Abstract:
Untethered micro or nanoscale probes may be dispersed within tissue to be individually addressed through external electromagnetic radiation to create local electrical currents used for direct stimulation, alteration of cellular potentials, or the release or modification of contained or attached chemical compounds.

Description:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
   This invention was made with United States government support awarded by the following agency: NSF 0520527. The United States has certain rights in this invention. 

   CROSS REFERENCE TO RELATED APPLICATION 
   1. Background of the Invention 
   The present invention relates to a method and apparatus for the manipulation of cellular-level activity and in particular to a small-scale wireless probe for electrical and/or chemical stimulation of living tissue. 
   The study of cellular activity, for example, the operation of neurons, may require localized stimulation of individual or small clusters of cells. This may be done through the use of fine electrodes inserted into or near the cells and connected by leads to external equipment. The cells may be stabilized with the tip of a pipette or by using an on-chip cell measurement system such as is commercially available from Nanion Technologies (www.nanion.de) or using a technology such as Neurochips offered by Infineon (www.infineon.com). 
   Such stabilization systems can be intrusive and may require a path through adjacent tissue to the stimulation site, and/or immobilization of the tissue, which, in some applications, may adversely affect the desired results. Interference between support structure for adjacent electrodes or pipettes can prevent close electrode spacing or placement of the electrodes in multiple dimensions. Such electrode systems can be impractical for long-term placement in living organisms. 
   2. Summary of the Invention 
   The present invention provides freely dispersible micro- or nano-scale probes that may be activated without a direct wired connection. The probes may receive electromagnetic radiation, for example light, and convert that light to a local electric potential and subsequently to an electrical-current flow. The current flow may be used directly for electrical stimulation or to trigger a chemical or mechanical release of chemical compounds held by the probes or activation of a biological system such as ion channels. In one embodiment the probes may be small tubes of strained semiconductor material that overlap upon themselves to form a heterojunction semiconductor device. In another embodiment the probes can be formed by junctions between different semiconducting nanoparticles. 
   Specifically then, the present invention provides a wirelessly stimulatable probe having a dispersible element attachable to tissue structure and substantially less than 100 micrometers in size. The dispersible element accepts electromagnetic radiation to produce an electrical current local to the dispersible element. 
   It is thus one aspect of the invention to provide for the local delivery of electrical current at extremely small scales for use in cellular research or novel therapies providing long-term electrical stimulation of neural cells. 
   The dispersible element may be a strain curved semiconductor membrane. 
   It is thus another aspect of the invention to provide both a material and a topology that provide at least one of: a sufficient surface area for the construction of integrated-circuit type microelectronics, a sufficient surface area or curved-surface area that may provide a carrier for bioactive chemical compounds, a desirable shape for an antenna or optical conduit, and/or an aspect ratio allowing the generation of an electrical dipole for electrical manipulation of the dispersible element in tissue. 
   The strain curved semiconductor membrane may be a tube having an overlap region comprising n and p type doped regions to form a heterojunction device. 
   It is thus one object of the invention to provide a simple method of fabricating a versatile p-n junction. 
   The p-n junction may be a photodiode. 
   It is thus another object of the invention to provide a simple method of fabricating a photodiode for the generation of local electrical currents. 
   The dispersible element may further include adhered chemicals activated by the electrical current. 
   It is another aspect of the invention to provide remotely controlled delivery of chemicals as triggered by the generated electrical current flow. 
   The dispersible elements may further include semiconductor circuitry activated by the electrical current. 
   It is thus another object of the invention to provide for a remote wireless triggering of more complex electrical circuitry contained on the dispersible element. 
   The dispersible element may include surface adhered biologically active chemicals. 
   It is another object of the invention to provide a dispersible element that is chemically compatible with the tissue or chemically targeted to particular tissue structures or types. 
   The invention may be used to produce a cellular scale addressable probe array having a plurality of dispersible elements attachable to spatially separated tissue structure, each dispersible element accepting an electromagnetic radiation to produce an electrical current local to the dispersible element. An electromagnetic radiation source may be steered to produce the electrical current local to a predetermined subset of the dispersible elements. 
   It is another object of the invention to provide for multidimensional stimulation of tissue without the need for connecting wires or interference among the connecting wires or connecting wires and tissue structure. 
   The electromagnetic radiation source is a laser. 
   It is another aspect of the invention to provide for a highly localized electromagnetic stimulation that may stimulate as few as a single dispersible element. 
   These particular objects and advantages may apply to only some embodiments falling within the claims and thus do not define the scope of the invention. 

