The nuclear pore complex (NPC) is a proteinaceous nanopore that solely and selectively regulates the molecular transport between the cytoplasm and nucleus of a eukaryotic cell. The ∼50 nm-diameter pore of the NPC perforates the double-membrane nuclear envelope to mediate both passive and facilitated molecular transport, thereby playing paramount biological and biomedical roles. Herein, we visualize single NPCs by scanning electrochemical microscopy (SECM). The high spatial resolution is accomplished by employing ∼25 nm-diameter ion-selective nanopipets to monitor the passive transport of tetrabutylammonium at individual NPCs. SECM images are quantitatively analyzed by employing the finite element method to confirm that this work represents the highest-resolution nanoscale SECM imaging of biological samples. Significantly, we apply the powerful imaging technique to address the long-debated origin of the central plug of the NPC. Nanoscale SECM imaging demonstrates that unplugged NPCs are more permeable to the small probe ion than are plugged NPCs. This result supports the hypothesis that the central plug is not an intrinsic transporter, but is an impermeable macromolecule, e.g., a ribonucleoprotein, trapped in the nanopore. Moreover, this result also supports the transport mechanism where the NPC is divided into the central pathway for RNA export and the peripheral pathway for protein import to efficiently mediate the bidirectional traffic.

A photo/electrochemical coupling interface of Ru[dcbpy]32+-AMT/Au (AMT; 5-Amino-1,3,4-thiadiazole-2-thiol) was fabricated using a dehydration condensation sulfhydrating method. For the interface functional properties, a combined dual-signal recording (CDSR) method was applied to characterize the response characteristics, and a scanning electrochemical microscopy-electrochemiluminescence (SECM-ECL) imaging was developed to assess the interface distribution uniformity. The interface biosensing compatibility was validated by constructing a simple DNA sensor. The research results show that the interaction between the two functional parameters follows a synergistic effect mechanism in the coupling conditions and an interference effect mechanism in the detection condition. Under optimized conditions, the saturation dual-signal response values are 156.0 and 86.8 μA, respectively. The statistics and imaging comparison analysis validate good interface distribution uniformity and stability performance. The DNA sensor\'s dual-signal detection limits to the signal probe (SP) are ∼30 fM and 0.3 pM with linear ranges of 100.0 fM ∼ 1.0 nM and 1.0 pM ∼ 10.0 nM, respectively. The fabricated interface exhibits an effective bi-functional response performance compatible with biosensing. The proposed imaging method has a high technical fit for studying photo/electrochemical coupling interfaces and can also provide a reference for other similar coupling interface analyses.

Scanning electrochemical microscopy (SECM) has emerged as a powerful method to quantitatively investigate the transport of molecules and ions across various biological membranes as represented by living cells. Advantageously, SECM allows for the in situ and non-destructive imaging and measurement of high membrane permeability under simple steady-state conditions, thereby facilitating quantitative data analysis. The SECM method, however, has not provided any information about the interactions of a transported species, i.e., a permeant, with a membrane through its components, e.g., lipids, channels, and carriers. Herein, we propose theoretically that SECM enables the quantitative investigation of membrane-permeant interactions by employing transient conditions. Specifically, we model the membrane-permeant interactions based on a Langmuir-type isotherm to define the strength and kinetics of the interactions as well as the concentration of interaction sites. Finite element simulation predicts that each of the three parameters uniquely affects the chronoamperometric current response of an SECM tip to a permeant. Significantly, this prediction implies that all three parameters are determinable from an experimental chronoamperometric response of the SECM tip. Complimentarily, the steady-state current response of the SECM tip yields the overall membrane permeability based on the combination of the three parameters. Interestingly, our simulation also reveals the optimum strength of membrane-permeant interactions to maximize the transient flux of the permeant from the membrane to the tip.

