Mario Castaño-Álvarez

Electroactive intercalators for DNA analysis on microchip electrophoresis

Miniaturized analytical systems, especially microchip CE (MCE), are becoming a promising tool for analytical purposes including DNA analysis. These microdevices require a sensitive and miniaturizable detection system such as electrochemical detection (ED). Several electroactive DNA intercalators, including the organic dye methylene blue (MB), anthraquinone derivatives, and the metal complexes Fe(phen)32+ and Ru(phen)32+, have been tested for using in combination with thermoplastic olefin polymer of amorphous structure (Topas) CE‐microchips and ED. Two end‐channel approaches for integration of gold wire electrodes in CE‐ED microchip were used. A 250 μm diameter gold wire was manually aligned at the outlet of the separation channel. A new approach based on a guide channel for integration of 100 and 50 μm diameter gold wire has been also developed in order to reduce the background current and the baseline noise level. Modification of gold wire electrodes has been also tested to improve the detector performance. Application of MCE‐ED for ssDNA detection has been studied and demonstrated for the first time using the electroactive dye MB. Electrostatic interaction between cationic MB and anionic ssDNA was used for monitoring the DNA on microchips. Thus, reproducible calibration curves for ssDNA were obtained. This study advances the feasibility of direct DNA analysis using CE‐microchip with ED.

Fabrication of SU-8 based microchip electrophoresis with integrated electrochemical detection for neurotransmitters.

A new SU-8 based microchip capillary electrophoresis (MCE) device has been developed for the first time with integrated electrochemical detection. Embedded electrophoretic microchannels have been fabricated with a multilayer technology based on bonding and releasing steps of stacked SU-8 films. This technology has allowed the monolithic integration in the device of the electrochemical detection system based on platinum electrodes. The fabrication of the chips presented in this work is totally compatible with reel-to-reel techniques, which guarantee a low cost and high reliability production. The influence of relevant experimental variables, such as the separation voltage and detection potential, has been studied on the SU-8 microchip with an attractive analytical performance. Thus, the effective electrical isolation of the end-channel amperometric detector has been also demonstrated. The good performance of the SU-8 device has been proven for separation and detection of the neurotransmitters, dopamine (DA) and epinephrine (EP). High efficiency (30,000-80,000 N/m), excellent precision, good detection limit (450 nM) and resolution (0.90-1.30) has been achieved on the SU-8 microchip. These SU-8 devices have shown a better performance than commercial Topas (thermoplastic olefin polymer of amorphous structure) microchips. The low cost and versatile SU-8 microchip with integrated platinum film electrochemical detector holds great promise for high-volume production of disposable microfluidic analytical devices.

New analytical portable instrument for microchip electrophoresis with electrochemical detection

A new portable instrument that includes a high voltage power supply, a bipotentiostat, and a chip holder has been especially developed for using microchips electrophoresis with electrochemical detection. The main unit of the instrument has dimensions of 150×165×70 mm (w×d×h) and consists of a four‐outputs high voltage power supply with a maximum voltage of ±3 KV and an acquisition system with two channels for dual amperometric (DC or pulsed amperometric detection) detection. Electrochemical detection has been selected as signal transduction method because it is relatively easily implemented, since nonoptical elements are required. The system uses a lithium‐ion polymer battery and it is controlled from a desktop or laptop PC with a graphical user interface based on LabVIEW connected by serial RS232 or Bluetooth®. The last part of the system consists of a reusable chip holder for housing the microchips, which contain all the electrical connections and reservoirs for making the work with microchips easy. The performance of the new instrument has been evaluated and compared with other commercially available apparatus using single‐ and dual‐channel pyrex microchips for the separation of the neurotransmitters dopamine, epinephrine, and 3,4‐dihydroxy‐L‐phenyl‐alanine. The reduction of the size of the instrument has not affected the good performance of the separation and detection using microchips electrophoresis with electrochemical detection. Moreover, the new portable instrument paves the way for in situ analysis making the use of microchips electrophoresis easier.

