Stochastic Electrical Detection of Single Ion-Gated Semiconducting Polymers.

Semiconducting polymer chains constitute the building blocks for a wide range of electronic materials and devices. However, most of their electrical characteristics at the single-molecule level have received little attention. Elucidating these properties can help understanding performance limits and enable new applications. Here, coupled ionic-electronic charge transport is exploited to measure the quasi-1D electrical current through long single conjugated polymer chains as they form transient contacts with electrodes separated by ≈10 nm. Fluctuations between internal conformations of the individual polymers are resolved as abrupt, multilevel switches in the electrical current. This behavior is consistent with the theoretical simulations based on the worm-like-chain (WLC) model for semiflexible polymers. In addition to probing the intrinsic properties of single semiconducting polymer chains, the results provide an unprecedented window into the dynamics of random-coil polymers and enable the use of semiconducting polymers as electrical labels for single-molecule (bio)sensing assays.

Utilizing the Oxygen Reduction Reaction in Particle Impact Electrochemistry: A Step toward Mediator-Free Digital Electrochemical Sensors.

The current blockade particle impact method opens a route toward highly parallelized single-entity electrochemical assays. An important limitation is, however, that a redox mediator must be present in the sample, which can detrimentally interfere with molecular recognition processes. Dissolved O2 that is naturally present in aqueous solutions under ambient conditions can in principle serve as a suitable mediator via the oxygen reduction reaction (ORR). Here, we demonstrate the validity of this concept by performing current blockade experiments to capture and detect individual microparticles at Pt microelectrodes using solely the ORR. The readout modality is independent of the absolute O2 concentration, allowing operation under varying conditions. We further determine how the trajectories of individual microparticles are influenced by the combination of electrophoresis and electroosmotic flows and how these can be utilized to provide continuous detection of cationic particles in water for environmental monitoring.

Multiplex In Situ Hybridization in the Study of Acute Kidney Injury : Multiplex In Situ Hybridization in AKI.

Recently developed in situ hybridization (ISH) methods, such as RNAscope™, have greatly expanded the accessibility and usefulness of ISH in biomedical research. Among many other advantages over traditional ISH, these newer methods enable the simultaneous use of multiple probes, including combination with antibody or lectin staining. We herein illustrate the application of RNAscope™ multiplex ISH in the study of the adapter protein Dok-4 in acute kidney injury (AKI). Specifically, we used multiplex ISH to define the expression of Dok-4 and some of its putative binding partners, together with nephron segment markers, as well as markers of proliferation and tubular injury. We also illustrate the use of QuPath image analysis software to perform quantitative analyses of multiplex ISH. Furthermore, we describe how these analyses can exploit the uncoupling of mRNA and protein expression in a knockout (KO) mouse created by CRISPR/CAS9-mediated frame shift to carry out highly focused molecular phenotyping studies at the single-cell level.

Integrated Glass Microfluidics with Electrochemical Nanogap Electrodes.

We present a framework for the fabrication of chip-based electrochemical nanogap sensors integrated with microfluidics. Instead of polydimethylsiloxane (PDMS), SU-8 aided adhesive bonding of silicon and glass wafers is used to implement parallel flow control. The fabrication process permits wafer-scale production with high throughput and reproducibility. Additionally, the monolithic structures allow simple electrical and fluidic connections, alleviating the need for specialized equipment. We demonstrate the utility of these flow-incorporated nanogap sensors by performing redox cycling measurements under laminar flow conditions.

Multiple pathways promote microtubule stabilization in senescent intestinal epithelial cells.

Intestinal epithelial cells are critical for gastrointestinal homeostasis. However, their function declines during aging. The aging-related loss of organ performance is largely driven by the increase in senescent cells. To date, the hallmarks and molecular mechanisms related to cellular senescence are not fully understood. Microtubules control epithelial functions, and we identified microtubule stabilization as a phenotypic marker of senescent intestinal epithelial cells. The senescence inducer determined the pathway to microtubule stabilization. Specifically, enhanced microtubule stability was associated with α-tubulin hyperacetylation or increased abundance of the microtubule-binding protein tau. We show further that overexpression of MAPT, which encodes tau, augmented microtubule stability in intestinal epithelial cells. Notably, pharmacological microtubule stabilization was sufficient to induce cellular senescence. Taken together, this study provides new insights into the molecular mechanisms that control epithelial cell homeostasis. Our results support the concept that microtubule stability serves as a critical cue to trigger intestinal epithelial cell senescence.

Involving Patient Partners in the KRESCENT Peer Review: Intent, Process, Challenges, and Opportunities.

