized TEMPO and show that its decrease is associated with the greater acidity of the cationic (oxidized) rather than radical (reduced) form of TEMPO. The protons that drive the decrease in pH arise from hydrolysis of the conjugated imidazolium-linker functional group of 4-[2-(N-methylimidazolium)acetoxy]-2,2,6,6-tetramethylpiperidine-1-oxyl chloride (MIMAcO-T), which was studied in comparison with 4-hydroxyl-TEMPO (4-OH-T). Voltammetric hysteresis is observed during the electrode oxidation of 4-OH-T and MIMAcO-T at a Pt UME in an unbuffered aqueous solution. The hysteresis arises from the pH-dependent formation and dissolution of Pt oxides, which interact with pHlocal in the vicinity of the UME. We find that electrogenerated MIMAcO-T+ significantly influences pHlocal, whereas 4-OH-T+ does not. Finite element analysis reveals that the thermodynamic and kinetic acid-base properties of MIMAcO-T+ are much more favorable than those of its reduced counterpart. Imidazolium-linker functionalized TEMPO molecules comprising different linking groups were also investigated. Reduced TEMPO molecules with carbonyl linkers behave as weak acids, whereas those with alkyl ether linkers do not. However, oxidized TEMPO+ molecules with alkyl ether linkers exhibit more facile acid-base kinetics than those with carbonyl ones. Density functional theory calculations confirm that OH- adduct formation on the imidazolium-linker functional group of TEMPO is responsible for the difference in the acid-base properties of the reduced and oxidized forms.

Electrochemical Detection of Single Aqueous Droplets in Organic Solvents via Pitting Collisions.

We report a novel detection method for single aqueous droplets in organic solvents by the collisional contact of the droplet, inducing the partial deformation of the ultramicroelectrode (UME) surface. For various chemical reactions in organic solvents, water impurities affect the catalytic activity, leading to a loss of productivity and selectivity. Therefore, it is necessary to monitor the water content of organic solvents in real time between many chemical production processes, from the laboratory to the industrial scale. Our method enables the detection of water contamination by real-time monitoring of the electrochemical signals or observing morphological changes in the microelectrode. When an aqueous droplet collides with the UME, the contact area of the electrode is electrolyzed, forming pits on the surface where the droplet falls. Current transient analysis shows a unique current spike corresponding to the reaction inside the adsorbed single aqueous droplet, which differs from those detected by the faradaic/nonfaradaic reaction of collision of other particles. Moreover, this analytical method can record the history of collision events from pits on the UME surface, implying that inspecting the UME surface could be a quick screening method for solvent contamination. Based on a comparison of the electrochemical signals and morphological changes of the electrode after each event, the sizes of the pits and droplets are related. A COMSOL simulation is performed to explain the shape of the peak current and pit formation during collision events. This experimental concept elucidates the dynamic behavior of aqueous droplets on a positively biased metal electrode.

Electrocatalytic Reduction of Benzyl Bromide during Single Ag Nanoparticle Collisions.

Previous reports of the electrocatalytic activity of Ag nanoparticles (AgNPs) toward the reduction of organic halides have been limited to measurements of immobilized nanoparticle ensembles. Here, we have investigated the electrochemical reduction of benzyl bromide (PhCH2Br) occurring at single AgNPs (4.2 to 37 nm radius) in methanol, where the effects of nanoparticle size on catalytic behavior can be more thoroughly examined and rigorously quantified. AgNP collisions at a 6.3 μm radius Au ultramicroelectrode (UME) result in measurable electrocatalytic amplification currents from the reduction of PhCH2Br, where collision events are indicated by a sudden step increase in the reduction current recorded in the current-time trace. The dependence of the height of these steps on the applied potential allowed for an analysis of reaction kinetics based on the Butler-Volmer model, resulting in an estimation of the standard rate constant (k0) as a function of AgNP size. Measured values of k0 range from 4.0 × 10-4 to 8.0 × 10-4 cm/s on AgNPs with radii of 14, 29, and 37 nm, whereas k0 was found to be 6.2 × 10-4 cm/s at a 12.3 μm radius Ag disk UME. The results indicate that the kinetics of PhCH2Br reduction are independent of AgNP size and are similar to the reaction kinetics observed at a Ag UME. The frequency of observed particle collisions was found to be dependent on particle size, where 14 nm radius AgNPs resulted in the highest-frequency collisions. The potential- and size-dependent interactions of AgNPs with the Au UME are discussed in terms of the DLVO theory.

