Single Entity Level Research Articles

Detection of Secretion of Exosomes from Individual Cell in Real-Time by Multifunctional Nanoelectrode-Nanopore Nanopipettes

In recent years, exosomes as the intercellular vehicles have received extensive attention for their fundamental roles in biosystem and as novel biomarkers for early detection and therapeutic applications. It has been demonstrated that anti-CD63 can effectively target exosomes from cancer cells. In this work, multifunctional nanoelectrode-nanopore nanopipettes (MNNPs) were prepared to detect exosomes secreted from human cervical cancer cells (HeLa cells) at the single-entity level using the nanopore- based resistive pulse sensing technique. Synthetic liposomes were also measured for comparison. By modifying the anti-CD63 on the carbon nanoelectrode surface near the nanopore, the detected current signals were very different from the signals from the non-modified MNNPs. The differences were induced by the specific interactions between anti-CD63 and the membrane protein CD63 at the surface of exosomes. No obvious differences were observed when liposomes were tested. Furthermore, by utilizing micromanipulation technology, the position of the nanopipette tip could be accurately located, which enabled monitoring the secretion of exosomes from individual cells in a real-time manner. In the control experiment, the probe-to-cell distance was optimized and the HeLa cells incubated with exosomes inhibitor (GW4869) were tested by MNNPs. These significantly reduced translocation signals proved that anti-CD63 modified MNNPs were competent for exosomes detection in complex cellular environments. A glass nanopipette based extracellular exosome detection method was developed. Multifunctional nanopore-nanoelectrodes nanoprobes (MNNPs) were prepared with the modification of antibody at the nanoelectrode surface for the specific detection of exosomes. The focus of this research is that MNNPs can ensure cell viability and detect exosomes secreted from individual human cervical cancer (HeLa) cells in real time by nanopore-based resistance pulse sensing method. The reported results demonstrate that the MNNPs can selectively enrich and detect exosomes in a complex cellular environment, and have the potential to be applied to the study of various nanosale biological entities.

Single-entity electrochemistry at confined sensing interfaces

Measurements at the single-entity level provide more precise diagnosis and understanding of basic biological and chemical processes. Recent advances in the chemical measurement provide a means for ultra-sensitive analysis. Confining the single analyte and electrons near the sensing interface can greatly enhance the sensitivity and selectivity. In this review, we summarize the recent progress in single-entity electrochemistry of single molecules, single particles, single cells and even brain analysis. The benefits of confining these entities to a compatible size sensing interface are exemplified. Finally, the opportunities and challenges of single entity electrochemistry are addressed.

Optical monitoring of the electrochemical nucleation and growth of silver nanoparticles on electrode: From single to ensemble nanoparticles inspection

The electrochemical nucleation and growth of nanoparticles (NPs) on an electrode surface at a constant potential is traditionally followed by recording the resulting current density during the experiment. The obtained chronoamperometric transients are average measurements, making it difficult to separate individual NP behaviors and to study their cross-talks. Herein, the recently developed Backside Absorbing Layer Microscopy (BALM) is employed to monitor optically in situ and operando the electrodeposition of silver NPs. This latter technique exploits a pseudo-antireflective and metallic contrast layer and allows both sub-nanometer vertical and sub-micrometer spatial resolutions. The information from the recorded movies is readily exploited to study the NP electrodeposition at the single entity level. The image sequences allow quantifying the local electrodeposition of nanomaterials onto the electrode surface, probing the NP dynamics through the extraction of single optical NP growth transients, and analyzing the effect of the neighboring nuclei on the growth of individual NPs.

Nanoimpact Based Single-Entity Detection of Proteins using a Nanopore-Nanoelectrode Nanopipette

Nanopores and nanoelectrodes are two promising electrochemical methods for single entities analysis. Combining these two methods in one nanopipette apex allows us to simultaneously monitor the signal changes at nanopore and nanoelectrode when a biomolecule enters the nanopore or collides at the nanoelectrode. Here, I present a facile electrical method of detecting proteins at the single-molecule level in the solution based on the nanoimpact events at the nanoelectrode. Proteins such as Horse Spleen Ferritin, Bovine Hemoglobin, and Lysozyme are used to test the method. Open circuit potential (OCP) changes are detected from the nanoimpact events due to the collision of individual proteins at or near the vicinity of the floating nanoelectrode. Compared to the commonly used amperometric method, the detected OCP changes are more sensitive showing a bigger detection range and a higher time resolution. The new nanopipette based potentiometric method provides new opportunities to study various biological entities at a single-entity level with close to physiological conditions.

