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.

Estimating Average Velocities of Particle Arrival Using the Time Duration of the Current Signal in Stochastic Blocking Electrochemistry.

In stochastic blocking electrochemistry, microparticles generate individual current steps when they adsorb on a microelectrode and decrease the current and flux of a redox mediator reacting at the surface. The amplitude of the current step informs on particle size and landing locus, while step frequency correlates with particle transport. Here, we report a new method to estimate the average arrival velocities of single rod-shaped bacteria (bacilli). The method relies on simulating the nearby threshold distance from the surface where the bacillus no longer perturbs mediator flux and the current step approaches zero. We estimated the average velocities of bacillus arrival by dividing the threshold distance over the current step duration, a parameter that here we detect for the first time and increases with bacillus length. By comparing diffusional fluctuations to bacillus average velocity, we estimated diffusion and migration contributions as a function of bacterium size. Average arrival velocities increase with bacillus length at the same time as migration intensifies and diffusion weakens. Our analysis is universal and more effective in determining transport mode contributions than the present approach of comparing theoretical and experimental step frequencies. Uncertainty in landing locus is inconsequential because the step duration used to calculate the average arrival speed already contains such information and knowing bacillus electrophoretic mobility or ζ-potential is not needed. Additionally, by simulating and assigning edge landings to the most repeated values of current steps in a recording, we obtain bacilli lengths and widths similar to scanning electron microscopy, from which we infer landing orientation.

Formation and Photoinduced Electron Transfer in Porphyrin- and Phthalocyanine-Bearing N-Doped Graphene Hybrids Synthesized by Click Chemistry.

Graphene doped with heteroatoms such as nitrogen, boron, and phosphorous by replacing some of the skeletal carbon atoms is emerging as an important class of two-dimensional materials as it offers the much-needed bandgap for optoelectronic applications and provides better access for chemical functionalization at the heteroatom sites. Covalent grafting of photosensitizers onto such doped graphenes makes them extremely useful for light-induced applications. Herein, we report the covalent functionalization of N-doped graphene (NG) with two well-known electron donor photosensitizers, namely, zinc porphyrin (ZnP) and zinc phthalocyanine (ZnPc), using the simple click chemistry approach. Covalent attachment of ZnP and ZnPc at the N-sites of NG in NG-ZnP and NG-ZnPc hybrids was confirmed by using a range of spectroscopic, thermogravimetric and imaging techniques. Ground- and excited-state interactions in NG-ZnP and NG-ZnPc were monitored by using spectral and electrochemical techniques. Efficient quenching of photosensitizer fluorescence in these hybrids was observed, and the relatively easier oxidations of ZnP and ZnPc supported excited-state charge-separation events. Photoinduced charge separation in NG-ZnP and NG-ZnPc hybrids was confirmed by using the ultrafast pump-probe technique. The measured rate constants were of the order of 1010  s,-1 thus indicating ultrafast electron transfer phenomena.

Dynamics of Collisions and Adsorption in the Stochastic Electrochemistry of Emulsion Microdroplets.

Current-time recordings of emulsified toluene microdroplets containing 20 mM Ferrocene (Fc), show electrochemical oxidation peaks from individual adsorption events on disk microelectrodes (5 µm diameter). The average droplet diameter (∼0.7 µm) determined from peak area integration was close to Dynamic Light Scattering measurements (∼1 µm). Random walk simulations were performed deriving equations for droplet electrolysis using the diffusion and thermal velocity expressions from Einstein. The simulations show that multiple droplet-electrode collisions, lasting ∼0.11 µs each, occur before a droplet wanders away. Updating the Fc-concentration at every collision shows that a droplet only oxidizes ∼0.58% of its content in one collisional journey. In fact, it would take ∼5.45 × 106 collisions and ∼1.26 h to electrolyze the Fc in one droplet with the collision frequency derived from the thermal velocity (∼0.52 cm/s) of a 1 µm-droplet. To simulate adsorption, the droplet was immobilized at first contact with the electrode while the electrolysis current was computed. This approach along with modeling of instrumental filtering, produced the best match of experimental peaks, which were attributed to electrolysis from single adsorption events instead of multiple consecutive collisions. These results point to a heightened sensitivity and speed when relying on adsorption instead of collisions. The electrochemical current for the former is limited by the probability of adsorption per collision, whereas for the latter, the current depends on the collision frequency and the probability of electron transfer per collision (J. Am. Chem. Soc. 2017, 139, 16923-16931).

Interplay of proton and electron transfer to determine concerted behavior in the proton-coupled electron transfer of glutathione oxidation.