Purpose of review: The Kidney Research Scientist Core Education and National Training (KRESCENT) is a national Canadian training program for kidney scientists, funded by the Kidney Foundation of Canada (KFOC), the Canadian Institutes of Health Research (CIHR), and the Canadian Society of Nephrology (CSN). We describe our first year of incorporating patient partners into a scientific peer-review committee, the 2017 committee to select senior research trainees and early-career kidney researchers for funding and training, in the hope that it will be helpful to others who wish to integrate the perspective of people with lived experience into the peer-review process. Sources of information: Other peer-review committees, websites, journal articles, patient partners, Kidney Foundation of Canada Research Council, Canadians Seeking Solutions and Innovations to Overcome Chronic Kidney Disease (Can-SOLVE CKD) Patient Council, participants in the 2017 Kidney Foundation of Canada KRESCENT peer-review panel. Methods: We describe our motivation, rationale, guiding principles, plans, feedback, implementation, and response. Key findings: We disseminated a "call for patient partners" 8 weeks before the meeting, seeking patients or their care givers to partner with the KRESCENT peer-review panel; we defined these people with lived experience of kidney disease as patient partners. Eight patient partners came forward and all participated as reviewers. Patient partners first participated in a webinar to learn about the function, structure, and processes of a peer-review committee. They practiced reviewing plain language summaries and giving feedback. In a subsequent teleconference, they shared and discussed their reviews. Plain language summaries were scored, overall, on the same 0-5 quality scale used by scientific reviewers. Three patient reviewers participated in some or all of the 6-hour meeting, which was conducted as usual, for this panel, by teleconference (initially audio only; from 2020 onwards by videoconference). In the meeting, the 2 assigned scientific reviewers first gave their scores, followed by the patient reviewers giving their scores, and discussion (mostly scientific, and conducted in usual scientific language). Scientific reviewers then negotiated a consensus score based on their initial scores, the discussion, patient reviewers' scores and statements, and the scientific officer's notes. Patient reviewers, scientific reviewers, and the Kidney Foundation of Canada (KFOC) were generally positive about the process. The increased length of the meeting (estimated at 1 hour) was generally thought to be acceptable. Patient reviewers also provided feedback on the methods used to incorporate patients into the research under review. These comments were concrete, insightful, and helpful. The patients did not uniformly recommend that basic scientists involve patients in their work. We did not detect bias against preclinical science, work that did not involve patients, or rarer diseases. Some patients found participation inspiring and enlightening. All participants appreciated the idea of patient partners as community witnesses to a group process committed to fairness and supportiveness. We discussed assigning formal meaningful weight to patient reviewers' assessments. Most, but not all, patients thought that the scientific reviewers were ultimately the best judges of the allocation of scarce research resources. Limitations: Patient participants tended to be Caucasian, middle class, and well educated. Because of the difficulties of travel for some people living with or supporting those living with kidney disease, our findings may not generalize fully to peer-review meetings that are conducted face to face. This is explicitly a supportive panel, committed to reviewing junior scientists with kindness as well as rigor; our findings may not generalize to panels conducted differently. We did not use formal qualitative methodology. Implications: Inclusion of patient partners as patient reviewers for the KRESCENT program peer-review panel was feasible, added value for scientific and patient reviewers, and for the funding stakeholders (CIHR, KFOC, and CSN). We were glad that we had taken this step and continue to refine the process with each successive competition.

