Craig A Aspinwall

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• Saavedra, S. S., Aspinwall, C. A., Christie, H. S., Teran Arce, P. F., & Fonseka, M. N. (2022). Nanomechanical properties of artificial lipid bilayers composed of fluid and polymerizable lipids. Langmuir, 11(38(1)), 100 - 111. doi:10.1021/acs.langmuir.1c02098
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  Polymerization enhances the stability of a planar supported lipid bilayer (PSLB) but it also changes its chemical and mechanical properties, attenuates lipid diffusion, and may affect the activity of integral membrane proteins. Mixed bilayers composed of fluid lipids and poly(lipids) may provide an appropriate combination of polymeric stability coupled with the fluidity and elasticity needed to maintain the bioactivity of reconstituted receptors. Previously (Langmuir, 2019, 35, 12483-12491) we showed that binary mixtures of the polymerizable lipid bis-SorbPC and the fluid lipid DPhPC form phase-segregated PSLBs composed of nanoscale fluid and poly(lipid) domains. Here we used atomic force microscopy (AFM) to compare the nanoscale mechanical properties of these binary PSLBs with single-component PSLBs. The elastic (Young's) modulus, area compressibility modulus, and bending modulus of bis-SorbPC PSLBs increased upon polymerization. Before polymerization, breakthrough events at forces below 5 nN were observed, but after polymerization, the AFM tip could not penetrate the PSLB up to an applied force of 20 nN. These results are attributed to the polymeric network in poly(bis-SorbPC), which increases the bilayer stiffness and resists compression and bending. In binary DPhPC/poly(bis-SorbPC) PSLBs, the DPhPC domains are less stiff, more compressible, and are less resistant to rupture and bending compared to pure DPhPC bilayers. These differences are attributed to bis-SorbPC monomers and oligomers present in DPhPC domains that disrupt the packing of DPhPC molecules. In contrast, the poly(bis-SorbPC) domains are stiffer and less compressible relative to pure PSLBs; this difference is attributed to DPhPC filling the nm-scale pores in the polymerized domains that are created during bis-SorbPC polymerization. Thus, incomplete phase segregation increases the stability of poly(bis-SorbPC) but has the opposite, detrimental effect for DPhPC. Overall, these results provide guidance for the design of partially polymerized bilayers for technological uses.
• Fonseka, N. M., Teran Arce, P. F., Aspinwall, C. A., Saavedra, S. S., & Christie, H. S. (2022). Nanomechanical Properties of Artificial Lipid Bilayers Composed of Fluid and Polymerizable Lipids. Langmuir, 38, 100-111. doi:https://doi.org/10.1021/acs.langmuir.1c02098
• Christie, H. S., Aspinwall, C. A., Saavedra, S. S., Fonseka, M. N., Liang, B., Orosz, K. S., Jones, I. W., & Hall Jr, H. (2019). Nanodomain Formation in Planar Supported Lipid Bilayers Composed of Fluid and Polymerized Dienoyl Lipids. Langmuir, 35, 12483-12491. doi:10.1021/acs.langmuir.9b02101
• Agasid, M. T., Wang, X., Huang, Y., Janczak, C. M., Bränström, R., Saavedra, S. S., & Aspinwall, C. A. (2018). Expression, purification, and electrophysiological characterization of a recombinant, fluorescent Kir6.2 in mammalian cells. Protein expression and purification, 146, 61-68.
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  The inwardly rectifying K(Kir) channel, Kir6.2, plays critical roles in physiological processes in the brain, heart, and pancreas. Although Kir6.2 has been extensively studied in numerous expression systems, a comprehensive description of an expression and purification protocol has not been reported. We expressed and characterized a recombinant Kir6.2, with an N-terminal decahistidine tag, enhanced green fluorescent protein (eGFP) and deletion of C-terminal 26 amino acids, in succession, denoted eGFP-Kir6.2Δ26. eGFP-Kir6.2Δ26 was expressed in HEK293 cells and a purification protocol developed. Electrophysiological characterization showed that eGFP-Kir6.2Δ26 retains native single channel conductance (64 ± 3.3 pS), mean open times (τ = 0.72 ms, τ = 15.3 ms) and ATP affinity (IC = 115 ± 25 μM) when expressed in HEK293 cells. Detergent screening using size exclusion chromatography (SEC) identified Fos-choline-14 (FC-14) as the most suitable surfactant for protein solubilization, as evidenced by maintenance of the native tetrameric structure in SDS-PAGE and western blot analysis. A two-step scheme using Co-metal affinity chromatography and SEC was implemented for purification. Purified protein activity was assessed by reconstituting eGFP-Kir6.2Δ26 in black lipid membranes (BLMs) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), l-α-phosphatidylinositol-4,5-bisphosphate (PIP) in a 89.5:10:0.5 mol ratio. Reconstituted eGFP-Kir6.2Δ26 displayed similar single channel conductance (61.8 ± 0.54 pS) compared to eGFP-Kir6.2Δ26 expressed in HEK293 membranes; however, channel mean open times increased (τ = 7.9 ms, τ = 61.9 ms) and ATP inhibition was significantly reduced for eGFP-Kir6.2Δ26 reconstituted into BLMs (IC = 3.14 ± 0.4 mM). Overall, this protocol should be foundational for the production of purified Kir6.2 for future structural and biochemical studies.
• Janczak, C. M., Calderon, I. A., Mokhtari, Z., & Aspinwall, C. A. (2018). Polystyrene-Core, Silica-Shell Scintillant Nanoparticles for Low-Energy Radionuclide Quantification in Aqueous Media. ACS applied materials & interfaces, 10(5), 4953-4960.
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  β-particle emitting radionuclides are useful molecular labels due to their abundance in biomolecules. Detection of β-emission fromH,S, andP, important biological isotopes, is challenging due to the low energies (E≤ 300 keV) and short penetration depths (≤0.6 mm) in aqueous media. The activity of biologically relevant β-emitters is usually measured in liquid scintillation cocktail (LSC), a mixture of energy-absorbing organic solvents, surfactants, and scintillant fluorophores, which places significant limitations on the ability to acquire time-resolved measurements directly in aqueous biological systems. As an alternative to LSC, we developed polystyrene-core, silica-shell nanoparticle scintillators (referred to as nanoSCINT) for quantification of low-energy β-particle emitting radionuclides directly in aqueous solutions. The polystyrene acts as an absorber for energy from emitted β-particles and can be loaded with a range of hydrophobic scintillant fluorophores, leading to photon emission at visible wavelengths. The silica shell serves as a hydrophilic shield for the polystyrene core, enabling dispersion in aqueous media and providing better compatibility with water-soluble analytes. While polymer and inorganic scintillating microparticles are commercially available, their large size and/or high density complicates effective dispersion throughout the sample volume. In this work, nanoSCINT nanoparticles were prepared and characterized. nanoSCINT responds toH,S, andP directly in aqueous solutions, does not exhibit a change in scintillation response between pH 3.0 and 9.5 or with 100 mM NaCl, and can be recovered and reused for activity measurements in bulk aqueous samples, demonstrating the potential for reduced production of LSC waste and reduced total waste volume during radionuclide quantification. The limits of detection for 1 mg/mL nanoSCINT are 130 nCi/mL forH, 8 nCi/mL forS, and
• Agasid, M. T., Comi, T. J., Saavedra, S. S., & Aspinwall, C. A. (2017). Enhanced Temporal Resolution with Ion Channel-Functionalized Sensors Using a Conductance-Based Measurement Protocol. ANALYTICAL CHEMISTRY, 89(2), 1315-1322.
• Bright, L. K., Baker, C. A., Bränström, R., Saavedra, S. S., & Aspinwall, C. A. (2015). Methacrylate Polymer Scaffolding Enhances the Stability of Suspended Lipid Bilayers for Ion Channel Recordings and Biosensor Development. ACS biomaterials science & engineering, 1(10), 955-963.
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  Black lipid membranes (BLMs) provide a synthetic environment that facilitates measurement of ion channel activity in diverse analytical platforms. The limited electrical, mechanical and temporal stabilities of BLMs pose a significant challenge to development of highly stable measurement platforms. Here, ethylene glycol dimethacrylate (EGDMA) and butyl methacrylate (BMA) were partitioned into BLMs and photopolymerized to create a cross-linked polymer scaffold in the bilayer lamella that dramatically improved BLM stability. The commercially available methacrylate monomers provide a simple, low cost, and broadly accessible approach for preparing highly stabilized BLMs useful for ion channel analytical platforms. When prepared on silane-modified glass microapertures, the resulting polymer scaffold-stabilized (PSS)-BLMs exhibited significantly improved lifetimes of 23 ± 9 to 40 ± 14 h and > 10-fold increase in mechanical stability, with breakdown potentials > 2000 mV attainable, depending on surface modification and polymer cross-link density. Additionally, the polymer scaffold exerted minimal perturbations to membrane electrical integrity as indicated by mean conductance measurements. When gramicidin A and α-hemolysin were reconstituted into PSS-BLMs, the ion channels retained function comparable to conventional BLMs. This approach is a key advance in the formation of stabilized BLMs and should be amenable to a wide range of receptor and ion channel functionalized platforms.
• Baker, C. A., & Aspinwall, C. A. (2015). Emerging trends in precision fabrication of microapertures to support suspended lipid membranes for sensors, sequencing, and beyond. Analytical and bioanalytical chemistry, 407(3), 647-52.
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  Suspended lipid membranes, also called black lipid membranes (BLMs), are an important model system that approximates the lipid bilayer environment of cell membranes. Increasingly, BLMs are utilized in sensing strategies that harness high sensitivity measurements of ion flux across the membrane, typically facilitated by ion channel proteins. BLMs are suspended across microapertures that connect two otherwise isolated fluidic compartments, and the precision fabrication of such microapertures can contribute to the stability and performance of the resulting BLM. Here, we highlight two emerging trends in the precision fabrication of microapertures for BLM formation: microfabrication in silicon-based thin film substrates, and microfabrication in the negative photoresist material SU-8. Four unique fabrication strategies are outlined, and we project the impact that these microfabrication strategies will have for BLM-integrated bioanalytical technologies.
• Gallagher, E. S., Adem, S. M., Baker, C. A., Ratnayaka, S. N., Jones, I. W., Hall, H. K., Saavedra, S. S., & Aspinwall, C. A. (2015). Highly stabilized, polymer-lipid membranes prepared on silica microparticles as stationary phases for capillary chromatography. Journal of chromatography. A, 1385, 28-34.
