Nobuo Misawa

Highly sensitive and selective odorant sensor using living cells expressing insect olfactory receptors

This paper describes a highly sensitive and selective chemical sensor using living cells (Xenopus laevis oocytes) within a portable fluidic device. We constructed an odorant sensor whose sensitivity is a few parts per billion in solution and can simultaneously distinguish different types of chemicals that have only a slight difference in double bond isomerism or functional group such as ─OH, ─CHO and ─C(═O)─. We developed a semiautomatic method to install cells to the fluidic device and achieved stable and reproducible odorant sensing. In addition, we found that the sensor worked for multiple-target chemicals and can be integrated with a robotic system without any noise reduction systems. Our developed sensor is compact and easy to replace in the system. We believe that the sensor can potentially be incorporated into a portable system for monitoring environmental and physical conditions.

Construction and structural analysis of tethered lipid bilayer containing photosynthetic antenna proteins for functional analysis.

The construction and structural analysis of a tethered planar lipid bilayer containing bacterial photosynthetic membrane proteins, light-harvesting complex 2 (LH2), and light-harvesting core complex (LH1-RC) is described and establishes this system as an experimental platform for their functional analysis. The planar lipid bilayer containing LH2 and/or LH1-RC complexes was successfully formed on an avidin-immobilized coverglass via an avidin-biotin linkage. Atomic force microscopy (AFM) showed that a smooth continuous membrane was formed there. Lateral diffusion of these membrane proteins, observed by a fluorescence recovery after photobleaching (FRAP), is discussed in terms of the membrane architecture. Energy transfer from LH2 to LH1-RC within the tethered membrane was observed by steady-state fluorescence spectroscopy, indicating that the tethered membrane can mimic the natural situation.

Cell-Based Odorant Sensor Array for Odor Discrimination Based on Insect Odorant Receptors

The olfactory system of living organisms can accurately discriminate numerous odors by recognizing the pattern of activation of several odorant receptors (ORs). Thus, development of an odorant sensor array based on multiple ORs presents the possibility of mimicking biological odor discrimination mechanisms. Recently, we developed novel odorant sensor elements with high sensitivity and selectivity based on insect OR-expressing Sf21 cells that respond to target odorants by displaying increased fluorescence intensity. Here we introduce the development of an odorant sensor array composed of several Sf21 cell lines expressing different ORs. In this study, an array pattern of four cell lines expressing Or13a, Or56a, BmOR1, and BmOR3 was successfully created using a patterned polydimethylsiloxane film template and cell-immobilizing reagents, termed biocompatible anchor for membrane (BAM). We demonstrated that BAM could create a clear pattern of Sf21 sensor cells without impacting their odorant-sensing performance. Our sensor array showed odorant-specific response patterns toward both odorant mixtures and single odorant stimuli, allowing us to visualize the presence of 1-octen-3-ol, geosmin, bombykol, and bombykal as an increased fluorescence intensity in the region of Or13a, Or56a, BmOR1, and BmOR3 cell lines, respectively. Therefore, we successfully developed a new methodology for creating a cell-based odorant sensor array that enables us to discriminate multiple target odorants. Our method might be expanded into the development of an odorant sensor capable of detecting a large range of environmental odorants that might become a promising tool used in various applications including the study of insect semiochemicals and food contamination.

Microfluidic Odorant Sensor with Frog Eggs Expressing Olfactory Receptors

This study describes a membrane-protein based odorant sensor device consisting of microfluidic channels and frog eggs (Xenopus laevis oocyte) expressing olfactory receptors. These receptors are from silkmoths receptors, BmOR1 and BmOR3 excited by specific pheromones, bombykol and bombykal, respectively. We employ a conventional two electrode voltage clamp method for the signal measurement of the oocytes in our microfluidic system. We have succeeded in selectively detecting these two types of odorant-like chemicals and the two current traces can be recorded by parallel measurement in this system. It implies that our suggested devices can be applied to multichannel detection as a chemical sensor using biological reactions.

Odor sensing method using olfactory receptors and fluorescent instrumentation

A sensing system based upon a living bodys mechanism is useful to improve the performances of current available artificial sensors especially in terms of their selectivity and sensitivity. This paper presents the fundamental study of odor sensor based on insect olfactory receptors (ORs). Cells expressing ORs are responsible for odor detection. It is possible to monitor the increase in calcium ion concentration using the fluorescent protein inside the cells expressing ORs. In our research we developed an instrumentation system to monitor the fluorescent intensity change for the odor sensing. The system consists of fluidic device, sky blue laser for light excitation, lenses, optical filter, dichroic mirror, and a cooled complementary metal oxide semiconductor (CMOS) camera. Sf21 cells expressing two ORs called OR56a and BmOR3 with their specific response to geosmin and bombykal have been prepared for experiments. Measurement results show that the developed instrumentation was able to detect the odorant with concentration down to 5 μM for about 15 seconds.

