Abstract:
The invention provides microfabricated devices and methods for directing the growth of a cell process to form an artificial synapse. The devices are called artificial synapse chips. The artificial synapse comprises a nanofabricated aperture (about 50–100 nm in size) that connects the cell process to a chemical or electrical means of neuronal excitation. Such an aperture width mimics the length scales of a natural synapse and thus emphasizes the localized spatial relationship between a neuron and a stimulation source. The invention further provides devices and methods for regenerating a nerve fiber into an electrode. The invention thus provides a regeneration electrode that uses a novel neural interface for stimulation and that uses novel surface methods for directing neuronal growth making possible in vivo connection of the devices to neural circuitry in a retina and other anatomical locations.

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This application is related to and claims priority under 35 U.S.C. § 119(e) from U.S. Provisional Application Serial No. 60/301,934, entitled “ARTIFICIAL SYNAPSE CHIP INTERFACE FOR ELECTRONIC PROSTHETIC RETINA”, by Fishman et al., filed Jun. 29, 2001, which is hereby incorporated by reference in its entirety. 
    
    
     FIELD OF THE INVENTION 
     The invention pertains to the devices for controlling cell growth and for stimulating cells. In particular, the invention is directed to methods and devices for controlling neuronal growth to provide artificial synapses and neural prostheses. 
     BACKGROUND OF THE INVENTION 
     Light entering the eye through the cornea is focussed through the lens (which further focuses the light) onto the retina, a thin layer of cells in the back of the eye. Normal human vision depends upon signals generated by nerve cells in the retina. The visual signals originate with the photoreceptor cells in the retina, which sense and respond to light, generating signals that in turn create and shape nerve signals in retinal ganglion cells. Nerve cells often have extended cellular portions called cell processes, which may be specialized for receiving information and stimulation, or for transmitting information. For example, the specialized elongated processes that conduct nerve impulses are termed axons. The axons of the retinal ganglion cells carry the visual signals from the retina to the brain. In the brain, nerve cell networks process the visual signals further to provide the full visual experience of a normally-sighted person. Disturbances at any step in the process may lead to visual impairment or blindness. 
     Age-related macular degeneration (AMD) is one of the most common forms of blindness in people over the age of 65. Currently, there is no effective treatment for most patients with AMD, a disease that often results in permanent damage to photoreceptors, but spares most retinal ganglion cells (RGCs). Similarly, other diseases such as retinitis pigmentosa (RP) cause vision impairment and blindness due to loss of photoreceptors. 
     Inherent to the power of the human visual system is the ability to transduce light by individual photoreceptors, thus making it a high-resolution image capture system. Several groups worldwide have carried out clinical experiments to determine if stimulating retinal cells, the optic nerve bundle, or cells of the visual cortex with microelectrode arrays can generate phosphenes (i.e., sensations of light) in individuals blinded from AMD. The electrical fields produced by the microelectrode arrays stimulate relatively large regions containing numerous neuronal and glial cells. These trials have shown that by stimulating neurons with a microelectrode array, blind individuals can indeed recognize a simple pattern such as a horizontal or vertical line. Although these trials have demonstrated that vision is recoverable in a limited fashion, major challenges remain. Due to the size and difficulties in placement of most available electrodes, imprecise electric field stimulation extending over long distances (several cell-body diameters) is used to depolarize neurons. However, such methods often require excessive stimulation, which may be harmful, leading to inflammation of the stimulated region and even to excessive growth of glial cells, or gliosis. Thus, an unmet major challenge of these approaches is that of constructing a neural interface that stimulates localized retinal regions, individual neurons, and even specialized portions of neurons with specificity. 
     Neurons may be grown on artificial substrates. However, the synaptic connections of neurons grown on artificial substrates may not be controlled or precisely directed to defined locations, and do not provide for the specific stimulation characteristics found in vivo. 
     Accordingly, methods and devices are needed that improve the specificity of neural stimulation, and preferably improve the specificity of neural stimulation with low power delivery to avoid gliosis and inflammation. 
     SUMMARY OF THE INVENTION 
     The invention combines micropatterned neuronal growth with a microfabricated stimulation interface to form a new neuronal regeneration electrode that serves as an artificial synapse, the device being termed an artificial synapse chip (ASC). A “regeneration electrode” is made by regenerating a nerve fiber into an electrode. The artificial synapse provides a microfabricated aperture (a “nanoaperture”) that connects a neuronal cell process (a neurite) to a chemical or electrical means of neuronal excitation. The nanoaperture mimics the length scales of a real synapse and thus emphasizes the localized spatial relationship between the neuron and the stimulation source. 
     The inventors have recognized that the problem of the development of a neural interface that preserves the high resolution, one-to-one registry with individual neurons and with low power delivery can be broken down into two puts: first, bringing the nerve and the stimulation source together, second, stimulating the nerve cell itself. The ASC combines directed, micropatterned neuronal growth with a neural stimulation source to provide low-power stimulation (nanostimulation) of at least a part of a desired neuronal cell. Thus, the ASC is not only effective to act as a substrate on which to direct the growth of neurites from nerve cells to a stimulation source, but is effective to serve as a stimulation source as well. 
     The invention is thus directed to devices and methods for controlling the growth of a cell process, which include a substrate with a surface configured for growing cells and cell processes and a micropattern effective to control the growth of cells and cell processes in a desired direction to a desired location or locations on the surface. A desired location may be a nanoaperture, an electrical contact, or a micropattern feature. The micropattern may include chemo-attractant factors, adhesion molecules, repulsive molecules, surface contours, and/or at least one region enriched in particular atoms. The micropattern may be produced by contacting a substrate surface with a microcontact printing stamp. Devices having a surface with such a micropattern embodying features of the invention may be used to control the growth of cell processes by contacting a cell with the surface, effective to control the growth of a cell process in a desired manner. Contacting a cell with a surface having a micropattern, directing the growth of a cell process to a desired position on the surface, and providing a stimulus from the desired location to a cell process is effective to stimulate at least a portion of the cell. 
     A device for contacting and stimulating a cell may have a surface configured for contacting at least a portion of a cell, and may have at least one contact in electrical continuity with a circuit. Such a device is effective to stimulate at least a portion of a cell, and is particularly suitable for the stimulation of a neurite. For example, the devices may be used to stimulate a cell or cell process adjacent an electrical contact by way of stimulation of a neurite, stimulation of a cell through a neurite, or stimulation of a cell body. 
     The invention also provides methods for directing the growth of a cell process to a location adjacent a contact of a circuit. The growth of a cell process may be directed to a location adjacent the contact by contacting a cell capable of growing a cell process with a surface that has a circuit and a micropattern. The micropattern may include factors such as chemo-attractant factors, adhesion molecules, repulsive molecules, surface contours, and/or have at least one region enriched in particular atoms. The micropattern for directing the growth of a cell process may be produced by a method including contacting a surface with a microcontact printing stamp. The growth of a cell process may be directed by delivering a neuromodulatory agent to at least a portion of a cell. Desired locations towards which growth may be directed include a nanoaperture, a contact of an electrical circuit, and a surface feature. 
     Devices embodying features of the invention may include a surface with a nanoaperture, and a reservoir that may contain neuromodulatory agents, such as neurotransmitters, hormones, ions, messenger molecules, nucleic acids, nucleic acid vectors, drugs, cells, cell fragments, cell organelles, liposomes, or other biologically active materials. The nanoaperture is effective to provide a conduit for the delivery of the neuromodulatory agents from the reservoir to at least a portion of a cell. A device for delivering neuromodulatory agents may have an exterior surface with a micropattern effective to direct the growth of a cell process as described above. Thus, microfabricated artificial synapse chips include microfabricated devices having a nanoaperture, a surface with a micropattern effective to direct the growth of a cell process so as to contact the nanoaperture, and a reservoir which may contain neuromodulatory agents connected to the nanoaperture. 
     A reservoir may be directly in contact with a nanoaperture, or may be connected to a nanoaperture by a conduit effective to deliver neuromodulatory agents from the reservoir to the nanoaperture. Pumps or other fluid-directing mechanism may be operably connected to a reservoir and/or conduit effective to induce fluid flow which may, for example, be effective to aid the delivery of neuromodulatory agents to the nanoaperture. 
     Delivery of a neuromodulatory agent may be effective to provide a stimulus to a cell. Embodiments of the invention provide methods for providing a stimulus to a cell process effective to stimulate at least a portion of a cell. In embodiments of the invention, methods of cell stimulation include stimulating a cell process, stimulating a cell through a cell process, and stimulating a cell body. 
     In other embodiments, the invention provides a regeneration electrode assembly that includes a neurite-directing device and a circuit effective to contact and stimulate at least a portion of a cell. The neurite-directing device may include a device for directing the growth of the cell process, a device for delivering neuromodulatory agents to at least a portion of a cell, or both. The circuit may include a device for contacting and stimulating at least a portion of a cell, or a cell process, or a cell body. 
     The ability to direct neurite extension to form directed contacts with a circuit is useful in therapies for any disease of neural tissue. Thus, in one aspect of the invention, the devices and methods provide a neural interface that can bypass retinal photoreceptors and connect a digital camera to individual nerve cells in the retina. In this way, cells damaged in AMD and other blinding diseases can be bypassed and visual information sent to the brain. Thus, the artificial synapse chip provides methods for the restoration of visual function in patients suffering from blindness due to age-related macular degeneration (AMD), retinitis pigmentosa, and other photoreceptor blinding diseases. The devices and methods of the invention provide a neural prosthesis suitable for implantation in any location within the nervous system or body of a patient, for the treatment of spinal cord injuries, neuropathies, bladder dysfunction, and other diseases due to neuronal disorders. 
     Methods of the invention may be used to produce intra-ocular devices. Devices configured for implantation into an eye embodying features of the invention include a device for contacting and stimulating at least a portion of a cell, and a regeneration electrode assembly configured for implantation into an eye. In embodiments of the invention, devices are configured for implantation into regions of the eye, including the retina, the inner limiting membrane and the subretinal space. 