Fabrication and evaluation of single-and dual-channel (Π-design) microchip electrophoresis with electrochemical detection

A new dual-channel microchip capillary electrophoresis (MCE) has been developed on glass substrates for the first time with electrochemical detection. Dual-channel (called Pi-design) as well as single-channel microchips have been fabricated on soda-lime glass using photolithography, wet etching and thermal bonding. Moreover, a laser writing system has been applied for the fabrication of photomasks with the different microchip designs (single- and dual-channel configurations). The microfabricated channels have been characterized by optical, confocal and scanning electron microscopy. The resulting single- and dual-channel microchips have been evaluated using an end-channel amperometric detector based on one (single-channel) or two (dual-channel) 100-mum gold wires aligned at the outlet of the separation channel. Parameters affecting the separation of several phenolic compounds (dopamine, p-aminophenol and hydroquinone) have been studied in the glass microchips. Thus, the influence of separation voltage, detection potential and background electrolyte has been examined in the single-channel microchip. Different total length microchannel has been compared. Furthermore, the possibility of carrying out two simultaneous measurements has been demonstrated in the new dual-channel microchip electrophoresis. The injection format has been checked and resulted to be critical, in such a way that a special and new form is employed for obtaining simultaneous signals at both channels. Analytical characteristics, such as sensitivity and reproducibility have been evaluated and resulted very adequate.

Smart portable electrophoresis instrument based on multipurpose microfluidic chips with electrochemical detection

A second generation of a battery‐powered portable electrophoresis instrument for the use of ME with electrochemical detection was developed. As the first‐generation, the main unit of the instrument (150 mm × 165 mm × 95 mm) consists of four‐outputs high‐voltage power supply (HVPS) with maximum voltage of 3 KV and acquisition system (bipotentiostat) containing 2‐channels for dual electrochemical detection. A new reusable microfluidic platform was designed in order to incorporate the microchips with the portable instrument. In this case, the platform is integrated to the main unit of the instrument so that it is not necessary to have any external cable for the interconnection of both parts, making the use of the complete system easier. The new platform contains all the electrical connections for the HVPS and bipotentiostat, as well as fluidic ports for driving the solutions. The microfluidic electrophoresis instrument is controlled by means of a user‐friendly interface from a computer. The possibility of wireless connection (Bluetooth®) allows the use of the instrument without any external cable improving the portability. Therefore, the second generation brings a more compact and integrated electrophoresis instrument for “in situ” applications using microfluidic chips in an easy way. The performance of the electrophoresis system was initially evaluated using single‐ and dual‐channel SU‐8/Pyrex microchips with different models of integrated electrodes including microelectrodes and interdigitated arrays. The method was tested in different analytical applications such as separation of neurotransmitters, chlorophenols, purine derivatives, vitamins, polyphenolic acids, and flavones.

Fast and reliable urine analysis using a portable platform based on microfluidic electrophoresis chips with electrochemical detection

A novel ready-to-use portable microfluidic platform was adapted for analysis of uric acid and related compounds in urine samples. Microfluidic devices, especially microchips electrophoresis (ME), are very attractive for clinical and pharmaceutical analysis. Thus, a novel portable and easy-to-handle instrument, HVStat (165 × 150 × 85 mm), based on microfluidic electrophoresis chips with amperometric detection was used for the determination of uric acid and interfering compounds (ascorbic acid, paracetamol, epinephrine…) in urine samples. Moreover, the microfluidic platform performance is controlled by a user-friendly PC interface (MicruX® Manager) especially designed for the use of microchips electrophoresis with electrochemical detection. The adapted analysis methodology at portable microfluidic platform allows the separation and detection of uric acid and related compounds in less than 90s with minimal sample pre-treatment. Thus, the uric acid is directly detected without previous enzymatic based-reactions or other complex pretreatment. The urine sample is simply diluted in the buffer solution and injected directly in the microchip where the uric acid is separated and detected at the platinum electrode of a SU-8/Pyrex microfluidic chip. The microfluidic chips were used for several analyses with a good performance and precision, decreasing drastically the cost and time per analysis. Thus, the complete microfluidic platform, including the main instrument, reusable holder and microchips, has been demonstrated as an excellent analytical tool for fast and reliable urine analysis.

