Electrochemistry is ideally suited to serve as a detection mechanism in miniaturized analysis systems. A significant hurdle can, however, be the implementation of reliable micrometer-scale reference electrodes. In this tutorial review, we introduce the principal challenges and discuss the approaches that have been employed to build suitable references. We then discuss several alternative strategies aimed at eliminating the reference electrode altogether, in particular two-electrode electrochemical cells, bipolar electrodes and chronopotentiometry.

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.

In nanofluidic electrochemical sensors based on redox cycling, zeptomole quantities of analyte molecules can be detected as redox-active molecules travel diffusively between two electrodes separated by a nanoscale gap. These sensors are employed to study the properties of multiferrocenylic compounds in nonpolar media, 2,3,4-triferrocenylthiophene and 2,5-diferrocenylthiophene, which display well-resolved electrochemically reversible one-electron transfer processes. Using stochastic analysis, we are able to determine, as a function of the oxidation states of a specific redox couple, the effective diffusion coefficient as well as the faradaic current generated per molecule, all in a straightforward experiment requiring only a mesoscopic amount of molecules in a femtoliter compartment. It was found that diffusive transport is reduced for higher oxidation states and that analytes yield very high currents per molecule of 15 fA.

Electrochemistry provides a powerful sensor transduction and amplification mechanism that is highly suited for use in integrated, massively parallelized assays. Here, the cyclic voltammetric detection of flexible, linear poly(ethylene glycol) polymers is demonstrated, which have been functionalized with redox-active ferrocene (Fc) moieties and surface-tethered inside a nanofluidic device consisting of two microscale electrodes separated by a gap of <100 nm. Diffusion of the surface-bound polymer chains in the aqueous electrolyte allows the redox groups to repeatedly shuttle electrons from one electrode to the other, resulting in a greatly amplified steady-state electrical current. Variation of the polymer length provides control over the current, as the activity per Fc moiety appears to depend on the extent to which the polymer layers of the opposing electrodes can interpenetrate each other and thus exchange electrons. These results outline the design rules for sensing devices that are based on changing the polymer length, flexibility, and/or diffusivity by binding an analyte to the polymer chain. Such a nanofluidic enabled configuration provides an amplified and highly sensitive alternative to other electrochemical detection mechanisms.

In order to analyze DNA, it must usually be amplified, i.e. replicated into several copies. As illustrated in Figure 1.1b, this involves "unzipping" the molecule and splitting the two individual strands so that they can act as templates for new copies. In the presence of excess nucleotides, these individual strands can be grown via an enzyme-assisted reaction to form two DNA molecules consisting of one new and one old chain of nucleotides wound into a double helix once again. This forms the basic principle of the so-called polymerase chain reaction (PCR) that is used to amplify DNA and is a recurring theme in this chapter. Chapter 5 introduces a new generation of nanogap sensors that are integrated with microfluidic channels based on SU-8 and glass wafer bonding. The incorporation of flow is a prerequisite for the sequential detection of multiple analytes at the singlemolecule level (scheme 1 in Figure 1.5a), and the use of glass has the further benefit of minimizing contamination. In order to demonstrate the functioning of flowincorporated devices, dopamine measurements are described. Chapter 6 demonstrates the detection of flexible, linear poly(ethylene glycol) polymers which are functionalized with redox-active moieties inside the nanogap devices. This represents the key building block of scheme 2 discussed above (Figure 1.5b). Depending on the length of the polymer, the end groups allow the transfer of