     A photosensitive assembly for restoring vision in an eye having decreased photoreceptor function includes a photosensitive device effective to respond to light with photoactivated signals, an artificial synapse chip, a power source effective to power the photosensitive device, and an effective connection between the photosensitive device and the artificial synapse chip. Implantation of a photosensitive assembly provides a method for restoring vision in an eye having decreased photoreceptor function. An eye disorder may be treated by implanting a photosensitive assembly into an eye, directing the growth of retinal neuron cell processes to the photosensitive assembly and stimulating retinal neurons by photoactivated signals derived from the photosensitive assembly. 
     Devices of the invention, each termed an “artificial synapse chip” (ASC), comprise a high-resolution neural interface to the visual system that incorporates micropatterned neuronal growth to produce an artificial synapse. The nanoapertures of a very small size, guided contacts with desired portions of individual cells, and targeted stimulation of individual cells via these contacts provide a neural interface with high spatial resolution for connecting a circuit to individual neurons. Thus, unlike previous methods for stimulating retinal neurons that offer only poor spatial resolution, the ASC provides the advantages of specificity and control of stimulation at a cellular level to provide novel ways to influence the behavior of a cellular system. In addition, since the source is in direct contact with the neuron, the ASC uses less power for stimulation than prior art field stimulators. The ASC thus provides a new class of regeneration electrodes—one that uses a novel neural interface for stimulation and that uses modern surface science methods for directing neuronal growth so that connection to in vivo neural circuitry in a retina is possible. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1A  shows a perspective view of an artificial synapse chip embodying features of the invention. 
         FIG. 1B  is a plan view of the artificial synapse chip of  FIG. 1A . 
         FIG. 1C  is a cross-sectional view of the artificial synapse chip of  FIG. 1A  taken along plane  1 C— 1 C. 
         FIG. 1D  is a cross-sectional view of an artificial synapse chip as in  FIG. 1A  taken along plane  1 C— 1 C, illustrating an embodiment of the invention having electrodes. 
         FIG. 1E  is a cross-sectional elevation view of a system having a pump and a depot for holding a store of solution and including an artificial synapse chip. 
         FIG. 1F  is a cross-sectional elevation view of a portion of a system having a pump including an artificial synapse chip. 
         FIG. 2A  is a plan view scanning electron micrograph (SEM) of a nanoaperture of an artificial synapse chip embodying features of the invention. 
         FIG. 2B  is a plan view SEM of a reservoir of an artificial synapse chip embodying features of the invention. 
         FIG. 2C  illustrates patterned growth of retinal ganglion cells on a patterned substrate. 
         FIG. 2D  illustrates growth of PC12 cells around and over a 5 μm-diameter aperture in a silicon nitride substrate. 
         FIG. 3  is a plan view SEM of a stamp embodying features of the invention for making a micropattern on a surface. 
         FIG. 4  is a system for implantation of an artificial synapse chip into an animal comprising artificial synapse chip (ASC), a photosensitive device, a means of communication between the ASC and the photosensitive device, and a power source. 
         FIG. 5A  illustrates a cross-section of an eye of animal with an implanted ASC. 
         FIG. 5B  is a detailed cross-sectional view showing the eye of  FIG. 5A  and the ASC in place in the subretinal space of the eye. 
         FIG. 6A  shows current flow through α-hemolysin channels in an artificial bilayer membrane formed across a nanoaperture of an artificial synapse chip embodying features of the invention, with current shown along the vertical axis and time along the horizontal axis, the insert showing a short portion of the record at expanded scale along the time axis. 
         FIG. 6B  is a plot of the measured current flow through α-hemolysin channels in an artificial bilayer membrane formed across a nanoaperture of an artificial synapse chip embodying features of the invention, with current shown along the vertical axis and time along the horizontal axis. 
         FIG. 6C  shows current flow through two α-hemolysin channels in an artificial bilayer membrane formed across a nanoaperture of an artificial synapse chip embodying features of the invention, with current shown along the vertical axis and time along the horizontal axis. 
         FIG. 7A  is a schematic perspective view of a fluidic channel portion of a device embodying features of the invention. 
         FIG. 7B  is a schematic representation of the bonding together of a fluidic channel portion with a silicon aperture portion of a device embodying features of the invention, showing a combined device in cross-section. 
         FIG. 8A  illustrates the fluorescence intensity of cells growing on a substrate embodying features of the invention at a time just prior to the flow of a physiological solution containing bradykinin. 
         FIG. 8B  illustrates the fluorescence intensity of cells growing on a substrate embodying features of the invention 3 seconds following the beginning of the flow of a physiological solution containing bradykinin. 
         FIG. 8C  illustrates the fluorescence intensity of cells growing on a substrate embodying features of the invention 9 seconds after the flow of a physiological solution containing bradykinin begins. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     An artificial synapse chip  10  embodying features of the invention is shown in  FIG. 1 .  FIG. 1A  shows a perspective view, and  FIG. 1B  a plan view of an ASC. The cell-contacting surface of the ASC includes the substrate  12 , which may be made with any material or materials compatible with cell attachment and growth. For example, glass, ceramic, silicon, silicon compounds and mixtures, polyimide, polystyrene, polyethylene, polylactide, TEFLON® (Polytetrafluoroethylene—PTFE) or other polymer, are suitable materials. In preferred embodiments, substrate  12  includes polyimide. 
     A micropattern  14  is provided on substrate  12 , effective to direct and guide the growth of cells and cell processes in contact with the substrate  12 . The micropattern  14  may be etched into substrate  12 , may be deposited onto substrate  12 , or may be integral with substrate  12 . In preferred embodiments, the micropattern  14  is made by microcontact printing onto the substrate  12 . The micropattern  14  may include growth factors, cell adhesion molecules, antibodies specific to cell surface proteins of the neurite or cell body, or other molecules or atoms effective to guide or modulate the growth of a neurite or the attachment of a cell or cell process. 
     Underlying the substrate  12  is a supporting layer  16 . An intermediate layer  18 , preferably formed with silicon, is provided adjacent to and below the supporting layer. A base layer  20  is shown lying below the intermediate layer  18 , so that intermediate layer  18  is sandwiched between supporting layer  16  and base layer  20 . In embodiments of the invention, supporting layer  16  and base layer  20  are formed with silicon nitride. 
     Silicon and silicon nitride provide stable intermediate and substrate layers, and may be produced and formed with widely available tools and knowledge for fabrication. Techniques for silicon device production are highly reproducible and accurate at the sub-micron level. Additionally, silicon allows for greater control of aperture geometry and location, including the ability to create arrays of apertures. 
     The devices and methods of the invention may be used to direct the growth of cells and cell processes, and to modulate or stimulate such cells and cell processes. A “cell process” is an elongated portion of a cell extending out from a cell body, or soma, and may be an axon, a dendrite, a neurite, a growth cone, or other elongated growing portion of a cell. A “neurite” is an elongated portion, or process, of a neural cell often forming the leading portion of the neural cell in its growth on a substrate. A “growth cone” is a specialized tip of a neurite that leads the growth or movement of a cell in the direction of the tip. The term “neurite” is used herein to refer inclusively to all neuronal cell processes, including axons, dendrites, and neurites and growth cones together. 
     Neurites may be extended and retracted from a cell in a variety of directions and at different times. The direction and rate of their growth may be influenced by the substrate, chemical gradients in the environment and along the substrate, electrical fields, hormones, and other physical, chemical and biological influences. As used herein, “growth” of a cell process such as a neurite comprises the elongation and migration are normal actions of these cell processes and may occur spontaneously or may be artificially induced or enhanced. Such growth may be directed by the devices and methods of the invention. 
     Directed growth of a cell process on a device embodying features of the invention is shown in  FIG. 1A . A cell  26 , with a cell process (neurite  28  with a growth cone  30  at its tip) is shown in contact with substrate  12  and micropattern  14 . The path followed by neurite  28  and growth cone  30  on substrate  12  is guided by micropattern  14  so that neurite  28  and growth cone  30  are led to recess  22  and aperture  24 . Recess  22  in the substrate  12  leads to an aperture  24  which forms a passage across the supporting layer  16 . As shown in  FIG. 1B , the floor  32  of recess  22  is formed of supporting layer  16  free of overlying substrate  12 . Aperture rim  34 , in supporting layer  16  surrounding aperture  24 , defines the passageway through supporting layer  16 . Although only one cell and only one neurite is shown in  FIG. 1A , it will be understood that a plurality of cells, neurites and growth cones may be in contact with substrate  12 , recess  22  and aperture  24 . A neurite may be directed by the path of micropatterned growth factors to a microfabricated aperture  24 , as shown in  FIG. 1A . 
     As shown in  FIGS. 1C and 1D , which are cross-sectional views taken along plane  1 C— 1 C of  FIG. 1A , aperture  24  opens into reservoir  36  defined by wall  38  of the intermediate layer  18  and wall  40  of the base layer  20 . A membrane  42 , such as a lipid bilayer membrane, may be formed across aperture  24  to separate reservoir  36  from recess  22 . 
     A membrane  42 , in place across aperture  24 , may prevent substantially all passage of material between recess  22  and reservoir  36 . However, membrane  42  may be a semi-permeable membrane effective to regulate the passage of material through aperture  24  without completely preventing all passage of material. For example, membrane  42  may form a semi-permeable membrane that allows the passage of some atoms, molecules, and ions while restricting the passage of other atoms, molecules and ions. A lipid bilayer membrane has such properties; in particular, a lipid bilayer membrane containing molecules such as ion channels or carriers is able to readily pass specific ions while restricting or substantially preventing the passage of other ions. Lipid bilayer membranes may be formed by Langmuir-Blodgett techniques as is known in the art. See, for example Montal and Mueller,  Proc. Natl. Acad. Sci. USA.  69:3561–3566 (1972); Montal,  Meth. Enzymol.  32:545–556 (1974); and Lindstrom et al.,  J. Biol. Chem.  255:8340–8350 (1980). 
     Recess  22  and reservoir  36  may each contain a solution; the solution in recess  22  may be the same or different from the solution in reservoir  36 . The solutions are preferably physiological solutions, such as a saline solution, that is compatible with cell growth and proliferation. Examples of such solutions include phosphate-buffered saline, bicarbonate-buffered saline, HEPES-buffered saline, Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM, Sigma Chemical Co., St. Louis Mo., Cat. # D6546), and other solutions known in the art. 