Motif de la revue: Le KRESCENT (Kidney Research Scientist Core Education and National Training) est un programme national de formation pour les chercheurs en santé rénale financé par la Fondation canadienne du rein (FCR), les Instituts de recherche en santé du Canada (IRSC) et la Société canadienne de néphrologie (SCN). Nous décrivons notre première année d'intégration de partenaires patients dans un comité d'examen scientifique par les pairs, le comité de 2017, visant la sélection de stagiaires de recherche et de chercheurs en santé rénale en début de carrière pour le financement et la formation, dans l'espoir que cela sera utile à ceux qui souhaitent intégrer la perspective des personnes ayant une expérience vécue au processus d'examen par les pairs. Sources: Autres comités d'examen par les pairs, sites Web, articles de revues, partenaires patients, Conseil de recherche de la Fondation canadienne du rein, conseil des patients de Canadians Seeking Solutions and Innovations to Overcome Chronic Kidney Disease (CAN-SOLVE CKD), participants au comité d'examen par les pairs de la Fondation canadienne du rein de 2017. Méthodologie: Nous décrivons ce qui a motivé cette étude, notre raisonnement, nos principes directeurs, nos plans, la rétroaction, la mise en œuvre et les réponses. Principaux résultats: Nous avons diffusé un « appel à des partenaires patients ¼ huit semaines avant la réunion pour trouver des patients ou des soignants prêts à collaborer avec le comité d'examen par les pairs de KRESCENT; nous avons défini comme partenaires patients les personnes ayant une expérience vécue de maladie rénale. Huit partenaires patients ont répondu à l'appel et tous ont participé en tant qu'examinateurs. Les partenaires patients ont d'abord participé à un webinaire pour en apprendre davantage sur la fonction, la structure et les processus d'un comité d'examen par les pairs. Ils se sont ensuite entraînés à examiner des résumés en langage simple et à donner des commentaires. Lors d'une téléconférence ultérieure, ils ont partagé et discuté de leurs examens respectifs. Les résumés en langage clair ont été notés, dans l'ensemble, sur la même échelle de qualité de 0 à 5 utilisée par les examinateurs scientifiques. Trois patients examinateurs ont participé à une partie ou à la totalité de la réunion de 6 heures, qui s'est tenue comme d'habitude, pour ce panel, par téléconférence (initialement en audio seulement; par vidéoconférence à partir de 2020). Au cours de la réunion, les deux examinateurs scientifiques désignés ont d'abord donné leurs notes, puis les patients examinateurs ont donné leurs notes, et une discussion a suivi (principalement scientifique, et menée dans le langage scientifique habituel). Les examinateurs scientifiques ont ensuite négocié pour établir une note consensuelle en fonction de leurs notes initiales, de la discussion, des notes et des commentaires des patients examinateurs et des notes de l'agent scientifique.Les patients examinateurs, les examinateurs scientifiques et la Fondation canadienne du rein étaient généralement positifs à l'égard du processus. La durée accrue de la réunion (estimée à 1 heure) a généralement été jugée acceptable. Les patients examinateurs ont également fourni des commentaires sur les méthodes utilisées pour intégrer les patients à la recherche à l'étude. Ces commentaires étaient concrets, pertinents et utiles. Les patients ne recommandent pas uniformément que les scientifiques en recherche fondamentale impliquent les patients dans leur travail. Nous n'avons pas détecté de biais contre la science préclinique, les études qui n'impliquent pas de patients ou les maladies plus rares. Certains patients ont trouvé la participation inspirante et instructive. Tous les participants ont aimé l'idée des partenaires patients comme témoins communautaires d'un processus de groupe engagé dans l'équité et le soutien.Nous avons discuté de l'attribution d'un poids formel significatif aux évaluations des patients examinateurs. La plupart des patients, mais pas tous, étaient d'avis que les examinateurs scientifiques étaient en fin de compte les meilleurs juges de l'allocation des ressources limitées de la recherche. Limites: Les patients participants étaient pour la plupart de race blanche, de classe moyenne et bien éduqués. En raison des difficultés de déplacement pour certaines personnes vivant avec ou soutenant les personnes vivant avec une maladie rénale, nos résultats peuvent ne pas se généraliser entièrement aux réunions d'examen par les pairs menées en personne. Il s'agit essentiellement d'un groupe de soutien, qui s'est engagé à examiner les jeunes chercheurs avec bienveillance et rigueur; nos conclusions peuvent ne pas se généraliser à des groupes de travail menés différemment. Nous n'avons pas utilisé de méthodologie qualitative officielle. Résultats: L'inclusion de partenaires patients comme examinateurs dans un comité d'examen par les pairs du programme KRESCENT s'est avérée réalisable, et une valeur ajoutée pour les examinateurs scientifiques, les patients examinateurs et les parties responsables du financement (IRSC, FCR et SCN). Nous sommes heureux d'avoir franchi cette étape, nous continuons de raffiner le processus à chaque concours successif.

Ring Ultramicroelectrodes for Current-Blockade Particle-Impact Electrochemistry.

In current-blockade impact electrochemistry, insulating particles are detected amperometrically as they impinge upon a micro- or nanoelectrode via a decrease in the faradaic current caused by a redox mediator. A limit of the method is that analytes of a given size yield a broad distribution of response amplitudes due to the inhomogeneities of the mediator flux at the electrode surface. Here, we overcome this limitation by introducing microfabricated ring-shaped electrodes with a width that is significantly smaller than the size of the target particles. We show that the relative step size is somewhat larger and exhibits a narrower distribution than at a conventional ultramicroelectrode of equal diameter.

Electrochemical characterization of individual oil micro-droplets by high-frequency nanocapacitor array imaging.

CMOS-based nanocapacitor arrays allow local probing of the impedance of an electrolyte in real time and with sub-micron spatial resolution. Here we report on the physico-chemical characterization of individual microdroplets of oil in a continuous water phase using this new tool. We monitor the sedimentation and wetting dynamics of individual droplets, estimate their volume and infer their composition based on their dielectric constant. From measurements before and after wetting of the surface, we also attempt to estimate the contact angle of individual micron-sized droplets. These measurements illustrate the capabilities and versatility of nanocapacitor array technology.

