Addressing spatiotemporal signal variations in pair correlation function analysis.

Fluorescence correlation spectroscopy (FCS) is a cornerstone technique in optical microscopy to measure, for example, the concentration and diffusivity of fluorescent emitters and biomolecules in solution. The application of FCS to complex biological systems, however, is fraught with inherent intricacies that impair the interpretation of correlation patterns. Critical among these intricacies are temporal variations beyond diffusion in the quantity, intensity, and spatial distribution of fluorescent emitters.

Understanding Transient Ionic Diode Currents and Impedance Responses for Aquivion-Coated Microholes.

Ionic diode based devices or circuits can be applied, for example, in electroosmotic pumps or in desalination processes. Aquivion ionomer coated asymmetrically over a Teflon film (5 μm thickness) with a laser-drilled microhole (approximately 10 μm diameter) gives a cationic diode with a rectification ratio of typically 10-20 (measured in 0.01 M NaCl with ±0.

Amperometric Monitoring of Dissolution of pH-Responsive EUDRAGIT Polymer Film Coatings.

Electrochemical sensors are powerful tools for the detection and real-time monitoring of a wide variety of analytes. However, the long-term operation of Faradaic sensors in complex media is challenging due to fouling. The protection of the electrode surface during in vivo operation is a key element for improving the monitoring of analytes.

Probing DNA - Transcription Factor Interactions Using Single-Molecule Fluorescence Detection in Nanofluidic Devices.

Single-molecule fluorescence detection offers powerful ways to study biomolecules and their complex interactions. Here, nanofluidic devices and camera-based, single-molecule Förster resonance energy transfer (smFRET) detection are combined to study the interactions between plant transcription factors of the auxin response factor (ARF) family and DNA oligonucleotides that contain target DNA response elements. In particular, it is shown that the binding of the unlabeled ARF DNA binding domain (ARF-DBD) to donor and acceptor labeled DNA oligonucleotides can be detected by changes in the FRET efficiency and changes in the diffusion coefficient of the DNA.

Electrochemiluminescence reaction pathways in nanofluidic devices.

Nanofluidic electrochemical devices confine the volume of chemical reactions to femtoliters. When employed for light generation by electrochemiluminescence (ECL), nanofluidic confinement yields enhanced intensity and robust luminescence. Here, we investigate different ECL pathways, namely coreactant and annihilation ECL in a single nanochannel and compare light emission profiles.

Switching Anionic and Cationic Semipermeability in Partially Hydrolyzed Polyacrylonitrile: A pH-Tunable Ionic Rectifier.

Membrane materials with semipermeability for anions or for cations are of interest in electrochemical and nanofluidic separation and purification technologies. In this study, partially hydrolyzed polyacrylonitrile (phPAN) is investigated as a pH-switchable anion/cation conductor. When switching from anionic to cationic semipermeability, also the ionic current rectification effect switches for phPAN materials deposited asymmetrically onto a 5, 10, 20, or 40 μm diameter microhole in a 6 μm thick polyethylene-terephthalate (PET) film substrate.

Digestion-on-a-chip: a continuous-flow modular microsystem recreating enzymatic digestion in the gastrointestinal tract.

In vitro digestions are essential for determining the bioavailability of compounds, such as nutrients. We have developed a cell-free, miniaturized enzymatic digestive system, employing three micromixers connected in series to mimic the digestive functions of the mouth, stomach and small intestine. This system continuously processes samples, e.

Enhanced annihilation electrochemiluminescence by nanofluidic confinement.

Microfabricated nanofluidic electrochemical devices offer a highly controlled nanochannel geometry; they confine the volume of chemical reactions to the nanoscale and enable greatly amplified electrochemical detection. Here, the generation of stable light emission by electrochemiluminescence (ECL) in transparent nanofluidic devices is demonstrated for the first time by exploiting nanogap amplification. Through continuous oxidation and reduction of [Ru(bpy)] luminophores at electrodes positioned at opposite walls of a 100 nm nanochannel, we compare classic redox cycling and ECL annihilation.