An ultrasensitive photoelectrochemical platform for quantifying photoinduced electron-transfer properties of a single entity.

Understanding the photoinduced electron-transfer process is of paramount importance for realizing efficient solar energy conversion. It is rather difficult to clarify the link between the specific properties and the photoelectrochemical performance of an individual component in an ensemble system because data are usually presented as averages because of interplay of the heterogeneity of the bulk system. Here, we report a step-by-step protocol to fabricate an ultrasensitive photoelectrochemical platform for real-time detection of the intrinsic photoelectrochemical behaviors of a single entity with picoampere and sub-millisecond sensitivity. Using a micron-thickness nanoparticulate TiO2-filmed Au ultramicroelectrode (UME) as the electron-transport electrode, photocurrent transients can be observed for each individual dye-tagged oxide semiconductor nanoparticle collision associated with a single-entity photoelectrochemical reaction. This protocol allows researchers to obtain high-resolution photocurrent signals to quantify the photoinduced electron-transfer properties of an individual entity, as well as to precisely process the data obtained. We also include procedures for dynamic light scattering (DLS) analysis, transmission electron microscopy (TEM) imaging and collision frequency-concentration correlation to confirm that the photoelectrochemical collision events occur at an unambiguously single-entity level. The time required for the entire protocol is ~36 h, with a single-entity photoelectrochemical measurement taking <1 h to complete for each independent experiment. This protocol requires basic nanoelectrochemistry and nanotechnology skills, as well as an intermediate-level understanding of photoelectrochemistry.

Wireless nanopore electrodes for analysis of single entities

Measurements of a single entity underpin knowledge of the heterogeneity and stochastics in the behavior of molecules, nanoparticles, and cells. Electrochemistry provides a direct and fast method to analyze single entities as it probes electron/charge-transfer processes. However, a highly reproducible electrochemical-sensing nanointerface is often hard to fabricate because of a lack of control of the fabrication processes at the nanoscale. In comparison with conventional micro/nanoelectrodes with a metal wire inside, we present a general and easily implemented protocol that describes how to fabricate and use a wireless nanopore electrode (WNE). Nanoscale metal deposition occurs at the tip of the nanopipette, providing an electroactive sensing interface. The WNEs utilize a dynamic ionic flow instead of a metal wire to sense the interfacial redox process. WNEs provide a highly controllable interface with a 30- to 200-nm diameter. This protocol presents the construction and characterization of two types of WNEs-the open-type WNE and closed-type WNE-which can be used to achieve reproducible electrochemical measurements of single entities. Combined with the related signal amplification mechanisms, we also describe how WNEs can be used to detect single redox molecules/ions, analyze the metabolism of single cells, and discriminate single nanoparticles in a mixture. This protocol is broadly applicable to studies of living cells, nanomaterials, and sensors at the single-entity level. The total time required to complete the protocol is ~10-18 h. Each WNE costs ~$1-$3.

NADPH As a Sole Reducing Agent for Silver Nanoparticle Formation - TEM and Electrochemical Insight at the Ensemble and Single Entity Level

The ability of biomolecules to reduce metal salts to nanoparticles has been repeatedly demonstrated. However, the underlying mechanism of the occurring redox reaction is still vague. We present our recent results, demonstrating that NADPH, a major redox shuttle in organisms, can solely serve as a reducing agent, leading to the formation of monodispersed silver nanoparticles. We further show that this process does not depend on enzymatic catalysis which is the currently accepted route for biological synthesis of nanoparticles. We support our results with UV-vis spectroscopy, TEM images and electrochemical measurements at the ensemble and single entity level.

Advanced Nanoscale Approaches to Single-(Bio)entity Sensing and Imaging.

Individual (bio)chemical entities could show a very heterogeneous behaviour under the same conditions that could be relevant in many biological processes of significance in the life sciences. Conventional detection approaches are only able to detect the average response of an ensemble of entities and assume that all entities are identical. From this perspective, important information about the heterogeneities or rare (stochastic) events happening in individual entities would remain unseen. Some nanoscale tools present interesting physicochemical properties that enable the possibility to detect systems at the single-entity level, acquiring richer information than conventional methods. In this review, we introduce the foundations and the latest advances of several nanoscale approaches to sensing and imaging individual (bio)entities using nanoprobes, nanopores, nanoimpacts, nanoplasmonics and nanomachines. Several (bio)entities such as cells, proteins, nucleic acids, vesicles and viruses are specifically considered. These nanoscale approaches provide a wide and complete toolbox for the study of many biological systems at the single-entity level.

