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Biomedical subjects

Michael G Roper

Publications and source records attributed to Michael G Roper.

8 recordsLinked to original sources
PubMed2006

A fully integrated microfluidic genetic analysis system with sample-in-answer-out capability.

We describe a microfluidic genetic analysis system that represents a previously undescribed integrated microfluidic device capable of accepting whole blood as a crude biological sample with the endpoint generation of a genetic profile. Upon loading the sample, the glass microfluidic genetic analysis system device carries out on-chip DNA purification and PCR-based amplification, followed by separation and detection in a manner that allows for microliter samples to be screened for infectious pathogens with sample-in-answer-out results in < 30 min. A single syringe pump delivers sample/reagents to the chip for nucleic acid purification from a biological sample. Elastomeric membrane valving isolates each distinct functional region of the device and, together with resistive flow, directs purified DNA and PCR reagents from the extraction domain into a 550-nl chamber for rapid target sequence PCR amplification. Repeated pressure-based injections of nanoliter aliquots of amplicon (along with the DNA sizing standard) allow electrophoretic separation and detection to provide DNA fragment size information. The presence of Bacillus anthracis (anthrax) in 750 nl of whole blood from living asymptomatic infected mice and of Bordetella pertussis in 1 microl of nasal aspirate from a patient suspected of having whooping cough are confirmed by the resultant genetic profile.

DNA↗
PubMed2006

A simple, valveless microfluidic sample preparation device for extraction and amplification of DNA from nanoliter-volume samples.

A glass microdevice has been constructed for the on-line integration of solid-phase extraction (SPE) of DNA and polymerase chain reaction (PCR) on a single chip. The chromatography required for SPE in the microfluidic sample preparation device (muSPD) was carried out in a silica bead/sol-gel SPE bed, where the purified DNA was eluted directly into a downstream chamber where conventional thermocycling allowed for PCR amplification of specific DNA target sequences. Through rapid, simple passivation of the PCR chamber with a silanizing reagent, reproducible DNA extraction and amplification was demonstrated from complex biological matrixes in a manner amenable to any research laboratory, using only a syringe pump and a conventional thermocycler. The muSPD allowed for SPE concentration of DNA from 600 nL of blood coupled to subsequent on-chip amplification that yielded a detectable amplicon; this simple device can be applied to a variety of routine genetic analyses without the need for sophisticated instrumentation. In addition, the applicability of these developments to nonconventional thermocycling was demonstrated through the use of noncontact, IR-mediated heating. This was exemplified with the isolation of DNA from an anthrax spore-spiked nasal swab and the subsequent on-chip amplification of target DNA sequences in a total processing time of only 25 min.

DNA↗
PubMed2005

Glass microfluidic devices with thin membrane voltage junctions for electrospray mass spectrometry.

In this study a novel glass membrane was prepared for conducting high voltage (HV) to solution in the channel of a microfabricated device for generation of liquid electrospray. Taylor cone formation and mass spectra obtained from this microdevice confirmed the utility of the glass membrane, but voltage conduction through the membrane could not be successfully explained based solely on the conductivity of the glass itself. This novel method for developing a high-voltage interface for microdevices avoids direct metal/liquid contact eliminating bubble formation in the channel due to water hydrolysis on the surface of the metal. Further, this arrangement produces no dead volume as is often found with traditional liquid junctions. At the same time, preliminary investigations into the outlet design of glass microdevices for interfacing with electrospray mass spectrometry, was explored. Both the exit shape and the use of hydrophobic coatings at the channel exit of the microdevice electrospray interface were evaluated using standard proteins with results indicating the utility of this type of design after further optimization.

Equipment Design↗
PubMed2005

Extrinsic Fabry-Perot interferometry for noncontact temperature control of nanoliter-volume enzymatic reactions in glass microchips.

Optical fiber extrinsic Fabry-Perot interferometry (EFPI) was investigated as a noncontact temperature sensor and utilized for regulating the temperature of small-volume solutions in microchips. Interference pattern analysis determined the optical path lengths (OPL) associated with reflections from various surfaces on or in the microchip, in particular, from gold sputtered on the bottom of a microchannel. Since OPL is directly proportional to refractive index, which is dependent on solution temperature, the EFPI sensor was capable of noncontact monitoring of solution temperature simply from alterations in the measured path length. Calibration of the sensor against a thermocouple was performed while heating the microchip in a noncontact manner with an IR lamp. The combination of EFPI temperature sensor, IR-mediated heating, and air cooling allowed a fully noncontact system for small-volume temperature control in microchip structures, and its utility was illustrated by optimal digestion of DNA by a temperature-dependent restriction endonuclease in 320 nL. The functionality and simplicity of the microchip EFPI temperature sensor was enhanced by replacing the prebonding sputtered gold with a tunable, chemically plated semireflective silver coating created in situ after chip fabrication. This provided an 8-fold improvement in the lowest detectable temperature change (deltaT = 0.1 degrees C), facilitated primarily by enhanced reflection from both the bottom and top surfaces of the microchannel. This approach for controlling micro- and nanoscale reactions--with heating, cooling, and temperature control being carried out in a completely noncontact fashion--provides an accurate and sensitive method for executing chemical and biochemical reactions in microchips.