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  The ability to rapidly screen complex libraries of pharmacological modulators is paramount to modern drug discovery efforts. This task is particularly challenging for agents that interact with lipid bilayers or membrane proteins due to the limited chemical, physical, and temporal stability of conventional lipid-based chromatographic stationary phases. Here, we describe the preparation and characterization of a novel stationary phase material composed of highly stable, polymeric-phospholipid bilayers self-assembled onto silica microparticles. Polymer-lipid membranes were prepared by photochemical or redox initiated polymerization of 1,2-bis[10-(2',4'-hexadieoyloxy)decanoyl]-sn-glycero-2-phosphocholine (bis-SorbPC), a synthetic, polymerizable lipid. The resulting polymerized bis-SorbPC (poly(bis-SorbPC)) stationary phases exhibited enhanced stability compared to particles coated with 1,2-dioleoyl-sn-glycero-phosphocholine (unpolymerized) phospholipid bilayers when exposed to chemical (50mM triton X-100 or 50% acetonitrile) and physical (15min sonication) insults after 30 days of storage. Further, poly(bis-SorbPC)-coated particles survived slurry packing into fused silica capillaries, compared to unpolymerized lipid membranes, where the lipid bilayer was destroyed during packing. Frontal chromatographic analyses of the lipophilic small molecules acetylsalicylic acid, benzoic acid, and salicylic acid showed >44% increase in retention times (P
• Li, Z., Muhandiramlage, T. P., Keogh, J. P., Hall, H. K., & Aspinwall, C. A. (2015). Aptamer-functionalized porous phospholipid nanoshells for direct measurement of Hg(2+) in urine. Analytical and bioanalytical chemistry, 407(3), 953-60.
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  A porous phospholipid nanoshell (PPN) sensor functionalized with a specific aptamer sensor agent was prepared for rapid detection of Hg(2+) in human urine with minimal sample preparation. Aptamer sensors provide an important class of optical transducers that can be readily and reproducibly synthesized. A key limitation of aptamer sensors, and many other optical sensors, is the potential of biofouling or biodegradation when used in complex biological matrices such as serum or urine, particularly when high levels of nucleases are present. We prepared Hg(2+)-responsive, PPN-encapsulated aptamer sensors that overcome these limitations. PPNs provide a protective barrier to encapsulate the aptamer sensor in an aqueous environment free of diffusional restrictions encountered with many polymer nanomaterials. The unique porous properties of the PPN membrane enable ready and rapid transfer of small molecular weight ions and molecules into the sensor interior while minimizing the macromolecular interactions between the transducer and degradants or interferents in the exterior milieu. Using Hg(2+)-responsive, PPN-encapsulated aptamer sensors, we were able to detect sub-100 ppb (chronic threshold limit from urine test) Hg(2+) in human urine with no sample preparation, whereas free aptamer sensors yielded inaccurate results due to interferences from the matrix. The PPN architecture provides a new platform for construction of aptamer-functionalized sensors that target low molecular weight species in complex matrices, beyond the Hg(2+) demonstrated here.
• Otero-González, L., Field, J. A., Calderon, I. A., Aspinwall, C. A., Shadman, F., Zeng, C., & Sierra-Alvarez, R. (2015). Fate of fluorescent core-shell silica nanoparticles during simulated secondary wastewater treatment. Water research, 77, 170-8.
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  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.
• Cheng, Z., Zaki, A. A., Jones, I. W., Hall, H. K., Aspinwall, C. A., & Tsourkas, A. (2014). Stabilized porous liposomes with encapsulated Gd-labeled dextran as a highly efficient MRI contrast agent. Chemical Communications, 50(19), 2502-2504.
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  Abstract: A highly efficient contrast agent for magnetic resonance imaging was developed by encapsulating gadolinium within a stabilized porous liposome. The highly porous membrane leads to a high relaxivity of the encapsulated Gd. The stability of the liposome was improved by forming a polymer network within the bilayer membrane. © 2014 The Royal Society of Chemistry.
• Gallagher, E. S., Adem, S. M., Bright, L. K., Calderon, I. A., Mansfield, E., & Aspinwall, C. A. (2014). Hybrid phospholipid bilayer coatings for separations of cationic proteins in capillary zone electrophoresis. Electrophoresis, 35(8), 1099-105.
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  Protein separations in CZE suffer from nonspecific adsorption of analytes to the capillary surface. Semipermanent phospholipid bilayers have been used to minimize adsorption, but must be regenerated regularly to ensure reproducibility. We investigated the formation, characterization, and use of hybrid phospholipid bilayers (HPBs) as more stable biosurfactant capillary coatings for CZE protein separations. HPBs are formed by covalently modifying a support with a hydrophobic monolayer onto which a self-assembled lipid monolayer is deposited. Monolayers prepared in capillaries using 3-cyanopropyldimethylchlorosilane (CPDCS) or n-octyldimethylchlorosilane (ODCS) yielded hydrophobic surfaces with lowered surface free energies of 6.0 ± 0.3 or 0.2 ± 0.1 mJ m(-2) , respectively, compared to 17 ± 1 mJ m(-2) for bare silica capillaries. HPBs were formed by subsequently fusing vesicles comprised of 1,2-dilauroyl-sn-glycero-3-phosphocholine or 1,2-dioleoyl-sn-glycero-3-phosphocholine to CPDCS- or ODCS-modified capillaries. The resultant HPB coatings shielded the capillary surface and yielded reduced electroosmotic mobility (1.3-1.9 × 10(-4) cm(2) V(-1) s(-1) ) compared to CPDCS- and ODCS-modified or bare capillaries (3.6 ± 0.2 × 10(-4) cm(2) V(-1) s(-1) , 4.8 ± 0.4 × 10(-4) cm(2) V(-1) s(-1) , and 6.0 ± 0.2 × 10(-4) cm(2) V(-1) s(-1) , respectively), with increased stability compared to phospholipid bilayer coatings. HPB-coated capillaries yielded reproducible protein migration times (RSD ≤ 3.6%, n ≥ 6) with separation efficiencies as high as 200 000 plates/m.
• Gallagher, E. S., Mansfield, E., & Aspinwall, C. A. (2014). Stabilized phospholipid membranes in chromatography: toward membrane protein-functionalized stationary phases. Analytical and bioanalytical chemistry, 406(9-10), 2223-9.
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  Transmembrane protein (TMP)-functionalized materials have resulted in powerful new methods in chemical analysis. Of particular interest is the development of high-throughput, TMP-functionalized stationary phases for affinity chromatography of complex mixtures of analytes. Several natural and synthetic phospholipids and lipid mimics have been used for TMP reconstitution, although the resulting membranes often lack the requisite chemical and temporal stability for long-term use, a problem that is exacerbated in flowing separation systems. Polymerizable lipids with markedly increased membrane stability and TMP functionality have been developed over the past two decades. More recently, these lipids have been incorporated into a range of analytical methods, including separation techniques, and are now poised to have a significant impact on TMP-based separations. Here, we describe current methods for preparing TMP-containing stationary phases and examine the potential utility of polymerizable lipids in TMP affinity chromatography.
• Johnson, G. M., Chozinski, T. J., Gallagher, E. S., Aspinwall, C. A., & Miranda, K. M. (2014). Glutathione sulfinamide serves as a selective, endogenous biomarker for nitroxyl after exposure to therapeutic levels of donors. Free radical biology & medicine, 76, 299-307.
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  Nitroxyl (HNO) donors exhibit promising pharmacological characteristics for treatment of cardiovascular disorders, cancer, and alcoholism. However, whether HNO also serves as an endogenous signaling agent is currently unknown, largely because of the inability to selectively and sensitively detect HNO in a cellular environment. Although a number of methods to detect HNO have been developed recently, sensitivity and selectivity against other nitrogen oxides or biological reductants remain problematic. To improve selectivity, the electrophilic nature of HNO has been harnessed to generate modifications of thiols and phosphines that are unique to HNO, especially compared to nitric oxide (NO). Given high bioavailability, glutathione (GSH) is expected to be a major target of HNO. As a result, the putative selective product glutathione sulfinamide (GS(O)NH2) may serve as a high-yield biomarker of HNO production. In this work, the formation of GS(O)NH2 after exposure to HNO donors was investigated. Fluorescent labeling followed by separation and detection using capillary zone electrophoresis with laser-induced fluorescence allowed quantitation of GS(O)NH2 with nanomolar sensitivity, even in the presence of GSH and derivatives. Formation of GS(O)NH2 was found to occur exclusively upon exposure of GSH to HNO donors, thus confirming selectivity. GS(O)NH2 was detected in the lysate of cells treated with low-micromolar concentrations of HNO donors, verifying that this species has sufficient stability to server as a biomarker of HNO. Additionally, the concentration-dependent formation of GS(O)NH2 in cells treated with an HNO donor suggests that the concentration of GS(O)NH2 can be correlated to intracellular levels of HNO.
• Zhou, Y., Bright, L. K., Shi, W., Aspinwall, C. A., & Baker, L. A. (2014). Ion channel probes for scanning ion conductance microscopy. Langmuir : the ACS journal of surfaces and colloids, 30(50), 15351-5.
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  The sensitivity and selectivity of ion channels provide an appealing opportunity for sensor development. Here, we describe ion channel probes (ICPs), which consist of multiple ion channels reconstituted into lipid bilayers suspended across the opening of perflourinated glass micropipets. When incorporated with a scanning ion conductance microscope (SICM), ICPs displayed a distance-dependent current response that depended on the number of ion channels in the membrane. With distance-dependent current as feedback, probes were translated laterally, to demonstrate the possibility of imaging with ICPs. The ICP platform yields several potential advantages for SICM that will enable exciting opportunities for incorporation of chemical information into imaging and for high-resolution imaging.
• Adem, S. M., Mansfield, E., Keogh, J. P., Hall Jr., H. K., & Aspinwall, C. A. (2013). Practical considerations for preparing polymerized phospholipid bilayer capillary coatings for protein separations. Analytica Chimica Acta, 772, 93-98.
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  PMID: 23540253;PMCID: PMC3670586;Abstract: Phosphorylcholine (PC) based phospholipid bilayers have proven useful as capillary coating materials due to their inherent resistance to non-specific protein adsorption. The primary limitation of this important class of capillary coatings remains the limited long-term chemical and physical stability of the coatings. Recently, a method for increasing phospholipid coating stability in fused silica capillaries via utilization of polymerized, synthetic phospholipids was reported. Here, we expand upon these studies by investigating polymerized lipid bilayer capillary coatings with respect to separation performance including run-to-run, day-to-day and column-to-column reproducibility and long-term stability. In addition, the effects of pH and capillary inner diameter on polymerized phospholipid coated capillaries were investigated to identify optimized coating conditions. The coatings are stabilized for protein separations across a wide range of pH values (4.0-9.3), a unique property for capillary coating materials. Additionally, smaller inner diameter capillaries (≤50. μm) were found to yield marked enhancements in coating stability and reproducibility compared to wider bore capillaries, demonstrating the importance of capillary size for separations employing polymerized phospholipid coatings. © 2013 Elsevier B.V.
