Patent Application
20060021881
The present invention relates in generally to systems and methods for chemical and biological detection, and more specifically to systems and methods of detecting chemical and biological analytes in various phases utilizing nanotubes.
Nanobiosensor
Biosensors are devices, which incorporate a biological element (e.g., enzyme, antibody) to detect chemical, biological and organic species. Biosensors have potential applications including, but not limited to, extreme environments (Dong et al., Electroanalysis, 15, 157, 2003), detection of food and pathogenic bacteria (Ivnitski et al., Electroanalysis, 12, 317, 2000), glucose monitoring (Wang, Electroanalysis 13, 983, 2001) and the food industry.
Conventional sensing electrodes (e.g., gold, platinum, glass, carbon) that have been used to immobilize biological enzymes have shown limited sensitivity and selectivity. In addition, sensor performance has been limited due to possible interfering compounds.
Most electrochemical biosensors operate in the liquid phase. There are instances where the analyte may be in the gaseous form and the electrochemical biosensor would be unable to detect the gaseous species. The performance of the liquid electrolyte sensors are limited by saturation of the analyte in the liquid phase, electrode corrosion and other operation problems including the requirement of continuous stirring to ensure the effective interaction of the analyte with the sensing element.
Aligned carbon nanotubes grown in situ are useful in electrochemical biosensing applications but the difficulties in achieving reproducibility severely limits their use in such applications. High manufacturing costs associated with producing aligned nanotubes further limit their commercialization.
There have been previous reports of electrochemical gas biosensors using ionic conducting films like nafion and tetrabutylammonium toluene-4-sulphonate (TBATS) for the detection of hydrogen peroxide and phenol vapors (Saini et al., Biosensors and Bioelectronics 10, 945, 1995; EP0585113A2), which use specific enzymatic reactions. However, these sensors used an enzyme (horseradish peroxidase) and a mediator (potassium hexacyanoferrate (II)) for sensing hydrogen peroxide with enzyme mediator gels. The “drop and dry” process of the mediator, gel and enzymes did not yield a high sensitive and selective detection. There have been reports about biosensors using a thick film electrochemical device with an insulating substrate for the determination of ethanol vapors using alcohol dehydrogenase enzyme which also involved the “drop and dry” process (EP634488A2).
CNT-TFT
Chemical sensors are devices that detect chemical and biological species based upon an interaction between two molecules. These sensors can be used to detect various analytes in gas, liquid and solid phases. Sensors can be manufactured to operate in ambient or extreme environmental conditions. When optimized, chemical sensors can detect very low levels of a desired analyte, however, the drawback is usually the large amount of support equipment. This equipment usually prevents the sensors from being portable.
Conventional sensors can be made using a wide variety of techniques, each specific to the desired detectable analyte. A suitable technique would focus on the interactions between two molecules that result in signal generation. The signal produced could be light emission, electron transfer or other physical change. Every sensor needs a method of transduction, i.e., converting the chemical event to a measurable output signal.
Current methods of detection have limited selectivity when operating at the limits of detection. At these extremes of performance, separating a signal from the surrounding noise becomes extremely difficult. One method for increasing this signal to noise ratio is to have internal amplification of the desired signal. Internal amplification prevents additional noise being introduced into a detection system through outside electronics. A simple way to achieve amplification is to build the detector using transistor architecture. This architecture can take advantage of the inherent gain associated with a semiconducting material.
Carbon nanotube (CNT) transistors have been known for several years. (Tan et al., Nature, 1998 (393) 49, Martel et al., Appl. Phys. Lett. 1998 (73) 2447). Many examples of these devices rely on a single CNT placed between electrodes. These devices are difficult to prepare, requiring tedious placement of electrodes with respect to CNT position. These are advanced techniques that require highly specialized instrumentation including electron microscopes and electron beam writing. The use of this instrumentation required for characterization and fabrication prevents this from being a manufacturable technique.
An embodiment of the invention is a sensor for detecting an analyte comprising: a carbon nanotube; a polymer immobilized to the carbon nanotube; and a sensing element for detecting the analyte, wherein the sensing element is immobilized to the carbon nanotube.