   
     BRIEF DESCRIPTION OF THE DRAWINGS 
       FIG. 1  is a schematic representation of a nerve cell showing wireless probes of the present invention arrayed along a neural pathway; 
       FIG. 2  is a perspective view of a single wireless probe of  FIG. 1  having a tubular form with an overlapped axial seam; 
       FIG. 3  is a cross-section along line  3 - 3  of  FIG. 2  showing a heterojunction formed at the seam of the tube of  FIG. 2  such as may be used to create a photodiode; 
       FIG. 4  is a schematic representation of the tube of  FIG. 2  showing use of the electrical current generated by the photodiode to activate chemicals attached to or contained in the tube of the probe; 
       FIG. 5  is a simplified diagram of the tube of  FIG. 2  with a surface coating of chemicals used for targeting the probe to a particular structure; 
       FIG. 6  is a simplified diagram of the elements of a scanning laser system that may be used to selectively activate the probes of  FIG. 1 ; 
       FIG. 7  is a figure similar to that of  FIG. 2  showing an alternative configuration of the probe in which the seam is reduced to a point contact; and 
       FIG. 8  is a perspective view of one process for creating the tubes of  FIGS. 2 and 7  using strained semiconductor membranes released from a substrate by etching. 
   

   DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
   Referring now to  FIG. 1 , the direct stimulation of neural tissue  10  for research or therapeutic purposes may require electrical stimulation at various locations along an axon  12  of a neural cell  14 . Such stimulation may be accomplished by attaching a series of floating stimulating probes  16  to the axon  12  at the desired sites. The term floating, as used herein, means untethered by signal leads. 
   The probe  16  may be placed at the desired sites in the tissue by direct mechanical manipulation, for example with forceps or micromanipulators, or steered to the sites using an electrostatic field operating on an electrical dipole formed in the probes  16 . Importantly, however, once the probes  16  are positioned, they do not need electrical or mechanical connection to an external device. 
   As will be described in greater detail below, when the probes  16  are properly positioned, they may be stimulated by electromagnetic radiation  20 , including light or a radiofrequency electromagnetic field, to produce a local current flow. In the example of  FIG. 1 , this local current flow may provide a direct electrical stimulation to the axon  12  to which the probes  16  are attached. Because the probes  16  do not require supporting structure or connecting leads, they may be freely placed in three dimensions within the tissue and may allow free movement or growth of the tissue. 
   Referring to  FIG. 2 , in a preferred embodiment each of the probes  16  is formed of a tube  23  of semiconducting membrane rolled about an axis  22  to overlap along a seam  24  extending parallel to the axis  22 . Referring also to  FIG. 3 , the outer surface of the tube  23  may be doped with an electron donor to create a so-called n-material  21  while the inner surface of the tube may be doped with an electron acceptor to create so-called p-material  25 . The rolling of the semiconducting membrane into the tube  23  creates at the interface of the seam  24  an abutment of p-material  25  and n-material  21  producing a heterojunction p-n device  27 . 
   Referring now to  FIGS. 3 and 4 , as is generally understood in the art, the heterojunction p-n device  27  may be operated as a photodiode  27 ′. Photodiodes  27 ′ are well known devices that may be used either in a photoconductive mode, in which their conductivity changes as a function of received light or, in this embodiment, in a photovoltaic mode, in which they generate a current  26  flowing from an effective or constructed ohmic contact between the p-material  25  and surrounding tissue and an effective or constructed ohmic contact between the surrounding tissue and the n-material  21 . This current flow may be outside of the tube  23  or inside of the tube  23  as controlled by the addition of insulating materials to the inside and/or the outside of the tube  23  as may be desired. 
   In addition, or alternatively, conductive paths and ohmic contacts may be formed at particular locations on the surface of the tube  23 , for example, using standard photolithographic integrated circuit techniques, as will be described. These conductive paths may be used to channel the current flows to particular locations on the tubes  23 , for example axial ends of the tubes  23 , as may be desired. Referring again to  FIG. 2 , the electrical currents  26  as channeled may be used to power other electrical devices  28 , for example FET transistors, formed by integrated circuit techniques on the inner or outer surface of the tube  23  as may be desired. In this case, the effective surface area of the tube  23  provides larger fabrication areas for such integrated circuits such as may not be available in a solid structure of similar outside dimensions. 
   If a piezoelectric material is used together with the semiconductor membrane, then the electricity from generated by the heterojunction may be used to create mechanical motion or movement of the probe  16  to maneuver probe  16 , release chemicals therefrom, or provide mechanical stimulation on an extremely small and localized scale. 
   Referring now to  FIG. 4 , photodiode  27 ′ may alternatively or in addition provide for conductive pathways  30  conveying the current  26  to chemical substances  32  held on the surface of the tube  23  or within the tube  23 . The currents may activate the chemical substances  32  or generate new chemical substances (e.g., by promoting electrochemical reactions) or may release the chemical substances  32 , for example using electrophoresis techniques or by modification of mediating chemical compounds. Alternatively the electrical current can be used to create local heating of the tube  23  causing it to unroll to release its chemical contents. The chemicals conjugated to the probes  16  may include enzymes, antibodies, polysaccharides, and the like. 
   In this way the electromagnetic radiation  20  may be used not only for electrical stimulation but also for the release of chemical compounds  32  on a local basis. Because the probes  16  are wireless, they may be implanted within a cell membrane to interact with internal cellular mechanisms. 