MCE-electrochemical detection for following interactions of ssDNA and dsDNA with methylene blue

The interaction between the organic dye, methylene blue and DNA has been studied by MCE with electrochemical detection. Interaction produces two different signals, one corresponding to free methylene blue and other, for the complex methylene blue–DNA. The hybridization between a ssDNA and a complementary sequence, specific to the severe acute respiratory syndrome virus, has been performed and studied in a thermoplastic olefin polymer of amorphous structure CE‐microchip with an end‐channel gold wire detector. Moreover, studies with a longer dsDNA, an expression vector involved in the transitory or stable expression in mammals cells, pFLAG‐CMV4, has also been performed.

Multiple-point electrochemical detection for a dual-channel hybrid PDMS-glass microchip electrophoresis device

A new PDMS‐based dual‐channel MCE with multiple‐point amperometric detection has been evaluated. Electrophoresis has been optimised in a single‐channel device. Pretreatment with 0.1 M NaOH is very important for increasing and stabilising the EOF. The precision is adequate for a days work in terms of both peak current and migration time. The RSD of the peak current for five successive signals was 1.9, 2.4 and 3.1% for dopamine, p‐aminophenol and hydroquinone. RSD for the migration time was always less than 1.3%, which demonstrates the stability of the EOF and the possibility of running multiple experiments in the same microchip. The adequate inter‐microchip precision as well as the rapid and simple manufacturing procedure indicates the disposable nature of the PDMS microchips. A dual‐channel device with very simple multiple‐point amperometric detection is proposed here. Elasticity of the PDMS allows removing the polymer slightly and aligning gold wires working electrodes. Injection can be performed from each of the sample reservoirs or from both simultaneously. The distance between the separation channels is critical for obtaining adequate signals as well as the introduction of a high‐voltage electrode in the buffer reservoir. Simultaneous measurement of the same analytes in both channels is possible by applying the same potential. Moreover, since no cross‐separation is produced, different analytes or samples can be simultaneously measured.

Analytical Performance of CE Microchips with Amperometric Detection

Abstract Miniaturized Total Analysis Systems (µTAS), such as microchip electrophoresis devices are promising tools for analytical purposes. Capillary electrophoresis (CE) microchips made of various materials, e.g., poly(methylmethacrylate) (PMMA). Topas (thermoplastic olefin polymer of amorphous structure), and glass have been evaluated in this work. An electrochemical detection that combines adequately with these polymeric and glass microchips has been employed. The performance of amperometric detectors based on an end‐channel configuration was tested. Thus, integration and alignment of gold/platinum wire and thick‐film (screen‐printed carbon electrodes) working electrodes was studied. In order to verify the correct alignment of the end‐channel metal‐wire detector, a gold‐based dual electrode detector was also employed. The dual detector was based on a gold film within the separation channel and a gold wire at the end of the channel. The microchip pretreatment, which can affect the charge on the channel surface and electroosmotic flow (EOF), has been discussed, as well as the separation and injection performance.

Interdigitated ring electrodes: Theory and experiment

Abstract The oxidation of potassium ferrocyanide, K4Fe (CN)6, in aqueous solution under fully supported conditions is carried out at interdigitated band and ring electrode arrays, and compared to theoretical models developed to simulate the processes. Simulated data is found to fit well with experimental results using literature values of diffusion coefficients for Fe ( CN ) 6 4 - and Fe ( CN ) 6 3 - . The theoretical models are used to compare responses from interdigitated band and ring arrays, and the size of ring array required to approximate the response to a linear band array is investigated. An equation is developed for the radius of ring required for a pair of electrodes in a ring array to give a result with 5% of a pair of electrodes in a band array. This equation is found to be independent of the scan rate used over six orders of magnitude.