Another embodiment of the invention is a sensor for detecting an analyte comprising: a carbon nanotube; a polymer immobilized to the carbon nanotube; and a sensing element for detecting the analyte, wherein the sensing element is immobilized to the polymer.
Another embodiment of the invention is a sensor comprising a sensing element for detecting an analyte embedded in a polymer matrix interdispersed with carbon nanotubes.
Another embodiment of the invention is a sensor comprising a sensing element for detecting an analyte, wherein the sensor is coupled to a carbon nanotube.
Yet another embodiment of the inventions is a method of detecting an analyte comprising the steps of: immobilizing a polymer and sensing element to a carbon nanotube; and utilizing a transduction mechanism with the sensing element to detect the analyte.
For a more complete understanding of the present invention, and the advantages thereof, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which:
The present invention is directed to systems and methods for detecting chemical, organic and biological analytes.
While most of the terms used herein will be recognizable to those of skill in the art, the following definitions are nevertheless put forth to aid in the understanding of the present invention. It should be understood, however, that when not explicitly defined, terms should be interpreted as adopting a meaning presently accepted by those of skill in the art.
“Analyte,” as defined herein, refers to the substance being measured in an analytical procedure.
“Carbon nanotubes,” as defined herein, refers to sheets of graphite that form tubes.
“Chemical sensor,” as defined herein, refers to devices that detect chemical and biological species based upon an interaction between two molecules.
“Immobilized” as defined herein, refers to a substance being attached, directly or indirectly (e.g., through a polymer), to another substance.
“Multi-walled nanotube,” as defined herein, refers to nanotubes within nanotubes.
“Nanotube,” as defined herein, refers to any tube with nanoscale dimensions.
“Sensing element,” as defined herein, refers to material responsible for the detection of the substance of interest.
“Sensor,” as defined herein, refers to a system or device that measures or detects a substance.
“Single-walled nanotube,” as defined herein, refers to a nanotube that does not contain another nanotube.
“Thin film transistor,” as defined herein, refers to thin films deposited for metallic contacts.
The present invention incorporates a number of advantages over presently known devices, systems or processes. These advantages include:
The inventors have developed nanoscale composites of polymers (e.g., polyaniline, polypyrrole) and carbon nanotubes for electrochemical sensing applications. For example, the conducting polymer acts as the electron mediator while the carbon nanotubes provide enhanced surface area. The inventors have also immobilized an enzyme onto these structures through electrochemical polymerization to mediate a biological reaction specific to a desired analyte. The nanoscale composites of carbon nanotubes and conducting polymers promote enhanced electron transfer for the biochemical reactions. This yields higher sensitivity and selectivity compared to conventional sensing electrodes. The high surface ratio and three dimensional network of these composites tend to lower the oxidation potential of compounds generated as a result of reaction between the analyte and the biological enzymes. This helps to eliminate the interference from other oxidizable species in biochemical reactions. The inventors have also devised a simple method of preparing carbon nanotube electrodes for electrochemical sensing by screen printing and spraying methods which are feasible for device fabrication. This invention provides an easy and effective method towards the commercialization of carbon nanotube biosensors.
An embodiment of the present invention is a biosensor for detecting an analyte comprising a carbon nanotube, a polymer and a biological element attached to the nanotube to form an active sensing element. The polymer and the biological element can also be attached in situ to the nanotube to form the sensing element. The carbon nanotube can be used as an electrode in an electrochemical, TFT (thin film transistor), enzyme FET (field effect transistor) or other transduction mechanisms to detect analytes using a specific chemical or biological reaction schemes
The polymer can be comprised of a conjugated, redox, insulating or conducting polymer or of a similar kind that provides a mechanical support and/or promotes electron transfer, a recognition event between the analyte and the sensing element. The conducting polymer can comprise of polyaniline, polypyrrole, polythiophenes and substituted derivatives of these polymers etc.
The biological element can be an enzyme, antibody, nucleic acid or other biologically active entity. The enzyme may be a member of a group including, but not limited to, Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases, Ligases, Dehydrogenases, Glycosidases, Kinases, Luciferases, Lysing Enzymes, Oxidases, Proteases, and their substituents.