Precise Placement of Microbubble Templates at Single Entity Resolution.

Microbubbles have been used as a soft template to produce hollow structures for diverse applications in chemistry, materials science, and biomedicine. It is a challenge, however, to control their size and position at single-entity level. We report on an on-demand method to produce and place a single microbubble with programmed size and position. The method exploits scanning an electrolyte-filled micropipette to place a hydrogen (H2) bubble, generated by water electrolysis, on the desired position. The bubble growth is self-limited after the bubble size fits to the pipet aperture, yielding well-controlled bubble size. The bubble growth dynamics within the pipet is successfully investigated by a methodology that combines phase-contrast X-ray imaging and electric-current measurement. We show that the microbubbles, accurately controlled in size and position, can be used for the fabrication of various polypyrrole microcontainer arrays. We expect the scanning-pipet strategy could be generalized for manipulating various soft materials at will.

Simultaneous Opto- and Spectro-Electrochemistry: Reactions of Individual Nanoparticles Uncovered by Dark-Field Microscopy.

Despite the frequent use of silver nanoparticles in consumer products and medical treatments, their reactivity and degradation in aqueous suspensions are still under debate. Here we elucidate this reactivity by an in situ opto- and spectro-electrochemical approach. Using dark-field microscopy coupled to a spectrophotometer and to an electrochemical cell, redox reactions of individual silver nanoparticles are studied in the presence of chloride. The intensity and spectral position of the plasmon resonance of an individual particle are tracked simultaneously in real time during cyclic voltammetry. They both change almost instantaneously with the detected current in a chemically reversible way. Thus, it is evidenced that the intensity decrease of the optical signal at the silver peak position is caused by the reversible formation of silver chloride and not by dissolution of silver. Moreover, at large positive potentials, further transformation to silver oxide or chlorite is revealed spectroscopically, although the electrochemical current is hidden by water and chloride oxidation. Thus, the combination of electrochemistry with dark-field microscopy and hyperspectral imaging is introduced as a new tool for real-time analysis of (electro-)chemical reactions of nanoparticles on a single-entity level.

Modelling Banking Commissions: An Application to the Italian Banking System

This paper presents a procedure for the modelling of net bank commissions (Non Interest Rate Income) as a function of macroeconomic variables. The methodology used to estimate the models is based on a Bayesian approach (BACE) method which provides the degree of relevance for each macroeconomic variable, determining its impact in explaining the evolution of the commissions. Uses of the procedure described above range from internal management purposes to regulatory and supervisory stress testing. A quantitative analysis of the commissions is presented for the Italian banking system, at an aggregate level, and for a sample of Italian banks, at single entity level.

A materials driven approach for understanding single entity nano impact electrochemistry

Summary We discuss here the design of functional nanoparticles and their study at the single entity level using nanoparticle impact electroanalysis (NIA). In particular, developments in electrochemical amplification (ECA) and transformative nanoparticle impact analysis (T-NIA) will be described that enable a better characterization of functional nanomaterials and their reactivity in their native (liquid) environment. This review highlights some outstanding works from the past two years that demonstrate the rapid progression in single entity nano-impact electrochemistry and discusses the remaining challenges to be overcome.

Catalytic activity of catalase-silica nanoparticle hybrids: from ensemble to individual entity activity.

We demonstrate the electrochemical detection and characterization of individual nanoparticle-enzyme hybrids. Silica nanoparticles were functionalized with catalase enzyme and investigated spectroscopically and electrochemically. The catalytic activity of the hybrids towards hydrogen peroxide decomposition was comparable to the activity of a freely diffusing enzyme in solution, exhibiting a Michaelis-Menten constant of KM = 74 mM and a turnover number of kcat = 8 × 107 s-1 per NP. The fast turnover number of the hybrid further enabled the electrochemical detection of individual nanoparticle-enzyme hybrid via a novel method: the hydrogen peroxide substrate was generated at a microelectrode which enabled enzymatic activity exclusively within the diffusion layer of the electrode. The method is the first electrochemical approach for measuring hybrid nanoparticles, at the single entity level.