     The solutions may further contain bioactive agents  44 , so that recess  22  and/or reservoir  36  contain bioactive agents. Bioactive agents present within recess  22  and/or reservoir  36  may thus have access to aperture  24  and membrane  42 . For example, reservoir  36  may contain hormones, neurotransmitters in liposomes, actual cells, or simply an ionic solution able to be held at an electric potential to stimulate the neuron. Aperture  22  may thus be a stimulation site effective to stimulate a cell by chemical, hormonal, cellular, electronic, or other interactions. In all cases, the stimulation site is very specific to a single cell  26 , such as a neuron, and mimics the length scales of chemical synapses or gap junctions in the body. 
     Bioactive agents  44  may regulate the permeability of the membrane  42 , or may be capable of contacting and fusing with membrane  42  effective to deliver agents to the recess  24  from the reservoir  36  or from the recess  24  to the reservoir  36 . The bioactive agents are preferably present in reservoir  36  where the bioactive agents  44  are present in only one of recess  24  and reservoir  36 . Bioactive agents  44  may include channel forming molecules, such as α-hemolysin, gramicidin, alamethicin, or other channel former; substances such as drugs, neurotransmitters, chemoattractants, hormones, growth factors, adhesion molecules, amino acids, sugars, antibodies, and so forth; dyes; sources of cellular energy; or other compounds. Bioactive agents  44  may be micelles, liposomes, or biological membrane preparations containing ion channels, receptors, or other biologically active molecules that may fuse with and insert molecules into membrane  42 . Such bioactive agents may be effective to stimulate cell  26  or to modulate its activity. 
     An embodiment of the invention having electrodes  46  is shown in  FIG. 1D . Electrodes  46  may be made from any of a variety of materials, including silver, silver chloride, chromium, tin, indium, indium tin oxide, zinc oxide, colloidal stamped carbon, platinum, palladium, gold, aluminum, and other elements, oxides and materials known in the art. Electrodes  46  may be used to carry electrical signals from power source  48  to supply current or impose a voltage between electrodes  46  and to stimulate cell  26  or modulate its activity. 
     A cell, portion of a cell, or cells growing on an artificial synapse chip having features of the invention may be stimulated by neuromodulators delivered to a reservoir  36  and aperture  24  via a microfluidic delivery system. An artificial synapse chip  10  shown in  FIG. 1E  is part of a system including a fluid conduit  41  configured to carry a fluid  39  (with fluid flow optionally induced by a pump  43 ) to a microfluidic channel  45  for delivery to reservoir  36  and aperture  24 . A fluid  39  is preferably a biocompatible fluid, such as a saline, preferably including pH buffers to maintain its pH near levels compatible with maintaining cellular health, and may include bioactive agents  44 , such as neurotransmitters, neuromodulators, liposomes including neurotransmitters, and other agents that may affect a cell. A supply of fluid  39  may be stored in a depot  47  operably connected to pump  43  and microfluidic channel  45  by fluid conduit  41  or by other means. A fluid  49  may be used to drain or remove excess or waste fluid. A pump effective to cause fluid  39  to flow in a desired direction may be any mechanism suitable for inducing fluid flow. A mechanism for inducing fluid flow may force fluid to flow due to a pressure differential, an osmotic differential, may induce flow by electrical means, including electro-osmotic means, or in other ways. 
     For example, a pump  43  may include a mechanical pump mechanism, such as a piezoelectric, pneumatic, peristaltic, electrostatic, or electromagnetic pump. Alternatively, or in addition, a pump  43  may include a non-mechanical pump mechanism, in which, for example, fluid force is generated by thermal, chemical (including osmotic), acoustic, magnetic, electric, or electrosomotic, means or mechanisms. Pumps suitable for use with microfabricated devices, particularly electroosmotic pumps, are discussed in Andersson et al., Sensors and Actuators B 72:259–265 (2001); Morf et al., Sensors and Actuators B 72:266–272 (2001); Morf et al., Sensors and Actuators B 72:273–282 (2001); and Zeng et al., Sensors and Actuators B 82:209–212 (2002). 
     For example, a portion of a system with a pump  43  is illustrated in  FIG. 1F . The system includes an artificial synapse chip  10  having a cell with growth cone  30  growing over a pattern  14  on a silicon nitride substrate  16 , and a fluid conduit  41  comprised of two-parts, a buffer inlet  41 A and a transmitter inlet  41 B. Not shown are a depot  47  containing buffer connected to buffer inlet  41 A and a depot  47  containing transmitter solution connected to transmitter inlet  41 B. The pump  43  illustrated in  FIG. 1F  is a micro-electro-mechanical (MEM) pump similar to those used in ink-jet printers to eject drops of fluid. Such pumps are described in, for example, U.S. Pat. No. 5,734,395 to Kamisuki et al. A MEM pump as illustrated in  FIG. 1F  includes of a silicon diaphragm  51 , a counter electrode  53 , and a microfluidic channel  55  built over the diaphragm structure. The region of the microfluidic channel  55  above the diaphragm  51  is filled with fluid  39  and in fluid continuity with a depot  47  (not shown). The fluid contains bioactive agents  44 , which may be, for example, neurotransmitter agents, neuromodulatory agents, synaptosomes, or liposomes containing bioactive agents of any kind. Initially, the diaphragm  51  is in a horizontal (undeflected) configuration. The application of a minute bias voltage between the diaphragm  51  and the counter electrode  53  is effective to deflect the diaphragm  51  downward as shown in  FIG. 1F , thereby increasing the volume of the microfluidic channel  55  region above the diaphragm  51  and drawing fluid  39  from the depot  47  along transmitter inlet  41 B. Removal of the bias voltage allows the diaphragm  51  to relax back to its initial position, forcing fluid out of microfluidic channel  55  and towards reservoir  36  and aperture  24 . Neurotransmitter agents  44  in fluid  39  thus are transported near to reservoir  36 , and can diffuse into reservoir  36  and aperture  24  to contact growth cone  30  and affect the cell. In this way, for example, a brief pulse of neurotransmitter agent may be delivered to a cell having a portion growing across an aperture  24 . In embodiments of artificial synapse chips, conduit  41  would include simply transmitter inlet  41 B; in other embodiments, such as the one illustrated in  FIG. 1F , conduit  41  also includes a buffer inlet  41 A. Flow of buffer solution through buffer inlet serves to flush out the microfluidic conduit  45  with buffer, carrying away neurotransmitter agents  44 , reducing or ending the effect of these agents. Such flushing prepares the system for a subsequent pulse of neurotransmitter agents  44  as well as acting to end the effects of a prior pulse. 
     Diffusion of neurotransmitter agents  44  through aperture  24  can be very rapid due to the thinness of the aperture, which may be, for example, only about 500 nm thick. The diaphragm  51  of a MEM pump  43  may flex at high frequency so as to eject fluid  39  at high frequency. Pulses of bioactive agents  44  (e.g., neuromodulatory or neurotransmitter agents  44 ) may be delivered at high frequency, including frequencies ranging from between only a few cycles per second, or Herz (Hz) to about hundreds of kHz. Such rapid signaling matches the rapid signaling rates found in vivo in the brain and retina. 
     The concentration of bioactive agents  44  is determined by several factors, including the MEM ejector pulsing frequency, the flow rate of fluid through the microfluidic conduit  45 , and, where electro-osmotic flow may also be induced, the voltage on the optional buffer chamber electrodes. The concentration of bioactive agents  44  at the aperture  24  is determined in part by the diffusion rate, which is affected by the concentration. The size of a pump  43 , such as the ejector diameter determined by the diameter of the outlet  57  of transmitter inlet  41  B, can range from between a few microns (μm) to hundreds of μm. The size may depend on the required capacity of a microfluidic channel. 
     The performance of a pump  43  and a system as illustrated in  FIG. 1F  depends on the design and materials used, and on the fluids employed during its use. For example, the damping experienced by the system is related to several factors, including fluid viscosity and the geometry of the microfluidic conduit  45 , the geometry of the microfluidic channel  55 , and the geometry of other components. In order to obtain the desired performance, preferred systems are configured with a diaphragm  51  comprised of polysilicon, a narrow microfluidic channel  55  and a small initial separation between the diaphragm  51  and the counter electrode  53 . Since there is no threshold voltage for activating the motion of a polysilicon diaphragm, a MEM ejector pump can deliver small volumes as small as attoliter to zeptoliter volumes. The power required to charge a capacitor of the size of a diaphragm  51  to a fraction of a volt is about a picowatt. A single photodiode, such as an avalanche photodiode capable of generating nanoWatts of power, is thus able to charge hundreds or even thousands of such MEM pumps to deliver bioactive agents to cells. 
     The power to actuate a pump  43  may thus come from a photodiode in a photodiode array  59  as illustrated in  FIG. 1F . Light contacting such an array  59  is thus effective to actuate a pump  43  configured to pump a fluid  39  containing bioactive agents  44  into a microfluidic conduit  45  where the bioactive agents  44  may flow and diffuse through an aperture  24  and into contact with, for example, a growth cone  30  growing across an aperture  24 . In this way, for example, an artificial synapse chip  10  may be used to transduce a light signal into a biological signal. An array of artificial synapse chips  10 , or an array of systems including such chips, or an artificial synapse chip or chips having an array of apertures, may also be used in similar ways to transduce light signals into biological signals. Alternatively, or in addition, electrical signals may be used to stimulate a cell or cells grown on an artificial synapse chip configured to direct the growth of cells, such as to direct cell growth towards electrodes. 
     The components and features necessary to construct devices such as an artificial synapse chip  10  may be made using methods commonly termed “microfabrication” or “nanofabrication” techniques. Methods for microfabrication useful for practice of the invention may be found in, e.g., U.S. Pat. No. 5,776,748 to Singhvi et al.; U.S. Pat. No. 5,900,160 to Whitesides et al.; U.S. Pat. No. 6,060,121 to Hidber et al.; U.S. Pat. No. 6,180,239 to Whitesides et al.; “Patterning of a Polysiloxane Precursor to Silicate Glasses by Microcontact Printing”, Marzolin, et al., Thin Solid Films 1998,315, 9–12; “Microfabrication, Microstructures and Microsystems”, Qin, et al.; In Microsystem Technology in Chemistry and Life Sciences, vol.194, Manz, A. and Becker, H., Eds.; Springer-Verlag, Berlin, 1998 , 1–20; “Unconventional Methods for Fabricating and Patterning Nanostructures,” Xia et al., Chem. Rev. 99:1823–1848 (1999). All patents and publications, both supra and infra, are hereby incorporated by reference in their entirety. The sophisticated microstructures that may be constructed using such microfabrication methods may be used to make devices such as artificial synapse chips  10  and to modify substrates. The structures shown in the following Figures were made using the Stanford Nanofabrication facility (Leland Stanford Junior University, Stanford, Calif. 94305). 
     An aperture  24  formed in a silicon nitride supporting layer  16  of an ASC embodying features of the invention is shown in  FIG. 2A . The view in  FIG. 2A  is in the same orientation as the view shown in  FIG. 1B , showing the aperture  24  facing the cell-contacting surface of the ASC. The aperture is approximately 10 μm in diameter (scale bar represents 1 μm). Aperture  24  is bounded by rim  34  in exposed floor  32  of recess  22 . At the small scale shown in  FIG. 2A , the aperture  24  is quite smooth, both in terms of shape and surface. To improve resolution, the device was first coated in gold. Note that the plasma etching used for this processing does not create vertical sidewalls in the aperture. The aspect ratio of the sidewalls is roughly 2.5:1. Although the example of the aperture  24  shown in  FIG. 2A  forms a passageway through a supporting layer  16  made from silicon nitride, other materials may also be used, such as polymers and glass. 