High-throughput, non-equilibrium studies of single biomolecules using glass-made nanofluidic devices.

Single-molecule detection schemes offer powerful means to overcome static and dynamic heterogeneity inherent to complex samples. However, probing biomolecular interactions and reactions with high throughput and time resolution remains challenging, often requiring surface-immobilized entities. Here, we introduce glass-made nanofluidic devices for the high-throughput detection of freely-diffusing single biomolecules by camera-based fluorescence microscopy.

Electrochemical sensing with single nanoskived gold nanowires bisecting a microchannel.

We suspended a single nanoskived gold nanowire in a microfluidic channel. In this preliminary report, a 200 nm-diameter nanowire was used as an electrode to perform hydrodynamic voltammetry in the center of solution flow. Suspended nanowires exhibit superior current response due to highly efficient mass transport in the area of fastest flow.

Potential-Controlled Adsorption, Separation, and Detection of Redox Species in Nanofluidic Devices.

Nanoscale channels and electrodes for electrochemical measurements exhibit extreme surface-to-volume ratios and a correspondingly high sensitivity to even weak degrees of surface interactions. Here, we exploit the potential-dependent reversible adsorption of outer-sphere redox species to modulate in space and time their concentration in a nanochannel under advective flow conditions. Induced concentration variations propagate downstream at a species-dependent velocity.

Electrochemical Amplification in Side-by-Side Attoliter Nanogap Transducers.

We report a strategy for the fabrication of a new type of electrochemical nanogap transducer. These nanogap devices are based on signal amplification by redox cycling. Using two steps of electron-beam lithography, vertical gold electrodes are fabricated side by side at a 70 nm distance encompassing a 20 attoliter open nanogap volume.

A Cationic Diode Based on Asymmetric Nafion Film Deposits.

A thin film of Nafion, of approximately 5 μm thickness, asymmetrically deposited onto a 6 μm thick film of poly(ethylene terephthalate) (PET) fabricated with a 5, 10, 20, or 40 μm microhole, is shown to exhibit prominent ionic diode behavior involving cation charge carrier ("cationic diode"). The phenomenon is characterized via voltammetric, chronoamperometric, and impedance methods. Phenomenologically, current rectification effects are comparable to those observed in nanocone devices where space-charge layer effects dominate.

Single-molecule electrochemistry in nanochannels: probing the time of first passage.

The diffusive mass transport of individual redox molecules was probed experimentally in microfabricated nanogap electrodes. The residence times for molecules inside a well-defined detection volume were extracted and the resulting distribution was compared with quantitative analytical predictions from random-walk theory for the time of first passage. The results suggest that a small number of strongly adsorbing sites strongly influence mass transport at trace analyte levels.

Bisecting Microfluidic Channels with Metallic Nanowires Fabricated by Nanoskiving.

This paper describes the fabrication of millimeter-long gold nanowires that bisect the center of microfluidic channels. We fabricated the nanowires by nanoskiving and then suspended them over a trench in a glass structure. The channel was sealed by bonding it to a complementary poly(dimethylsiloxane) structure.

Handling and Sensing of Single Enzyme Molecules: From Fluorescence Detection towards Nanoscale Electrical Measurements.

Classical methods to study single enzyme molecules have provided valuable information about the distribution of conformational heterogeneities, reaction mechanisms, and transients in enzymatic reactions when individual molecules instead of an averaging ensemble are studied. Here, we highlight major advances in all-electrical single enzyme studies with a focus on recent micro- and nanofluidic tools, which offer new ways of handling and studying small numbers of molecules or even single enzyme molecules. We particularly emphasize nanofluidic devices, which enable the integration of electrochemical transduction and detection.

Challenges of biomolecular detection at the nanoscale: nanopores and microelectrodes.

The interest in analytical devices, which typically rely on the reactivity of a biological component for specificity, is growing rapidly. In this Perspective, we highlight current challenges in all-electrical biosensing as these systems shrink toward the nanoscale and enable the detection of analytes at the single-molecule level. We focus on two sensing principles: nanopores and amperometric microelectrode devices.