Three-dimensional (3D)-cultured cells have attracted the attention of researchers in tissue engineering- and drug screening-related fields. Among them, 3D cellular fibers have attracted significant attention because they can be stacked to prepare more complex tissues and organs. Cellular fibers are widely fabricated using extrusion 3D bioprinters. For these applications, it is necessary to evaluate cellular activities, such as the oxygen consumption rate (OCR), which is one of the major metabolic activities. We previously reported the use of scanning electrochemical microscopy (SECM) to evaluate the OCRs of cell spheroids. However, the SECM approach has not yet been applied to hydrogel fibers prepared using the bioprinters. To the best of our knowledge, this is the first study to evaluate the OCR of cellular fibers printed by extrusion 3D bioprinters. First, the diffusion theory was discussed to address this issue. Next, diffusion models were simulated to compare realistic models with this theory. Finally, the OCRs of MCF-7 cells in the printed hydrogel fibers were evaluated as a proof of concept. Our proposed approach could potentially be used to evaluate the OCRs of tissue-engineered fibers for organ transplantation and drug screening using in-vitro models.

Extracellular matrix (ECM) stiffness modulates a variety of cellular processes, including ferroptosis, a process with significant potential implications for hepatocellular carcinoma (HCC) fibrosis and cirrhosis. However, the exact relationship between ECM stiffness and HCC ferroptosis is yet unclarified, partially due to the lack of in situ information on key parameters of the ferroptosis process of living HCC cells. This study pioneers the use of in vitro mechanical microenvironment models of HCC and the scanning electrochemical microscopy (SECM) technique for understanding this interplay. We first cultured HuH7 cells on 4.0, 18.0, and 44.0 kPa polyacrylamide (PA) gels to simulate early, intermediate, and advanced HCC ECM stiffness, respectively. Then, we used SECM to in situ monitor changes in cell membrane permeability, respiratory activity, and reactive oxygen species (ROS) levels of erastin-induced HuH7 cells on PA gels, finding that increasing ECM stiffness potentiates ferroptosis, including increased membrane permeabilization and H2O2 release as well as reduced respiratory activity. Through further transcriptome sequencing and molecular biology measurements, we identified a critical role for focal adhesion kinase (FAK)-mediated yes-associated protein (YAP) in regulating the ferroptosis process dependent on ECM stiffness, which provides novel insights into the mechanical regulation of ferroptosis in HCC cells and may pave the way for innovative therapeutic strategies.

Cytochrome c oxidase deficiency (COXD) is an inherited disorder characterized by the absence or mutation in the genes encoding for the cytochrome c oxidase protein (COX). COX deficiency results in severe muscle weakness, heart, liver, and kidney disorders, as well as brain damage in infants and adolescents, leading to death in many cases. With no cure for this disorder, finding an efficient, inexpensive, and early means of diagnosis is essential to minimize symptoms and long-term disabilities. Furthermore, muscle biopsy, the traditional detection method, is invasive, expensive, and time-consuming. This study demonstrates the applicability of scanning electrochemical microscopy to quantify COX activity in living human fibroblast cells. Taking advantage of the interaction between the redox mediator N, N, N\', N\'-tetramethyl-para-phenylene-diamine, and COX, the enzymatic activity was successfully quantified by monitoring current changes using a platinum microelectrode and determining the apparent heterogeneous rate constant k0 using numerical modeling. This study provides a foundation for developing a diagnostic method for detecting COXD in infants, which has the potential to increase treatment effectiveness and improve the quality of life of affected individuals.

Blood is one of the most frequent and valuable traces encountered at crime scenes, where knowing the time since deposition (TSD) of bloodstains tremendously assists forensic experts to screen out crime-related evidence and aids in the reconstruction of the event sequence. Although increasing proof-of-concept methodologies for investigating the TSD of bloodstains have been reported, there is still no accepted strategy in forensic practice as the aging mechanism involves complex components, leading to the inaccuracy of the estimation results. Herein, an endogenous biomarker of alkaline phosphatase (ALP) was chosen to investigate the TSD by scanning electrochemical microscopy (SECM). Results demonstrate that the ALP activity acquired via SECM lateral scan assay exhibited a clear decrease over time, and a similar trend was observed on both poly(vinylidene fluoride) (PVDF) membrane and glass, with the aging kinetics on PVDF membrane being faster than glass. By means of quantitatively calculating the flux of generated p-aminophenol (PAP), we established the aging curve and realized the TSD estimation of blood fingerprints (BFPs) that was unable to be distinguished via optical measurements. Intriguingly, the as-obtained estimation accuracy ranged from 74.6 to 93.7%, proving the possibility of using an ALP biomarker and SECM. More appealingly, the predicted TSDs were capable of accurately differentiating the deposition sequence of overlapping BFPs, which was hardly achieved by optical means. Therefore, this proof-of-concept strategy demonstrates the value of SECM as a forensic tool and opens possibilities for revealing multidimensional information about crime.