• Aspinwall, C., Baker, C. A., Bright, L. K., & Aspinwall, C. A. (2013). Photolithographic fabrication of microapertures with well-defined, three-dimensional geometries for suspended lipid membrane studies. Analytical chemistry, 85(19).
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  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.
• Baker, C. A., Bright, L. K., & Aspinwall, C. A. (2013). Photolithographic fabrication of microapertures with well-defined, three-dimensional geometries for suspended lipid membrane studies. Analytical Chemistry, 85(19), 9078-9086.
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  PMID: 23987300;PMCID: PMC3822043;Abstract: 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. © 2013 American Chemical Society.
• Berglund, E., Berglund, D., Akcakaya, P., Ghaderi, M., Daré, E., Berggren, P., Köhler, M., Aspinwall, C. A., Lui, W., Zedenius, J., Larsson, C., & Bränström, R. (2013). Evidence for Ca²⁺-regulated ATP release in gastrointestinal stromal tumors. Experimental Cell Research, 319(8), 1229-1238.
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  PMID: 23499741;PMCID: PMC3628080;Abstract: Gastrointestinal stromal tumors (GISTs) are thought to originate from the electrically active pacemaker cells of the gastrointestinal tract. Despite the presence of synaptic-like vesicles and proteins involved in cell secretion it remains unclear whether GIST cells possess regulated release mechanisms. The GIST tumor cell line GIST882 was used as a model cell system, and stimulus-release coupling was investigated by confocal microscopy of cytoplasmic free Ca2+ concentration ([Ca2+]i), flow cytometry, and luminometric measurements of extracellular ATP. We demonstrate that GIST cells have an intact intracellular Ca2+-signaling pathway that regulates ATP release. Cell viability and cell membrane integrity was preserved, excluding ATP leakage due to cell death and suggesting active ATP release. The stimulus-secretion signal transduction is at least partly dependent on Ca2+ influx since exclusion of extracellular Ca2+ diminishes the ATP release. We conclude that measurements of ATP release in GISTs may be a useful tool for dissecting the signal transduction pathway, mapping exocytotic components, and possibly for the development and evaluation of drugs. Additionally, release of ATP from GISTs may have importance for tumor tissue homeostasis and immune surveillance escape. © 2013 Elsevier Inc.
• Bright, L. K., Baker, C. A., Agasid, M. T., Lin, M. a., & Aspinwall, C. A. (2013). Decreased aperture surface energy enhances electrical, mechanical, and temporal stability of suspended lipid membranes. ACS Applied Materials and Interfaces, 5(22), 11918-11926.
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  Abstract: The development of next-generation transmembrane protein-based biosensors relies heavily on the use of black lipid membranes (BLMs); however, electrical, mechanical, and temporal instability of BLMs poses a limiting challenge to biosensor development. In this work, micrometer-sized glass apertures were modified with silanes of different chain length and fluorine composition, including 3-cyanopropyldimethychlorosilane (CPDCS), ethyldimethylchlorosilane (EDCS), n-octyldimethylchlorosilane (ODCS), (tridecafluoro-1, 1, 2, 2-tetrahydrooctyl)dimethylchlorosilane (PFDCS), or (heptadecafluoro-1,1,2,2- tetrahydrodecyl)dimethylchlorosilane (PFDDCS), to explore the effect of substrate surface energy on BLM stability. Low energy silane-modified surfaces promoted enhanced lipid-substrate interactions that facilitate the formation of low-leakage, stable BLMs. The surface energies of silane-modified substrates were 30 ± 3, 16 ± 1, 14 ± 2, 11 ± 1, and 7.1 ± 2 mJ m-2 for CDCS, EDCS, ODCS, PFDCS, and PFDDCS, respectively. Decreased surface energy directly correlated to improved electrical, mechanical, and temporal BLM stability. Amphiphobic perfluorinated surface modifiers yielded superior performance compared to traditional hydrocarbon modifiers in terms of stability and BLM formation, with only marginal effects on BLM membrane permeability. Leakage currents obtained for PFDCS and PFDDCS BLMs were elevated only 10-30%, though PFDDCS modification yielded >5-fold increase in electrical stability as indicated by breakdown voltage (> 2000 mV vs 418 ± 73 mV), and >25-fold increase in mechanical stability as indicated by air-water transfers (> 50 vs 2 ± 0.2) when compared to previously reported CPDCS modification. Importantly, the dramatically improved membrane stabilities were achieved with no deleterious effects on reconstituted ion channel function, as evidenced by α-hemolysin activity. Thus, this approach provides a simple, low cost, and broadly applicable alternative for BLM stabilization and should contribute significantly toward the development of next-generation ion-channel-functionalized biosensors. © 2013 American Chemical Society.
• Aspinwall, C., Gallagher, E. S., Comi, T. J., Braun, K. L., & Aspinwall, C. A. (2012). Online photolytic optical gating of caged fluorophores in capillary zone electrophoresis utilizing an ultraviolet light-emitting diode. Electrophoresis, 33(18).
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  Photolytic optical gating (POG) facilitates rapid, on-line and highly sensitive analyses, though POG utilizes UV lasers for sample injection. We present a low-cost, more portable alternative, employing an ultraviolet light-emitting diode (UV-LED) array to inject caged fluorescent dyes via photolysis. Utilizing the UV-LED array, labeled amino acids were injected with nanomolar limits of detection (270 ± 30 nM and 250 ± 30 nM for arginine and citrulline, respectively). When normalized for the difference in light intensity, the UV-LED array provides comparable sensitivity to POG utilizing UV lasers. Additionally, the UV-LED array yielded sufficient beam quality and stability to facilitate coupling with a Hadamard transform, resulting in increased sensitivity. This work shows, for the first time, the use of an UV-LED for online POG with comparable sensitivity to conventional laser sources but at a lower cost.
• Aspinwall, C., Janczak, C. M., & Aspinwall, C. A. (2012). Composite nanoparticles: the best of two worlds. Analytical and bioanalytical chemistry, 402(1).
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  Nanomaterials have rapidly moved into the mainstream for chemical and biological analysis. Nanoparticle probes enhance signal intensity, increase the chemical and physical stability of the probe, and facilitate surface modification for specific targeting. Unfortunately, common problems are encountered with many nanoparticle probes, e.g., poor solubility, poor biocompatibility, and leakage of encapsulated components, that severely restrict the application of probes to ex vivo samples under carefully controlled conditions. A wide range of recently developed multifunctional nanomaterials are poised to make significant contributions to molecular analysis of biological systems. Composite nanoparticle geometries, including composites, hybrids, and core-shell nanoparticles prepared using two or more materials, e.g., silica/inorganic, silica/polymer, or polymer/inorganic combinations, offer improved solubility, easier functionalization, and decreased toxicity compared with the related single-component materials. Furthermore, composite nanomaterials present substantial signal amplification, and improved multiplexing for higher-sensitivity and higher-resolution measurements. Further development and integration of composite nanomaterials into the quantitative sciences will play a key role in the future of functional probes for imaging, quantitative analysis, and biological manipulation.
• Aspinwall, C., Muhandiramlage, T. P., Cheng, Z., Roberts, D. L., Keogh, J. P., Hall, H. K., & Aspinwall, C. A. (2012). Determination of pore sizes and relative porosity in porous nanoshell architectures using dextran retention with single monomer resolution and proton permeation. Analytical chemistry, 84(22).
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  Unilamellar phospholipid vesicles prepared using the polymerizable lipid bis-sorbylphosphatidylcholine (bis-SorbPC) yield three-dimensional nanoarchitectures that are highly permeable to small molecules. The resulting porous phospholipid nanoshells (PPNs) are potentially useful for a range of biomedical applications including nanosensors and nanodelivery vehicles for cellular assays and manipulations. The uniformity and size distribution of the pores, key properties for sensor design and utilization, have not previously been reported. Fluorophore-assisted carbohydrate electrophoresis (FACE) was utilized to assess the nominal molecular weight cutoff limit (NMCL) of the PPN via analysis of retained dextran with single monomer resolution. The NMCL of PPNs prepared from pure bis-SorbPC was equivalent to a 1800 Da linear dextran, corresponding to a maximum pore diameter of 2.6 nm. Further investigation of PPNs prepared using binary mixtures of bis-SorbPC and dioleoylphosphatidylcholine (DOPC) revealed a similar NMCL when the bis-SorbPC content exceeded 30 mol %, whereas different size-dependent permeation was observed below this composition. Below 30 mol % bis-SorbPC, dextran retention provided insufficient mass resolution (162 Da) to observe porosity on the experimental time scale; however, proton permeability showed a marked enhancement for bis-SorbPC ≥ 10 mol %. Combined, these data suggest that the NMCL for native pores in bis-SorbPC PPNs results from an inherent property within the lipid assembly that can be partially disrupted by dilution of bis-SorbPC below a critical value for domain formation. Additionally, the analytical method described herein should prove useful for the challenging task of elucidating porosity in a range of three-dimensional nanomaterials.
• Gallagher, E. S., Comi, T. J., Braun, K. L., & Aspinwall, C. A. (2012). Online photolytic optical gating of caged fluorophores in capillary zone electrophoresis utilizing an ultraviolet light-emitting diode. Electrophoresis, 33(18), 2903-2910.
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  PMID: 22911376;PMCID: PMC3716455;Abstract: Photolytic optical gating (POG) facilitates rapid, on-line and highly sensitive analyses, though POG utilizes UV lasers for sample injection. We present a low-cost, more portable alternative, employing an ultraviolet light-emitting diode (UV-LED) array to inject caged fluorescent dyes via photolysis. Utilizing the UV-LED array, labeled amino acids were injected with nanomolar limits of detection (270 ± 30 nM and 250 ± 30 nM for arginine and citrulline, respectively). When normalized for the difference in light intensity, the UV-LED array provides comparable sensitivity to POG utilizing UV lasers. Additionally, the UV-LED array yielded sufficient beam quality and stability to facilitate coupling with a Hadamard transform, resulting in increased sensitivity. This work shows, for the first time, the use of an UV-LED for online POG with comparable sensitivity to conventional laser sources but at a lower cost. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
• Muhandiramlage, T. P., Cheng, Z., Roberts, D. L., Keogh, J. P., Hall, H. K., & Aspinwall, C. A. (2012). Determination of pore sizes and relative porosity in porous nanoshell architectures using dextran retention with single monomer resolution and proton permeation. Analytical Chemistry, 84(22), 9754-9761.