Glutathione (GSH), whose thiol group dictates its redox chemistry, is oxidized to the thiyl radical (GS˙), which rapidly dimerizes to GSSG. Previously, we found that the oxidation rate of GSH by IrCl62- depends on the base (B) concentration and the pKa of its conjugate acid BH+, so that collateral to a stepwise mechanism, the concerted pathway GSH + IrCl62- + B = GS˙ + IrCl63- + BH+ was proposed as the rate determining step. Herein, this investigation is extended to include oxidant-base pairs that render exothermic and endothermic conditions of ΔG°' for electron transfer (ET) and proton transfer (PT). Experiments were conducted by the reaction of GSH with an electrogenerated oxidant M+ and using digital simulations to infer the mechanism. Data analysis shows that despite parallel mechanisms, the concerted one seems to predominate for the oxidant-base pair that renders the most isoenergetic coupled state, whereby a PT with is capable of producing an ET with , as a result of the Nernstian shift of with pKa. In contrast, the stepwise PT-ET appears to dominate when GS- grows in stability as becomes more negative. Understanding the interplay between ET and PT will help in the design of catalysts for energy harvesting processes that rely on proton-coupled electron transfer.

Fabrication of surface charge gradients in open-tubular capillaries and their characterization by spatially resolved pulsed streaming potential measurements.

Surface charge gradients have been formed on the inside surface of 75 µm i.d. silica capillaries via controlled rate infusion using 3-aminopropyltriethoxysilane as the reactive precursor. These 400 mm length gradients have been characterized using spatially resolved streaming potential measurements, from which the zeta potential as a function of distance was determined. The gradient capillaries exhibited a gradual variation in zeta potential from top to bottom, whereas uniformly modified and as-received capillaries were relatively homogeneous along their length. For a gradient prepared with a relatively high concentration of aminosilane, the zeta potential changed over 60 mV from one end of the capillary to the other, yielding a variation in the magnitude of the apparent surface charge of ~7 fold. By changing the concentration of the aminoalkoxysilane and/or the rate of infusion, both the value of the zeta potential (and hence surface charge) and its spatial profile (i.e., rate of change with distance) could be manipulated.

Real time detection of lysozyme by pulsed streaming potentials using polyclonal antibodies immobilized on a renewable nonfouling surface inside plastic microfluidic channels.

A composite surface was prepared on cyclic olefin copolymer (COC) microchannels by UV-photografting of polyethylene glycol acrylate (PEGA) and poly(acrylic acid) (PAA) films. A PEGA layer of globular particles with average thickness of 60 nm was formed after 15 min of polymerization. Real time monitoring by pulsed streaming potentials demonstrated the ability of the PEGA layer to inhibit the adhesion of five different nonspecific adsorbing proteins when compared with pristine COC. Roughness determined by atomic force microscopy (AFM) after PAA grafting on COC-PEGA at different UV illumination times suggests that PAA formation is initiated at the free space in between the PEGA particles. Carboxylic groups activated with N-hydroxysuccinimide and N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide were used to bind anti-lysozyme polyclonal antibodies. The composite COC-PEGA-PAA-anti-lysozyme surface demonstrated its ability to detect lysozyme with a dynamic range between 140 and 860 nM. Linearity was maintained even when samples were spiked with 250 nM of cytochrome as interfering species. The equilibrium constant K(eq) for the adsorption of Ly on COC-PEGA-PAA-anti-Ly was estimated to be 2.7 × 10(6) M(-1), and it shows that this kinetic approach of monitoring the surface charge is also useful to estimate affinity interactions for proteins in label free fashion. The regeneration of the surface exhibited an average percentage of recovery of ∼97% for each of six adsorption-regeneration cycles. This feature enables curve calibration on a single microfluidic chip because each point of the curve has a reproducible and renewable surface.

Quantifying surface-accessible quaternary charge for surface modified coatings via streaming potential measurements.

Prior research established that P[AB]-copolyoxetane polyurethanes with soft blocks having A = trifluoroethoxy (CF(3)CH(2)-O-CH(2)-, 3FOx) and B = dodecylammonium-butoxy (C12) are highly effective as polymer surface modifiers (PSMs). These PSMs displayed high contact antimicrobial efficiency against spray challenge that was attributed to surface concentration of quaternary charge. Herein, using a novel cell design and polymer coating process, streaming potential (SP) measurements are reported for estimating accessible surface charge density. Fused-silica capillaries were embedded in flat polypropylene sheets, and the inner capillary walls were coated with neat HMDI-BD(30)-P[(3FOx)(C12)-87:13-5100] (PU-1) and 1 wt % PU-1 in HMDI-BD(50)-PTMO-1000 (base polyurethane 2). Effects of annealing (60 degrees C) and electrolyte flow cycles on near-surface quaternary charge concentration were determined. Neat PU-1 had a constant SP that was cycle-independent and actually increased on annealing. As-cast 1 wt % PU-1 showed initial SPs about half those for neat PU-1, with substantial attenuation over 16 measurement cycles. SPs for annealed 1 wt % PU-1 displayed lower initial values that attenuated rapidly over multiple cycles. Zeta potentials and surface charge densities were calculated from SPs and discussed relative to contact antimicrobial properties. Tapping mode atomic force microscopy (TM-AFM) imaging was employed for investigation of 1 wt % PU-1 surface morphology. Microscale phase separation occurs on annealing 1 wt % PU-1 for 24 h at 60 degrees C. Surprisingly, phase separation was also observed after short immersion of 1 wt % PU-1 coatings in water. The morphological changes are correlated with instability of near-surface charge found by SP measurements. A model is proposed for near-surface spinodal decomposition of metastable as-cast 1 wt % PU-1. The formation of a fluorous modifier rich phase apparently sequesters near-surface quaternary charge and accounts for temporal instability of antimicrobial properties. The results are important in providing a facile method for screening polycation-based, contact antimicrobial coatings for accessible charge density and in assessing durability.