Electroformation from patterned single-layered supported lipid bilayers for formation of giant vesicles with narrow size distribution

We describe a method of producing solvent-free giant vesicles (GVs) with a narrow size distribution from patterned supported lipid bilayers (SLBs). The SLBs were prepared on a patterned SiO2 surface by vesicle fusion, and the GVs were formed from the SLBs by electroformation. Fluorescence observation showed the formation of single-layered SLBs on circular patterns of SiO2 with a diameter of 20 µm, the area of which corresponded to that of a unilamellar vesicle of 10 µm diameter. The electroformation from the patterned SLBs produced the GVs with a median diameter of 8.53 µm and a coefficient of variation of 10.9%.

Nano-depth grooves formed through O2 plasma etching in the presence of PTFE

In this paper, we describe a simple method for fabricating nano-depth grooves in glass. In our method, the depth of the grooves can be easily controlled at the several tens of nanometers scale for the vertical features by simply applying O2 plasma in the presence of polytetrafluoroethylene (PTFE). Using atomic force microscopy, we found that (1) the etching rate varies in different types of glasses; (2) the etched glass surface is flat (root-mean-square roughness ≈1 nm); (3) the etched depth of the glass almost linearly depends on the output power of the plasma equipment; and (4) the etching is influenced by the surface area of PTFE that is exposed to O2 plasma in the etching chamber. Furthermore, using these nano-depth groove structures, we made nano-depth (≈60 nm) fluidic channels as a demonstration of micro/nanofabrication. The channels are composed of a silicon substrate and etched glass anodically bonded together. This simple method is a useful technique for the production of nano-depth fluidic channels.

Membrane protein-based biosensors

This review highlights recent development of biosensors that use the functions of membrane proteins. Membrane proteins are essential components of biological membranes and have a central role in detection of various environmental stimuli such as olfaction and gustation. A number of studies have attempted for development of biosensors using the sensing property of these membrane proteins. Their specificity to target molecules is particularly attractive as it is significantly superior to that of traditional human-made sensors. In this review, we classified the membrane protein-based biosensors into two platforms: the lipid bilayer-based platform and the cell-based platform. On lipid bilayer platforms, the membrane proteins are embedded in a lipid bilayer that bridges between the protein and a sensor device. On cell-based platforms, the membrane proteins are expressed in a cultured cell, which is then integrated in a sensor device. For both platforms we introduce the fundamental information and the recent progress in the development of the biosensors, and remark on the outlook for practical biosensing applications.

Fluorescence detection of submicromolar concentration using a filter-free fluorescence sensor

In this paper, the detection of fluorescence from the solution of a fluorescent dye with a submicromolar concentration by the filter-free fluorescence sensor is reported. The filter-free fluorescence sensor has a photo-gate structure and the intensities of excitation and fluorescent lights can be calculated from the photocurrents for different gate voltages. It makes it possible to measure various wavelengths even without optical filters and mirrors. The separation ability of excitation light (470 nm) and fluorescence (530 nm) in the developed filter-free fluorescence sensor was 1200:1 for LED light source. The fluorescent measurements were carried out for the dye of fluorescein isothiocyanate isomer. The excitation light with the wavelength of 470 nm was used, and the photocurrents were measured for the gate voltages of 1 V and 3 V, respectively. Based on the calculation, the fluorescence from fluorescein isothiocyanate isomer was detected for the solution with the concentration of as low as 100 nM (12 bases).

FROG EGG-Array device integrated with fluidic channel and microelectrodes for chemical sensing

This paper describes a membrane protein-based chemical sensor that is consisted of fluidic channels and cells (Xenopus laevis oocytes) expressing chemical receptors. The fluidic device has Si-based microfabricated electrodes to measure the Xenopus oocytes response to chemicals using two-electrode voltage clamping. The fluidic device was fabricated by combining fluidic channel made of acrylic resin and the electrode substrate. After the cell installing, the fluidic device can be separated to each fluidic channel that can measure an individual oocyte membrane potential change. We succeeded to array each oocyte in the device and to detect an individual Xenopus oocytes responses to chemical stimulus.