An enzyme-modified capillary as a platform for simultaneous fluorometric detection of d-glucose and l- lactate.

The preparation of a glass capillary pattered with lipid layers　on which lactate dehydrogenase (LDH) and glucose dehydrogenase (GDH) were regionally adsorbed and its application for simultaneous detection of d-glucose and l-lactate in human serum is described. A lipid layer was formed on the surface of BSA-unabsorbed octadecyltrichlorosilane (OTS) inner wall of a glass capillary. The electrostatic charge of the lipid layer was a key factor for adsorbing the enzymes on the lipid layer. The fluorescence intensities were observed at each enzyme site in the presence of diaphorase (DIA), ß-nicotinamide-adenine dinucleotide oxidized (NAD), resazurin, d-glucose and l-lactate. The fluorescence intensities at each enzyme site increased with an increase in the concentration of d-glucose and l-lactate=with the detection limits of 32 µM and 4.9 µM, respectively.

Transmembrane Signaling with Lipid-Bilayer Assemblies as a Platform for Channel-Based Biosensing.

Artificial and natural lipid membranes that elicit transmembrane signaling is are useful as a platform for channel-based biosensing. In this account we summarize our research on the design of transmembrane signaling associated with lipid bilayer membranes containing nanopore-forming compounds. Channel-forming compounds, such as receptor ion-channels, channel-forming peptides and synthetic channels, are embedded in planar and spherical bilayer lipid membranes to develop highly sensitive and selective biosensing methods for a variety of analytes. The membrane-bound receptor approach is useful for introducing receptor sites on both planar and spherical bilayer lipid membranes. Natural receptors in biomembranes are also used for designing of biosensing methods.

A Planar Bilayer Lipid Membrane Sensor Using a Miniaturized Auto-patch System.

A bilayer lipid membrane sensor was constructed with a miniaturized auto-patch system. The performance of the patch system was optimized to obtain an analytically relevant signal. A biosensor based on an anti-BSA-antibody as a receptor and gramicidin as a signal transduction element was demonstrated. The sensor for BSA exhibited a detection limit of pg/mL level for BSA.

Inhibitory assay for degradation of collagen IV by cathepsin B with a surface plasmon resonance sensor.

We describe a simple method for evaluating the inhibition of collagen IV degradation by cathepsin B with a surface plasmon resonance (SPR) biosensor. The change in the SPR signal decreased with an increase in the concentration of cathepsin B inhibitors. The order of the inhibitory constant (Ki) obtained by the SPR method was CA074Me≈Z-Phe-Phe-FMK < leupeptin. This order was different from that obtained by benzyloxycarbonyl-Phe-Phe-Fluoromethylketone (Z-Phe-Phe-FMK) as a peptide substrate. The comparison of Ki suggested that CA074 and Z-Phe-Phe-FMK inhibited exopeptidase activity, and leupeptin inhibited the endopeptidase activity of cathepsin B more strongly.

Monitoring of cholesterol oxidation in a lipid bilayer membrane using streptolysin O as a sensing and signal transduction element.

Streptolysin O (SLO), which recognizes sterols and forms nanopores in lipid membranes, is proposed as a sensing element for monitoring cholesterol oxidation in a lipid bilayer. The structural requirements of eight sterols for forming nanopores by SLO confirmed that a free 3-OH group in the ß-configuration of sterols is required for recognition by SLO in a lipid bilayer. The extent of nanopore formation by SLO in lipid bilayers increased in the order of cholestanol

Giant unilamellar vesicles containing Rhodamine 6G as a marker for immunoassay of bovine serum albumin and lipocalin-2.

Functionalized giant unilamellar vesicles (GUVs) containing a fluorescence dye Rhodamine 6G is proposed as a marker in sandwich-type immunoassay for bovine serum albumin (BSA) and lipocalin-2 (LCN2). The GUVs were prepared by the electroformation method and functionalized with anti-BSA antibody and anti-LCN2 antibody, respectively. The purification of antibody-modified GUVs was achieved by conventional centrifugation and a washing step in a flow system. To antigen on an antibody slip, antibody-modified GUVs were added as a marker and incubated. After wash-out of excess reagents and lysis of the bound GUVs with Triton X-100, the fluorescence image was captured. The fluorometric immunoassays for BSA and LCN2 exhibited lower detection limits of 4 and 80 fg ml(-)(1), respectively.

Visual Assay of Total Iron in Human Serum with Bathophenanthrolin Disulfonate-accommodated MCM-41.