In an embodiment of the invention, the inventors have incorporated a membrane that is permeable to gaseous species into the electrochemical sensing mechanisms in order to sense gaseous compounds. Any gas which can undergo an electrochemical reaction with the sensing electrode can be sensed effectively.
This invention may be used in sensing gases and liquids that can undergo an electrochemical reaction at the sensing electrode. This invention can be used for, but not limited to, the detection of hydrogen peroxide, which undergoes the following reaction in the electrolytic cell.
2H2O2→2H2O+O2+2e−
The electrolytic cell consists of a working electrode (carbon nanotubes) where the oxidation of hydrogen peroxide occurs. The cell also consists of a counter electrode (e.g., platinum wire), which counters the reaction in the working electrode (reduction) and a reference electrode (e.g., standard calomel electrode, SCE). The electrolytic reaction produces electrons which can be read by a current response at a characteristic potential. The characteristic potential represents the oxidation potential of the analyte (e.g., hydrogen peroxide). This current will produce an amperometric oxidation peak in a cyclic voltammogram. When the concentration of the analyte (e.g., hydrogen peroxide) is increased, there is a corresponding increase in the peak current (
In another embodiment, the enzyme glucose oxidase is immobilized into the sensing electrode along with the conducting polymer to sense glucose in the solution based on the following electrochemical reaction.
Glucose is oxidized to form hydrogen peroxide, which can be sensed by the amperometric oxidation peak at a characteristic potential. An main advantage of employing carbon nanotubes in the sensing process is that the oxidation of hydrogen peroxide is lowered to a range of 0.3 V to 0.45 V (depending on the nature of nanotubes). This eliminates interference by compounds such as ascorbic acid, urea, uric acid, acetametaphenone etc. which oxidize around 0.6 V. The conventional electrodes (e.g., gold, platinum) have an oxidation potential of 0.6 V wherein the characteristic peak is influenced by the interfering compounds. The carbon nanotube sensor also provides enhanced sensitivity due to higher surface area in comparison to the conventional electrodes sensing electrodes (e.g., gold, platinum).
The present invention also improves the operation of the sensor in a wide range of scan rates as shown in
The present invention also provides an effective way to determine wide ranges of concentration of gases and liquids electrochemically.
The present invention also provides a sensing mechanism which is not affected by interfering compounds. The response of the hydrogen peroxide sensor is unaffected by the addition of other conducting electrolytes like, potassium phosphate buffer, methanol, isopropyl alcohol, sulfuric acid, hydrochloric acid (
Table 1 shows a comparison of different nanotube electrodes for electrochemical sensing applications. The sensitivity of different nanotube electrodes has been determined. The multi-walled nanotube (MWNT) spray electrode has around three times higher sensitivity as the aligned carbon nanotubes prepared by the chemical vapor deposition method, which are desired for sensing applications. Generally, the multi-walled nanotube electrodes (MWNT) were found to be better candidates for electrochemical sensing in comparison with the single walled nanotubes (SWNT). The firing and activation process removed around 40% nanotubes from the substrate which decreased the sensitivity of the biosensor.
Previous efforts of using carbon nanotube paste electrodes for biosensing did not yield good sensitivity due to the insulating components like mineral oil (Rubianes and Rivas, Electrochemistry Communications, 5, 689, 2003) and prussian blue (Ricci et al., Analytical Letters, 36, 1921, 2003) that were used to bind the nanotubes. The present invention utilizes a conducting matrix for enzyme immobilization. Analytes and enzymes that may be used in the present invention include but are not limited to those listed in Table 2.
The polymers utilized may include, but are not limited to, insulating, conducting, or redox polymers. Insulating polymers are used for mechanical support when immobilizing enzymes. The conjugated and conducting conjugated polymers are used as electron mediators. The redox polymers are used to promote reduction—oxidation reactions between the analyte and the sensing element. The polymers used may include, but are not limited to any one or more of the following; polyaniline, polypyrrole, polythiophenes and substituted derivatives of these polymers.