     A microfluidic reservoir  36  may be connected to the other side of recess  22 . A reservoir  36  may be configured to be of a size able to contain neuromodulatory agents in aqueous solution or aqueous suspension. Aperture  24  provides a conduit for the delivery of the neuromodulatory agents from the reservoir  36  to at least a portion of a cell  26 . In addition, other conduits and fluidic delivery systems may be used to transport fluid and neuromodulatory agents to desired locations at or adjacent the aperture  24 , reservoir  36 , or other location. For example, where a depot containing a reserve of fluid and/or neuromodulatory agents is located at a position away from an aperture, a conduit may operably connect the depot with a reservoir  36  and with an aperture  24 . 
       FIG. 2B  is a scanning electron micrograph (SEM) of a microfabricated well that has a microaperture in the bottom, showing reservoir  36  of an artificial synapse chip embodying features of the invention, viewed from the face opposite to the cell-contacting substrate surface  12  of the ASC. Shown at a larger scale than  FIG. 2A , this micrograph of the reservoir  36  viewed from the trans side of the artificial synapse chip  10  shows the smooth silicon nitride surface surrounding the aperture  24 . Wall  38  of the intermediate layer  18  and wall  40  of the base layer  20  are shown, with a small amount of base layer  20  shown framing the walls  38  and  40 . The black spot indicates the aperture  24  configured for cell attachment and stimulation (not clearly visible at this magnification). The reservoir  36  is designed for holding the culture solution for the cells. The size of the bottom of the well is 1 mm across. 
     As shown in  FIGS. 1A and 1B , a substrate  12  of ASC  10  may have a micropattern  14  effective to guide and direct the growth of a cell process, such as neurite  28  with growth cone  30 . Such directed cell growth is shown in  FIG. 2C , which is a scanning electron micrograph showing rat P7 retinal ganglion cells (RGCs) grown on a plastic substrate that was patterned with a laminin pattern. The insert at the bottom left of  FIG. 2C  illustrates the sawtooth pattern microfabricated onto the substrate before addition of the RGCs. As shown in the electron micrograph, both the cell bodies and the cellular processes follow the pattern quite closely. The scale bar represents a length of 100 μm. 
     Cells are also able to grow over a microfabricated aperture  24  through a supporting layer  16  of an ASC  10 .  FIG. 2D  shows PC12 cells growing around and over a 5 μm-diameter aperture in a silicon nitride surface. The boundary of a reservoir  36  under the aperture  24  may be seen at the margins of  FIG. 2D . 
     A preferred method of producing a micropattern  14  is to contact substrate  12  with a microcontact printing stamp having an ordered assemblage of molecules, which may be a discontinuous assemblage, for deposition on to substrate  12 . Microfabrication methods are suitable for making microcontact stamps.  FIG. 3  is a plan view SEM of a stamp embodying features of the invention for making a micropattern  14  on a surface. The surface topology is given by the array of squares. Deposition of material onto the surface of a stamp, and contacting a substrate  12  of a device, such as an artificial synapse chip  10  shown in  FIG. 1 , with the stamp is effective to form a micropattern on a substrate  12 . The formation of a micropattern in this way is one method of microcontact printing. Micropatterns formed by such microcontact printing methods are effective to align the position and growth of cells on a substrate. Shown in  FIG. 3  is a scanning electron micrograph (SEM) picture of a poly(dimethylsiloxane) (PDMS) stamp that was made from a master that was micromachined from a silicon wafer. The microcontact stamp shown in  FIG. 3  has a surface topology given by an array of squares. Other patterns, including circles, ovals, stripes, and other shapes, may be made on the surface of a microcontact stamp. 
     Microstamps such as the one shown in  FIG. 3  may be fabricated using photolithography techniques. For example, the stamp shown in  FIG. 3  was formed from a thin (1–7 μm) photoresist layer on a silicon wafer that was patterned to create a master for the microcontact printing. The mask and stamp master was fabricated at the Stanford Nanofabrication Facility. The master pattern consists of arrays of lines configured for cell attachment and neuron growth. The master was prepared by ultra-violet (UV) etching of a mask on positive photoresist on silicon, and PDMS stamps were generated in situ on the master using Sylgard 184 silicone elastomer followed by thermal curing. Stamps were also prepared by pouring an elastomer and curing agent together to form PDMS on a silicon master, degassed and allowed to set at room temperature. Stamps were then made by cutting a portion of the PDMS followed by plasma treatment to increase hydrophobicity for enhanced protein adsorption and imaged using SEM. 
     A variety of different stamp patterns may be produced by the methods, and adapted to the optimal line width or thickness, length and spacing for neurite growth. For example, line widths ranging from a few nanometers (nm) wide to several hundreds of micrometers (μm) wide may be used; preferably, line widths range from about 10 nm to about 20 μm. Lines may be as short as a few nm and may be as long as several millimeters; preferably line length is within the range of about 10 nm to about 100 μm long. The spacing between lines in a pattern may range from about 1 μm to several hundreds of μm; preferably line spacing is between about 2 μm to about 100 μm. 
     Following microfabrication of the stamps, the stamps were coated with molecules desired to be deposited onto a substrate  12  to provide a micropattern  14 . Micropatterns may include biologically active molecules and agents such as neurotransmitters, hormones, growth factors such as nerve growth factors, epidermal growth factor, and insulin-like growth factor, co-stimulatory molecules, antibodies, and other biomolecules known in the art. For example, stamps may be coated with adhesion agents that promote call adhesion. Adhesion agents include poly-L-lysine, CELL TAK™ (Cell and tissue adhesive: Becton Dickinson, Franklin Lakes, N.J.), cell adhesion molecules such as neural cell adhesion molecule (NCAM), lectins, and other adhesion agents known in the art. The adhesion agents may also be labeled with fluorescein for visualization. The pattern may be stamped on glass, silicon, silicon nitride, polyimide, polystyrene, polyethylene, polylactide, TEFLON® (Polytetrafluoroethylene—PTFE), other polymer, or any substrate suitable for use as a substrate for cell growth. For example, a coated stamp may be contacted with a polyimide substrate on a silicon nitride supporting layer to provide a substrate to facilitate cell adhesion and growth. Cell adhesion and growth may be monitored with a fluorescence microscope. A mercury arc lamp may be used to excite the fluorescent dye conjugated to the poly-L-lysine or other micropattern molecule to provide fluorescence signal for visualization of adhesion agents. 
     A system  50  for implantation into an animal is shown in  FIG. 4 . In embodiments, the system is implanted into the retina of an animal. The system  50  includes an ASC  52 , a photosensitive device  54 , a communication link  56  between the ASC and the photosensitive device, and a power source  58 . The photosensitive device  54  may be separate from the ASC  52 , or may be in contact with the ASC  52 , or may comprise part of the ASC  52 . The photosensitive device  54  may be a photomultiplier, a semiconductor photosensor, a chemical photosensor, a metallic photosensor such as a selenium or other photocell, or other photosensor known in the art. The communications link  56  may be any electrical conductor, such as a wire, tracing, or other electrical link. In embodiments, the communications link  56  is a chemical communications link, whereby a photosensor alters the chemical environment so that a chemical signal is delivered to at least a portion of the ASC  52 . The power source  58  may be any power source, such as a battery, a thermal power source capable of producing power by a temperature gradient, or a photocell capable of producing energy from light. 
       FIG. 5A  illustrates an eye  60  of animal into which an ASC  62  has been implanted. The ASC  62  is shown implanted in the subretinal space  64  of the animal, so that it occupies a position between the retinal photoreceptors  66  and the retinal pigment epithelium  68 . In embodiments of the invention, the ASC  62  may be implanted near the ganglion cell layer  70  on the inner limiting membrane  72  near the boundary of the vitreous humor  74 . A detailed view of the subretinal space  64  and implanted ASC is shown in  FIG. 5B . 
     ASCs may be used for implantation into the nervous system of an animal. For example, ASCs embodying features of the invention may be implanted into a retina of an animal to provide a neural prostheses where the retina suffers from traumatic injury, disease or degeneration. Patterns may include one or a combination of molecules such as neurotrophins and growth factors including nerve growth factor, brain-derived growth factor (BDGF), epidermal growth factor (EGF), ciliary neurotrophic factor (CNTF), glial-derived neurotrophic factor (GDNF), NT-3, fibroblast growth factors (FGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), vascular endothelial growth factors (VEGF) and others; cyclic nucleotides such as cyclic adenosine monophosphate, cyclic guanosine monophosphate and others; extracellular matrix molecules such as laminin, tenascin, collagen, fibronectin, integrins, immunoglobins (including molecules such cell adhesion molecules N-CAM and L-CAM, axonin, cadherins, and so forth), proteglycans, anosmin-1, thrombospondin and others; myelin and myelin associated inhibitors such as myelin-associated glycoprotein and nogo; tyrosine kinase receptors such as ephrins; netrins; inflammatory cytokines such as transforming growth factor δ, leukemia inhibitory factor (LIF), tumor necrosis factors (TNF), interleukins, and others; neurotransmitter such as acetylcholine and others; stimulatory molecules such as potassium chloride, insulin, and others; co-stimulatory molecules, antibodies, and other growth and modulatory factors known in the art. 
     It is critical to optimize the retention of the pattern for transfer from the stamp to the cellular system for use of the substrate in implantation, such as retinal implantation. The line width and concentration of biomolecules may be used to control the number of neurites per microprinted line. The degree of pattern transfer may be determined using microscopy. 
     As shown in  FIG. 1 , the recesses  22  and reservoirs  36  of the devices of the present invention are suitable for the storage of neuromodulatory agents, and for the delivery of neuromodulatory agents to at least a portion of a cell. The present invention provides the ability to direct the delivery of neuromodulatory agents to single cells, in particular to localized portions of such cells, by directing the growth of cell processes to nanoapertures, and delivering neuromodulatory agents to the cell processes via the nanoapertures. Suitable neuromodulatory agents include any agent effective to stimulate a cell, or to modulate the effects of other agents effective to stimulate a cell. For example, the neuromodulatory agents may be neurotransmitters, hormones, ions, messenger molecules, nucleic acids, nucleic acid vectors, drugs, cells, cell fragments, cell organelles, liposomes, or other biologically active materials. Neuromodulatory agents such as neurotransmitters include amino acids such as glutamate, aspartate, and glycine, and related neurotransmitters and stimulatory agents such as N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxalone propionic acid (AMPA), quisqualate, and kainate, and analogs thereof, and other glutaminergic and glycinergic agents known in the art; cholinergic agents such as acetylcholine, suberyldicholine, analogs thereof and other cholinergic agents known in the art; adrenergic agents such as dopamine, epinephrine, norepinephrine, analogs thereof, and other adrenergic agents known in the art; serotinin, and serotonergic agents known in the art; gamma-amino butryic acid (GABA) and other GABA-ergic agents known in the art; taurine, octopamine, nucleotide phosphates such as adenosine triphosphate (ATP), adenosine diphosphate (ADP) and guanosine diphosphate (GDP) and triphosphate (GTP), cyclic nucleotides such as cyclic adenosine monoposphate (cAMP) and cyclic guanosine monophosphate (cGMP), and other neurotransmitter and neuromodulator molecules known in the art. In addition, neurotransmitters include all agents active at neurotransmitter receptors, such as glutamate receptors, NMDA-receptors, AMPA-receptors, glycine receptors, dopamine receptors, acetylcholine receptors, and others known in the art. Neuromodulatory agents also include messenger agents including peptide hormones and neuromodulators such as enkephalins, endorphins, adrenocorticotrophin hormone (ACTH), vasoactive intestinal peptide (VIP), and other peptides known in the art, steroid hormones, second messengers such as inositol phosphates, and ions such as calcium, potassium, zinc and salts thereof. These agents may be free in aqueous solution or aqueous suspension, may be present in micelles, or may be carried by liposomes. 
