AMPA-type glutamate receptor (AMPAR) is one of neurotransmitter receptors at excitatory synapses in neuronal cell. For realizing the artificial control of synaptic transmission, we have applied optical trapping of quantum-dot (QD) conjugated AMPARs on neuronal cells. Here, we demonstrate simultaneous measurement combined with optical trapping and patch-clamp recordings to evaluate the neuronal electrical activity. The relationship between optical trapping dynamics of QD-AMPARs located on neuronal cells and the neuronal electrical activity was discussed.
AMPA-type glutamate receptor (AMPAR) is one of the major neurotransmitter receptors at excitatory synapses. The initial assembling states of AMPARs at cell surface are essential for synaptic transmission, which is related with learning and memory in living neural systems. To realize artificial control of synaptic transmission, we demonstrate to modulate the initial assembling states of quantum-dot conjugated AMPARs (QD-AMPARs) with optical trapping. The optical trapping dynamics of QD-AMPARs on living neurons was evaluated with fluorescence imaging and fluorescence correlation spectroscopy (FCS). The transit time at laser focus of QD-AMPARs on neurons estimated from FCS analysis increased with the culturing days and addition of neurotransmitter, which suggests that QD-AMPARs are confined at the focal spot due to optical trapping.
Molecular dynamics of glutamate receptor, which is major neurotransmitter receptor at excitatory synapse located on neuron, is essential for synaptic plasticity in the complex neuronal networks. Here we studied molecular dynamics in an optical trap of AMPA-type glutamate receptor (AMPAR) labeled with quantum-dot (QD) on living neuronal cells with fluorescence imaging and fluorescence correlation spectroscopy (FCS). When a 1064-nm laser beam for optical trapping was focused on QD-AMPARs located on neuronal cells, the fluorescence intensity of QD-AMPARs gradually increased at the focal spot. Using single-particle tracking of QD-AMPARs on neurons, the average diffusion coefficient decreased in an optical trap. Moreover, the decay time obtained from FCS analysis increased with the laser power and the initial assembling state of AMPARs depended on culturing day, suggesting that the motion of QD-AMPAR was constrained in an optical trap.
Molecular dynamics at synaptic terminals in neuronal cells is essential for synaptic plasticity and subsequent modulation
of cellular functions in a neuronal network. For realizing artificial control of living neuronal network, we demonstrate
laser-induced perturbation into molecular dynamics in the neuronal cells. The optical trapping of cellular molecules such
as synaptic vesicles or neural cell adhesion molecules labeled with quantum dots was evaluated by fluorescence imaging
and fluorescence correlation spectroscopy. The trapping and assembling dynamics was revealed that the molecular
motion was constrained at the focal spot of a focused laser beam due to optical trapping force. Our method has a
potential to manipulate synaptic transmission at single synapse level.
KEYWORDS: Neurons, Electrodes, Signal detection, Brain, Action potentials, Data processing, In vitro testing, Brain mapping, Logic, Intelligence systems
Neurons form complex networks and it seems that the living neuronal network can perform certain type of information processing. We are interested in intelligence autonomously formed in vitro. The most important features of the two-dimensional culture neural network are that it is a system in which the information processing is autonomously carries out. We reported previously that the functional connections were dynamically modified by synaptic potentiation and the process may be required for reorganization of the functional group of neurons. Such neuron assemblies are critical for information processing in brain. Certain types of feedback stimulation caused suppression of spontaneous network electrical activities and drastic re-organization of functional connections between neurons, when these activities are initially almost synchronized. The result suggests that neurons in dissociated culture autonomously re-organized their functional neuronal networks interacted with their environment. The spatio-temporal pattern of activity in the networks may be a reflection of their external environment. We also interfaced the cultured neuronal network with moving robot. The planar microelectrodes can be used for detecting neuronal electrical signals from the living neuronal network cultured on a 2-dimensional electrode array. The speed of actuators of moving robot was determined by these detected signals. Our goal is reconstruction of the neural network, which can process "thinking" in the dissociated culture system.