Single-Entity Electrochemistry for Digital Biosensing at Ultralow Concentrations.

Quantifying ultralow analyte concentrations is a continuing challenge in the analytical sciences in general and in electrochemistry in particular. Typical hurdles for affinity sensors at low concentrations include achieving sufficiently efficient mass transport of the analyte, dealing with slow reaction kinetics, and detecting a small transducer signal against a background signal that itself fluctuates slowly in time. Recent decades have seen the advent of methods capable of detecting single analytes ranging from the nanoscale to individual molecules, representing the ultimate mass sensitivity to these analytes. However, single-entity detection does not automatically translate into a superior concentration sensitivity. This is largely because electrochemical transducers capable of such detection are themselves miniaturized, exacerbating mass transport and binding kinetic limitations. In this Perspective, we discuss how these challenges can be tackled through so-called digital sensing: large arrays of separately addressable single-entity detectors that provide real-time information on individual binding events. We discuss the advantages of this approach and the barriers to its implementation.

Self-Assembly of Viral Capsid Proteins Driven by Compressible Nanobubbles.

Colloidal nanobubbles occur in gas-saturated aqueous solutions following high power water electrolysis. Here the influence of nanobubble solutions on the self-assembly properties of viral capsid proteins (CP) was investigated. Interestingly, we found that gas solutions were able to trigger the self-assembly of CP of cowpea chlorotic mottle virus (CCMV) in the absence of the viral genome, most likely by acting as a negatively charged template. The process was demonstrated by three distinct techniques, namely, dynamic light scattering (DLS), atomic force microscopy (AFM), and transmission electron microscopy (TEM). Furthermore, nanobubble-induced self-assembly of viral CP was found to depend on protein concentration. Low CP concentrations led to assembly of 18 nm virus-like particles (VLPs), comparable to T = 1 (Casper and Klug triangulation number) virus capsids, whereas high CP concentrations led to 28 nm VLPs (similar to T = 3 capsids). This paves a new route for self-assembly of VLPs.

Self-Induced Convection at Microelectrodes via Electroosmosis and Its Influence on Impact Electrochemistry.

Faradaic reactions at low supporting electrolyte concentrations induce convection via electroosmotic flows. Here we combine finite-element simulations and electrochemical measurements on microparticles at ultramicroelectrodes to explore this effect. We show that convection becomes the dominant form of mass transport for experiments at low salt concentrations, violating the common assumption that convection can be neglected.

Electrochemistry in Micro- and Nanochannels Controlled by Streaming Potentials.

Fluid and charge transport in micro- and nanoscale fluidic systems are intrinsically coupled via electrokinetic phenomena. While electroosmotic flows and streaming potentials are well understood for externally imposed stimuli, charge injection at electrodes localized inside fluidic systems via electrochemical processes remains to a large degree unexplored. Here, we employ ultramicroelectrodes and nanogap electrodes to study the subtle interplay between ohmic drops, streaming currents, and faradaic processes in miniaturized channels at low concentrations of supporting electrolyte. We show that electroosmosis can, under favorable circumstances, counteract the effect of ohmic losses and shift the apparent formal potential of redox reactions. This interplay can be described by simple circuit models, such that the results described here can be adapted to other micro- and nanofluidic electrochemical systems.

Electrochemical Collisions of Individual Graphene Oxide Sheets: An Analytical and Fundamental Study.

We propose an analytical method based on electrochemical collisions to detect individual graphene oxide (GO) sheets in an aqueous suspension. The collision rate is found to exhibit a complex dependence on redox mediator and supporting electrolyte concentrations. The analysis of multiple collision events in conjunction with numerical simulations allows quantitative information to be extracted, such as the molar concentration of GO sheets in suspension and an estimate of the size of individual sheets. We also evidence by numerical simulation the existence of edge effects on a 2D blocking object.

Quasi-One-Dimensional Generator-Collector Electrochemistry in Nanochannels.

Mass transport in fluidic channels under conditions of pressure-driven flow is controlled by a combination of convection and diffusion. For electrochemical measurements the height of a channel is typically of the same order of magnitude as the electrode dimensions, resulting in complex two- or three- dimensional concentration distributions. Electrochemical nanofluidic devices, however, can have such a low height-to-length ratio that they can effectively be considered as one-dimensional. This greatly simplifies the modeling and quantitative interpretation of analytical measurements. Here we study mass transport in nanochannels using electrodes in a generator-collector configuration. The flux of redox molecules is monitored amperometrically. We observe the transition from diffusion-dominated to convection-dominated transport by varying both the flow velocity and the distance between the electrodes. These results are described quantitatively by the one-dimensional Nernst-Planck equation for mass transport over the full range of experimentally accessible parameters.