Redox cycling without reference electrodes.

The reference electrode is a key component in electrochemical measurements, yet it remains a challenge to implement a reliable reference electrode in miniaturized electrochemical sensors. Here we explore experimentally and theoretically an alternative approach based on redox cycling which eliminates the reference electrode altogether. We show that shifts in the solution potential caused by the lack of reference can be understood quantitatively, and determine the requirements for accurate measurements in miniaturized systems in the absence of a reference electrode.

Integrated biodetection in a nanofluidic device.

The sensing of enzymatic processes in volumes at or below the scale of single cells is challenging but highly desirable in the study of biochemical processes. Here we demonstrate a nanofluidic device that combines an enzymatic recognition element and electrochemical signal transduction within a six-femtoliter volume. Our approach is based on localized immobilization of the enzyme tyrosinase in a microfabricated nanogap electrochemical transducer.

Nanoscale methods for single-molecule electrochemistry.

The development of experiments capable of probing individual molecules has led to major breakthroughs in fields ranging from molecular electronics to biophysics, allowing direct tests of knowledge derived from macroscopic measurements and enabling new assays that probe population heterogeneities and internal molecular dynamics. Although still somewhat in their infancy, such methods are also being developed for probing molecular systems in solution using electrochemical transduction mechanisms. Here we outline the present status of this emerging field, concentrating in particular on optical methods, metal-molecule-metal junctions, and electrochemical nanofluidic devices.

Noise phenomena caused by reversible adsorption in nanoscale electrochemical devices.

We theoretically investigate reversible adsorption in electrochemical devices on a molecular level. To this end, a computational framework is introduced, which is based on 3D random walks including probabilities for adsorption and desorption events at surfaces. We demonstrate that this approach can be used to investigate adsorption phenomena in electrochemical sensors by analyzing experimental noise spectra of a nanofluidic redox cycling device.

Reversible Adsorption of Outer-Sphere Redox Molecules at Pt Electrodes.

Adsorption often dominates the response of nanofluidic systems due to their high surface-to-volume ratios. Here we harness this sensitivity to investigate the reversible adsorption of outer-sphere redox species at electrodes, a phenomenon that is easily overlooked in bulk measurements. We find that even though adsorption does not necessarily play a role in the electron-transfer process, such adsorption is nevertheless ubiquitous for the widely used outer-sphere species.

Electrochemical single-molecule detection in aqueous solution using self-aligned nanogap transducers.

Electrochemical detection of individual molecular tags in nanochannels may enable cost-effective, massively parallel analysis and diagnostics platforms. Here we demonstrate single-molecule detection of prototypical analytes in aqueous solution based on redox cycling in 40 nm nanogap transducers. These nanofluidic devices are fabricated using standard microfabrication techniques combined with a self-aligned approach that minimizes gap size and dead volume.

Redox couples with unequal diffusion coefficients: effect on redox cycling.

Redox cycling between two electrodes separated by a narrow gap allows dramatic amplification of the faradaic current. Unlike conventional electrochemistry at a single electrode, however, the mass-transport-limited current is controlled by the diffusion coefficient of both the reduced and oxidized forms of the redox-active species being detected and, counterintuitively, by the redox state of molecules in the bulk solution outside the gap itself. Using a combination of finite-element simulations, analytical theory, and experimental validation, we elucidate the interplay between these interrelated factors.

Single-molecule electrochemistry: present status and outlook.

The development of methods for detecting and manipulating matter at the level of individual macromolecules represents one of the key scientific advancements of recent decades. These techniques allow us to get information that is largely unobtainable otherwise, such as the magnitudes of microscopic forces, mechanistic details of catalytic processes, macromolecular population heterogeneities, and time-resolved, step-by-step observation of complex kinetics. Methods based on optical, mechanical, and ionic-conductance signal transduction are particularly developed.