In-channel simplified decoupler with renewable electrochemical detection

Microchip CE is a useful separation technique that enables rapid detection of analytes within small sample volumes. Since the introduction of miniaturized CE systems over a decade ago [140], microchip CE has become increasingly popular, especially for high-throughput analyses, because of its low consumption of reagents, low generation of waste, high disposability and portability, the ability to integrate pre-treatment and post-separation processes, and the ability to perform multiplexed analysis [133,141-144].

Although laser-induced fluorescence (LIF) is the most commonly utilized optical detection

achieved only when the analyte is derivatized prior to analysis. Unlike optical methods, the performance of electrochemical (EC) detection systems does not decline upon miniaturizing the electrodes [131]; in addition, EC allows opaque materials to be used in the microchips [132]. Thus, EC detection for microchip CE analyses is becoming increasingly popular [133,142,148]. Of the three EC methods—amperometry [149,150], conductimetry [125,151], and voltammetry [152]—that have been applied to microchip CE detection, amperometric detection is the most popular. Examples of microchip CE-EC applications are found in enzyme assays, immunoassays, neurotransmitter analysis, clinical diagnostics, and environmental monitoring [153-156]. Many of the methods used to fabricate microfluidic channels can also be employed to construct microchip CE-EC systems. A fully integrated microchip containing electrodes for both electrophoresis and EC detection has been demonstrated [157].

There are three main approaches to integrating EC with microchip CE: end-channel, in-channel, and off-channel detection [158]. For end-channel detection, the working electrode is positioned in the exit of microchannel. The effect of the separation field on the working electrode is minimized through use of extremely low separation currents. Although the detection noise due to the separation current decreases when the working electrode is positioned further from the microchannel outlet, the detection sensitivity also decreases as a result of the loss of analytes through diffusion in the detection cell [159]. In-channel and off-channel detection systems can be employed to prevent such loss of analytes, but interference from the high electric field and electrophoretic current on the EC measurement can be problematic, leading to a larger EC background current and a shifted redox potential [158]. An in-channel amperometric detection system lacking a decoupler has been developed;

it uses an electrically isolated potentiostat [160]. In the off-channel arrangement, the separation potential must be grounded before the analyte reaches the microchannel outlet to

eliminate interference from the separated electric field and to protect the EC detector from damage resulting from surges in current. A decoupler placed in front of the working electrode can be used to protect the EC detection system from interference [106,161-164]. Currently, several types of decoupler have been fabricated, including thin film [106,161-164] and microwire [163] electrodes. A solid Pd film integrated directly into the CE microchip across the separation channel has been prepared using microfabrication techniques; it replaced the decoupler in a joint connection-type system [161]. The reason why Pd metal was adopted is that platinum (Pt)-group metals, such as Pd and Pt, effectively reduce and absorb hydrogen ions. Because molecular hydrogen diffuses faster on a Pd surface, it is eliminated from the Pd decoupler before the development of hydrogen bubbles in the electroosmotic flow (EOF), maintaining the efficiency of the decoupler [161,162]. A Pd microelectrode wire decoupler and metal wire working electrode have been integrated within a microchip CE-EC system to eliminate the microfabrication steps used to construct the electrodes [163].

The fouling of metal working electrodes is a serious concern affecting microchip CE-EC [165]. Inactivation of electrodes upon repeated injection or varying analyte concentrations affects the sensitivity, limit of detection, and reproducibility of microchips. Although surface regeneration of an electrode can improve its performance, an optimized cleaning procedure applied to a specific electrode might not be applicable to routine retuning of all electrodes.

Most CE microchips have been fabricated from glass or quartz because such materials are suitable for optical detection and readily generate EOFs. Unfortunately, the fabrication of glass substrate microchips requires clean-room processing and high-temperature thermal bonding; thus, it is difficult to perform in most general laboratories [136]. A more amenable approach is the use of polymers, such as PMMA, polycarbonate, polyethylene, and PDMS, rather than glass or silicon, to form CE microfluidic channels [137].

A simple method for integrating decouplers and working electrodes into PDMS-based

microchip CE-EC devices was presented. The procedures used to place the metal wires and assemble the microchip are illustrated in Fig. II-4. The Pt wire electrode serving as a decoupler and the Cu working electrode were incorporated into the PDMS sheet. Catechol and dopamine as analytes to demonstrate the separation efficiency of the microchip CE-EC system and to evaluate the performance of the renewable working electrode were employed.

The performance of this renewable working electrode microchip CE-EC system is shown in Fig. II-5. The extended reuse of such renewable working electrodes coupled with these simple microchip fabrication techniques will increase the usability of microchip CE-EC systems.

Figure II-4. Procedure for the fabrication of the in-channel decoupler/renewable electrode microchip CE-EC system [166].

Figure II-5. Using the working electrode to detect 250 μM dopamine during 12 sequential analyses. Electropherograms displaying the first, 10th, and 12th runs, and the first run using the renewed electrode [166].

II.2 Integrating a dry-film photoresist-based microchip with an electrochemical sensor