A six-channel microfluidic immunoassay device with a scanned fluorescence detection system is described. Six independent mixing, reaction, and separation manifolds are integrated within one microfluidic wafer, along with two optical alignment channels. The manifolds are operated simultaneously and data are acquired using a singlepoint fluorescence detector with a galvano-scanner to step between separation channels. A detection limit of 30 pM was obtained for fluorescein with the scanning detector, using a 7.1-Hz sampling rate for each of the reaction manifolds and alignment channels (57-Hz overall sampling rate). Simultaneous direct immunoassays for ovalbumin and for anti-estradiol were performed within the microfluidic device. Mixing, reaction, and separation could be performed within 60 s in all cases and within 30 s under optimized conditions. Simultaneous calibration and analysis could be performed with calibrant in several manifolds and sample in the other manifolds, allowing a complete immunoassay to be run within 30 s. Careful chip conditioning with methanol, water, and 0.1 M NaOH resulted in peak height RSD values of 3-8% (N = 5 or 6), allowing for cross-channel calibration. The limit of detection (LOD) for an anti-estradial assay obtained in any single channel was 4.3 nM. The LOD for the cross-channel calibration was 6.4 nM. Factors influencing chip and detection system design and performance are discussed in detail.

We present three generations of microchip-based "in-space" sample fractionators and collectors for use in proteomics. The basic chip design consisted of a single channel for CE separation of analytes that then intersects a fractionation zone feed into multiple high aspect ratio microchannels for fractionation of separated components. Achievements of each generation are discussed in relation to important design criteria. CE-separated samples were electrokinetically driven to multiple collection channels in sequence without cross-contamination under the protection of sheath streams. A 36-channel fractionator demonstrated the efficacy of a high-throughput fractionator with no observed cross-contamination. A mixture of IgG and BSA was used to test the efficiency of the fractionator and collector. CE of the fractionated samples was performed on the same device to verify their purity. Our demonstration proved to be efficient and reproducible in obtaining non-contaminated samples over 15 sample injections. Experimental results were found to be in close agreement with PSpice simulation in terms of flow behavior, contamination control and device performance. The design presented here has a great potential to be integrated in proteomic platforms.

Using a high-affinity antibody for estradiol, thermodynamic and experimental limitations on detection limits for competitive capillary electrophoretic immunoassays were examined. Theoretical modeling of the dose-response curves for such assays allowed for optimization of experimental conditions. Through the examination of experimental and theoretical results generalizations could be made as to the ability of capillary electrophoretic immunoassays to achieve low detection limits. An experimental detection limit of 310 pM, corresponding to 2100 molecules, was achieved. A minimum theoretical detection limit for the antibody of interest was approximated to be 125-525 pM depending on the standard deviation. An overview of the optimization process is given as well as commentary on theoretical predictions.

Due to the broad impact of microfabrication technology on chemistry and biology, new methods to pattern and etch a variety of materials are being explored in a number of laboratories. Here, we report the design, fabrication, and operation of a glassy carbon (GC) microchip interfaced to a nanoelectrospray ionization source and a quadrupole mass spectrometer. The method involves standard photolithographic pattern transfer to a photoresist layer and anodization of the exposed GC substrate in basic electrolyte to produce a series of channels with well-defined wall structure. The performance of the microchip was evaluated with standard polymer and peptide samples.

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