Craig A Aspinwall

PMID: 10037726;Abstract:

Functional insulin receptors are known to occur in pancreatic beta cells; however, except for a positive feedback on insulin synthesis, their physiological effects are unknown. Amperometric measurements at single, primary pancreatic beta cells reveal that application of exogenous insulin in the presence or absence of nonstimulatory concentrations of glucose evokes exocytosis mediated by the beta cell insulin receptor. Insulin also elicits increases in intracellular Ca²⁺ concentration in beta cells but has minimal effects on membrane potential. Conditions where the insulin receptor is blocked or cell surface concentration of free insulin is reduced during exocytosis diminishes secretion induced by other secretagogues, providing evidence for direct autocrine action of insulin upon secretion from the same cell. These results indicate that the beta cell insulin receptor can mediate positive feedback for insulin secretion. The presence of a positive feedback mechanism for insulin secretion mediated by the insulin receptor provides a potential link between impaired insulin secretion and insulin resistance.

PMID: 9551792;Abstract:

An automated on-line competitive immunoassay based on capillary electrophoresis (CE) was utilized to monitor secretion of insulin from single islets of Langerhans stimulated by glucose and tolbutamide. In the instrument, fluorescein isothiocyanate-labeled insulin (FITC-insulin), monoclonal anti-insulin and perifusate of single islets were mixed on-line while islets were exposed to different levels of glucose and tolbutamide. Insulin released from single islets competed with FITC-insulin for antibody binding sites. Therefore, the amounts of bound and free FITC-insulin were modulated by insulin released from islets. The bound and the free FITC-insulin were separated by CE every 3 s and the bound over free ratio (B/F) was measured. Insulin levels were obtained by comparing B/F with calibration curves obtained under the same conditions except that the islet perfusate was replaced with various concentrations of insulin. Patterns of insulin secretion stimulated by glucose and tolbutamide observed were comparable to what has been seen previously using radioimmunoassay or enzyme-linked immunoassay. This on-line competitive immunoassay system provided a fast and direct way to measure insulin release from single islets. The effects of temperature on antibody-antigen reaction rate and binding equilibrium were also studied.

Increasing use of silica nanoparticles (SiO2 NPs) in consumer products and industrial processes leads to SiO2 NP discharge into wastewater. Thus, there is a need to understand the fate of SiO2 NPs during wastewater treatment. However, the detection of SiO2 NPs in environmental systems is hindered by the elevated background levels of natural silicon. In this work, laboratory-synthesized fluorescent core-shell SiO2 NPs were used to study the fate of these NPs during secondary wastewater treatment. Fluorescent measurements provided an easy and fast method for SiO2 NP tracking. A laboratory-scale activated sludge system consisting of an aeration tank and a settler was fed with synthetic wastewater containing ca. 7.5 mg L(-1) of fluorescent SiO2 NPs for 30 days. SiO2 NPs were effectively removed from the wastewater (>96%) during the first 6 days, however the concentration of SiO2 NPs in the effluent gradually increased afterwards and the NP discharge was as high as 65% of the input after 30 days of NP dosing. The poor removal of the SiO2 NPs was related to the high colloidal stability of the NPs in the wastewater and their limited propensity to biosorption. Although some degree of NP adsorption on the biomass was observed using fluorescence microscopy, the affinity of SiO2 NPs for the activated sludge was not enough for a sustained and effective removal of the SiO2 NPs from the wastewater.

We have developed a chemical derivatization scheme for primary amines that couples the fast kinetic properties of o-phthaldialdehyde (OPA) with the photophysical properties of visible, high quantum yield, fluorescent dyes. In this reaction, OPA is used as a cross-linking reagent in the labeling reaction of primary amines in the presence of a fluorescent thiol, 5-((2-(and-3)-S-(acetylmercapto)succinoyl)amino)fluorescein (SAMSA fluorescein), thereby incorporating fluorescein (epsilon = 78 000 M(-1), quantum yield of 0.98) into the isoindole product. Detection is based on excitation and emission of the incorporated fluorescein using the 488 nm laser line of an Ar(+) laser rather than the UV-excited isoindole, thereby eliminating the UV light sources for detection. Using this method, we have quantitatively labeled biologically important primary amines in less than 10 s. Detection limits for analysis of glutamate, glycine, GABA, and taurine were less than 2 nM. We present the characterization of OPA/SAMSA-F reaction and the potential utility of the derivatization reaction for dynamic chemical monitoring of biologically relevant analytes using CE.

Robust and high-density biosensors incorporating suspended lipid membranes require microfabricated apertures that can be readily integrated into complex analysis systems. Apertures with well-defined, three-dimensional geometries enable the formation of suspended lipid membranes and facilitate reduced aperture size compared to vertical-walled apertures. Unfortunately, existing methods of producing apertures with well-defined, three-dimensional geometries are based on complex and expensive fabrication procedures, some of which yield apertures in excessively fragile thin-film materials. Here, we describe a microfabrication method utilizing incline and rotate lithography that achieves sloped-wall microapertures in SU-8 polymer substrates with precision control of the aperture diameter, substrate thickness, and wall angle. This approach is simple, is of low cost, and is readily scaled up to allow highly reproducible parallel fabrication. The effect of the incident angle of UV exposure and the size of photomask features on the aperture geometry were investigated, yielding aperture diameters as small as 7 μm and aperture wall angles ranging from 8° to 36° measured from the normal axis. Black lipid membranes were suspended across the apertures and showed normalized conductance values of 0.02-0.05 pS μm(-2) and breakdown voltages of 400-600 mV. The functionality of the resulting sloped-wall microapertures was validated via measurement of reconstituted α-hemolysin activity and the voltage-gated channel activity of alamethicin.