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  PMID: 23083108;PMCID: PMC3502667;Abstract: Unilamellar phospholipid vesicles prepared using the polymerizable lipid bis-sorbylphosphatidylcholine (bis-SorbPC) yield three-dimensional nanoarchitectures that are highly permeable to small molecules. The resulting porous phospholipid nanoshells (PPNs) are potentially useful for a range of biomedical applications including nanosensors and nanodelivery vehicles for cellular assays and manipulations. The uniformity and size distribution of the pores, key properties for sensor design and utilization, have not previously been reported. Fluorophore-assisted carbohydrate electrophoresis (FACE) was utilized to assess the nominal molecular weight cutoff limit (NMCL) of the PPN via analysis of retained dextran with single monomer resolution. The NMCL of PPNs prepared from pure bis-SorbPC was equivalent to a 1800 Da linear dextran, corresponding to a maximum pore diameter of 2.6 nm. Further investigation of PPNs prepared using binary mixtures of bis-SorbPC and dioleoylphosphatidylcholine (DOPC) revealed a similar NMCL when the bis-SorbPC content exceeded 30 mol %, whereas different size-dependent permeation was observed below this composition. Below 30 mol % bis-SorbPC, dextran retention provided insufficient mass resolution (162 Da) to observe porosity on the experimental time scale; however, proton permeability showed a marked enhancement for bis-SorbPC ≥ 10 mol %. Combined, these data suggest that the NMCL for native pores in bis-SorbPC PPNs results from an inherent property within the lipid assembly that can be partially disrupted by dilution of bis-SorbPC below a critical value for domain formation. Additionally, the analytical method described herein should prove useful for the challenging task of elucidating porosity in a range of three-dimensional nanomaterials. © 2012 American Chemical Society.
• Heitz, B. A., Xu, J., Jones, I. W., Keogh, J. P., Comi, T. J., Hall, H. K., Aspinwall, C. A., & Saavedra, S. S. (2011). Polymerized planar suspended lipid bilayers for single ion channel recordings: comparison of several dienoyl lipids. Langmuir : the ACS journal of surfaces and colloids, 27(5), 1882-90.
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  The stabilization of suspended planar lipid membranes, or black lipid membranes (BLMs), through polymerization of mono- and bis-functionalized dienoyl lipids was investigated. Electrical properties, including capacitance, conductance, and dielectric breakdown voltage, were determined for BLMs composed of mono-DenPC, bis-DenPC, mono-SorbPC, and bis-SorbPC both prior to and following photopolymerization, with diphytanoyl phosphocholine (DPhPC) serving as a control. Poly(lipid) BLMs exhibited significantly longer lifetimes and increased the stability of air-water transfers. BLM stability followed the order bis-DenPC > mono-DenPC ≈ mono-SorbPC > bis-SorbPC. The conductance of bis-SorbPC BLMs was significantly higher than that of the other lipids, which is attributed to a high density of hydrophilic pores, resulting in relatively unstable membranes. The use of poly(lipid) BLMs as matrices for supporting the activity of an ion channel protein (IC) was explored using α-hemolysin (α-HL), a model IC. Characteristic i-V plots of α-HL were maintained following photopolymerization of bis-DenPC, mono-DenPC, and mono-SorbPC, demonstrating the utility of these materials for preparing more durable BLMs for single-channel recordings of reconstituted ICs.
• Aspinwall, C., Rauf, F., Huang, Y., Muhandiramlage, T. P., & Aspinwall, C. A. (2010). Analysis of protein kinase A activity in insulin-secreting cells using a cell-penetrating protein substrate and capillary electrophoresis. Analytical and bioanalytical chemistry, 397(8).
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  A cell-penetrating, fluorescent protein substrate was developed to monitor intracellular protein kinase A (PKA) activity in cells without the need for cellular transfection. The PKA substrate (PKAS) was prepared with a 6xhistidine purification tag, an enhanced green fluorescent protein (EGFP) reporter, an HIV-TAT protein transduction domain for cellular translocation and a pentaphosphorylation motif specific for PKA. PKAS was expressed in Escherichia coli and purified by metal affinity chromatography. Incubation of PKAS in the extracellular media facilitated translocation into the intracellular milieu in HeLa cells, betaTC-3 cells and pancreatic islets with minimal toxicity in a time and concentration dependent manner. Upon cellular loading, glucose-dependent phosphorylation of PKAS was observed in both betaTC-3 cells and pancreatic islets via capillary zone electrophoresis. In pancreatic islets, maximal PKAS phosphorylation (83 +/- 6%) was observed at 12 mM glucose, whereas maximal PKAS phosphorylation (86 +/- 4%) in betaTC-3 cells was observed at 3 mM glucose indicating a left-shifted glucose sensitivity. Increased PKAS phosphorylation was observed in the presence of PKA stimulators forskolin and 8-Br-cAMP (33% and 16%, respectively), with corresponding decreases in PKAS phosphorylation observed in the presence of PKA inhibitors staurosporine and H-89 (40% and 54%, respectively).
• Heitz, B. A., Jones, I. W., Hall, H. K., Aspinwall, C. A., & Saavedra, S. S. (2010). Fractional polymerization of a suspended planar bilayer creates a fluid, highly stable membrane for ion channel recordings. Journal of the American Chemical Society, 132(20), 7086-93.
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  Suspended planar lipid membranes (or black lipid membranes (BLMs)) are widely used for studying reconstituted ion channels, although they lack the chemical and mechanical stability needed for incorporation into high-throughput biosensors and biochips. Lipid polymerization enhances BLM stability but is incompatible with ion channel function when membrane fluidity is required. Here, we demonstrate the preparation of a highly stable BLM that retains significant fluidity by using a mixture of polymerizable and nonpolymerizable phospholipids. Alamethicin, a voltage-gated peptide channel for which membrane fluidity is required for activity, was reconstituted into mixed BLMs prepared using bis-dienoyl phosphatidylcholine (bis-DenPC) and diphytanoyl phosphatidylcholine (DPhPC). Polymerization yielded BLMs that retain the fluidity required for alamethicin activity yet are stable for several days as compared to a few hours prior to polymerization. Thus, these polymerized, binary composition BLMs feature both fluidity and long-term stability.
• Aspinwall, C., Roberts, D. L., Ma, Y., Bowles, S. E., Janczak, C. M., Pyun, J., Saavedra, S. S., & Aspinwall, C. A. (2009). Polymer-stabilized phospholipid vesicles with a controllable, pH-dependent disassembly mechanism. Langmuir : the ACS journal of surfaces and colloids, 25(4).
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  In this letter, we report a facile method to prepare robust phospholipid vesicles using commonly available phospholipids that are stabilized via the formation of an interpenetrating, acid-labile, cross-linked polymer network that imparts a site for controlled polymer destabilization and subsequent vesicle degradation. The polymer network was formed in the inner lamella of the phospholipid bilayer using 2,2-di(methacryloyloxy-1-ethoxy)propane (DMOEP) and butyl methacrylate (BMA). Upon exposure to acidic conditions, the highly cross-linked polymer network was partially converted to smaller linear polymers, resulting in substantially reduced vesicle stability upon exposure to chemical and physical insults. Isolated polymers had pH-dependent-solubility in THF. Transmission electron microscopy and dynamic light scattering revealed time-dependent enhanced vesicle stability in high concentrations of surfactant and vacuum conditions at elevated pH, whereas exposure to acidic pH rapidly decreased the vesicle stability, with complete destabilization observed in less than 24 h. The resultant transiently stabilized vesicles may prove useful for enhanced drug delivery and chemical sensing applications and allow for improved physiological clearance.
• Heitz, B. A., Xu, J., Hall, H. K., Aspinwall, C. A., & Saavedra, S. S. (2009). Enhanced long-term stability for single ion channel recordings using suspended poly(lipid) bilayers. Journal of the American Chemical Society, 131(19), 6662-3.
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  Black lipid membranes (BLMs) are widely used for recording the activity of incorporated ion channel proteins. However, BLMs are inherently unstable structures that typically rupture within a few hours after formation. Here, stabilized BLMs were formed using the polymerizable lipid bis-dienoyl phosphatidylcholine (bis-DenPC) on glass pipettes of approximately 10 microm (I.D.). After polymerization, these BLMs maintained steady conductance values for several weeks, as compared to a few hours for unpolymerized membranes. The activity of an ion channel, alpha-hemolysin, incorporated into bis-DenPC BLMs prior to polymerization, was maintained for 1 week after BLM formation and polymerization. These lifetimes are a substantial improvement over those achievable with conventional BLM technologies. Polymerized BLMs containing functional ion channels may represent an enabling technology for development of robust biosensors and drug screening devices.
• Aspinwall, C., Braun, K. L., Hapuarachchi, S., Fernandez, F. M., & Aspinwall, C. A. (2007). High-sensitivity detection of biological amines using fast Hadamard transform CE coupled with photolytic optical gating. Electrophoresis, 28(17).
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  Here, we report the first utilization of Hadamard transform CE (HTCE), a high-sensitivity, multiplexed CE technique, with photolytic optical gating sample injection of caged fluorescent labels for the detection of biologically important amines. Previous implementations of HTCE have relied upon photobleaching optical gating sample injection of fluorescent dyes. Photolysis of caged fluorescent labels reduces the fluorescence background, providing marked enhancements in sensitivity compared to photobleaching. Application of fast Hadamard transform CE (fHTCE) for fluorescein-based dyes yields a ten-fold higher sensitivity for photolytic injections compared to photobleaching injections, due primarily to the reduced fluorescent background provided by caged fluorescent dyes. Detection limits as low as 5 pM (ca. 18 molecules per injection event) were obtained with on-column LIF detection using fHTCE in less than 25 s, with the capacity for continuous, online separations. Detection limits for glutamate and aspartate below 150 pM (1-2 amol/injection event) were obtained using photolytic sample injection, with separation efficiencies exceeding 1 x 10(6) plates/m and total multiplexed separation times as low as 8 s. These results strongly support the feasibility of this approach for high-sensitivity dynamic chemical monitoring applications.