Average collision velocity of single yeast cells during electrochemically induced impacts.

We recorded current-time (i-t) profiles for oxidizing ferrocyanide (FCN) while spherical yeast cells of radius (rc ≈ 2 μm) collided with disk ultramicroelectrodes (UMEs) of increasing radius (re ≈ 12-45 μm). Collision signals appear as minority steps and majority blips of decreased current overlayed on the i-t baseline when cells block ferrocyanide flux (JFCN). We assigned steps to adsorption events and blips to bouncing collisions or contactless passages. Yeast cells exhibit impact signals of long duration (Δt ≈ 15-40 s) likely due to sedimentation. We assume cells travel a threshold distance (T) to generate collision signals of duration Δt. Thus, T represents a distance from the UME surface, at which cell perturbations on JFCN blend in with the UME noise level. To determine T, we simulated the UME current, while placing the cell at increasing distal points from the UME surface until matching the bare UME current. T-Values at 90°, 45°, and 0° from the UME edge and normal to the center were determined to map out T-regions in different experimental conditions. We estimated average collision velocities using the formula T/Δt, and mimicked cells entering and leaving T-regions at the same angle. Despite such oversimplification, our analysis yields average velocities compatible with rigorous transport models and matches experimental current steps and blips. We propose that single-cells encode collision dynamics into i-t signals only when cells move inside the sensitive T-region, because outside, perturbations of JFCN fall within the noise level set by JFCN and rc/re (experimentally established). If true, this notion will enable selecting conditions to maximize sensitivity in stochastic blocking electrochemistry. We also exploited the long Δt recorded here for yeast cells, which was undetectable for the fast microbeads used in early pioneering work. Because Δt depends on transport, it provides another analytical parameter besides current for characterizing slow-moving cells like yeast.

Savitzky-Golay processing and bidimensional plotting of current-time signals from stochastic blocking electrochemistry to analyze mixtures of rod-shaped bacteria.

In stochastic blocking electrochemistry, adsorptive collisions of nano and micro-particles with an ultramicroelectrode (UME) generate steps of decreasing current overlaid on the current-time (i-t) baseline of an electroactive mediator reacting at the UME. The step amplitude (Δi) induced by particle blockage informs about its size, while collision frequency correlates with particle transport. However, because most particles arrive at the UME faster than the acquisition speed of conventional electrochemical instruments, current steps appear vertical. Recently, when analyzing rod-shape bacteria (bacilli), we detected slanted steps of duration Δt (∼0.6 to 1.1 s) that were found to scale up with bacillus length (∼1 to 5 μm, respectively). In this work, we apply a Savitzky-Golay (SG) algorithm coded in MATLAB to convert experimental i-t recordings into derivative plots of Δi/Δt versus t. As a result, current steps become peaks on a flat baseline. Unlike the original values of Δi and Δt that require manual gauging, the coded SG-algorithm generates both parameters automatically from peak integration. We then display Δi and Δt in bidimensional scatter plots comparing mixtures of A. erythreum (∼1 μm) and B. subtilis (∼5 μm). The spread of Δi and Δt values complies with the size distribution observed using scanning electron microscopy. By introducing SG-processing and bidimensional plotting of i-t recordings from stochastic blocking data we broaden the scope of the technique. The approach facilitates distinguishing bacilli in mixtures because both Δt and Δi increase with bacillus length and now they can be displayed in a single graph along with adsorption frequency. Moreover, density distribution and proportion of data points from groups of bacteria are also discernible from the plots.

Exploring single-entity electrochemistry beyond conventional potential windows: mechanistic insights into hydrazine/hydrazinium ion oxidation.

