Carbon-fiber amperometry has been extensively used to monitor the time course of catecholamine release from cells as individual secretory granules discharge their contents during the process of quantal exocytosis, but microfabricated devices offer the promise of higher throughput. Here we report development of a microchip device that uses transparent indium tin oxide (ITO) electrodes to measure quantal exocytosis from cells in microfluidic channels. ITO films on a glass substrate were patterned as 20-mum-wide stripes using photolithography and wet etching and then coated with polylysine to facilitate cell adherence. Microfluidic channels (100 mum wide by 100 mum deep) were formed by molding poly(dimethylsiloxane) (PDMS) on photoresist and then reversibly sealing the PDMS slab to the ITO-glass substrate. Bovine adrenal chromaffin cells were loaded into the microfluidic channel and adhered to the ITO electrodes. Cells were stimulated to secrete by perfusing a depolarizing "high-K" solution while monitoring oxidation of catecholamines on the ITO electrode beneath the cell using amperometry. Amperometric spikes with charges ranging from 0.1 to 1.5 pC were recorded with a signal-to-noise ratio comparable to that of carbon-fiber electrodes. Further development of this approach will enable high-throughput measurement of quantal catecholamine release simultaneously with optical cell measurements such as fluorescence.

This paper describes an indium tin oxide (ITO) electrode-based Ru(bpy)3(2+) electrochemiluminecence (ECL) detector for a microchip capillary electrophoresis (CE). The microchip CE-ECL system described in this article consists of a poly(dimethylsiloxane) (PDMS) layer containing separation and injection channels and an electrode plate with an ITO electrode fabricated by a photolithographic method. The PDMS layer was reversibly bound to the ITO electrode plate, which greatly simplified the alignment of the separation channel with the working electrode and enhanced the photon-capturing efficiency. In our study, the high separation electric field had no significant influence on the ECL detector, and decouplers for isolating the separation electric field were not needed in the microchip CE-ECL system. The ITO electrodes employed in the experiments displayed good durability and stability in the analytical procedures. Proline was selected to perform the microchip device with a limit of detection of 1.2 microM (S/N = 3) and a linear range from 5 to 600 microM.

Immunoaffinity monolith pretreatment columns have been coupled with capillary electrophoresis separation in poly(methyl methacrylate) (PMMA) microchips. Microdevices were designed with 8 reservoirs to enable the electrically controlled transport of selected analytes and solutions to carry out integrated immunoaffinity extraction and electrophoretic separation. The PMMA microdevices were fabricated reproducibly and with high fidelity by solvent imprinting and thermal bonding methods. Monoliths with epoxy groups for antibody immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene dimethacrylate in a porogenic solvent consisting of 70% dodecanol and 30% hexanol. Anti-fluorescein isothiocyanate (FITC) was utilized as a model affinity group in the monoliths, and the immobilization process was optimized. A mean elution efficiency of 92% was achieved for the monolith-based extraction of FITC-tagged human serum albumin. FITC-tagged proteins were purified from a contaminant protein and then separated electrophoretically using these devices. The developed immunoaffinity column/capillary electrophoresis microdevices show great promise for combining sample pretreatment and separation in biomolecular analysis.

A new method for the fabrication of an integrated microelectrode for electrochemical detection (ECD) on an electrophoresis microchip is described. The pattern of the microelectrode was directly made on the surface of a microscope slide through an electroless deposition procedure. The surface of the slide was first selectively coated with a thin layer of sodium silicate through a micromolding in capillary technique provided by a poly(dimethylsiloxane) (PDMS) microchannel; this left a rough patterned area for the anchoring of catalytic particles. A metal layer was deposited on the pattern guided by these catalytic particles and was used as the working electrode. Factors influencing the fabrication procedure were discussed. The whole chip was built by reversibly sealing the slide to another PDMS layer with electrophoresis microchannels at room temperature. This approach eliminates the need of clean room facilities and expensive apparatus such as for vacuum deposition or sputtering and makes it possible to produce patterned electrodes suitable for ECD on microchip under ordinary chemistry laboratory conditions. Also once the micropattern is ready, it allows the researchers to rebuild the electrode in a short period of time when an electrode failure occurs. Copper and gold microelectrodes were fabricated by this technique. Glucose, dopamine, and catechol as model analytes were tested.

Summary Biomarkers in human body fluids have great potential for use in screening for diseases such as cancer and diabetes, diagnosis, determining the effectiveness of treatments, and detecting recurrence. Present 96-well immunoassay technology effectively analyzes large numbers of samples; however, this approach is more expensive and less time effective on single or a few samples. In contrast, microfluidic systems are well suited for assaying small numbers of specimens in a point-of-care setting, provided suitable procedures are developed to work within peak capacity constraints when analyzing complex mixtures like human blood serum. Here, we developed integrated microdevices with an affinity column and capillary electrophoresis channels to isolate and quantitate a panel of proteins in complex matrices. To form an affinity column, a thin film of a reactive polymer was photopolymerized in a microchannel, and four antibodies were covalently immobilized to it. The retained protein amounts were consistent from chip to chip, demonstrating reproducibility. Furthermore, the signals from four fluorescently labeled proteins captured on-column were in the same range after rinsing, indicating the column has little bias toward any of the four antibodies or their antigens. These affinity columns have been integrated with capillary electrophoresis separation, enabling us to simultaneously quantify four protein biomarkers in human blood serum in the low ng/mL range using either a calibration curve or standard addition. Our systems provide a fast, integrated and automated platform for multiple biomarker quantitation in complex media such as human blood serum.