     Liposomes, as is known in the art, are small membranous vesicles suitable for delivery of both hydrophilic and hydrophobic compounds. Pharmaceutical administration systems based on liposomes are described in, e.g., Gregoriadis, G. (editor) Liposome Technology, Vol. II, Incorporation of Drugs, Proteins and Genetic Material, CRC Press 1984, and in Knight, C. G. (editor), Liposomes: From Physical Structure to Therapeutic Applications, Elsevier 1981. Neuromodulatory agents suitable for the practice of the invention further include biological membrane preparations containing ion channels, receptors, or other biologically active molecules, as described in, e.g., Coronado et al. J. Gen. Phys. 76:424–446 (1980). Such biological membrane preparations may fuse with and insert molecules into a membrane  42  across an aperture  24 , or into the membrane of a cell  26 , neurite  28  or growth cone  30 . For example, gramicidin, alamethicin, and other molecules known in the art are suitable pore-forming molecules for the practice of this embodiment of the invention. Ion channel molecules suitable for the practice of the invention include multi-subunit macromolecule assemblies such as ligand-gated ion channels including cyclic nucleotide-gated channels, calcium-activated channels, ACHR ion channels, glutamate receptor ion channels, including all NMDA, AMPA, quisqualate, kainate subtypes, glycine receptor ion channels, and voltage-gated ion channel molecules and multi-subunit macromolecular assemblies such as sodium channels, potassium channels, calcium channels, chloride channels, and other channels, including gap junction channels, mechanosensitive channels, non-gated, and non-selective channels. Carrier molecules such as amphotericin are also suitable. Alternatively, membranes may be formed with proteins, such as pore-formers and carriers, already incorporated as part of the membrane-forming material. See, e.g., Schindler, Methods Enzymol. 1989:171:225–253. 
     EXAMPLE 1 
     Microfabrication of Apertures 
     Microfabricated apertures on a silicon chip surfaces have been made using the Stanford Nanofabrication Facility. Microfabricated wells with microfabricated apertures are shown in  FIG. 2 . Standard silicon processing techniques were adapted for producing micrometer and nanometer-sized apertures in a silicon nitride membrane. Using low-pressure chemical vapor deposition (LPCVD), silicon nitride was grown on the surface of &lt;100&gt; orientation silicon wafers. A combination of lithography to define the structures in a photosensitive polymer was followed by plasma etching to pattern the structures in the silicon nitride creates apertures on one side of the wafer and an etchant masking layer on the other side. An anisotropic etchant, such as tetramethylammonium hydroxide (TMAH), was used to remove the silicon along the {111} crystal plane, but leave the silicon nitride unaffected. This produced a via hole (a connecting passageway) beneath the aperture, exposing the silicon nitride membrane and completing the processing. 
     Shown in  FIG. 2A  is a SEM of the microfabricated container. Note the black spot indicated by the arrow is the microetched aperture adapted for cell attachment and stimulation. The well was designed for holding the culture solution for the cells. The size of the bottom of the well is 1 mm across.  FIG. 2B  shows the microaperture in the bottom of the container shown in  FIG. 2A . The microaperture is approximately 10 μm in diameter. Although not shown, the other side of the aperture is connected to a microchannel reservoir that is made by sealing a PDMS stamp with microchannels to the underside of this substrate. 
     The conduit, or via, opens into a microfluidic channel that serves as a reservoir for neuromodulatory agents that may be applied to cells adherent to the substrate. The microfluidic channel was made from a standard PDMS stamp as described above and sealed to the wafer. Such a microfluidic channel can be readily sealed to the wafer with excellent sealant properties. For example, a PDMS stamp having a channel may be bonded to a silicon nitride surface after acid cleaning (e.g., HCl) and plasma treating, forming an irreversible bond. The microfluidic channel described has wide ranging ramifications for use including (1) acting as general purpose buffer reservoir for constant replenishing/exchanging waste products from the other side of the cell (2) delivery of transmitters, liposomes, voltage/current clamping of the cell, or (3) for sampling released products from the cell. 
     Apertures may be formed in sizes ranging from a few nm to a few tens of μm over which cells may be grown. For example, cells may be grown directly over 50 nm apertures. Use of an aperture smaller than the length scale of the neuron is effective to insure that only a single cell is stimulated. 
     EXAMPLE 2 
     Device Fabrication and Optimization 
     This Example describes the manufacture and optimization of devices embodying features of the invention for use in forming bilayer membranes across the microfabricated apertures of the devices. Chips were made with surface areas of about 1 cm 2  and with a final thickness of roughly 0.5 mm. Circular apertures of 25 μm through 250 μm (diameter) were plasma etched in 500 nm thick silicon nitride. The chips were covered in a thick polyimide, except for a square region of exposed silicon nitride 500 μm on a side. 
     Fabrication was done at the Stanford Nanofabrication Facility (SNF) with 4 inch, &lt;100&gt; orientation, boron-doped, double-polished silicon wafers, nominally 500 μm thick. Using low-pressure chemical vapor deposition (LPCVD), a thin layer (500 nm) of silicon nitride was grown on the surface of the wafers. Standard contact photolithography and plasma etching of the silicon nitride was used to define the small features (e.g., the apertures). The larger features on the backside of the wafer were similarly defined using backside alignment, contact photolithography, and plasma etching. 
     The silicon was etched anisotropically along the {111} plane at an angle of 54.7° to the wafer surface. The square hole in the backside of the wafer was chosen to yield a square 180 μm larger than, and centered on, the aperture. This left a thin silicon nitride membrane freely spanning the region without any silicon support. Because of the high tensile strength of silicon nitride, this nitride membrane was quite strong and stable, and was able to readily withstand the forces generated during processing. 
     With the features defined in the silicon nitride, the wafers were placed in 20% tetramethylammonium hydroxide (TMAH) at 100° C. for approximately 6 hours. The silicon nitride acted as a mask, allowing the TMAH to etch anisotropically through the wafer along the {111} crystal plane. 
     Since the exposed silicon is conductive, it was necessary to oxidize the surface to reduce capacitance and noise. This was accomplished with a steam oxidation at 1100° C. for 4 hours, providing ˜1.1 μm of oxide. Finally, to reduce the capacitance further, a photosensitive polyimide (Durimide 7520, Arch Chemicals, Zwijndrecht, Belgium) was spun on 30–70 μm thick, exposed under a contact aligner, developed, and cured, yielding a coating 15–35 μm thick. 
     To create a hydrophobic surface, the chips were then soaked in a mixture of hexadecane (Sigma, St. Louis, Mo.), chloroform, and octyltrichlorosilane (Aldrich, Milwaukee, Wis.) in a ratio 80:19:1 (by volume) for 15 minutes per side. Two rinses in chloroform for 5 minutes each completed the processing. The coating was tested by applying ˜5 μl droplets of water and verifying that the contact angle was greater than 90°. 
     One advantage of silicon is the ability to control the thickness of the bilayer supporting partition (BSP). The thickness of the silicon nitride BSP was chosen to be an order of magnitude smaller than TEFLON® (Polytetrafluoroethylene—PTFE) partitions used to form apertures for bilayer formation (6–25 μm), with the expectation that thinner partitions provide a smaller solvent torus and a larger bilayer area. The partition is still a couple of orders of magnitude larger than a 2–4 nm bilayer, so bending of the lipids from the edges of the partition to the bilayer is still necessary. However, this bending distance is smaller, yielding a larger bilayer area relative to the aperture size. The impact of this upon stability is unknown, but it does allow more area for protein insertion and the ability to create bilayers across smaller apertures. 
     An ASC is to able to provide precise stimulation of neurons and the making of sensitive electrical measurements. As with any electronic circuit, excessive capacitance may present a problem by increasing electrical noise. Excessive capacitance is a problem for two reasons: (1) electrical noise due to the access resistance in series with this capacitance, and (2), where a lipid bilayer is to be made across the aperture of an ASC, the inability to observe the membrane capacitance over the background. Since silicon is a conductor at room temperature, any contact of the bath to the silicon effectively connects the entire area of the chip to the system. A 1 cm 2  chip with 500 nm of silicon nitride (ε≈7.5) has a capacitance of 13 nF, three orders of magnitude greater than the capacitance of a 25 μm diameter bilayer. 
     However, a thin BSP in a solution containing charge-carriers has a large capacitance, which may present a problem where precise electrical measurements or precise electrical stimulation of a cell are desired. The solution to this problem was found to be two-fold. First, to remove the electrical connection between the silicon and bath, the wafers were exposed to steam at 1100° C., yielding just over a micron of oxide on all exposed silicon surfaces. This reduced the capacitance by a factor of two, since the system effectively becomes two nitride capacitors in series connected by a silicon conductor. It does, however, simplify the capacitative model of the system by removing discontinuities that would occur as the bath contacts the silicon. 
     Second, capacitance was reduced by addition of a polyimide layer. A negative, photosensitive polyimide (ε=3.5) was chosen that can be processed using standard lithography. An application of 30 to 100 μm of polyimide becomes 15 to 50 μm when cured. In addition, the cured polyimide is highly resistant to solvent degradation. The design leaves 500 μm by 500 μm of nitride uncovered over the aperture. Manipulation of the solution level so that only 5 mm by 5 mm of the chip was exposed to solution, reducing the capacitance from 35 μm of polyimide to only 22 pF. 
     EXAMPLE 3 
     Bilayer Formation Across Silicon Nitride Apertures 
     Lipid bilayers were formed by the method of Montal and Mueller (1972). In practicing the Langmuir-Blodgett technique, one raises two lipid monolayers across an aperture, allowing the lipids to align their hydrophobic tail portions across the aperture so as to form a lipid bilayer. Because of the hydrophobic nature of the lipid tails, in order to form stable bilayers the surface of a BSP must also be hydrophobic. If the substrate is hydrophobic, the lipids can smoothly transition from coating the substrate to spanning the aperture. To reverse the wetting properties of the naturally hydrophilic silicon nitride, the silicon nitride was coated with an alkylsilane (octyltrichlorosilane). Application of this coating was quite simple and very effective. It was not found to be possible to form a bilayer with untreated devices. Use of longer carbon chain silanes or alternative materials to make the surface even more hydrophobic would further increase bilayer stability. 
     The characteristics of the devices are shown in Table 1. The thickness of the polyimide was varied to verify our background capacitance model for the chips. The model is based upon our chambers, where the baths contact 5 mm by 5 mm of chip. The intrinsic capacitance of the baths and amplifier was measured to be 7.2 pF, and is included in this number. For a 50 μm aperture device, where the polyimide is 32 um thick, the model yields a background capacitance of 45 pF, compared to 77 pF for 6 μm thick Teflon®. 
     
