In this report, we show that a novel capillary-based photopolymerized monolith offering unprecedented efficiency (approximately 80%) for DNA extraction from submicroliter volumes of whole blood (Wen, J.; Guillo, C.; Ferrance, J. P.; Landers, J. P. Anal. Chem. 2006, 78, 1673-1681) can be translated to microfluidic devices. However, owing to the large mass of protein present in blood, both DNA binding capacity and extraction efficiency were significantly decreased when extraction of DNA was carried out directly from whole blood (38+/-1%). To circumvent this, a novel two-stage microdevice was developed, consisting in a C18 reversed-phase column for protein capture (stage 1) in series with a monolithic column for DNA extraction (stage 2). The two-stage, dual-phase design improves the capability of the monolith for whole blood DNA extraction by approximately 100-fold. From a 10-microL load of whole blood containing 350 ng of DNA, 99% (340+/-10 ng) traverses the C18 phase while approximately 70% (1020+/-45 ug) of protein is retained. A total of 240+/-2 ng of DNA was eluted from the second-stage monolith, resulting in an overall extraction efficiency of 69+/-1%. This provided not only an improvement in extraction efficiency over other chip-based DNA extraction solid phases but also the highest extraction efficiency reported to-date for such sample volumes in a microfluidic device. As an added bonus, the two-stage, dual-phase microdevice allowed the 2-propanol wash step, typically required to remove proteins from the DNA extraction phase for successful PCR, to be completely eliminated, thus streamlining the process without affecting the PCR amplifiability of the extracted DNA.

A novel high-capacity, high-efficiency DNA extraction method is described using a photopolymerized silica-based monolithic column in a fused-silica capillary. Development involved investigation of the composition of the sol-gel monomer, fabrication conditions, and surface modifications in order to optimize the binding capacity. Extraction capacity and efficiency with the 3-(trimethoxysilyl)propyl methacrylate (TMSPM) monolith formulations fabricated in capillaries were investigated using a simple three-step procedure consisting of sample loading, washing of the solid phase, and elution of the DNA using a low ionic strength Tris buffer at pH 8. Once the TMSPM monomer concentration was optimized to yield a monolith with maximum test stability (robustness) and minimum back pressure, the monolith surface was modified by the grafting of tetramethyl orthosilicate (TMOS) for increased DNA binding capacity. After the examination of a variety of TMOS concentrations, 85% v/v TMOS was found to be optimal for DNA extraction without any obvious changes to the monolith structure. The reduction of time allowed for TMSPM hydrolysis prior to UV polymerization from 20 to 5 min led to a lower back pressure of the monolith, enabling better TMOS derivatization and therefore higher binding capacity. Minimal buffer volume (as low as 1 muL) was required to elute DNA from the solid phase, providing a DNA concentrating effect potentially important for downstream processes. While experimentation employed monolithic columns that were 12 cm in length, reduction of the length to 2 cm still allowed for a DNA binding capacity of at least 100 ng of prepurified human genomic DNA and extraction efficiencies greater than 85%. Extraction of low sample volumes (submicroliter) of human whole blood were successfully performed, with extraction efficiencies from the 2-cm monolithic column higher than those obtained from a commercial DNA extraction kit. These results position this novel matrix as an attractive alternative for solid-phase extraction of DNA and other biologically active molecules in microscale devices.

A multianalyte CE competitive immunoassay using two-color detection was developed to measure insulin and glucagon in islets of Langerhans. Insulin was quantified with FITC-insulin (Ins*) and anti-insulin antibodies (Ins Ab) and glucagon was quantified with Cy5-glucagon (Glu*) and anti-glucagon antibodies (Glu Ab). A 3 mW Ar(+) laser at 488 nm and a 25 mW laser diode at 635 nm were used to excite FITC and Cy5, respectively. Fluorescence was split with a half-silvered mirror and passed through a 520 +/- 20 nm bandpass filter or a 663 nm longpass filter for the detection of insulin and glucagon, respectively. The two-color detection format enabled independent quantitation of both analytes even with concentrations of insulin immunoassay reagents 20-fold higher than glucagon reagents. Simultaneous calibration curves were generated and used to determine insulin and glucagon content in islets of Langerhans. Amounts of insulin and glucagon were 56.6 +/- 3.2 and 1.0 +/- 0.5 ng/islet, respectively. LODs were 7 nM insulin and 3 nM glucagon. The assay will be applicable to fast monitoring of multiple peptides secreted from islets of Langerhans and can be applied to other systems for the quantitation of multiple analytes with large differences in concentrations.

A capillary electrophoresis competitive immunoassay was developed for simultaneous quantitation of insulin, glucagon, and islet amyloid polypeptide (IAPP) secretion from islets of Langerhans. Separation buffers and conditions were optimized for resolution of fluorescein isothiocyanate (FITC)-labeled glucagon and IAPP immunoassay reagents, which were excited with the 488 nm line of an Ar+ laser and detected at 520 nm with a photomultiplier tube (PMT). Cy5-labeled insulin immunoassay reagents were excited by a 635 nm laser diode module and detected at 700 nm with a separate PMT. Optimum resolution was achieved with a 20 mM carbonate separation buffer at pH 9.0 using a 20 cm effective separation length with an electric field of 500 V/cm. Limits of detection for insulin, glucagon, and IAPP were 2, 3, and 3 nM, respectively. This method was used to monitor simultaneous secretion of these peptides from as few as 14 islets after incubation in 4, 11, and 20 mM glucose for 6 hours. For insulin and IAPP, a statistically significant increase in secretion levels was observed, while glucagon levels were significantly reduced in the 4 and 11 mM glucose conditions. To further demonstrate the utility of the assay, the Ca2+-dependent secretion of these peptides was demonstrated which agreed with published reports. The ability to examine the secretion of multiple peptides may allow for determination of regulation of secretory processes within islets of Langerhans.

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