Scanning electrochemical microscopy (SECM) is being used increasingly to monitor electrochemical processes at the interface of living cells and electrodes. This allows the detection and quantification of biomarkers that further the understanding of various diseases. Rapid SECM experiments are often carried out without monitoring the analyte solution temperature or are performed at room temperature. The reported research demonstrates that temperature control is crucial during SECM imaging of living cells to obtain reliable data. In this study, a SECM-integrated thermostatic ring on the sample stage enabled imaging of living biological cells in a constant height mode at various temperatures. Two-dimensional line scans were conducted while scanning single Adenocarcinoma Cervical cancer (HeLa) cells. Numerical modeling was carried out to evaluate the effect of the temperature on the electrochemical current response of living cells to compare the apparent heterogeneous rate constant (k0), representing cellular reaction kinetics. This study reveals that even slight temperature variations of approximately 2 °C affect the reaction kinetics of single living cells, altering the measured current during SECM.

The electron-transfer (ET) reaction of cytochrome c (Cytc) protein with biomolecules is a cutting-edge research area of interest in understanding the functionalities of natural systems. Several electrochemical biomimicking studies based on Cytc-protein-modified electrodes prepared via electrostatic interaction and covalent bonding approaches have been reported. Indeed, natural enzymes involve multiple types of bonding, such as hydrogen, ionic, covalent, and π-π, etc. In this work, we explore a Cytc-protein chemically modified glassy carbon electrode (GCE/CB@NQ/Cytc) prepared via π-π bonding using graphitic carbon as an underlying surface and an aromatic organic molecule, naphthoquinone (NQ), as a cofactor for an effective ET reaction. A simple drop-casting technique-based preparation of GCE/CB@NQ showed a distinct surface-confined redox peak at a standard electrode potential (E°) = -0.2 V vs Ag/AgCl (surface excess = 21.3 nmol cm-2) in pH 7 phosphate buffer solution. A control experiment of modification of NQ on an unmodified GCE failed to show any such unique feature. For the preparation of GCE/CB@NQ/Cytc, a dilute solution of Cytc-pH 7 phosphate buffer was drop-cast on the GCE/CB@NQ surface, wherein the protein folding and denaturalization-based complication and its associated ET functionalities were avoided. Molecular dynamics simulation studies show the complexation of NQ with Cytc at the protein binding sites. The protein-bound surface shows an efficient and selective bioelectrocatalytic reduction performance of H2O2, as demonstrated using cyclic voltammetry and amperometric i-t techniques. Finally, the redox-competition scanning electrochemical microscopy (RC-SECM) technique was adopted for in situ visualization of the electroactive adsorbed surface. The RC-SECM images clearly show the regions of highly bioelectrocatalytic active sites of Cytc-proteins bound to NQ molecules on a graphitic carbon surface. The binding of Cytc with NQ has significant implications for studying the biological electron transport mechanism, and the proposed method provides the requisite framework for such a study.

The electrochemical visualization of proteins in the plasma membrane of single fixed cells was achieved with a spatial resolution of 160 nm using scanning electrochemical cell microscopy. The model protein, the carcinoembryonic antigen (CEA), is linked with a ruthenium complex (Ru(bpy)32+)-tagged antibody, which exhibits redox peaks in its cyclic voltammetry curves after a nanopipette tip contacts the cellular membrane. Based on the potential-resolved oxidation or reduction currents, an uneven distribution of membrane CEAs on the cells is electrochemically visualized, which could only be achieved previously using super-resolution optical microscopy. Compared with current electrochemical microscopy, the single-cell scanning electrochemical cell microscopy (SECCM) strategy not only improves the spatial resolution but also utilizes the potential-resolved current from the antibody-antigen complex to increase electrochemical imaging accuracy. Eventually, the electrochemical visualization of cellular proteins at the nanoscale enables the super-resolution study of cells to provide more biological information.