• Aspinwall, C., Hapuarachchi, S., & Aspinwall, C. A. (2007). Design, characterization, and utilization of a fast fluorescence derivatization reaction utilizing o-phthaldialdehyde coupled with fluorescent thiols. Electrophoresis, 28(7).
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  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.
• Aspinwall, C., Mansfield, E., Ross, E. E., & Aspinwall, C. A. (2007). Preparation and characterization of cross-linked phospholipid bilayer capillary coatings for protein separations. Analytical chemistry, 79(8).
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  Analysis of protein and peptide mixtures via capillary electrophoresis is hindered by nonspecific adsorption of analytes to the capillary walls, resulting in poor separations and quantitative reproducibility. Phospholipid bilayer (PLB) coatings are very promising for improving protein and peptide separations due to the native resistance to nonspecific protein adsorption offered by PLBs; however, these coatings display limited chemical and temporal stability. Here, we show the preparation and characterization of a highly cross-linked, polymerized phospholipid capillary coating prepared using bis-SorbPC. Poly(bis-SorbPC) PLB coatings are prepared in situ within fully enclosed fused silica capillaries via self-assembly and radical polymerization. Polymerization of the PLB coating stabilizes the membrane against desorption from the surface and migration in an electric field, improves the temporal and chemical stability, and allows for the separation of both cationic and anionic proteins, while preserving the native resistance to nonspecific protein adsorption of natural PLBs.
• Aspinwall, C., Mansfield, E., Ross, E. E., D'Ambruoso, G. D., Keogh, J. P., Huang, Y., & Aspinwall, C. A. (2007). Fabrication and characterization of spatially defined, multiple component, chemically functionalized domains in enclosed silica channels using cross-linked phospholipid membranes. Langmuir : the ACS journal of surfaces and colloids, 23(22).
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  The utilization of photopolymerized phospholipids for the preparation of spatially defined, chemically functionalized, micron-sized domains within enclosed fluidic channels was recently reported (Ross, E. E.; et al. J. Am. Chem. Soc. 2005, 127, 16756-7). Fabrication of the phospholipid patterns is achieved via self-assembly of photoreactive phospholipid membranes that are subsequently cross-linked via UV-irradiation through a photomask. In this work, we have characterized the chemical and physical stability of the self-assembled, chemically functionalized, cross-linked phospholipid patterns and extended this approach to the preparation of cross-linked phospholipid patterns with multiple chemical functionalities. Poly(bis-SorbPC) patterns were found to withstand a number of chemical and physical challenges, including drying/rehydration, solvent or surfactant rinse, and extended storage without compromising the size or morphology of the cross-linked phospholipid patterns. Nonspecific adsorption of proteins was found to be markedly reduced in the presence of UV-photopolymerized poly(bis-SorbPC) compared to bare silica capillaries. The resulting barcode-like patterns were used to prepare protein-functionalized domains via covalent attachment of fluorescent proteins and active enzymes to chemically functionalized lipid headgroups. We also demonstrate multiple component polymer lipid patterns with adjacent chemically functionalized polymer lipid regions. The unique combination of stability, biocompatibility, reduced nonspecific protein adsorption, and the availability of numerous chemically functionalized lipid headgroups suggests the utility of this approach for preparing a widely applicable platform for multicomponent, high-throughput chemical sensing and screening applications.
• Braun, K. L., Hapuarachchi, S., Fernandez, F. M., & Aspinwall, C. A. (2007). High-sensitivity detection of biological amines using fast Hadamard transform CE coupled with photolytic optical gating. Electrophoresis, 28(17), 3115-3121.
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  PMID: 17674422;Abstract: Here, we report the first utilization of Hadamard transform CE (HTCE), a high-sensitivity, multiplexed CE technique, with photolytic optical gating sample injection of caged fluorescent labels for the detection of biologically important amines. Previous implementations of HTCE have relied upon photobleaching optical gating sample injection of fluorescent dyes. Photolysis of caged fluorescent labels reduces the fluorescence background, providing marked enhancements in sensitivity compared to photobleaching. Application of fast Hadamard transform CE (fHTCE) for fluorescein-based dyes yields a ten-fold higher sensitivity for photolytic injections compared to photobleaching injections, due primarily to the reduced fluorescent background provided by caged fluorescent dyes. Detection limits as low as 5 pM (ca. 19 molecules per injection event) were obtained with on-column LIF detection using fHTCE in less than 25 s, with the capacity for continuous, online separations. Detection limits for glutamate and aspartate below 150 pM (1-2 amol/ injection event) were obtained using photolytic sample injection, with separation efficiencies exceeding 1 × 106 plates/m and total multiplexed separation times as low as 8 s. These results strongly support the feasibility of this approach for high-sensitivity dynamic chemical monitoring applications. © 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
• Bränström, R., Leibiger, I. B., Leibiger, B., Klement, G., Nilsson, J., Århem, P., Aspinwall, C. A., Corkey, B. E., Larsson, O., & Berggren, P. -. (2007). Single residue (K332A) substitution in Kir6.2 abolishes the stimulatory effect of long-chain acyl-CoA esters: Indications for a long-chain acyl-CoA ester binding motif. Diabetologia, 50(8), 1670-1677.
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  PMID: 17522836;Abstract: Aims/hypothesis: The pancreatic beta cell ATP-sensitive potassium (K ATP) channel, composed of the pore-forming α subunit Kir6.2, a member of the inward rectifier K+channel family, and the regulatory β subunit sulfonylurea receptor 1 (SUR1), a member of the ATP-binding cassette superfamily, couples the metabolic state of the cell to electrical activity. Several endogenous compounds are known to modulate KATP channel activity, including ATP, ADP, phosphatidylinositol diphosphates and long-chain acyl coenzyme A (LC-CoA) esters. LC-CoA esters have been shown to interact with Kir6.2, but the mechanism and binding site(s) have yet to be identified. Materials and methods: Using multiple sequence alignment of known acyl-CoA ester interacting proteins, we were able to identify four conserved amino acid residues that could potentially serve as an acyl-CoA ester-binding motif. The motif was also recognised in the C-terminal region of Kir6.2 (R311-332) but not in SUR1. Results: Oocytes expressing Kir6.2ΔC26 K332A repeatedly generated K+currents in inside-out membrane patches that were sensitive to ATP, but were only weakly activated by 1 μmol/l palmitoyl-CoA ester. Compared with the control channel (Kir6.2ΔC26), Kir6.2ΔC26 K332A displayed unaltered ATP sensitivity but significantly decreased sensitivity to palmitoyl-CoA esters. Coexpression of Kir6.2ΔC26 K332A and SUR1 revealed slightly increased activation by palmitoyl-CoA ester but significantly decreased activation by the acyl-CoA esters compared with the wild-type KATP channel and Kir6.2ΔC26+SUR1. Computational modelling, using the crystal structure of KirBac1.1, suggested that K332 is located on the intracellular domain of Kir6.2 and is accessible to intracellular modulators such as LC-CoA esters. Conclusions/interpretation: These results verify that LC-CoA esters interact at the pore-forming subunit Kir6.2, and on the basis of these data we propose an acyl-CoA ester binding motif located in the C-terminal region. © 2007 Springer-Verlag.
• Mansfield, E., Ross, E. E., & Aspinwall, C. A. (2007). Preparation and characterization of cross-linked phospholipid bilayer capillary coatings for protein separations. Analytical Chemistry, 79(8), 3135-3141.
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  PMID: 17373774;PMCID: PMC2665996;Abstract: Analysis of protein and peptide mixtures via capillary electrophoresis is hindered by nonspecific adsorption of analytes to the capillary walls, resulting in poor separations and quantitative reproducibility. Phospholipid bilayer (PLB) coatings are very promising for improving protein and peptide separations due to the native resistance to nonspecific protein adsorption offered by PLBs; however, these coatings display limited chemical and temporal stability. Here, we show the preparation and characterization of a highly cross-linked, polymerized phospholipid capillary coating prepared using bis-SorbPC. Poly(bis-SorbPC) PLB coatings are prepared in situ within fully enclosed fused silica capillaries via self-assembly and radical polymerization. Polymerization of the PLB coating stabilizes the membrane against desorption from the surface and migration in an electric field, improves the temporal and chemical stability, and allows for the separation of both cationic and anionic proteins, while preserving the native resistance to nonspecific protein adsorption of natural PLBs. © 2007 American Chemical Society.
• Mansfield, E., Ross, E. E., D'Ambruoso, G. D., Keogh, J. P., Huang, Y., & Aspinwall, C. A. (2007). Fabrication and characterization of spatially defined, multiple component, chemically functionalized domains in enclosed silica channels using cross-linked phospholipid membranes. Langmuir, 23(22), 11326-11333.
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  PMID: 17892310;PMCID: PMC2654225;Abstract: The utilization of photopolymerized phospholipids for the preparation of spatially defined, chemically functionalized, micron-sized domains within enclosed fluidic channels was recently reported (Ross, E. E.; et al. J. Am. Chem. Soc. 2005, 127, 16756-7). Fabrication of the phospholipid patterns is achieved via self-assembly of photoreactive phospholipid membranes that are subsequently cross-linked via UV-irradiation through a photomask. In this work, we have characterized the chemical and physical stability of the self-assembled, chemically functionalized, cross-linked phospholipid patterns and extended this approach to the preparation of cross-linked phospholipid patterns with multiple chemical functionalities. Poly(bis-SorbPC) patterns were found to withstand a number of chemical and physical challenges, including drying/ rehydration, solvent or surfactant rinse, and extended storage without compromising the size or morphology of the cross-linked phospholipid patterns. Nonspecific adsorption of proteins was found to be markedly reduced in the presence of UV-photopolymerized poly(bis-SorbPC) compared to bare silica capillaries. The resulting barcode-like patterns were used to prepare protein-functionalized domains via covalent attachment of fluorescent proteins and active enzymes to chemically functionalized lipid headgroups. We also demonstrate multiple component polymer lipid patterns with adjacent chemically functionalized polymer lipid regions. The unique combination of stability, biocompatibility, reduced nonspecific protein adsorption, and the availability of numerous chemically functionalized lipid headgroups suggests the utility of this approach for preparing a widely applicable platform for multicomponent, high-throughput chemical sensing and screening applications. © 2007 American Chemical Society.
• Senarath-Yapa, M. D., Phimphivong, S., Coym, J. W., Wirth, M. J., Aspinwall, C. A., & Saavedra, S. S. (2007). Preparation and characterization of poly(lipid)-coated, fluorophore-doped silica nanoparticles for biolabeling and cellular imaging. Langmuir : the ACS journal of surfaces and colloids, 23(25), 12624-33.