Single-entity electrochemistry (SEE) enables research into the electrochemical properties of nanoparticles (NPs) at the individual NP level. Recent studies on active particle-active electrode systems have expanded the scope of SEE measurements, moving beyond the limitations of inert electrode-based methods that rely on distinct NP-electrode catalytic differences, thereby enhancing mechanistic understanding of catalytic reactions. In this study, we investigated SEE signals from Pt NPs colliding with Au ultramicroelectrodes (UME) at elevated potentials where both Pt and Au UME exhibit electrocatalytic activity. Under conditions where Au UME is activated for hydrazine oxidation, distinctive combined spike and staircase current responses were observed. SEE signals exhibited varied shapes depending on pH and hydrazine concentration. Analyzing these variations provided insights into changes in reaction mechanisms according to pH and hydrazine concentration.

Single-Impact Electrochemistry for Bacterial Sensing

The main objective in the detection and analysis of bio-entities such as bacteria is a low detection limit, a short assay time and a high selectivity in identification, ideally combined with easy-handling and cheap instrumentation. In this way, the development of innovative analytical methods in terms of high sensitivity and spatial-temporal resolution allowing both qualitative and quantitative analysis at single-cell and subcellular levels is becoming a technological requirement. The main advantage of electrochemistry in biosensing applications is to combine high sensitivity and ease of use with the possibility of miniaturization at low cost. Especially, new electrochemical techniques and various types of electrodes have been recently developed offering the feasibility of analysis at the single-cell level with high sensitivity and selectivity. Nano-electrochemistry has been widely extended to various applications such as biosensing thanks to the continuous improvement of the instrumentation sensitivity, especially for individual entity electrochemical detection. Single-impact electrochemistry provides a low limit of detection (in principle, one single species) inherent to this electro-analytical method and the ability to study single entities (cells, viruses, bacteria, nanoparticles...) in real-time through a dynamic measurement [1]. The electrochemical nano-impacts method or discrete collisions technique (stochastic events) consists in detecting single impacts of various entities such as nanoparticles, cells, bacteria, vesicles, viruses, proteins in solution at a polarized ultramicroelectrode (UME) [2]. For each collision event, a specific signal is recorded in the chronoamperometry curve (current as a function of time) corresponding to an “impact” of the entity onto the UME surface. Crucial information can be reached from the analysis of the electrochemical impact event in the amperometry measurement, such as the concentration and the size of the colliding entity.Electrochemical nano-impacts (or collisions) of single bacteria are usually detected by recording a chronoamperometry measurement (i–t curve) at a UME biased at a redox potential of an electroactive species in an aqueous electrolyte containing between 106 and 109 bacteria per milliliter [1, 3]. Since 2015, most of the reports in this area concern Escherichia coli (E. coli) as a model bacterium but this sensitive electro-analytical technique has not proved to be efficient for selective detection yet, except for the discrimination between Gram-positive and Gram-negative bacteria. In order to develop single-impact electrochemistry toward the identification of bacterial cells, several studies deal with the use of redox mediators for probing the electrochemical activity of the bacterium. Also, an original and recent approach is to detect the virulence factors released by pathogenic bacteria rather than the cells themselves, based on redox liposomes single-impact electrochemistry [2, 4].

Observing Discrete Blocking Events at a Polarized Micro- or Submicro-Liquid/Liquid Interface.

Single-entity collisional electrochemistry (SECE), a subfield of single-entity electrochemistry, enables directly characterizing entities and particles in the electrolyte solution at the single-entity resolution. Blockade SECE at the traditional solid ultramicroelectrode (UME)/electrolyte interface suffers from a limitation: only redox-inactive particles can be studied. The wide application of the classical Coulter counter is restricted by the rapid translocation of entities through the orifice, which results in a remarkable proportion of undetected signals. In response, the blocking effect of single charged conductive or insulating nanoparticles (NPs) at low concentrations for ion transfer (IT) at a miniaturized polarized liquid/liquid interface was successfully observed. Since the particles are adsorbed at the liquid/liquid interface, our method also solves the problem of the Coulter counter having a too-fast orifice translocation rate. The decreasing quantal staircase/step current transients are from landings (controlled by electromigration) of either conductive or insulating NPs onto the interface. This interfacial NP assembly shields the IT flux. The size of each NP can be calculated by the step height. The particle size measured by dynamic light scattering (DLS) is used for comparison with that calculated from electrochemical blocking events, which is in fairly good agreement. In short, the blocking effect of IT by single entities at micro- or submicro-liquid/liquid interface has been proven experimentally and is of great reference in single-entity detection.