The neurons in dissociation culture autonomously re-organized their functional neuronal networks, after the process for elongating neurites and establishing synaptic connections. The spatio-temporal patterns of activity in the networks might be a reflection of functional neuron assemblies. The functional connections were dynamically modified by synaptic potentiation and the process may be required for reorganization of the functional group of neurons. Such neuron assemblies are critical for information processing in brain. To visualize the functional connections between neurons, we have analyzed the autonomous activity of synaptically induced action potentials in the living neuronal networks on a multi-electrode array, using "connection map analysis" that we developed for this purpose. Moreover, we designed aan original wide area covering electrode array and succeeded in recording spontaneous action potentials from wider area than commercial multi electrode arrays.
It is well-known that electrically controllable soft actuators can be formed by using the composite composed of ionic-conductive polymer electrolyte membranes and precious metals such as platinum or gold. The response of the polymer electrolyte actuator is quick and soft. This paper describes the preparing method of the composites, the principle of the electric response, the response kinetics of the composite, and the medical application of the polymer actuator.
Protein patterns were printed using conventional microlithographic materials in a bilayer arrangement and unconventional exposure tools. The bilayer resist stack consists of a bottom Poly(tertButylMethAcrylate) layer and a top DNQ-novolak layer. The protein features were printed in 'step & repeat' mode, that is a flow-cell, 'real-time' process, as follows: (1) the exposure step is carried out by the focused beam of a confocal microscope tuned to 488 nm wavelength; (2) the development step is performed flowing the photoresist developer through the cell; (3) the selective deposition of the protein (a green fluorescent protein, FITC avidin for visualization) is achieved via the flow of the protein solution through the cell until a desired contrast has been reached; (4) the control step consists of an on-line monitoring of the red fluorescence for the control of the development of the top layer, and of the green fluorescence for the control of the protein patterning. respectively. The techniques have of a seamless portability in a biomedical environment, and for 'step & repeat' protein patterning the advantage of a high and controllable resolution. The process can be applied for the in-house fabrication of model biomolecular and cellular devices. Examples for the patterning of neuronal cells are also given.
The fluorescence of fluorescently tagged proteins on functionalized polymer surfaces shifts towards higher wavelengths. Two types of polymers for light-assisted surface manipulation have been used, namely diazo-naphto- quionone/novolak (DNQ/N) resist and poly)tert-butyl- methacrylate). The proteins were either hydrophobicity- attached; covalently linked; or specific protein-protein recognition. We observed that on hydrophilic surfaces the fluorescence is shifted towards lower wavelengths. This parasitic effect has to be taken into account when 'reading' biochips but it can be also used for the 'alignment' of the fluorescence of the fluorescently tagged proteins on the same wavelengths via the manipulation of the properties of the substrate polymer.
KEYWORDS: Polymers, Photoresist materials, Spectroscopy, Glasses, Independent component analysis, Signal processing, Picture Archiving and Communication System, Magnetism, Optical lithography, Chemical species
We propose a method for the monitoring of the glass transition temperature of the resist used in semiconductor lithography based on the broad-band Nuclear Magnetic Resonance technique. The method is capable to trace the evolution of the mobility of several major chemical species present in a resist system versus processing parameters, e.g. exposure energy and bake temperature. The most important components, namely the lower molecular weight photoactive compound and the higher molecular weight base resin, were characterized, in accordance with their signals, as mobile and the rigid component, respectively. The method has the potential for being used for process optimization and for on-line monitoring.
The contribution proposes relationships for the diffusivity of small molecular species in resist systems; then reviews the possible mechanisms in thin polymeric films, namely simple Fickian, Case II, and diffusion accompanied by chemical reaction, with relevance to microlithography processes. The review of the kinetics reveals the inconsistencies in the models advanced for two important microlithographic processes: (i) silylation in Surface Imaging patterning; and (ii) the deactivation of the Chemical Amplification resist due to the parasitic diffusion of N-methyl-pyrolid-one. These inconsistencies can be easily and elegantly resolved only if the polymeric thin film system is supposed to exhibit a kinetics consistent with the diffusion accompanied by chemical reaction.
Protein patterns were printed using conventional microlithographic materials in a bilayer and a top DNQ/novolak layer. The protein features were printed in both 'contact printing', and 'step and repeat' mode. The latter printing mode can be managed in a single-cell, 'real time' process, as follows: (i) the exposure step is carried out in a cell by focused beam of a confocal microscope tuned to 488nm exposure; (ii) the development step is performed flowing the photoresist developer through the cell; (iii) the selective deposition of the protein is achieved via the flow of the protein solution through the cell until a desired contrast has been reached; (iv) the control step consists of an on-line monitoring of the red fluorescence for the control of the development of the top layer, and of the green fluorescence for the control of the protein patterning, respectively. The techniques have a seamless portability in a biomedical environment, and for 'step and repeat' protein patterning the advantage of a high and controllable resolution. The process can be applied in the fabrication of medical microanalysis devices.