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  The fabrication, characterization, and implementation of poly(lipid)-coated, highly luminescent silica nanoparticles as fluorescent probes for labeling of cultured cells are described. The core of the probe is a sol-gel-derived silica nanoparticle, 65-100 nm in diameter, in which up to several thousand dye molecules are encapsulated (Lian, W.; et al. Anal. Biochem. 2004, 334, 135-144). The core is coated with a membrane composed of bis-sorbylphosphatidylcholine, a synthetic polymerizable lipid that is chemically cross-linked to enhance the environmental and chemical stability of the membrane relative to a fluid lipid membrane. The poly(lipid) coating has two major functions: (i) to reduce nonspecific interactions, based on the inherently biocompatible properties of the phosphorylcholine headgroup, and (ii) to permit functionalization of the particle, by doping the coating with lipids bearing chemically reactive or bioactive headgroups. Both functions are demonstrated: (i) Nonspecific adsorption of dissolved proteins to bare silica nanoparticles and of bare nanoparticles to cultured cells is significantly reduced by application of the poly(lipid) coating. (ii) Functionalization of poly(lipid)-coated nanoparticles with a biotin-conjugated lipid creates a probe that can be used to target both dissolved protein receptors as well as receptors on the membranes of cultured cells. Measurements performed on single nanoparticles bound to planar supported lipid bilayers verify that the emission intensity of these probes is significantly greater than that of single protein molecules labeled with several fluorophores.
• Aspinwall, C., Braun, K. L., Hapuarachchi, S., Fernandez, F. M., & Aspinwall, C. A. (2006). Fast Hadamard transform capillary electrophoresis for on-line, time-resolved chemical monitoring. Analytical chemistry, 78(5).
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  We report a new approach for collecting and deconvoluting the data in Hadamard transform capillary electrophoresis, referred to as fast Hadamard transform capillary electrophoresis (fHTCE). Using fHTCE, total analysis times can be reduced by up to 48% per multiplexed separation compared to conventional Hadamard transform capillary electrophoresis (cHTCE) while providing comparable signal-to-noise ratio enhancements. In fHTCE, the sample is injected following a pseudorandom pulsing sequence derived from the first row of a simplex matrix (S-matrix) in contrast to cHTCE, which utilizes a sequence of twice the length. In addition to the temporal savings provided by fHTCE, a 50% reduction in sample consumption is also realized due to the decreased number of sample injections. We have applied fHTCE to the analysis of mixtures of neurotransmitters and related compounds to yield improved signal-to-noise ratios with a total analysis time under 10 s. In addition, we demonstrate the capability of fHTCE to perform time-resolved monitoring of changes in the concentration of model neurochemical compounds.
• Aspinwall, C., Cheng, Z., & Aspinwall, C. A. (2006). Nanometre-sized molecular oxygen sensors prepared from polymer stabilized phospholipid vesicles. The Analyst, 131(2).
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  Nanometre-sized, chemically-stabilized phospholipid vesicle sensors have been developed for detection of dissolved molecular oxygen. Sensors were prepared by forming 150 nm phospholipid vesicles from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or DOPC doped with small (
• Aspinwall, C., Cheng, Z., D'Ambruoso, G. D., & Aspinwall, C. A. (2006). Stabilized porous phospholipid nanoshells. Langmuir : the ACS journal of surfaces and colloids, 22(23).
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  Chemically stabilized, porous phospholipid nanoshells (PPNs) were prepared via copolymerization of reactive monomers with unilamellar bis-Sorbyl phosphatidylcholine vesicles. The resulting PPN vesicular assemblies possess a highly porous membrane structure that allows passage of small molecules, which can react with encapsulated proteins and reporters. The unique combination of membrane stability and porosity will prove useful for preparing nanometer-sized sensor, container, and reactor platforms stable in harsh chemical and biological environments.
• Aspinwall, C., Hapuarachchi, S., Janaway, G. A., & Aspinwall, C. A. (2006). Capillary electrophoresis with a UV light-emitting diode source for chemical monitoring of native and derivatized fluorescent compounds. Electrophoresis, 27(20).
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  We report the utilization of a high power UV light-emitting diode for fluorescence detection (UV-LED-IF) in CE separations. CE-UV-LED-IF allows analysis of a range of environmentally and biologically important compounds, including PAHs and biogenic amines, including neurotransmitters, amino acids, proteins, and peptides, that have been derivatized with UV-excited fluorogenic labels, e.g., o-phthalic dicarboxaldehyde/beta-mercaptoethanol (OPA/beta-ME). The 365 nm UV-LED was used as a stable, low cost source for detection of UV-excited fluorescent compounds. UV-LED-IF was used with both zonal CE separations and MEKC. Native fluorescence detection of PAHs was accomplished with detection limits ranging from 10 nM to 1.3 microM. Detection limits for OPA/beta-ME-labeled glutamic acid and aspartic acid were 11 and 10 nM, respectively, for off-line labeling, and 47 and 47 nM, respectively, for on-line labeling, comparable to UV-laser-based systems. Analysis of OPA/beta-ME-labeled proteins and peptides was performed with 28 and 47 nM detection limits for BSA and myoglobin, respectively.
• Aspinwall, C., Hapuarachchi, S., Premeau, S. P., & Aspinwall, C. A. (2006). High-speed capillary zone electrophoresis with online photolytic optical injection. Analytical chemistry, 78(11).
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  We report an online, optical injection interface for capillary zone electrophoresis (CZE) based upon photophysical activation of a caged, fluorogenic label covalently attached to the target analyte. This injection interface allows online analysis of biomolecular systems with high temporal resolution and high sensitivity. Samples are injected onto the separation capillary by photolysis of a caged-fluorescein label using the 351-364 nm irradiation of an Ar+ laser. Following injection, the sample is separated and detected via laser-induced fluorescence detection at 488 nm. Detection limits for online analysis of arginine, glutamic acid, and aspartic acid were less than 1 nM with separation times less than 5 s and separation efficiencies exceeding 1,000,000 plates/m. Rapid injection of proteins was demonstrated with migration times less than 500 ms and 0.5 nM detection limits. Online monitoring was performed with response times less than 20 s, suggesting the feasibility of this approach for online, in vivo analysis for a range of biologically relevant analytes.
• Braun, K. L., Hapuarachchi, S., Fernandez, F. M., & Aspinwall, C. A. (2006). Fast Hadamard transform capillary electrophoresis for on-line, time-resolved chemical monitoring. Analytical Chemistry, 78(5), 1628-1635.
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  PMID: 16503616;Abstract: We report a new approach for collecting and deconvoluting the data in Hadamard transform capillary electrophoresis, referred to as fast Hadamard transform capillary electrophoresis (fHTCE). Using fHTCE, total analysis times can be reduced by up to 48% per multiplexed separation compared to conventional Hadamard transform capillary electrophoresis (cHTCE) while providing comparable signal-to-noise ratio enhancements. In fHT-CE, the sample is injected following a pseudorandom pulsing sequence derived from the first row of a simplex matrix (S-matrix) in contrast to cHTCE, which utilizes a sequence of twice the length. In addition to the temporal savings provided by fHTCE, a 50% reduction in sample consumption is also realized due to the decreased number of sample injections. We have applied fHTCE to the analysis of mixtures of neurotransmitters and related compounds to yield improved signal-to-noise ratios with a total analysis time under 10 s. In addition, we demonstrate the capability of fHTCE to perform time-resolved monitoring of changes in the concentration of model neurochemical compounds. © 2006 American Chemical Society.
• Cheng, Z., & Aspinwall, C. A. (2006). Nanometre-sized molecular oxygen sensors prepared from polymer stabilized phospholipid vesicles. Analyst, 131(2), 236-243.
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  PMID: 16440088;Abstract: Nanometre-sized, chemically-stabilized phospholipid vesicle sensors have been developed for detection of dissolved molecular oxygen. Sensors were prepared by forming 150 nm phospholipid vesicles from 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC) or DOPC doped with small (
• Cheng, Z., D'Ambruoso, G. D., & Aspinwall, C. A. (2006). Stabilized porous phospholipid nanoshells. Langmuir, 22(23), 9507-9511.
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  PMID: 17073472;PMCID: PMC3500630;Abstract: Chemically stabilized, porous phospholipid nanoshells (PPNs) were prepared via copolymerization of reactive monomers with unilamellar bis-Sorbyl phosphatidylcholine vesicles. The resulting PPN vesicular assemblies possess a highly porous membrane structure that allows passage of small molecules, which can react with encapsulated proteins and reporters. The unique combination of membrane stability and porosity will prove useful for preparing nanometersized sensor, container, and reactor platforms stable in harsh chemical and biological environments. © 2006 American Chemical Society.
• Gooding, J. J., Spence, D., Aspinwall, C. A., Benson, D. E., Cliffel, D. E., Culbertson, C., Desaire, H., Garcia, C. D., Garno, J., Håkansson, K., Hilder, E., Hobbs, J., Lingjun, L. i., Martin, R. S., Minteer, S. D., Nordon, A., Reid, G., Shippy, S., Stevenson, K. J., , Szunerits, S., et al. (2006). Emerging investigators special issue. Analyst, 131(2), 179-.
• Hapuarachchi, S., Janaway, G. A., & Aspinwall, C. A. (2006). Capillary electrophoresis with a UV light-emitting diode source for chemical monitoring of native and derivatized fluorescent compounds. Electrophoresis, 27(20), 4052-4059.
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  PMID: 16983640;Abstract: We report the utilization of a high power UV light-emitting diode for fluorescence detection (UV-LED-IF) in CE separations. CE-UV-LED-IF allows analysis of a range of environmentally and biologically important compounds, including PAHs and biogenic amines, including neurotransmitters, amino acids, proteins, and peptides, that have been derivatized with UV-excited fluorogenic labels, e.g., o-phthalic dicarbox-aldehyde/ β-mercaptoethanol (OPA/β-ME). The 365 nm UV-LED was used as a stable, low cost source for detection of UV-excited fluorescent compounds. UV-LED-IF was used with both zonal CE separations and MEKC. Native fluorescence detection of PAHs was accomplished with detection limits ranging from 10 nM to 1.3 μM. Detection limits for OPA/ β-ME-labeled glutamic acid and aspartic acid were 11 and 10 nM, respectively, for off-line labeling, and 47 and 47 nM, respectively, for on-line labeling, comparable to UV-laser-based systems. Analysis of OPA/ β-ME-labeled proteins and peptides was performed with 28 and 47 nM detection limits for BSA and myoglobin, respectively. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
• Hapuarachchi, S., Premeau, S. P., & Aspinwall, C. A. (2006). High-speed capillary zone electrophoresis with online photolytic optical injection. Analytical Chemistry, 78(11), 3674-3680.