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Characteristics of the devices used in this study. The polyimide thickness and 
               
               
                 total capacitance are measured, while the other properties are calculated. 
               
             
          
           
               
                   
                   
                 Chip 
                   
                 Bilayer 
                   
               
               
                 Aperture 
                 Polyimide 
                 Background 
                   
                 Capacitance 
                 Expected 
               
               
                 Size (μm) 
                 Thickness (μm) 
                 Capacitance (pF) 
                 Total Capacitance (pF) 
                 (μF/cm 2 ) 
                 Noise (pA) 
               
               
                   
               
             
          
           
               
                 25 
                 9.6 
                 100.1 
                 103.4 +/− 1.8 (n = 6) 
                 0.67 +/− 0.36 
                 3.4 
               
               
                 50 
                 32.1 
                 45.0 
                  57.7 +/− 2.1 (n = 10) 
                 0.65 +/− 0.11 
                 1.4 
               
               
                 75 
                 19.8 
                 60.6 
                  91.1 +/− 2.0 (n = 9) 
                 0.69 +/− 0.04 
                 1.8 
               
               
                 100  
                 17.5 
                 66.4 
                 117.4 +/− 1.1 (n = 2) 
                 0.65 +/− 0.01 
                 2.0 
               
               
                 150  
                 21.7 
                 59.4 
                 182.6 +/− 5.3 (n = 6) 
                 0.70 +/− 0.03 
                 2.6 
               
               
                 250  
                 19.6 
                 65.9 
                 379.8 +/− 6.3 (n = 5) 
                 0.64 +/− 0.01 
                 4.4 
               
               
                   
               
             
          
         
       
     