Electron-beam lithography employing poly(tert-Buthyl- Methacrylate)-co-(Methyl-Methacrylate) as radiation sensitive system was used to pattern bioactive molecules at super-high resolution. Positive and negative tone lithography succeed in printing fluorescent avidin into the range of 100 nm resolution. Two mechanisms were used for protein attachment, namely: (1) the linkage of the amino-end of the protein to the radiation-induced carboxylic acid sites, via NH2-to-COOH crosslinking mediated by carbodiimide; and (2) hydrophobic interaction between the patterned proteins and unexposed surfaces, in contrast to hydrophilic-repulsive interaction with exposed one. The first mechanism produces positive tone, half-tone images, while the second produces negative tone, sharp contrast images. On this basis, we assume that the first mechanism is concentration-controlled, while the second is an on-off one. This study proves that e-beam lithography materials and techniques can be easily transferred in bio- microlithography, with impact on biodevices fabrication and combinatorial chemistry.
KEYWORDS: Polymers, Polymeric sensors, Resistance, Sensors, Chemical fiber sensors, Electrodes, Human-machine interfaces, Chemical elements, Artificial intelligence, Biological and chemical sensing
The discovery of new sensing materials and electrodes has greatly expanded the range of scientific methods including electrochemical techniques. Conducting polymer such as polypyrrole and polyaniline represent a new class of organic polymers that are capable of molecular interactions and being able to interact, chemically or electrochemically, with the species of interest for detection. Although these conductive materials have unique properties they have their specific problems with respect to their reproducibility and reusability. Problems exist due to the dynamic nature of these polymers thereby mitigating against their successful applications as novel sensors. This has also hindered the production of analytical useful, sensitive, and reversible signals using these polymers. This paper has sought to examine the problems due to the lack of useful analytical, sensitive, reversible and reusable signals through the introduction of new series of integrated artificial intelligence/conducting polymer based sensors. In these types of sensors analytical responses, which look irreversible and nonreproducible, are combined by an artificial intelligence trained computer by which reproducible output can be predicted based on the created model and pattern by the computerized system.
This study attempts to assess the feasibility of building purposefully designed molecular motor arrays, the proteins responsible for the movements of the living organisms and cells. The 'building' process used high-resolution e-beam patterning, originating in semiconductor technology, upgraded to make biomicrolithography compatible with the patterning of bioactive molecules. The material used as a scaffold for the array [a copolymer of poly(tert-butyl-methacrylate/methyl- methacrylate)] was tailored to exhibit large difference in hydrophobicity when exposed to e-beam exposure. The e-beam patterning exposure-induced difference in hydrophobicity is responsible for the selective attachment of the myosin molecules on the patterned deep-submicron 'tracks,' and the higher concentration of 'guiding' molecules selectively confines the movement of the actin filaments.
The surface of photosensible diazo-naphto-quinone/novolak film was chemically manipulated through UV exposure and subsequent functionalization processes to obtain surfaces with different chemistries (DNQ, carboxylic, and peptidic groups). The neuronal cell attachment is controlled by three pairs of antagonistic surface variables: charged/uncharged species, amino/carboxylic groups and hydrophilic/hydrophobic balance, in which the former promote the adhesion. The study proves that microlithographic techniques in connection with surface functionalization with specific neuro-peptides can be used to build artificial networks with neuronal cells.
Photochemically induced surface functionality manipulation of Diazo-Naphto-Quinone/novolak polymeric films was used for controlling the specificity of the attachment and growth of neuronal cells and biologically active molecules (proteins and peptides) patterning. Different microlithographic techniques (standard positive tone, negative tone image reversal based on catalyzed decarboxylation, positive tone of image reversal resist, and surface imaging based on silylation), controlling the surface hydrophobicity and surface concentration of carboxylic groups, were assessed in the view of the suitability as microlithographic techniques for patterning biologically active molecules and cells. It was found that DNQ-based materials and techniques can be easily transferred in bio-microlithography, which is the building of laterally ordered architectures with biological structural elements.
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