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  PMID: 16737223;Abstract: We report an online, optical injection interface for capillary zone electrophoresis (CZE) based upon photophysical activation of a caged, fluorogenic label covalently attached to the target analyte. This injection interface allows online analysis of biomolecular systems with high temporal resolution and high sensitivity. Samples are injected onto the separation capillary by photolysis of a caged-fluorescein label using the 351-364 nm irradiation of an Ar+ laser. Following injection, the sample is separated and detected via laser-induced fluorescence detection at 488 nm. Detection limits for online analysis of arginine, glutamic acid, and aspartic acid were less than 1 nM with separation times less than 5 s and separation efficiencies exceeding 1 000 000 plates/m. Rapid injection of proteins was demonstrated with migration times less than 500 ms and 0.5 nM detection limits. Online monitoring was performed with response times less than 20 s, suggesting the feasibility of this approach for online, in vivo analysis for a range of biologically relevant analytes. © 2006 American Chemical Society.
• Aspinwall, C. A., & Yeung, E. S. (2005). Screening populations of individual cells for secretory heterogeneity. Analytical and Bioanalytical Chemistry, 381(3), 660-666.
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  PMID: 15609009;Abstract: Many common metabolic and neurological disorders are related to defective regulation of exocytosis at the level of single cells. In exocytosis, vesicles containing the secretory product of a given cell type fuse with the plasma membrane allowing release of the vesicular contents into the extracellular environment where the physiological action can be exerted. The typical secretory vesicle contains between 0.15 and 10 attomoles of material that is released on a millisecond timescale. Hence, detection of this process presents several chemical and analytical challenges. In this work, we utilize the native ATP, stored at high concentrations within the secretory vesicles of most neuroendocrine cells and co-released during exocytosis and during cell lysis, as a universal tracer of cellular secretion events. Organisms studied include pancreatic islets, mast cells, and Escherischia coli. Cellular processes investigated include exocytotic release, stimulated cell lysis, and programmed cell lysis. © Springer-Verlag 2004.
• Aspinwall, C., Ross, E. E., Mansfield, E., Huang, Y., & Aspinwall, C. A. (2005). In situ fabrication of three-dimensional chemical patterns in fused silica separation capillaries with polymerized phospholipids. Journal of the American Chemical Society, 127(48).
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  We report a new molecular approach for in situ generation of micron scale, chemically and biochemically functionalized patterns inside three-dimensional, completely enclosed fluidic channels. The formation of chemical patterns is based upon a combination of lipid bilayer self-assembly and UV photopolymerization of photoreactive, cross-linkable phospholipids. Using this approach, we have functionalized capillaries of varying inner diameters with a range of chemistries useful for protein and peptide immobilization. Here, we demonstrate the ability to produce small molecule and protein-based chemical patterns.
• Ross, E. E., Mansfield, E., Huang, Y., & Aspinwall, C. A. (2005). In situ fabrication of three-dimensional chemical patterns in fused silica separation capillaries with polymerized phospholipids. Journal of the American Chemical Society, 127(48), 16756-16757.
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  PMID: 16316200;Abstract: We report a new molecular approach for in situ generation of micron scale, chemically and biochemically functionalized patterns inside three-dimensional, completely enclosed fluidic channels. The formation of chemical patterns is based upon a combination of lipid bilayer self-assembly and UV photopolymerization of photoreactive, cross-linkable phospholipids. Using this approach, we have functionalized capillaries of varying inner diameters with a range of chemistries useful for protein and peptide immobilization. Here, we demonstrate the ability to produce small molecule and protein-based chemical patterns. Copyright © 2005 American Chemical Society.
• Berggren, P., Yang, S., Murakami, M., Efanov, A. M., Uhles, S., Köhler, M., Moede, T., Fernström, A., Appelskog, I. B., Aspinwall, C. A., Zaitsev, S. V., Larsson, O., Moitoso, L., Fecher-Trost, C., Weißgerber, P., Ludwig, A., Leibiger, B., Juntti-Berggren, L., Barker, C. J., , Gromada, J., et al. (2004). Removal of Ca²⁺ channel β₃ subunit enhances Ca²⁺ oscillation frequency and insulin exocytosis. Cell, 119(2), 273-284.
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  PMID: 15479643;Abstract: An oscillatory increase in pancreatic β cell cytoplasmic free Ca 2+ concentration, [Ca2+]i, is a key feature in glucose-induced insulin release. The role of the voltage-gated Ca2+ channel β3 subunit in the molecular regulation of these [Ca 2+]i oscillations has now been clarified by using β3 subunit-deficient β cells. β3 knockout mice showed a more efficient glucose homeostasis compared to wild-type mice due to increased glucose-stimulated insulin secretion. This resulted from an increased glucose-induced [Ca2+]i oscillation frequency in β cells lacking the β3 subunit, an effect accounted for by enhanced formation of inositol 1,4,5-trisphosphate (InsP3) and increased Ca2+ mobilization from intracellular stores. Hence, the β3 subunit negatively modulated InsP3-induced Ca 2+ release, which is not paralleled by any effect on the voltage-gated L type Ca2+ channel. Since the increase in insulin release was manifested only at high glucose concentrations, blocking the β3 subunit in the β cell may constitute the basis for a novel diabetes therapy.
• Bränström, R., Aspinwall, C. A., Välimäki, S., Östensson, C., Tibell, A., Eckhard, M., Brandhorst, H., Corkey, B. E., Berggren, P. -., & Larsson, O. (2004). Long-Chain CoA esters activate human pancreatic beta-cell K_ATP channels: Potential role in Type 2 diabetes. Diabetologia, 47(2), 277-283.
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  PMID: 14740158;Abstract: Aims/hypothesis. The ATP-regulated potassium (KATP) channel in the pancreatic beta cell couples the metabolic state to electrical activity. The primary regulator of the KATP channel is generally accepted to be changes in ATP/ADP ratio, where ATP inhibits and ADP activates channel activity. Recently, we showed that long-chain CoA (LC-CoA) esters form a new class of potent KATP channel activators in rodents, as studied in inside-out patches. Methods. In this study we have investigated the effects of LC-CoA esters in human pancreatic beta cells using the inside-out and whole-cell configurations of the patch clamp technique. Results. Human K ATP channels were potently activated by acyl-CoA esters with a chain length exceeding 12 carbons. Activation by LC-CoA esters did not require the presence of Mg2+ or adenine nucleotides. A detailed characterization of the concentration-dependent relationship showed an EC50 of 0.7±0.1 μmol/l. Furthermore, in the presence of an ATP/ADP ratio of 10 (1.1 mmol/l total adenine nucleotides), whole-cell KATP channel currents increased approximately six-fold following addition of 1 μmol/l LC-CoA ester. The presence of 1 μmol/l LC-CoA in the recording pipette solution increased beta-cell input conductance, from 0.5±0.2 nS to 2.5±1.3 nS. Conclusion/interpretation. Taken together, these results show that LC-CoA esters are potent activators of the KATP channel in human pancreatic beta cells. The fact that LC-CoA esters also stimulate K ATP channel activity recorded in the whole-cell configuration, points to the ability of these compounds to have an important modulatory role of human beta-cell electrical activity under both physiological and pathophysiological conditions.
• Aspinwall, C. A., Qian, W., Roper, M. G., Kulkarni, R. N., Kahn, C. R., & Kennedy, R. T. (2000). Roles of insulin receptor substrate-1, phosphatidylinositol 3-kinase, and release of intracellular Ca²⁺ stores in insulin-stimulated insulin secretion in β-cells. Journal of Biological Chemistry, 275(29), 22331-22338.
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  PMID: 10764813;Abstract: The signaling pathway by which insulin stimulates insulin secretion and increases in intracellular free Ca2+ concentration ([Ca2+](i)) in isolated mouse pancreatic β-cells and clonal β-cells was investigated. Application of insulin to single β-cells resulted in increases in [Ca2+](i) that were of lower magnitude, slower onset, and longer lifetime than that observed with stimulation with tolbutamide. Furthermore, the increases in [Ca2+](i) originated from interior regions of the cell rather than from the plasma membrane as with depolarizing stimuli. The insulin-induced [Ca2+](i) changes and insulin secretion at single β-cells were abolished by treatment with 100 nM wortmannin or 1 μM thapsigargin; however, they were unaffected by 10 μM U73122, 20 μM nifedipine, or removal of Ca2+ from the medium. Insulin-stimulated insulin secretion was also abolished by treatment with 2 μM bisindolylmaleimide I, but [Ca2+](i) changes were unaffected. In an insulin receptor substrate-1 gene disrupted β-cell tumor line, insulin did not evoke either [Ca2+](i) changes or insulin secretion. The data suggest that autocrine-activated increases in [Ca2+](i) are due to release of intracellular Ca2+ stores, especially the endoplasmic reticulum, mediated by insulin receptor substrate-1 and phosphatidylinositol 3-kinase. Autocrine activation of insulin secretion is mediated by the increase in [Ca2+](i) and activation of protein kinase C.
• Qian, W., Aspinwall, C. A., Battiste, M. A., & Kennedy, R. T. (2000). Detection of secretion from single pancreatic β-cells using extracellular fluorogenic reactions and confocal fluorescence microscopy. Analytical Chemistry, 72(4), 711-717.
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  PMID: 10701254;Abstract: Confocal microscopy with Zinquin, a fluorogenic Zn2+-specific indicator, was used for spatially and temporally resolved measurement of Zn2+ efflux from single pancreatic β-cells. When cells were incubated in buffer containing Zinquin, application of insulin secretagogues evoked an increase in fluorescence around the surface of the cell, indicative of detection of Zn2+ efflux from the cell. The fluorescence increases corresponded spatially and temporally with measurements of exocytosis obtained simultaneously by amperometry. When images were taken at 266-ms intervals, the detection limit for Zn2+ was ~0.5 μM. With this image frequency, it was possible to observe bursts of fluorescence which were interpreted as fluctuations of Zn2+ level due to exocytosis. The average intensity of these fluorescence bursts corresponded to a Zn2+ concentration of ~7 μM. Since insulin is co-stored with Zn2+ in secretory vesicles, it was concluded that the Zn2+ efflux corresponded to exocytosis of insulin/Zn2+-containing granules from the β-cell. Exocytosis sites identified by this technique were frequently localized to one portion of the cell, indicative of active areas of release.