A simple visual method for determining the total iron in human serum is proposed based on color development in the nanospace of mesoporous silica MCM-41 and a chromogenic ligand bathophenathroline disulfonate (BPS). Observing the color intensity of a complex between iron(II) and BPS devloped on the MCM-41 material by the naked eye enabled us to quntify iron(II) with a detection limit of 0.5 µM. The BPS-loaded MCM-41 was successfully applied for quantifying the total iron in human serum.

An enzyme-entrapped agarose gel for visualization of ischemia-induced L-glutamate fluxes in hippocampal slices in a flow system.

An agarose gel slip containing L-glutamate oxidase (GluOx), horseradish peroxidase (HRP) and a dye DA-64 is proposed as a tool for visualizing ischemia-induced L-glutamate release in hippocampal slices in a flow system. The agarose slip with a detection limit of 6.0 ± 0.8 µmol L(-1) for L-glutamate enabled us to visualize L-glutamate fluxes in a flow system. The leak of a dye from the agarose gel was negligible and a diffusion blur due to spreading of Bindshedler's Green (BG) within the gel was suppressed. Monitoring the time-dependent change of ischemia-induced L-glutamate fluxes at neuronal regions CA1, DG and CA3 of hippocampal slices is demonstrated.

Evaluation of cathepsin B activity for degrading collagen IV using a surface plasmon resonance method and circular dichroism spectroscopy.

Evaluation of cathepsin B activities for degrading collagen IV and heat-denatured collagen IV (gelatin) were performed by surface plasmon resonance (SPR) and circular dichroism (CD) measurements. The optimal pH of cathepsin B activity for degrading each substrate was around 4.0. The ΔRU(15 min), which is a decrease in the SPR signal at 15 min after injection of cathepsin B, was smaller for collagen IV than for heat-denatured collagen IV owing to the presence of triple-helical conformation. An unstable nature of the triple-helical conformation of collagen IV at pH 4.0 was shown by the CD study. Degrading collagen IV by cathepsin B was facilitated owing to a local unwinding of the triple-helical conformation caused by proteolytic cleavage of the non-helical region. The concentration dependence of the initial velocity for degrading collagen IV by cathepsin B at pH 4.0 was biphasic, showing that cathepsin B at low concentration exhibits exopeptidase activity, while the enzyme at high concentration exhibits endopeptidase activity. The kinetic parameters for the exopeptidase activity of cathepsin B toward collagen IV and heat-treated collagen IV were evaluated and discussed in terms of the protease mechanism.

Pore-forming compounds as signal transduction elements for highly sensitive biosensing.

Pore-forming compounds are attracting much attention due to the signal transduction ability for the development of highly sensitive biosensing. In this review, we describe an overview of the recent advances made by our group in the design of molecular sensing interfaces of spherical and planar lipid bilayers and natural bilayers. The potential uses of pore-forming compounds, such as gramicidin and MCM-41, in lipid bilayers and natural glutamate receptor channels in biomembrane are presented.

Highly sensitive and rapid assay of substance P and streptolysin O in human serum using immuno-liposomes and gramicidin channels.

A highly sensitive and rapid method for the determination of substance P (SP) and streptolysin O (SLO) in human serum is described. The assay is based on enriching the analyte by agglutination/precipitation of immuno-liposomes and enhancing the fluorescence intensity by gramicidin A channels. A mixture of the immuno-liposomes encapsulating a pH-sensitive fluorescence dye BCECF, gramicidin A and a given concentration of SP (or SLO) is preincubated in a solution and captured on anti-SP (or anti-SLO)-modified cover slips, followed by measuring fluorescence images after removing excess liposomes. The method allowed quantifying SP and SLO in the range from sub-pg mL(-1) to pg mL(-1), with detection limits of 0.32 pg mL(-1) and 8 fg mL(-1), respectively. The present method could determine SP and SLO in 50-125 times diluted human serum without any extraction steps. The assay can be completed within 60 min.

Implantable patch sensor for L-glutamate in hippocampal slices.

We describe an implantable patch sensor for monitoring L-glutamate in hippocampal slices in submerged and interface preparations. This patch sensor is prepared by excising the cell membrane in the CA3 region of a hippocampal slice in a submerged preparation, and then implanted in the target neuronal region (CA1) of mouse hippocampal slices. The lifetime of the sensor was 3-8 min for the interface slice and 40-50 min for the submerged slice. The calibration of an implanted sensor can be achieved by adding an L-glutamate solution to a bath (ACSF) solution. The monitoring of L-glutamate release in the CA1 region of mouse hippocampal slices under chemical stimulation with γ-aminobutyric acid (GABA) and potassium chloride (KCl) was demonstrated.

