PRESENTATION LINK: The field of microfluidic lab-on-a-chip systems is rapidly maturing and moving in the direction of further integration. When considering integrated or parallel analyses, it is important to evaluate how many analysis elements can be accommodated on a microfluidic chip while maintaining a reasonable footprint, both for fabrication and operation concerns. Much of the work in the field has been to simply translate conventional analysis techniques (e.g., capillary zone electrophoresis; CZE) onto a planar chip format with minimal modifications to the basic mode of operation of the technique. An alternative paradigm would be development of methods that are by design able to be implemented in short separation lengths and in minimal areas. We have developed a novel technique for performing electrophoretic separations using this bottom-up approach termed Gradient Elution Moving Boundary Electrophoresis (GEMBE). The technique utilizes the electrophoretic migration of chemical species in combination with variable hydrodynamic bulk counter-flow of the solution through a separation capillary or microfluidic channel. Continuous sample introduction is used, eliminating the need for a sample injection mechanism. Only analytes with an electrophoretic velocity greater than the counter-flow velocity enter the separation channel. The counter-flow velocity is varied over time so that each analyte is brought into the separation column sequentially, allowing for high-resolution separations in very short channels. The new variable of bulk flow acceleration affords a new selectivity parameter to electrophoresis analogous to gradient elution compositions in chromatography. Non-linear gradients can easily be imposed, allowing for optimization of the separation space. GEMBE separations can be implemented in much smaller areas on a microfluidic chip as compared with conventional CZE because extra channels or access ports to form an injection zone are eliminated and because high-resolution separations can be performed in very short channels. The basic principles of GEMBE will be discussed, with emphasis made on the specific advantages of the technique over CZE. Examples of GEMBE separations of small dye molecules and amino acids will be presented, as well as GEMBE performed with a sieving matrix for DNA separations. In addition, results will be presented of the use of a low-cost polymeric GEMBE device with eight separation channels in less than one square inch of area. The results will include the use of the device to generate a calibration curve for a homogeneous insulin immunoassay using each of the eight simultaneous measurements as a calibration point.

POSTER LINK: Microfluidic lab-on-a-chip devices employing capillary electrophoresis are currently constrained by limitations of on-chip separation lengths and injection schemes. These limits are exacerbated in multiplexed devices, with the number of access ports and off-chip equipment needed for injections increasing with the number of parallel analyses. We describe a novel injection-less separation method, Gradient Elution Moving-Boundary Electrophoresis (GEMBE), which can provide the highest number of electrophoretic separations possible for a given, limited chip area. The reduction in chip area is accomplished by combining moving boundary electrophoresis, which does not require the formation of an injected plug, with a temporally varying hydrodynamic counter-flow, which provides high resolution separations in very short microchannels.

PRESENTATION LINK: A microfluidic device utilizing an immunoassay technique for monitoring the chemical environment around living cells has been developed. The competitive immunoassay was implemented using capillary zone electrophoresis (CZE) with laser induced fluorescence detection. CZE allowed for both highly sensitive and rapid assays to be performed. Each immunoassay was performed within 6 seconds with limits of detections as low as 0.8 nM, allowing for the study of cellular secretion kinetics. Use of a microfluidic platform, fabricated using standard photolithographic and wet etching techniques of glass, permitted a high degree of automation and integration to the system. Real-time detection of glucose-stimulated insulin secretion from single islets of Langerhans (microorgans comprised of a few thousand cells, the majority of which are insulin secreting beta-cells) was demonstrated. Traditionally, these measurements are performed in large-volume perfusion chambers utilizing off-line radioimmunoassay detection with temporal resolution on the order of minutes. Islets were housed in a microchamber on the device while perfusing biological media of varying glucose levels via a pressure driven system. Perfusate containing secreted insulin was sampled via electroosmotic flow and mixed on-line with fluorescein isothiocyanate-labeled insulin (FITC-insulin) and anti-insulin immunoglobulin (Ab). The reaction stream was then sampled and injected onto an electrophoresis channel via flow gated injection for the separation of FITC-insulin:Ab complex and free FITC-insulin. Insulin secretion from islets expressing beta-cell specific null mutations of insulin receptor, insulin growth factor receptor, and double null mutants were compared to wild type islets. All three mutants exhibited elevated fasting levels of insulin secretion and decreased secretion upon a glucose challenge compared to the control islets. The mutant phenotypes were similar to characteristics displayed by diabetic types, and the results provide further evidence of the importance of the two receptors in normal regulation of insulin secretion. This device could be applicable to chemical monitoring of other compounds and tissues as well.