Detection and accurate quantitation of biomarkers such as alpha-fetoprotein (AFP) can be a key aspect of early stage cancer diagnosis. Microfluidic devices provide attractive analysis capabilities, including low sample and reagent consumption, as well as short assay times. However, to date microfluidic analyzers have relied exclusively on calibration curves for sample quantitation, which can be problematic for complex mixtures such as human serum. We have fabricated integrated polymer microfluidic systems that can quantitatively determine fluorescently labeled AFP in human serum, using either the method of standard addition or a calibration curve. Our microdevices couple an immunoaffinity purification step with rapid microchip electrophoresis separation with laserinduced fluorescence detection system, all under automated voltage control in a miniaturized polymer microchip. In conjunction with laser-induced fluorescence detection, these systems can quantify AFP at ~1 ng/mL levels in ~10 µL of human serum in a few tens of minutes. Our polymer microdevices have been applied in determining AFP in spiked serum samples. These integrated microsystems offer excellent potential for rapid, simple and accurate biomarker quantitation in a point-of-care setting.The two most widely used quantitation tools in traditional analytical chemistry are the calibration curve and the method of standard addition. 1 Micromachined devices for chemical analysis 2, 3 that integrate multiple processes, 4 reduce sample and reagent consumption, 5 and decrease analysis time 6, 7 and instrument footprint, 8, 9 are becoming an attractive alternative to classical separation-based analysis approaches. Although calibration curves have been used in microchip-based chemical analysis,10 , 11 the method of standard addition, which is especially desirable for addressing matrix effects in complex samples 1 such as blood, has seen extremely limited use. Very recently, a serial dilution microfluidic device was applied in standard addition quantitation of mM concentrations of Fe(CN) 6 4− , a model analyte, although the aqueous KCl solution was not one for which matrix effects were anticipated. 12 Alpha-fetoprotein (AFP) is a diagnostic biomarker for Hepatocellular carcinoma (HCC),13 with a reported specificity of 65% to 94%.14 In general, patients with an elevated serum AFP concentration have a higher risk for HCC. Currently, enzyme linked immunosorbent assay (ELISA) is used in the clinical analysis of AFP in human serum.15 With trained personnel, ELISA can provide reliable results, although the multi-hour assay times and microplate format make ELISA best suited for clinical, rather than point-of-care (POC) diagnostics. In contrast, rapid analysis6 , 7 and the ability to combine multiple processing steps4 , 16 on a single device make a microfluidic-based approach very attractive for POC AFP analysis. The analysis and separation of AFP in spiked buffer solutions in a microdevice platform have been reported, [17][18][19] 11,20, 21 However, only calibr...

Developments in biology are increasing demands for rapid, inexpensive, and sensitive biomolecular analysis. In this study, polymer microdevices with monolithic columns and electrophoretic channels were used for biological separations. Glycidyl methacrylate-co-ethylene dimethacrylate monolithic columns were formed within poly(methyl methacrylate) microchannels by in situ photopolymerization. Flow experiments in these columns demonstrated retention and then elution of amino acids under conditions optimized for sample preconcentration. To enhance analyte selectivity, antibodies were immobilized on monoliths, and subsequent lysozyme treatment blocked nonspecific adsorption. The enrichment capability and selectivity of these affinity monoliths were evaluated by purifying fluorescently tagged amino acids from a mixture containing green fluorescent protein (GFP). Twenty-fold enrichment and 91% recovery were achieved for the labeled amino acids, with a <25,000-fold reduction in GFP concentration, as indicated by microchip electrophoresis analysis. These devices should provide a simple, inexpensive, and effective platform for trace analysis in complex biological samples.

We have developed a method for rapid prototyping of hard polymer microfluidic systems using solvent imprinting and bonding. We investigated the applicability of patterned SU-8 photoresist on glass as an easily fabricated template for solvent imprinting. Poly(methyl methacrylate) (PMMA) exposed to acetonitrile for 2 min then had an SU-8 template pressed into the surface for 10 min, which provided appropriately imprinted channels and a suitable surface for bonding. After a PMMA cover plate had also been exposed to acetonitrile for 2 min, the imprinted and top PMMA pieces could be bonded together at room temperature with appropriate pressure. The total fabrication time was less than 15 min. Under the optimized fabrication conditions, nearly 30 PMMA chips could be replicated using a single patterned SU-8 master with high chip-to-chip reproducibility. Relative standard deviations were 2.3% and 5.4% for the widths and depths of the replicated channels, respectively. Fluorescently labeled amino acid and peptide mixtures were baseline separated using these PMMA microchips in <15s. Theoretical plate numbers in excess of 5000 were obtained for a approximately 3 cm separation distance, and the migration time relative standard deviation for an amino acid peak was 1.5% for intra-day and 2.2% for inter-day analysis. This new solvent imprinting and bonding approach significantly simplifies the process for fabricating microfluidic structures in hard polymers such as PMMA.