Planar lipid bilayers containing gramicidin A as a molecular sensing system based on an integrated current.

The channel activity of gramicidin A in free-standing planar lipid bilayers with different charges of polar head groups and various lengths of hydrocarbon tails were analyzed in terms of the channel conductance, the lifetime of channel events and the magnitude of integrated currents. The channel activity of gramicidin A in lipid bilayers is tunable by adjusting the membrane composition. The in situ coupling of the anti-BSA antibody as a model protein to the amine moiety of phosphatidylethanolamine (PE) in a lipid bilayer by the amine coupling method allowed us to design an antigen (BSA)-sensitive interface, in which the integrated current, rather than the frequency of channel event, can be used as an analytical signal. The potential of the present system for highly sensitive and selective detection of BSA at 10(-9) g/mL level is demonstrated.

Real-time monitoring of matrix metalloproteinase-9 collagenolytic activity with a surface plasmon resonance biosensor.

We describe a simple method for real-time monitoring of matrix metalloproteinase-9 (MMP-9) collagenolytic activity for native triple helical collagen IV with a surface plasmon resonance (SPR) biosensor. The proteolytic activity of MMP-9 is measured as a decrease in the SPR signal resulting from the cleavage of collagen IV immobilized on the sensor surface. The kinetic parameters of full-length MMP-9 and its catalytic domain-catalytic constant (k(cat)), association rate constant (k(a)), and dissociation rate constant (k(d))-were estimated by the SPR method. The presence of sodium chloride and a nonionic detergent Brij-35 in a reaction solution led to the lower collagenolytic activity of MMP-9, whereas they suppressed the nonspecific interaction between MMP-9 and a cysteamine-modified chip. The comparison of kinetic parameters between MMP-9 and its catalytic domain revealed that the association constant of MMP-9 is much larger than that of the catalytic domain, suggesting that the interplay among hemopexin-like domain, fibronectin type II repeats motif, and linker region (O-glycosylated domain) plays an important role in recognizing collagen IV.

Simultaneous monitoring of excitatory postsynaptic potentials and extracellular L-glutamate in mouse hippocampal slices.

Simultaneous monitoring of amperometric currents at a glass capillary sensor based on recombinant GluOx and field excitatory postsynaptic potentials (fEPSPs) were performed in region CA1 of mouse hippocampal slices. A transient increase in the glutamate current relative to the basal one at control stimulation (0.052Hz) was evoked by stimulation at 2 Hz for 2 min. The magnitude of the glutamate current was dependent on the intensity (current) of a 2 Hz stimulus and reflected the slope of the fEPSP. The in situ calibration of the L-glutamate sensor revealed that the extracellular concentration of L-glutamate released by 2 Hz stimulation before tetanus is in the range from 0.8 to 2.2 µM and it is enhanced after tetanic stimulation. The L-glutamate level at a test stimulus (0.052 Hz) was estimated to be 32 nM. The recombinant GluOx-based sensor exhibited weak responses to glutamine above 300 µM and L-aspartic acid above 200 µM. The potential use of a glass capillary sensor in combination with fEPSP measurements for electrophysiological study is discussed.

In vitro measurements of extracellular L-glutamate level in region CA3 of mouse hippocampal slices under chemical stimulation.

The concentration level of extracellular L-glutamate released from region CA3 of mouse hippocampal slices under tetraethylammonium (TEA) chloride and KCl stimulation was measured with independent methods, i.e., a capillary-based enzyme sensor, a patch sensor, and an enzyme-based imaging method. The L-glutamate level was compared with those at regions CA1 and DG. It was found that the enhanced concentration level at CA3 by TEA stimulation is very similar to that at CA1, but it is much lower than that at DG. The order of the regional distribution of L-glutamate, i.e., DG > CA1 ≈ CA3, was the same as that obtained by K(+) stimulation. However, in the presence of an uptake inhibitor, DL-TBOA, KCl stimulation showed the strongest L-glutamate flux at CA1, while TEA stimulation exhibited the strongest flux at CA3. The usefulness of the present approach for knowing the extracellular L-glutamate level in acute hippocampal slices is discussed.

A reusable liposome array and its application to assay of growth-hormone-related peptides.