Biosensing Techniques↗
PubMed2004

Islet secretory defect in insulin receptor substrate 1 null mice is linked with reduced calcium signaling and expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)-2b and -3.

Mice with deletion of insulin receptor substrate (IRS)-1 (IRS-1 knockout [KO] mice) show mild insulin resistance and defective glucose-stimulated insulin secretion and reduced insulin synthesis. To further define the role of IRS-1 in islet function, we examined the insulin secretory defect in the knockouts using freshly isolated islets and primary beta-cells. IRS-1 KO beta-cells exhibited a significantly shorter increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)) than controls when briefly stimulated with glucose or glyceraldehyde and when l-arginine was used to potentiate the stimulatory effect of glucose. These changes were paralleled by a lower number of exocytotic events in the KO beta-cells in response to the same secretagogues, indicating reduced insulin secretion. Furthermore, the normal oscillations in intracellular Ca(2+) and O(2) consumption after glucose stimulation were dampened in freshly isolated KO islets. Semiquantitative RT-PCR showed a dramatically reduced islet expression of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)-2b and -3 in the mutants. These data provide evidence that IRS-1 modulation of insulin secretion is associated with Ca(2+) signaling and expression of SERCA-2b and -3 genes in pancreatic islets and provides a direct link between insulin resistance and defective insulin secretion.

Animals↗
PubMed2003

Microfluidic chip for continuous monitoring of hormone secretion from live cells using an electrophoresis-based immunoassay.

A microfluidic device has been developed for the determination of insulin secreted from islets of Langerhans by a capillary electrophoresis competitive immunoassay. Online assays were performed by electrophoretically sampling anti-insulin antibody (Ab), fluorescein isothiocyanate-labeled insulin (FITC-insulin), and insulin from separate reservoirs and allowing them to mix as they traveled through a 4-cm reaction channel heated to 38 degrees C. From the reaction channel, samples were injected onto a 1.5-cm-long electrophoresis channel where the FITC-insulin and FITC-insulin-Ab complex were separated in 5 s using an electric field of 500 V/cm. Detection limits for insulin were 3 nM in this mode of operation. Assays could be collected at 15-s intervals with continuous sampling and online mixing for up to 30 min with no intervention. Relative standard deviation was 2-6% depending on the insulin concentration. Response time to a step change in insulin concentration was 30 s. For live cell monitoring, single islets were placed into a reservoir on the chip and fluid in the immediate vicinity was continuously sampled to detect insulin secretion from the islet. Monitoring of insulin secretion with electropherograms taken at 15-s intervals resolved secretory profiles characteristic of first- and second-phase insulin secretion. The method should be amenable to other cell or tissue types for measurements of release with high temporal resolution.

Animals↗
PubMed2002

Effect of the insulin mimetic L-783,281 on intracellular Ca2+ and insulin secretion from pancreatic beta-cells.

L-783,281, an antidiabetic fungal metabolite that has previously been shown to activate insulin signaling in CHO cells, was tested for its effect on intracellular Ca(2+) ([Ca(2+)](i)) and insulin secretion in single mouse pancreatic beta-cells. Application of 10 micromol/l L-783,281 for 40 s to isolated beta-cells in the presence of 3 mmol/l glucose increased [Ca(2+)](i) to 178 +/- 10% of basal levels (n = 18) as measured by fluo-4 fluorescence. L-767,827, an inactive structural analog of the insulin mimetic, had no effect on beta-cell [Ca(2+)](i). The L-783,281-evoked [Ca(2+)](i) increase was reduced by 82 +/- 4% (n = 6, P < 0.001) in cells incubated with 1 micromol/l of the SERCA (sarco/endoplasmic reticulum calcium ATPase) pump inhibitor thapsigargin and reduced by 33 +/- 6% (n = 6, P < 0.05) in cells incubated with 20 micromol/l of the L-type Ca(2+)-channel blocker nifedipine. L-783,281-stimulated [Ca(2+)](i) increases were reduced to 31 +/- 3% (n = 9, P < 0.05) and 48 +/- 10% (n = 6, P < 0.05) of control values by the phosphatidylinositol 3-kinase (PI3-K) inhibitors LY294002 (25 micromol/l) and wortmannin (100 nmol/l), respectively. In beta-cells from IRS-1-/- mice, 10 micromol/l L-783,281 had no significant effect on [Ca(2+)](i) (n = 5). L-783,281 also resulted in insulin secretion at single beta-cells. Application of 10 micromol/l L-783,281 for 40 s resulted in 12.2 +/- 2.1 (n = 14) exocytotic events as measured by amperometry, whereas the inactive structural analog had no stimulatory effect on secretion. Virtually no secretion was evoked by L-783,281 in IRS-1-/- beta-cells. LY294002 (25 micromol/l) significantly reduced the effect of the insulin mimetic on beta-cell exocytosis. It is concluded that L-783,281 evokes [Ca(2+)](i) increases and exocytosis in beta-cells via an IRS-1/PI3-K-dependent pathway and that the [Ca(2+)](i) increase involves release of Ca(2+) from intracellular stores.

Animals↗