Electrochemical Red Blood Cell Counting: One at a Time

We demonstrate that the concentration of a red blood cell solution under physiological conditions can be determined by electrochemical voltammetry. The magnitude of the oxygen reduction currents produced at an edge-plane pyrolytic graphite electrode was diagnosed analytically at concentrations suitable for a point-of-care test device. The currents could be further enhanced when the solution of red blood cells was exposed to hydrogen peroxide. We show that the enhanced signal can be used to detect red blood cells at a single entity level. The method presented relies on the catalytic activity of red blood cells towards hydrogen peroxide and on surface-induced haemolysis. Each single cell activity is expressed as current spikes decaying within a few seconds back to the background current. The frequency of such current spikes is proportional to the concentration of cells in solution.

Electrochemical Red Blood Cell Counting: One at a Time

AbstractWe demonstrate that the concentration of a red blood cell solution under physiological conditions can be determined by electrochemical voltammetry. The magnitude of the oxygen reduction currents produced at an edge‐plane pyrolytic graphite electrode was diagnosed analytically at concentrations suitable for a point‐of‐care test device. The currents could be further enhanced when the solution of red blood cells was exposed to hydrogen peroxide. We show that the enhanced signal can be used to detect red blood cells at a single entity level. The method presented relies on the catalytic activity of red blood cells towards hydrogen peroxide and on surface‐induced haemolysis. Each single cell activity is expressed as current spikes decaying within a few seconds back to the background current. The frequency of such current spikes is proportional to the concentration of cells in solution.

Enhancement of static incubation time in microfluidic cell culture platforms exploiting extended air–liquid interface

Microfluidics based cell culture applications have facilitated the study of cellular dynamics at the single entity level. Yet, long term versions of such applications in a static framework suffer from the fast exhaustion of available oxygen, dissolved in the limited media volume available per cell, within the microconfined environment. In order to circumvent such drawbacks, we have improvised a microfluidic cell culture platform for prolonged sustenance of adherent mammalian cells by formation of an air-liquid interface through functionalizing inner surfaces of a polydimethylsiloxane (PDMS) based microdevice. We have demonstrated an augmented static incubation time for different cell lines using this approach.

Applying a network level in environmental impact assessments

Researchers and society devote increasing interest to environmental impact assessments. The study here discusses and questions current assessment models by relating them to inter-organizational network analyses, and demonstrates that single entities as the basis for environmental impact assessments may not be in the best interests of society. Three case studies focusing on logistical solutions illustrate environmental effects on a single-entity and a network level. The paper concludes that considering environmental impacts on a single-entity level disregards indirect effects, which in turn has consequences for the environment. The paper points to the importance of identifying the appropriate level for analysis of environmental impacts since the single entity as the basis for assessments may undermine environmentally friendly intentions.

Quantum dots as single-photon sources: Antibunching via two-photon excitation

Two-photon excitation induced photoluminescence (2PE-PL) microscopy of CdSe colloidal quantum dots at the single-entity level is demonstrated. We provide evidence for single nanoparticle microscopy in the two-photon excitation regime by varying the laser excitation average power, as well as by measuring the confocal point spread function in three dimensions with a single quantum dot. Model calculations of the point spread function are in good agreement with our experimental findings in the 2PE-PL nonsaturation regime. Ultimately, we observe photon antibunching and triggered single-photon emission at room temperature from those quantum nanostructures under two-photon excitation in a well-defined three-dimensional photonic volume.

Nonlinear nonlocal classical field theory of quantum phenomena

It is generally thought that there is a mysterious dividing line between classical physics and quantum physics: one objective, deterministic and continuous; the other discrete, indeterminate and subjective even at the single entity level. Such a division seems to be provisional and unsatisfactory in natural philosophy, and physicists never stopped to search for a deterministic and objective substratum of quantum phenomena. There is a recent revival of thought that matter and light and their interactions can be described by the ideas of classical field theories of continuous media and yet all the quantum effects can be reproduced, when the Dirac field for the electron is treated as a classical complex field. The discreteness in quantum physics is on the same footing as the discrete frequencies of a membrane due to the boundary conditions, and this discreteness disappears when radiative effects are included. The nonlinear nature of coupled matter and field equations plays a crucial role. Furthermore, the particle mechanics is a limit of a deterministic quantum theory of single events which is distinct from the quantum theory of repeated events for which we use probabilistic concepts.