     The bilayer specific capacitance was determined simply by dividing the difference between the measured total capacitance and the calculated background capacitance by the area of the aperture. This number is in the range of 0.64 to 0.70 μF/cm 2  and corresponds well with that found in other artificial bilayer experiments. The total capacitance was measured within a few minutes after bilayer formation to avoid changes due to bilayer thinning. Note that as the aperture area decreased, the bilayer capacitance became quite small compared to the background, yielding a large error in the specific capacitance. 
     The empirical evidence for the formation of a bilayer on any chip was threefold. For the largest size apertures, the change in capacitance due to the bilayer was readily observable. For a typical specific capacitance value of 0.65 μF/cm 2 , a bilayer on a 100 μm aperture would have a capacitance of 51 pF, which is easily observed over a 65 pF background. In addition, a resistance through the aperture greater than 1 GΩ indicates the presence of a bilayer. For all aperture sizes, a “gigaseal” of at least 2.5 GΩ was observed, indicating the formation of a bilayer. 
     For smaller apertures, it was more difficult to observe the capacitance change over the background. In this case, membrane-bound proteins that affect the electrical properties of the bilayer, such as carriers and ion channels, offered the best proof of the formation of a bilayer. The ion channel peptide Gramicidin D (gD), was chosen for ease of use and large conductance change. A lipid bilayer membrane is required in order for Gramicidin D to increase current flow. After adding 5 to 20 μl of 2 mg/ml gD (Sigma, St. Louis, Mo.) in ethanol to each bath, the conductance of the bilayer dramatically increased within minutes, while the capacitance remained constant. The addition of ethanol by itself had no effect. Thus, the increase in current flow in response to an applied potential (measured by an increase in conductance) indicated that a true lipid bilayer had been formed. 
     Observing individual ion channels or pores requires that the electrical noise to be as small as possible. In addition to environmental sources and capacitative noise, there are two major sources of electrical noise: photocarriers in the silicon, and access resistance. The first noise source, light, is produced when light incident upon the chip excites carriers across the band gap, creating a fluctuating charge between the two layers of nitride. Depending on the source and intensity of light, the noise produced may measure tens to hundreds of picoamperes peak-to-peak. Simply shutting off room lights or enclosing the setup in a light proof box was sufficient to reduce the electrical noise contributed by from noise source. 
     The other source of electrical noise was due to the access resistance of the baths in series with the bilayer capacitance. The total access resistance (R a ) contains three components: the bulk bath resistivity (32 A-cm), the bath resistivity in the aperture, and the access resistance to the aperture. For a small access resistance, the expected noise in amperes rms was √{square root over (4kTR α (2πf 2 C) 2 )} where f is the measurement bandwidth. 
     The results of this calculation for each tested chip are shown in Table 1. For the 50 μm aperture, the expectation from this calculation was 1.4 pA rms, while the actual measured values were between 1.8 pA and 2.4 pA. The difference was attributed to local environmental noise. 
     EXAMPLE 4 
     Bilayer Stability and Lifetime 
     Bilayers were formed by the technique of Montal and Mueller (1972). The aperture was first pretreated with ˜5 μl of 1:9 (v:v) hexadecane:hexane (Burdick &amp; Jackson, Muskegon, Mich.). The chip was mounted between two Teflon® baths with silicone high vacuum grease (Dow Corning, Midland, Mich.). Each bath was filled with 1 M KCl to just below the aperture. A solution of 5 μl at 10 mg/ml of 1,2-diphytanoyl-sn-glycerophosphocholine (Avanti Polar Lipids, Alabaster, Ala.) in chloroform was applied to each bath and allowed to evaporate. When the water level in each bath was raised, a lipid bilayer formed across the aperture, as evidenced by the capacitance and conductance of the device. 
     The ability to support the formation of a bilayer that will be stable for an extended period of time is an important property for any supporting substrate. Lipid bilayer membranes formed on ASCs were found to be very stable. ASCs were found to have two advantages over TEFLON® (Polytetrafluoroethylene—PTFE) partitions in terms of stability. First, lipid bilayer membranes formed on ASCs were thinner than those formed on TEFLON® (Polytetrafluoroethylene—PTFE) partitions, but are also more rigid. TEFLON® (Polytetrafluoroethylene—PTFE) partitions flex under changes in pressure, whereas nitride is comparatively inflexible. Second, the nitride surface and aperture edge are smooth at the nanometer level (see  FIG. 2 ), unlike mechanically formed apertures in TEFLON® (Polytetrafluoroethylene—PTFE) partitions, which have micron-scale defects along the aperture edge. 
     Membrane stability was demonstrated by observing lipid bilayer membrane lifetime. Roughly half of the bilayers broke within the first few minutes, but some were stable for much longer. The longest bilayer lifetime observed was 8 hours. No attempts to measure systematically for longer than this time scale were made. It was found that the number of stable bilayers that were formed depended heavily on the cleanliness of the chip. It was rather easy to form a stable bilayer membrane using a fresh ASC device that had just completed processing. However, it was more difficult to form a stable bilayer membrane on an ASC that was reused after cleaning. Lipid bilayer membrane formation was found to be impossible following use of a cleaning process that left a residue across the aperture. 
     EXAMPLE 5 
     Single-Channel Recordings with α-hemolysin 
     Ion channel activity due to staphylococcal α-hemolysin (αHL) channels was studied in lipid bilayer membranes formed across ASC apertures. This 293-amino acid heptameric pore forms 2 nm channels through the lipid bilayer. Single-channel recordings were performed with a patch clamp amplifier (Heka EPC-8, Heka Elektronik, Lambrecht, Germany) and an analog-to-digital converter (Instrutech ITC-18, Port Washington, N.Y.) sampled at 10 kHz. Filtering was performed with a built-in 7-pole low-pass Bessel filter at 5 kHz. The data was collected on computer using Pulse 8.4 (Heka) and analyzed with Igor Pro 4.0 (WaveMetrics, Lake Oswego, Oreg.). The αHL pores were added to the cis chamber (1 to 10 μl at 321 ng/ml), and held at −40 mV (trans side grounded). Addition of αHL to the trans side also yielded channels, but the diffusion time was longer due to the relatively long, narrow cavity. 
       FIGS. 6A and 6C  illustrate α-hemolysin (αHL) single-channel currents recorded from artificial bilayer membranes across 100 μm apertures in microfabricated devices exposed to β-cyclodextrin (ACD) in the trans bath. The holding potential was +40 mV (the cis bath was at ground potential). Shown in  FIG. 6A  are representative single-channel data for αHL channels. In similar experiments, voltage pulses of −40 mV were applied for 750 ms, and currents were recorded, giving measured currents of 31.0 +/−3.2 pA per channel). The calculated pore conductance of 811 +/−55 pS was typical for αHL channels. The added β-cyclodextrin (βCD), which inserts reversibly in the trans side of the channel, causes fluctuations in the current flow through the channel as the molecules move in and out of the protein. This effect is found at micromolar concentrations (e.g., 40 μM to 300 μM of βCD). When a channel becomes blocked, a clearly observable current reduction occurs, as shown in  FIG. 6A . Partial blocking events from βCD appear as downward spikes. The βCD events are more clearly shown in the inset at higher sampling rates (100 kHz) and expanded time scales. These results were in agreement with previous results for such channel recordings obtained with bilayers formed across TEFLON® (Polytetrafluoroethylene—PTFE) partitions.  FIG. 6B  shows a current voltage plot of αHL single-channel currents in 1M KCl, 10 mM Kpi at pH 7.4. The fit (solid line) is through the points at −40 mV and +40 mV.  FIG. 6C  shows current as a function of time for two aHL channels in artificial bilayer membranes across 100 μm apertures in microfabricated devices at +/−200 mV and at +/−300 mV. 
     EXAMPLE 6 
     Use of the Artificial Synapse for Single Cell Stimulation and Excitation 
     Methods for stimulating cells through the nanoaperture and measuring their activity using fluorescence from Ca 2+  sensitive dyes include the following: (1) voltage clamping the cell to the aperture (applying suction via the microchannel) and varying the voltage of the buffer in the microfluidic channel; (2) chemical stimulation of the cell by pulsing a bolus of neurotransmitter to the under side of the cell; (3) microfluidic bolus of liposomes containing transmitters to the aperture opening; (4) microfluidic reservoir of engineered cells that would stimulate the neurite through the release of transmitters. 
     A subconfluent layer of PC12 cells is cultured on an array of microapertures fabricated. Cell activity is measured by fluorescence microscopy with the cells loaded with Ca 2+  sensitive dyes (such as, e.g., Indo-1, fura −2, fluo- 3, calcium green, aequorin). The fluorescence serves both to monitor the activity of the cell directly above the aperture and to see the effect on neighboring cells. The surface may be modified around the aperture to achieve a good “seal” to the cell membrane (where a good seal is mechanically stable and has an electrical resistance near to or in excess of one gigaΩ). Surface modifiers may include different extracellular matrix proteins and “cell Tak” (Becton Dickinson). Different stimulation techniques suitable for use with the devices and methods of the invention include temporal and spatial resolution and chronic stimulation. The size of the aperture may be varied as well. In addition, the aperture may be coated with a single lipid bilayer with preloaded ion channels or artificial pore-forming molecules, including proteins that can form pores. These lipid bilayer membranes can be formed by as described in previous Examples. The ion channel or pore-forming molecules may be are already part of the membrane if they were part of the material used to form the membrane, or are then incorporated into the bilayer. 
     A microstamp, such as a PDMS stamp, is used to make a micropattern to overlay onto an array of microfabricated apertures. The micropattern is effective to direct the growth of cells cultured on the ASC substrate so that neurites of the cells grow to, adjacent to, or over ASC apertures. Any suitable alignment system may be used to align the microstamp pattern with the apertures on the chip. PC12 cells, retinal ganglion cells, or other cells grown on the substrate may be stimulated as described above on the array of microapertures connected the various microfluidics reservoirs. 
     Cells growing on ASC substrates are stimulated by voltage pulses from electrodes in contact with the solution in the recess and in the reservoir. The voltage pulses are effective to depolarize the cell process adjacent or across the aperture. Depolarization voltages range from about 1 mV to about 100 mV. Depolarizations of between about 10 mV to about 50 mV are found to be the most effective. 
     Liposomes containing the neurotransmitter acetylcholine and adenosine-tris-phosphate are placed in the reservoir. A lipid bilayer membrane spans the aperture. Cells with processes growing across or adjacent to the aperture are stimulated by contact with neurotransmitter released by liposomes fusing with the lipid bilayer membrane. Fusion is promoted by an osmotic gradient across the liposome membrane and across the lipid bilayer membrane. Fusion is also promoted by electrical gradients, optical methods, inclusion of fusion-promoting molecules in the liposomes and or membranes, and in other ways. 
     Neuronal excitation is measured using fluorescence with Ca 2+  sensitive dyes, electrical recording, and biochemical analysis to detect neurotransmitter release from the cultured cells into solutions in the recess or reservoir adjacent the aperture. 
