Chie Hosokawa

Carbon nanotube–liposome supramolecular nanotrains for intelligent molecular-transport systems

Biological network systems, such as inter- and intra-cellular signalling systems, are handled in a sophisticated manner by the transport of molecular information. Over the past few decades, there has been a growing interest in the development of synthetic molecular-transport systems. However, several key technologies have not been sufficiently realized to achieve optimum performance of transportation methods. Here we show that a new type of supramolecular system comprising of carbon nanotubes and liposomes enables the directional transport and controlled release of carrier molecules, and allows an enzymatic reaction at a desired area. The study highlights important progress that has been made towards the development of biomimetic molecular-transport systems and various lab-on-a-chip applications, such as medical diagnosis, sensors, bionic computers and artificial biological networks.

Bright, non-blinking, and less-cytotoxic SiO2 beads with multiple CdSe/ZnS nanocrystals

A method including surface silanization, phase transfer and self-assembly, and SiO2 shell growth has been developed to incorporate multiple hydrophobic CdSe/ZnS nanocrystals into SiO2 beads where they are well suited for bio-application due to their high brightness, less-cytotoxic, and non-blinking nature.

Resynchronization in neuronal network divided by femtosecond laser processing.

We demonstrated scission of a living neuronal network on multielectrode arrays (MEAs) using a focused femtosecond laser and evaluated the resynchronization of spontaneous electrical activity within the network. By an irradiation of femtosecond laser into hippocampal neurons cultured on a multielectrode array dish, neurites were cut at the focal point. After the irradiation, synchronization of neuronal activity within the network drastically decreased over the divided area, indicating diminished functional connections between neurons. Cross-correlation analysis revealed that spontaneous activity between the divided areas gradually resynchronized within 10 days. These findings indicate that hippocampal neurons have the potential to regenerate functional connections and to reconstruct a network by self-assembly.

Biomodeling System – Interaction Between Living Neuronal Networks and the Outer World

Rat hippocampal neurons reorganized into complexnetworks in a culture dish with 64 planar micro-electrodes and the electrical activity of neurons wererecorded from individual sites. Multi-site recordingsystem for extracellular action potentials was used forrecording the activity of living neuronal networks andfor applying input from the outer world to the net-work. The living neuronal network was able to dis-tinguish among patterns of evoked action potentialsbased on different input, suggesting that the livingneuronal network can express several pattern inde-pendently, meaning that it has fundamental mecha-nisms for intelligent information processing. We aredeveloping a “biomodeling system,” in which a livingneuronal network is connected to a moving robot withpremised control rules corresponding to a geneticallyprovided interface of neuronal networks to peripheralsystems. Premised rules are described in fuzzy logicandthe robot cangenerateinstinctive behavior,avoid-ing collision. Sensor input from the robot body wassenttoaneuronalnetwork,andtherobotmovedbasedon commands from the living neuronal network. Thisis a good modeling system to analyze interaction be-tween biological information processing and electricaldevices.Keywords: neuron, dissociated culture system, multi-electrode array (MED), fuzzy logic, moving robot

Enhancement of Biased Diffusion of Dye-Doped Nanoparticles by Simultaneous Irradiation with Resonance and Nonresonance Laser Beams

We propose and demonstrate the enhancement of the biased diffusion of dye-doped nanoparticles using resonance and nonresonance laser beams. The Brownian motion of nanoparticles in a laser focus is investigated by fluorescence correlation spectroscopy (FCS) and the time variation in fluorescence intensity. From the analysis of autocorrelation functions, it is demonstrated that the difference between the transit times of nanoparticles in the focal spot with and without resonance laser irradiation increases ~7-fold by the simultaneous irradiation of a near-infrared laser. This method is applicable to the selective optical manipulation of dye-stained nanomaterials and biomolecules in solution.

Vitroid – the robot system with an interface between a living neuronal network and outer world

We have developed a neuro-robot-hybrid system using a living neuronal network and a miniature moving robot. The living network of rat hippocampal neurons can distinguish patterns of action potentials evoked by different inputs, suggesting that a cultured neuronal network can represent particular states as symbols. We used a Khepera II robot and a robot made using a LEGO mindstorm NXT kit to interface with a living neuronal network and the outer world. We call the system ‘vitroid’. Vitroid has living neurons, a robot body, and direct coupling controllers to interface the neurons with the robot. Vitroid was able to perform obstacle avoidance behaviour with premised control rule sets.