We describe a reusable liposome array based on the formation of cleavable disulfide cross-links between liposomes and the surface of a glass slip. The N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP)-modified liposomes encapsulating a pH-sensitive fluorescence dye were immobilized on a 3-mercaptopropyltrimethoxysilane (MTS)-modified glass slip through the formation of disulfide bonds. The regeneration of a used slip was performed by the lysis of immobilized liposomes with Triton X-100 and the cleavage of disulfide bonds by reduction with TCEP, followed by immobilization of SPDP-modified liposomes. The regeneration steps did not affect the fluorescence intensity of re-immobilized liposomes. The liposome array was applied to simultaneous quantification of growth hormone related peptides, i.e., GHRF and somatostatin, in a mixture. After optimizing the assay condition, the method allowed quantification of GHRF and somatostatin in concentration ranges from 0.5 x 10(-9) to 0.5 x 10(-7) g/mL with detection limits of 2 x 10(-10) and 3 x 10(-10) g/mL, respectively.

Comparison of the L-glutamate level in mouse hippocampal slices under tetraethylammonium chloride stimulation as measured with a glass capillary sensor and a patch sensor.

The concentration level of L-glutamate released from the region CA1 of mouse hippocampal slices under tetraethylammonium chloride (TEA) stimulation was measured by two independent methods, i.e., a glass capillary-based enzyme sensor and a patch sensor, and compared with each other for different slice preparations. In a submerged slice preparation, the sensors were positioned in bath solutions several tens microm above CA1, respectively. The sensors exhibited almost the same level of extra-slice L-glutamate concentration. When a capillary sensor was implanted in region CA1 at a depth of approximately 10 microm, the TEA-induced L-glutamate release pattern was very similar to those observed with the capillary sensor in a bath use. The concentration level of intra-slice (extracellular) L-glutamate was found to be in the range from 6 to 10 microM, which was significantly larger than that of the extra-slice one. These results demonstrate that L-glutamate released from each neuronal region inevitably diffuses out of the slices, and the extra-slice L-glutamate level reflects the extracellular one.

Visualizing L-glutamate fluxes in acute hippocampal slices with glutamate oxidase-immobilized coverslips.

We used a glutamate oxidase (GluOx)-immobilized glass coverslip for reducing diffusional blur and improving the temporal resolution of visualizing L-glutamate fluxes in acute brain slices. The immobilization of GluOx on an avidin modified glass coverslips was achieved by optimized the amine coupling method. The GluOx coverslip was applied to the imaging of L-glutamate fluxes in acute hippocampal slices under hypoxia and KCl stimulation. A slice from mouse brain was loaded with horseradish peroxidase (HRP) and substrate DA-64, and placed on the GluOx coverslip for stimulation. The regional distribution of hypoxia-induced L-glutamate fluxes was analyzed. The maximum flux at 3 min after the onset of hypoxia increased in the order CA1>CA3>DG. The time-courses of the L-glutamate fluxes at CA1 and DG were biphasic, while that at CA3 decreased monotonously. The KCl-stimulated release of L-glutamate in the presence of the DL-TBOA uptake inhibitor was imaged. While no noticeable change was observed in the absence of DL-TBOA, L-glutamate fluxes in the presence of the inhibitor increased in the order CA1>CA3>DG, reflecting the effect of uptake processes. The present approach suppressed diffusional blur of the glutamate signal and improved the temporal resolution as compared with the BSA-HRP membrane method described earlier.

Methodological aspects of in vitro sensing of L-glutamate in acute brain slices.

L-Glutamate is a major amino acid neurotransmitter in the central neuronal system of the mammalian brain and plays a vital role in brain development, synaptic plasticity, neurotoxicity, and neuropathological disorders. Despite technical limitations, progress is being made in sensing L-glutamate in vivo and in vitro. Sophisticated microsensors with the necessary spatial and temporal resolution have recently been emerging, which enable us to discern regional distribution, concentration levels, and temporal changes of L-glutamate in acute brain slices. The L-glutamate sensors for in vitro sensing have different structures and sizes, such as glass capillary-based enzyme sensors, polymer-coated enzyme sensors, and patch sensors based on natural sensing probes. The concentration of L-glutamate released in brain slices by chemical stimulation is markedly dependent on neuronal regions, types of stimulation, and sensing methods. Real- and long-time monitoring of L-glutamate in acute hippocampal slices is beginning to shed light on L-glutamate release related to the molecular mechanisms of long-term potentiation. Progress is also being made toward the visualization of L-glutamate release in acute hippocampal slices. The methodological aspects of in vitro sensing of L-glutamate are discussed.