   Referring now to  FIG. 5 , the tube  23  may be further coated with chemical compounds, such as polymers, that improve its biocompatibility, or linkers to proteins, such as epoxide or aldehyde groups for chemical attachment of biomolecules such as DNA, or antibodies, or linkers to sugar components in phospholipid membranes. The compounds can target the probes  16  to particular locations or integrate the probes  16  into particular cellular processes. This functionalization of the surface of the tubes  23  may, for example, use the techniques described in U.S. Pat. No. 6,402,899 by Denes et al. entitled “Process for intercalation of spacer molecules between substrates and active biomolecules” issued Jun. 11, 2002, and hereby incorporated by reference. 
   Referring to  FIG. 6 , the probes  16  of the present invention may be employed with a scanning system  40  for selectively irradiating particular ones of the probes  16  to provide for selective activation of different probes  16 . In the embodiment depicted, cell structure, for example neural tissue  10 , may be exposed to a focused laser beam  42  from a laser source  44 . The laser beam  42  from the laser source  44  is steered to a particular probe  16  or group of probes  16  by a mirror assembly  46  providing for two axes of control, such mirror assemblies being well known in the art. The laser beam  42  may be modulated to be turned on only at the site of a probe  16  where activation is required, for a controllable amount of time. 
   Additional laser beams  42  may be used additively and/or at different angles to attain multiple dimensions of discrimination particularly for probes  16  that may be arrayed in a line from a mirror assembly  46  or that are blocked from one mirror assembly by tissue structure. The scanning system  40  in this way may permit three-dimensional dispersion of the probes  16  within a cell matrix limited only by the ability of the electromagnetic radiation to penetrate through the layers of the cells. 
   While light is considered to be the principal source of electromagnetic radiation for activating the probes  16 , it will be recognized that the tubular structure of the probe  16  also lends itself to the reception of lower-frequency electromagnetic radiation, in which case the tube  23  may form an antenna or resonant structure tuned to a particular frequency range. This tuning allows radiation to be steered to particular probes  16  by frequency multiplexing. Techniques of phased array beam steering may also be used. 
   Referring now to  FIG. 7 , many variations in the structures of the tube  23  may be envisioned, for example, in which the membrane  52  includes a tongue  48  at the seam  24  so that the area of overlap at the seam  24  is limited to the tongue  48 , and a channel  50  is opened along the axis  22  allowing improved or different flow of materials into and out of the center of the tube  23 . The tongue  48  may be used to localize and enhance the voltage drop across the heterojunction and may be placed alternatively at one end of the tube  23  to be proximate to the tissue to which the tube is attached. The outside of the tube  23  may be decorated with quantum dots  51  providing identification and improved location of the probes  16 . 
   Referring to  FIG. 8 , fabrication of the tubes of the probes  16  may be performed by standard integrated circuit techniques in which a semiconductor membrane  52  includes two layers of different materials  54  and  56 , for example Si and SiGe alloy. The different materials create stress at the interface of the material that is resisted by an underlying sacrificial layer  58  attaching the membrane  52  to a substrate  60 . In this planar state, the membrane  52  may be processed in a manner of standard integrated circuit construction including the doping of particular regions and the deposition of conductive pathways. When the sacrificial layer  58  is removed by etching, the natural stress between the materials  54  and  56  causes them to curl about axis  22 . An etched cut  62  allows the membrane  52  to be wholly released when the sacrificial layer  58  is fully etched away providing for the dispersible probe  16  of the present invention. A description of this process and citations to some techniques and applications for this process are described generally in Science, vol. 313, Jul. 14, 2006. The length of the tubes may be in the micro range (e.g., less than 100 micrometers) or nano range (e.g., less than 100 nanometers). 
   Alternatively, heterojunctions may be generated in individual semiconductor nanoparticles by doping the semiconductor to form different n- and p-regions, or by assembling two nanoparticles together, each having a different doping, so that the heterojunction is formed at their mechanical interface. 
   The present invention is not limited solely to electromagnetic stimulation but may also be used in combination with mechanical excitation, for example, ultrasonic stimulation. 
   The electromagnetic stimulation may also be used as part of a sensing process to power sensors in the probes  16  that return faint electrical echoes much in the manner of RFID tags to provide for information about cellular processes. In the case of the laser excitation of the probes  16 , local emission from the probes  16  may be detected such as may reveal information about the state of the probe  16  or its environment. The radiation will be highly directed and/or polarized as a result of the orientation and shape of probes  16 , providing information about the probes  16  and/or distinguishing the local emissions of the probes  16  from other sources. Embedding other materials in the walls of the probes  16 , e.g., quantum dots or fluorescent materials, can provide local radiator elements to enhance these effects. 
   It is specifically intended that the present invention not be limited to the embodiments and illustrations contained herein and the claims should be understood to include modified forms of those embodiments, including portions of the embodiments and combinations of elements of different embodiments as come within the scope of the following claims.