Optical trapping of synaptic vesicles in neurons

We demonstrate intracellular manipulation of synaptic vesicles in living neurons by optical trapping. When an infrared trapping laser is focused on synapses of a neuronal cell labeled with a fluorescent endocytic marker, fluorescence is observed at the focal spot. The fluorescence spectrum is attributed to fluorescent dye in the synaptic vesicles, indicating excitation by two-photon absorption of the trapping laser. The fluorescence intensity increases gradually within ∼100 s of laser irradiation, suggesting that trapping force causes vesicles assembly at the focus. Our method can be applied to manipulate synaptic transmission of a particular neuron in a neuronal network.

In situ sensitive fluorescence imaging of neurons cultured on a plasmonic dish using fluorescence microscopy.

A plasmonic dish was fabricated as a novel cell-culture dish for in situ sensitive imaging applications, in which the cover glass of a glass-bottomed dish was replaced by a grating substrate coated with a film of silver. Neuronal cells were successfully cultured over a period of more than 2 weeks in the plasmonic dish. The fluorescence images of their cells including dendrites were simply observed in situ using a conventional fluorescence microscope. The fluorescence from neuronal cells growing along the dish surface was enhanced using the surface plasmon resonance field. Under an epi-fluorescence microscope and employing a donut-type pinhole, the fluorescence intensity of the neuron dendrites was found to be enhanced efficiently by an order of magnitude compared with that using a conventional glass-bottomed dish. In a transmitted-light fluorescence microscope, the surface-selective fluorescence image of a fine dendrite growing along the dish surface was observed; therefore, the spatial resolution was improved compared with the epi-fluorescence image of the identical dendrite.

Detection of M-sequences from spike sequence in neuronal networks

In circuit theory, it is well known that a linear feedback shift register (LFSR) circuit generates pseudorandom bit sequences (PRBS), including an M-sequence with the maximum period of length. In this study, we tried to detect M-sequences known as a pseudorandom sequence generated by the LFSR circuit from time series patterns of stimulated action potentials. Stimulated action potentials were recorded from dissociated cultures of hippocampal neurons grown on a multielectrode array. We could find several M-sequences from a 3-stage LFSR circuit (M3). These results show the possibility of assembling LFSR circuits or its equivalent ones in a neuronal network. However, since the M3 pattern was composed of only four spike intervals, the possibility of an accidental detection was not zero. Then, we detected M-sequences from random spike sequences which were not generated from an LFSR circuit and compare the result with the number of M-sequences from the originally observed raster data. As a result, a significant difference was confirmed: a greater number of “0–1” reversed the 3-stage M-sequences occurred than would have accidentally be detected. This result suggests that some LFSR equivalent circuits are assembled in neuronal networks.

Enhanced fluorescence microscopy with the Bull’s eye-plasmonic chip

A Bulls eye-plasmonic chip composed of concentric circles was applied to enhanced fluorescence microscopy. Among one dimensional (1-D), 2-D, and Bulls eye periodic structures, the Bulls eye-plasmonic chip provided the most enhanced fluorescence intensity under the epi-fluorescence microscope, because incident light through the objective lens with all azimuthal angles can be effectively applied to the surface plasmon resonance- field (excitation field) and the plasmon-enhanced emission was also effectively collected. In the fluorescence observation of a single nanoparticle, the enhanced fluorescence images for a microsphere with ϕ 2 μm and a nanosphere with ϕ 200 nm were observed. For the nanospheres with ϕ 40 and 20 nm, the fluorescence image, which was undetectable on a glass slide, was observed in a spatial resolution of roughly diffraction limit on the Bulls eye-plasmonic chip. Furthermore, the use of an appropriate pinhole at the aperture stop in the incident optical system improved the fluorescence enhancement. The applicability of a Bulls eye-plasmonic chip to fluorescence imaging was demonstrated.