     EXAMPLE 7 
     Stimulation of Cells on an Artificial Synapse Chip 
     A device for localized fluid delivery  84  consists of two components, one for localization and one for fluid manipulation. Devices as illustrated in  FIGS. 1A–1D , with or without a substrate  12  or base layer  20 , combined with the device of  FIG. 7A , as shown in  FIG. 7B , are configured for localized fluid delivery.  FIG. 7A  illustrates a fluidic channel portion  76  embodying features of the invention, configured to provide fluid flow to and from a reservoir  36  and aperture  24  of an ASC  10 . A device for localized fluid delivery  84  is illustrated in  FIG. 7B .  FIG. 7B  illustrates the bonding process between an ASC  10  and a device for fluid manipulation  76  to provide a device for localized fluid delivery  84 . For localization, the devices use small apertures  24  (5 or 10 μm) in thin silicon nitride membranes  16  (e.g.,  FIG. 7B ). By providing an aperture  24  of small enough size, fluid delivery may be limited in both volume and location. The devices  84  used in these experiments were 1 cm 2  chips, with a thickness of roughly 0.5 mm. The silicon nitride layer  16  was patterned using plasma etching to create the aperture  24  and a square hole (a reservoir  36 ) in the backside of the wafer. The silicon was etched anisotropically along the (111) plane at an angle of 54.7° to the wafer surface, using the silicon nitride as an etch mask. The square hole  36  in the backside of the wafer was chosen to yield a region 100 μm larger than the aperture  24 . This left a thin silicon nitride membrane  16  freely spanning the region without any silicon support. Silicon nitride is transparent to the visible wavelengths of light, so cells were easily imaged through this membrane. Because of the high tensile strength of silicon nitride, this silicon nitride membrane was quite strong and stable; it readily withstood the forces generated during processing. 
     After localization, the other necessary component for a device for localized fluid delivery  84  is fluid delivery to the aperture. To accomplish this, a channel  78  made from PDMS ( FIG. 7A ), in fluid continuity with an inlet  80  and an outlet  82 , was attached beneath the aperture  24  of an ASC  10  ( FIG. 7B ). A master mold was made from 300 μm thick SU-8 photoresist on a silicon wafer using conventional photolithography and a mask made on a transparency using an office printer. The channels  78  were 900 μm wide, 150 μm deep, and 8 mm long, while the PDMS was poured approximately 5 mm deep. A cartoon depicting this design is shown in  FIG. 7A . 
     Once the PDMS cured, the channels  78  were attached to the ASC  10  as illustrated in  FIG. 7B . The PDMS was diced into 1 cm 2  pieces, with one channel per device. Both the silicon and the PDMS were cleaned in a dilute hydrochloric acid solution (1:4), followed by air plasma at 100 W for 60 seconds. The ACS  10  with its silicon aperture  24  was aligned and centered on top of the PDMS channel  78 , and bonded by squeezing the pieces together (˜0.2 N) and heating on a hot plate at 80° C. (see  FIG. 7B ). Once complete, this bond was irreversible; the PDMS will tear before separating from the silicon nitride. 
     Since rat pheochromocytoma cells (PC12) do not readily adhere to most substrates, including silicon/silicon nitride, it was therefore necessary to treat the devices  84  to modify its surface before seeding with cells. The devices  84  were first immersed in poly(D-lysine) at 50 μg/ml for 30 minutes at room temperature. The poly(D-lysine) provides a sticking layer for an application of mouse laminin, to which the PC12 cells adhered and spread. After rinsing the devices  84  in phosphate-buffered saline (PBS), the laminin was applied at 5 μg/ml in PBS for 8 hours in an incubator (37° C., 6.5% CO 2 ). The devices  84  were then rinsed in PBS and were ready for use. 
     Measurement of bradykinin stimulation was accomplished by observing changes in intracellular Ca 2+  levels using fluo-4 (Molecular Probes, Eugene, Oreg.). The loading solution was made from fluo-4 reconstituted in dimethylsulfoxide (DMSO) at 1 mM mixed in Ringer&#39;s solution (135 mM NaCl, 5 mM KCl, 10 mM D-glucose, 2 mM MgCl 2 , 2 mM CaCl 2 , 10 mM HEPES, pH 7.2) to a final fluo-4 concentration of 1 μM. 
     The stimulating solution was a mixture of bradykinin (Sigma, St. Louis, Mo.), Ringer&#39;s, and sulforhodamine 101 (Sigma). Bradykinin was reconstituted in Ringer&#39;s at 1 mg/ml (1 mM), and then diluted to the desired testing concentration. Sulforhodamine (Texas Red) was reconstituted in DMSO at 8 mM, and added to the stimulating solution to yield a final concentration of 4–8 μM. The Texas Red dye provided a means to visualize simultaneously the fluid flow and stimulation. 
     Changes in fluorescent levels were observed with either an inverted fluorescence microscope or an upright confocal microscope. The inverted microscope, used for the single-cell stimulation data was a Nikon TE300 (10x, 0.30 numerical aperture (NA)) with a Hamamatsu Orca ER CCD camera. The data was collected with Metamorph (Universal Imaging Corporation, Downingtown Pa.). The confocal microscope, used for the multi-cell and two-color experiments, was a Nikon E800 (10 x dipping objective, 0.30 NA) with a Nikon PCM 2000 confocal unit. Two lasers were used simultaneously to excite the fluo-4 (Argon ion, 488 nm) and Texas Red (HeNe, 543 nm). Images were sampled with two photomultiplier tubes simultaneously (515/30 bandpass and 605/32 bandpass filters), and analyzed using SimplePCl (Compix Inc., Cranberry Township, Pa.). 
     The microfluidic system, including devices  84  and associated fluid supply and syringes, supplied a small amount of stimulant through the aperture. The experimental design was to flow bradykinin through the channel  78  and to allow passage of bradykinin through the aperture  24 . While there are multiple methods for moving fluids in microchannels, and causing fluid to flow within a fluid delivery channel, including inducing flow by pumps, gravity, pressure (such as pressure produced by a piston moving within a cylinder), electroosmotic and other means, we chose a pressure-driven flow using a syringe. The bradykinin flow through the aperture  24  was due to a combination of the pressure gradient created by the syringe and chemical diffusion. 
     The fluid was supplied by inserting 24-gauge Teflon hoses into each access hole  80  and  82 . One-milliliter tuberculin syringes were used to drive the fluid through the hoses, at a rate of 10 to 30 μl/s. Injected volumes range from 250 μl through 1000 μl, taking 15 to 60 seconds to deliver. The average flow rate was 16 μl/s; when combined with the channel geometry, this yielded a Reynolds number of approximately 3100, above the limit for laminar flow. Being above the laminar limit is an advantage in this system. There is a 500 μm gap between the channel  78  and the aperture  24  due to the wafer thickness. Nonlaminar flow allows mixing to occur by methods other than diffusion, speeding the rate at which bradykinin reached the aperture. 
     With the fluidic delivery system in place, cell stimulation was accomplished by delivering an appropriate amount of stimulant to the cells growing on the supporting layer  16 . Rat pheochromocytoma cells (PC12) were chosen because of their usefulness as a neurobiological model, and because of the ease of their care and their ready availability. The PC12cell line changes its intracellular Ca 2+  levels upon stimulation by bradykinin, achieving a maximum change at an external bradykinin concentration of 1 μM. The cells were seeded on the devices  84  at least four hours before testing to allow them to adhere. 
     Two parameters of control over stimulation radius were concentration and volume. By adjusting either the concentration or the volume of bradykinin supplied, the distance from the aperture at which cells were stimulated was controlled. When a large total quantity of bradykinin was delivered to the aperture  24  (high concentration or large volume), many PC12 cells were stimulated. This is shown in  FIGS. 8A–8C , where time-lapse confocal micrographs of multi-cell stimulation show a wave of stimulated PC12 cells as bradykinin flows past PC12 cells adherent to the surface of a device  84 . The aperture  24  was 10 μm in diameter (half the size of a PC12 cell body) and is shown located at the center of the dotted circles in  FIGS. 8A–8C . 
     As shown in  FIGS. 8A–8C , bradykinin (100 μM) was driven through the channel  78  for approximately 21 seconds. Intensity cross-sections (arbitrary units, constant scale) indicate which PC12 cells were stimulated.  FIG. 8A  illustrates the control situation before application of bradykinin to the PC12 cells. The intensity plot in  FIG. 8A  shows two cross-sections at time zero, indicating that, at the starting time, no cells were stimulated. A Ringer&#39;s solution containing 100 μM bradykinin was applied to the channel  78  just after the frame displayed in  FIG. 8A  was taken. As the fluid radiated outward from the aperture  24 , PC12 cells were stimulated. Within 3 seconds, a PC12 cell 25 μm from the aperture was stimulated, as the bright cell to the left and below the aperture  24  in  FIG. 8B  shows ( FIG. 8B , arrow). After another 6 seconds, 9 seconds after the bradykinin began to flow, cells further away (100 μm) from the aperture  24  were stimulated ( FIG. 8C , dual arrows). Other PC12 cells in the region were also stimulated; the arrows indicate only representative events for which the intensity is displayed. 
     This example demonstrates the ability to stimulate cells locally using a chemical stimulus, providing a neurobiological system configured to stimulate cells with physiological stimuli and configured for use at desired location within an organ or tissue of animal. By varying the amount and concentration of neurotransmitter supplied through a microaperture, the stimulation distance and timing can be controlled, providing control compatible with normal animal physiological. 
     EXAMPLE 8 
     Implantation of an Artificial Synapse Chip 
     An artificial synapse chip is implanted into the subretinal space in the retina of a rabbit. A New Zealand white rabbit is anesthetized according to standard animal surgery techniques. An incision is made in the sclera near to the equator of the eye and a small scleral flap opened to provide access to the underlying choroid and retina. An incision is gently made in the choroid, choriocappilaris, Bruch&#39;s membrane and across the retinal pigment epithelium layer to provide access to the subretinal space facing the photoreceptors. Saline is gently infused into the subretinal space to separate the retinal pigment epithelium and the retinal photoreceptors. An ASC is placed into the subretinal space and slowly advanced towards the fovea from the point to entry near the equator of the eye. After the ASC is located at the desired location near to the fovea, a needle is inserted through the opening in the sclera, into the vitreous, and a small air bubble is injected into the vitreous to provide pressure against the retina to hold the retina in place over the implant. The incision is then closed. The air bubble shrinks and disappears within a few days as the gas is absorbed. 
     It will be apparent from the foregoing that, while particular forms of the invention have been illustrated and described herein primarily in terms of an artificial synapse chip, a device for localized fluid delivery, and similar devices and systems, various modifications can be made without departing from the spirit and scope of the invention. Moreover, those skilled in the art will recognize that features shown in one embodiment may be utilized in other embodiments. Terms such a “device”, “portion”, “section”, “steps” and words of similar import when used herein shall not be construed as invoking the provisions of 35 U.S.C. §112(6) unless the following claims expressly use the terms “means” or “step” followed by a particular function without specific structure or action. 
     While particular forms of the invention have been illustrated and described, it should be apparent that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, it is not intended that the invention be limited, except as by the appended claims.