• Aspinwall, C. A., Lakey, J. R., & Kennedy, R. T. (1999). Insulin-stimulated insulin secretion in single pancreatic beta cells. Journal of Biological Chemistry, 274(10), 6360-6365.
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  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.
• Jung, S., Aspinwall, C. A., & Kennedy, R. T. (1999). Detection of multiple patterns of oscillatory oxygen consumption in single mouse islets of Langerhans. Biochemical and Biophysical Research Communications, 259(2), 331-335.
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  PMID: 10362508;Abstract: A novel oxygen microsensor was used to measure oxygen levels in single mouse islets as a function of glucose concentration. Oxygen consumption of individual islets was 5.99 ± 1.17, 9.21 ± 2.15, and 12.22 ± 2.16 pmol/min at 3, 10, and 20 mM glucose, respectively (mean ± SEM, n = 10). Consumption of oxygen was islet-size dependent as larger islets consumed more oxygen than smaller islets but smaller islets consumed more oxygen per unit volume than larger islets. Elevating glucose levels from 3 to 10 mM induced pronounced fast oscillations in oxygen level (period of 12.1 ± 1.7 s, n = 6) superimposed on top of large slow oscillations (period of 3.3 ± 0.6 min, n = 6). The fast oscillations could be completely abolished by treatment with the L-type Ca2+-channel blocker nifedipine (40 μM) with a lesser effect on slow oscillations. Slow oscillations were almost completely dependent upon extracellular Ca2+. The oxygen patterns closely mimic those that have previously been reported for intracellular Ca2+ levels and are suggestive of an important role for Ca2+ in amplifying metabolic oscillations.
• Jung, S., Gorski, W., Aspinwall, C. A., Kauri, L. M., & Kennedy, R. T. (1999). Oxygen microsensor and its application to single cells and mouse pancreatic islets. Analytical Chemistry, 71(17), 3642-3649.
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  PMID: 10489519;Abstract: An oxygen microsensor with a
• R., J., Aspinwall, C. A., Cavanagh, T. J., & Kennedy, R. T. (1999). Secretion from islets and single islet cells following cryopreservation. Cell Transplantation, 8(6), 691-698.
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  PMID: 10701497;Abstract: The ability to cryopreserve pancreatic islets has allowed the development of low-temperature banks that permit pooling of islets from multiple donors and allows time for sterility and viability testing. However, previous studies have shown that during cryopreservation and thawing there is a loss of islet mass and a reduction in islet function. The aim of this study was to measure and compare insulin secretion from cultured nonfrozen and frozen-thawed canine islets and β-cells. Canine islets were isolated from mongrel dogs using intraductal collagenase distention, mechanical dissociation, and EuroFicoll purification. One group of purified islets was cultured overnight before dissociation into single cells and subsequent analysis. Remaining islets were cultured overnight (22°C) and then cryopreserved in 2 M dimethyl sulfoxide (DMSO) solution using a slow stepwise addition protocol with slow cooling to -40°C before storage in liquid nitrogen (-196°C). Frozen islets were rapidly thawed (200°C/min) and the DMSO removed using a sucrose dilution. From a series of seven consecutive canine islet isolations, islet recovery following posteryopreservation tissue culture was 81.5 ± 4.8% compared to precryopreservation counts. In vitro islet function was equivalent between cultured nonfrozen and frozen-thawed islets with a calculated stimulation index of 10.4 ± 1.5 (mean ± SEM) for the frozen-thawed islets, compared with 12.4 ± 1.2 for the cultured nonfrozen controls (p = ns, n = 7 paired experiments). Amperometric detection of secretion from single β-cells in vitro has the sensitivity and temporal resolution to detect single exocytotic events and allows secretion to be monitored from single β-cells in real time. Secretion from single β-cells elicited by chemical stimulation was detected using a carbon fiber microelectrode. The frequency of exocytosis events was equivalent between the cultured nonfrozen and frozen-thawed β-cells with an average of 7.0 ± 1.32 events per stimulation for the cultured nonfrozen group compared with 6.0 ± 1.45 events from the frozen then thawed preparations (minimum of 10 cells per run per paired experiment, p = ns) following stimulation with tolbutamide. The average amount of insulin released per individual exocytosis event was equivalent for the cultured nonfrozen and frozen-thawed islets. In addition, β-cells responded to both tolbutamide and muscarinic stimulation following cryopreservation. It was determined that β-cells recovered following cryopreservation are capable of secreting insulin at levels and frequencies comparable to those of cultured nonfrozen islet preparations.
• Lakey, J. R., Aspinwall, C. A., Cavanagh, T. J., & Kennedy, R. T. (1998). Effect of cryopreservation on canine islet insulin secretion as measured by amperometric techniques. Transplantation Proceedings, 30(2), 382-.
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  PMID: 9532091;
• Tao, L., Aspinwall, C. A., & Kennedy, R. T. (1998). On-line competitive immunoassay based on capillary electrophoresis applied to monitoring insulin secretion from single islets of Langerhans. Electrophoresis, 19(3), 403-408.
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  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.
• Aspinwall, C. A., Brooks, S. A., Kennedy, R. T., & R., J. (1997). Effects of intravesicular H⁺ and extracellular H⁺ and Zn²⁺ on insulin secretion in pancreatic beta cells. Journal of Biological Chemistry, 272(50), 31308-31314.
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  PMID: 9395458;Abstract: The effects of extracellular Zn2+ and pH and intravesicular pH on insulin and 5-hydroxytryptamine (5-HT) secretion from pancreatic beta cells were investigated. Insulin and 5-HT secretion from single cells was detected by amperometry as a series of current spikes corresponding to detection of multimolecular packets secreted by exocytosis. Spike width was used as a measure of the kinetics of clearance from the cell and the area of spikes as a measure of amount released. Changes in extracellular pH from 6.9 to 7.9 caused insulin spikes to become narrower with no change in area, whereas the same treatments had no effect on 5-HT secretion. Treatment of cells with Bafilomycin A1 or N-ethylmaleimide, both of which are expected to increase intravesicular pH by inhibiting V-type H+-ATPase, had no effect on 5-HT secretion but caused insulin spikes to become more narrow. These results indicate that exposure to high pH, whether intravesicular or extracellular, accelerates release of insulin during exocytosis without affecting the amount of insulin released. Increasing extracellular Zn2+ concentration from 0 to 25 μM increased the width and decreased the area of insulin spikes without affecting 5-HT secretion. Zn2+ effects were likely exerted through a common-ion effect on Zn2+-insulin dissociation. It was concluded that intravesicular storage conditions and extracellular ions can affect free insulin concentration in the vicinity of beta cells during secretion.
• Gorski, W., Aspinwall, C. A., Lakey, J. R., & Kennedy, R. T. (1997). Ruthenium catalyst for amperometric determination of insulin at physiological pH. Journal of Electroanalytical Chemistry, 425(1-2), 191-199.
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  Abstract: A ruthenium-oxide-type catalytic film (RuOx) was produced on carbon fiber microelectrodes by cycling the electrode potential between 0.65 and -0.85V vs. SSCE at 100 V s-1 in an air-equilibrated acidic solution of RuCl3. The film catalyzes oxidation of insulin in a saline buffer at pH7.4. The minimum number of electrons transferred during the insulin oxidation at 0.65 V is 6.7. The analytical performance of the modified electrode as an amperometric detector for insulin was characterized using flow injection analysis. Linear least squares calibration curves over the range 0.10 to 1.0 μM (five points) had slopes of 72 ± 2 pA μM-1 and correlation coefficients of 0.999 or greater. The detection limit, calculated as the concentration that would yield a signal equal to three times the root mean square noise, was 23 nM and response time (t90%) was 40ms or less. The electrode response to 0.2 μM insulin was stable for 3 days. The modified electrode was used for amperometric detection of exocytosis from individual pancreatic β-cells.
• Shen, H., Aspinwall, C. A., & Kennedy, R. T. (1997). Dual microcolumn immunoassay applied to determination of insulin secretion from single islets of Langerhans and insulin in serum. Journal of Chromatography B: Biomedical Applications, 689(2), 295-303.
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  PMID: 9080314;Abstract: A dual microcolumn immunoassay (DMIA) was developed and applied to determination of insulin in biological samples. The DMIA utilized a protein G capillary column (150 μm I.D.) with covalently attached anti-insulin to selectively capture and concentrate insulins in a sample. Insulins retained in the capillary immunoaffinity column were desorbed and injected onto a reversed-phase capillary column (150 μm I.D.) for further separation from interferences such as cross-reactive antigens and non-specifically adsorbed sample components. Bovine, porcine and rat insulin all cross-reacted with the antibody and could be determined simultaneously. Using a UV absorbance detector, the dual microcolumn system had a detection limit of 10 fmol or 20 pM for 500-μl sample volumes. The DMIA system was used to measure glucose-stimulated insulin secretion from single rat islets of Langerhans. Because of the separation in the second dimension, both rat I and rat II insulin could be independently determined. The system was also evaluated for determination of insulin in serum. Using microcolumns instead of conventional HPLC columns resulted in several advantages including use of less chromatographic material and improved mass detection limit.
• Kennedy, R. T., Huang, L., & Aspinwall, C. A. (1996). Extracellular pH is required for rapid release of insulin from Zn - insulin precipitates in β-cell secretory vesicles during exocytosis. Journal of the American Chemical Society, 118(7), 1795-1796.
• Carlin, R. T., Sullivan, T., Sherman, J. W., & Aspinwall, C. A. (1993). Asymmetric electrode kinetics induced by concurrent metal-ligand bond dissociation. Electrochimica Acta, 38(7), 927-934.
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  Abstract: The electrochemistry of the Cu(II)/Cu(I) couple under nitrogen and carbon monoxide has been investigated in the ambient-temperature molten salt AlCl3:MEICl (MEICl = 1-methyl-3-ethyl-imidazolium chloride) at a 250 μm tungsten disk electrode. Under nitrogen, the couple exhibits reversible electrode kinetics; however, under carbon monoxide, a Cu(I)CO complex is formed and the Cu(II)/Cu(I) couple displays asymmetric, quasi-reversible electrode kinetics. Pulse voltammetric data were fit with a nonlinear least-squares fitting program to give an apparent standard rate constant (k0a) of 1.5 × 10-3 cm s-1 and an anodic transfer coefficient (β) of 0.12-0.17 for the oxidation of the Cu(I)CO complex. The change from reversible to quasi-reversible electrode kinetics is attributed to the concurrent dissociation of the Cu(I)CO bond during the electron transfer process. © 1993.