PRESENTATION LINK: Microfabricated devices have made great strides in miniaturizing conventional analytical systems, as well as allowing for greater flexibility of fluid manipulation and the coupling of additional analyses of the sample. We have developed an automated microfluidic device for real-time monitoring of the chemical environment around living cells utilizing a capillary electrophoresis based immunoassay. The device, developed using standard photolithographic and wet etching techniques of glass, is used for real-time monitoring of insulin secretion from single pancreatic islets of Langerhans (microorgans comprised of a few thousand cells, the majority of which are insulin secreting beta-cells). Traditionally, these measurements are performed in large-volume perfusion chambers utilizing off-line radioimmunoassay detection with a temporal resolution on the order of minutes. Our device houses the islet in a microchamber while perfusing biological media via a pressure driven system; additionally, the perfusion system can be used to deliver various stimuli to the islet. Perfusate containing secreted hormones is then sampled via electroosmotic flow and mixed on-line with immunoreagants, followed by injection onto an electrophoresis channel. Analyses were performed within 6 s with limits of detection as low as 0.8 nM, allowing for the study of cellular secretion kinetics. Currently, the device is being used to evaluate insulin secretion phenotypes of various genetic mutants, such as insulin receptor and insulin-like growth factor receptor knock-out genotypes.

POSTER LINK: We describe the development of an automated microfluidic device for monitoring the chemical environment around live cells. Our device, developed using standard photolithographic and wet etching techniques of glass, was used to monitor in real-time insulin secretion from a single islet of Langerhans. The device houses the islet in a microchamber while perfusing biological media via a pressure driven system, which could be used to deliver various stimuli to the islet. Perfusate containing secreted insulin was then sampled via electroosmotic flow and mixed on-line with the immunoreagants. The reaction stream was then sampled and injected onto an electrophoresis channel via flow gated injection. Glucose stimulated patterns of insulin secretion could be monitored with 6 s temporal resolution, demonstrating the device's utility for fast and direct measurements while maintaining islet viability. This device could be applicable to other compounds and tissues as well.

POSTER LINK: Microfabricated devices have made great strides in miniaturizing conventional analytical systems, as well as allowing for greater flexibility of fluid manipulation and the coupling of additional analyses of the sample. We describe the development of an automated microfluidic device for monitoring the chemical environment around live cells. Our device, developed using standard photolithographic and wet etching techniques of glass, was used to monitor in real-time insulin secretion from a single islet of Langerhans housed within the chip. Traditionally, these measurements are performed in large-volume perfusion chambers utilizing off-line radioimmunoassay detection with temporal resolution on the order of minutes. The device described houses the islet in a microchamber while perfusing biological media via a pressure driven system. Additionally, the perfusion system could be used to deliver various stimuli to the islet. Perfusate containing secreted insulin was then sampled via electroosmotic flow and mixed on-line with the immunoreagants. The reaction reached steady-state as the perfusate traversed the reaction channel. The reaction stream was then sampled and injected onto an electrophoresis channel via flow gated injection. Separations were performed within 10 s across a 1.5 cm channel length with ca. 750 V/cm. Glucose stimulated patterns of insulin secretion could be monitored with a temporal resolution on the order of seconds, demonstrating the device's utility for fast and direct measurements while maintaining islet viability. This device could be applicable to other tissues as well.

PRESENTATION LINK: Abstract unavailable.

PRESENTATION LINK: Abstract unavailable.

PRESENTATION LINK: Abstract unavailable.