Shuo Kang

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. However, there is scope for new approaches that can broaden the range of molecular systems that we can study at this ultimate level of sensitivity and for developing new analytical methods relying on single-molecule detection. Approaches based on purely electrical detection are particularly appealing in the latter context, since they can be easily combined with microelectronics or fluidic devices on a single microchip to create large parallel assays at relatively low cost. A form of electrical signal transduction that has so far remained relatively underdeveloped at the single-molecule level is the direct detection of the charge transferred in electrochemical processes. The reason for this is simple: only a few electrons are transferred per molecule in a typical faradaic reaction, a heterogeneous charge-transfer reaction that occurs at the electrodes surface. Detecting this tiny amount of charge is impossible using conventional electrochemical instrumentation. A workaround is to use redox cycling, in which the charge transferred is amplified by repeatedly reducing and oxidizing analyte molecules as they randomly diffuse between a pair of electrodes. For this process to be sufficiently efficient, the electrodes must be positioned within less than 100 nm of each other, and the analyte must remain between the electrodes long enough for the measurement to take place. Early efforts focused on tip-based nanoelectrodes, descended from scanning electrochemical microscopy, to create suitable geometries. However, it has been challenging to apply these technologies broadly. In this Account, we describe our alternative approach based on electrodes embedded in microfabricated nanochannels, so-called nanogap transducers. Microfabrication techniques grant a high level of reproducibility and control over the geometry of the devices, permitting systematic development and characterization. We have employed these devices to demonstrate single-molecule sensitivity. This method shows good agreement with theoretical analysis based on the Brownian motion of discrete molecules, but only once the finite time resolution of the experimental apparatus is taken into account. These results highlight both the random nature of single-molecule signals and the complications that it can introduce in data interpretation. We conclude this Account with a discussion on how scientists can overcome this limitation in the future to create a new experimental platform that can be generally useful for both fundamental studies and analytical applications.

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. We demonstrate the detection of three common redox mediators at physiological salt concentrations.

Stochastic Amperometric Fluctuations as a Probe for Dynamic Adsorption in Nanofluidic Electrochemical Systems

Adsorption of analyte molecules is ubiquitous in nanofluidic channels due to their large surface-to-volume ratios. It is also difficult to quantify due to the nanometric scale of these channels. We propose a simple method to probe dynamic adsorption at electrodes that are embedded in nanofluidic channels or which enclose nanoscopic volumes. The amperometric method relies on measuring the amplitude of the fluctuations of the redox cycling current that arise when the channel is diffusively coupled to a bulk reservoir. We demonstrate the versatility of this new method by quantifying adsorption for several redox couples, investigating the dependence of adsorption on the electrode potential and studying the effect of functionalizing the electrodes with self-assembled monolayers of organothiol molecules bearing polar end groups. These self-assembled monolayer coatings are shown to significantly reduce the adsorption of the molecules on to the electrodes. The detection method is not limited to electrodes in nanochannels and can be easily extended to redox cycling systems that enclose very small volumes, in particular scanning electrochemical microscopy with nanoelectrodes. It thus opens the way for imaging spatial heterogeneity with respect to adsorption, as well as rational design of interfaces for redox cycling based sensors.

Response time of nanofluidic electrochemical sensors

Nanofluidic thin-layer cells count among the most sensitive electrochemical sensors built to date. Here we study both experimentally and theoretically the factors that limit the response time of these sensors. We find that the key limiting factor is reversible adsorption of the analyte molecules to the surfaces of the nanofluidic system, a direct consequence of its high surface-to-volume ratio. Our results suggest several means of improving the response time of the sensor, including optimizing the device geometry and tuning the electrode biasing scheme so as to minimize adsorption.

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. We investigate the physical factors driving adsorption and find that this counterintuitive behavior is mediated by the anionic species in the supporting electrolyte, closely following the well-known Hofmeister series. Our results provide foundations both for theoretical studies of the underlying mechanisms and for contriving strategies to control adsorption in micro/nanoscale electrochemical transducers where surface effects are dominant.

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. In so doing, we generalize previous results obtained in the context of scanning electrochemical microscopy and obtain simple analytical results that are generally applicable to experimental situations where efficient redox cycling takes place.

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.

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.

Response time of electrochemical nanofluidic sensors

Nanofluidic thin-layer cells count among the most sensitive electrochemical sensors built to date. Here we study both experimentally and theoretically the factors that limit the response time of these sensors. We find that the key limiting factor is reversible adsorption of the analyte molecules to the surfaces of the nanofluidic system, a direct consequence of its high surface-to-volume ratio. Our results suggest several means of improving the response time of the sensor, including optimizing the device geometry and tuning the electrode biasing scheme so as to minimize adsorption.