Label-free detection of heparin, streptavidin, and other probes by pulsed streaming potentials in plastic microfluidic channels.

In this paper, pulsed streaming potentials generated in plastic microfluidic channels are used for the label-free detection of some model analytes. The microchannels are fabricated with the commodity plastic cyclic olefin copolymer (COC), and the detection signal arises from a change in the surface charge upon analyte adsorption on the modified microchannel surface. The role of the surface modification is to confer the microchannel with a predetermined charge and a particular specificity toward the adsorption of the target analyte. In this work, several target probes displaying different levels of specificity were investigated. Heparin and streptavidin were detected by adsorption on microchannel surfaces modified with protamine and biotin, respectively, whereas bovine serum albumin (BSA) and methylene blue (MB) showed nonspecific adsorption on almost any modified or unmodified COC microchannel surface. The magnitude of the streaming potential was found to be proportional to the liquid pressure and the surface charge of the microchannel in accord with the Smoluchowski equation. Because the relative polarity of the streaming potential is determined by the surface charge, the most straightforward detection with this method occurs when the charge is reversed upon analyte adsorption. This strategy was used for the species described in this work, and the lowest concentrations detected were approximately 0.01 units/mL for heparin (below clinical relevance), approximately 10 (-9) M for BSA, and approximately 10 (-6) M for MB. Unlike the conventional method of steady flow, in this work, the streaming potentials were measured under pulsed conditions of flow and using nonreference electrodes. This approach removes the need of special electrolytes as it is usually required when using reference electrodes, and at the same time, it mitigates the interference of electrochemical drift from the electrodes. Relative standard deviations of approximately 1-2% and measuring times of approximately 10 s are readily attained with this experimental setup. The on-channel modification of the surface was carried out by UV-photografting methods given the significant UV transparency of COC.

Adsorption kinetics of proteins in plastic microfluidic channels: real-time monitoring of lysozyme adsorption by pulsed streaming potentials.

A proof-of-concept study is presented illustrating the real-time monitoring of lysozyme (Ly) adsorption on the surface of microchannels made with cyclic olefin copolymer (COC). The signal arises from changes in the surface charge upon adsorption of Ly which is monitored by pulsed streaming potentials. Because streaming potentials are electrochemical potentials resulting from the pressure-driven flow of the liquid and the surface charge of the microchannel, this approach is ideal for microfluidics. Initial adsorption rates showed a linear correlation with the bulk concentration of [Ly] in the range between 7.0 and 350 nM. Fitting of the adsorption isotherms allowed the estimation of equilibrium and rate constants of adsorption. The influence of phosphate ions on the adsorption kinetics of Ly was also investigated. Unlike the steady flow used in conventional streaming potential measurements, the present approach incorporates pulsed flow and the ability to inject finite samples into the microfluidic stream. The pulsed flow allows the use of non-reference electrodes which removes the need for special electrolytes to stabilize the electrode potentials. Likewise, the injection permits monitoring of adsorption and desorption events in real time. The label-free monitoring of these events and the high sensitivity of the adsorption kinetics of Ly to solution species found in this work indicates that this method could be applicable to study protein-protein interactions.

On-chip micropatterning of plastic (cylic olefin copolymer, COC) microfluidic channels for the fabrication of biomolecule microarrays using photografting methods.

This paper reports on the surface modification of plastic microfluidic channels to prepare different biomolecule micropatterns using ultraviolet (UV) photografting methods. The linkage chemistry is based upon UV photopolymerization of acryl monomers to generate thin films (0.01-6 microm) chemically linked to the organic backbone of the plastic surface. The commodity thermoplastic, cyclic olefin copolymer (COC) was selected to build microfluidic chips because of its significant UV transparency and easiness for microfabrication by molding techniques. Once the polyacrylic films were grafted on the COC surface using photomasks, micropatterns of proteins, DNA, and biotinlated conjugates were readily obtained by surface chemical reactions in one or two subsequent steps. The thickness of the photografted films can be tuned from several nanometers up to several micrometers, depending on the reaction conditions. The micropatterned films can be prepared inside the microfluidic channel (on-chip) or on open COC surfaces (off-chip) with densities of functional groups about 10(-7) mol/cm2. Characterization of these films was performed by attenuated-total-reflectance IR spectroscopy, fluorescence microscopy, profilometry, atomic force microscopy, and electrokinetic methods.