A multitude of biological elements may be used to detect a wide variety of analytes in addition to those listed in Table 2. Enzymes that may be used include but are not limited to, the classes Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases, Ligases, Dehydrogenases, Glycosidases, Kinases, Luciferases, Lysing Enzymes, Oxidases, Proteases, and their substituents.
Antibodies (e.g. immunoglobulins, recombinant antibodies, monoclonal antibodies) may be immobilized with the conducting polymer on the carbon nanotube to detect a corresponding antigen in the analyte through a specific antigen-antibody interaction.
A DNA hybridization event may be detected by immobizing a single stranded DNA onto the conducting polymer—carbon nanotube system and recording the hybridization event with the analyte.
The signal from the biological/chemical interaction between the analyte and the sensing element can be recorded using electrochemical techniques (amperometric, potentiometric etc.), using a thin film transistor (TFT) or enzyme field effect transistor (FET) technique based on the nature of application.
This invention also provides a simple and effective way to sense gases which can undergo an electrochemical reaction. Some examples include, but are not limited to, the electrochemical sensing using this invention are:
A hydrophobic gas permeable membrane was employed for the detection of gases through this invention. Fluoropolymers such as polytetrafluoroethylene (PTFE), trifluorinatedethylene (TFE) and fluorinated ethylene propylene (FEP) have very low gas absorption properties and are suitable for use in this invention. It is foreseen that any fluoropolymer with a low gas absorption property would be suitable for use in the present invention. The membrane (2) may be encapsulated near the working electrode (3) as shown in
This invention provides a method to detect hydrogen peroxide at lower anodic potentials (0.2 to 0.4 V) which eliminates the interference from other compounds like ascorbic acid, acetametaphenone, urea, uric acid which oxidize at 0.6-0.8 V.
CNT-TFT
In addition to electrochemical techniques, CNT based sensors may also be created utilizing a thin-film transistor technique. (Snow et al., App. Phys. Lett., 2003, 82 (13) 2145). This technique allows CNT films to be applied to a substrate independent of their position. It relies on low resistance tunnel junctions between intersecting CNTs to establish a sub-monolayer conducting film. This film can be patterned and is completely compatible with existing microelectronic processing techniques.
This thin-film transistor technique does not require a completely semiconducting CNT material. It relies on percolation pathways where the semiconducting CNTs in the mixture form the current limiting connections. This is in contrast to a single CNT device. If the single CNT is not semiconducting, the transistor will not work. At present, there are no methods for controlling the electronic structure while producing CNTs. This means that a statistical distribution of metallic and semiconducting CNTs will be present. The device yield for an individual CNT device will be, at a maximum, the percentage of semiconducting CNTs in the sample. At present this is nearly 60%.
The inventors have developed a nanoscale architecture for chemical sensing. This architecture uses carbon nanotube based thin-film transistors to provide amplification of signal for chemical sensors. The chemical sensor may have several forms depending on the desired application and intended results.
The invention is based on a combination of polymer coatings, specific chemical binders, and a carbon nanotube thin film transistor (CNT-TFT) device. Polymers may be used as a conductive pathway and/or a mechanical support for hosting the active chemical sensing element. The polymer may be applied using various techniques, including but not limited to, spin coating, electropolymerization, chemical vapor polymerization or printing techniques (i.e. ink jet printing). The polymer may be electro-polymerized in place. Insulating polymers may be used to support a sensing element (labeled “E” in the drawings), bypassing the need for direct chemical attachment to the TFT surface. These polymers may also be used to define specificity by allowing or excluding diffusion of analyte molecules.
The process of making the CNT-TFTs utilizes liquid deposition processes. These processes allow for manufacturing of large area substrates (>1 m2) and on rigid or flexible substrates, such as glass or plastics.
A gated TFT structure is depicted in
In one embodiment of the CNT-TFT sensor, the sensing element (E) is attached directly to the gate. The CNT film is then deposited onto this surface (
In another embodiment of the sensor, the sensing element (E) and polymer are attached to CNT's already on the surface. This can be a direct attachment or direct host in a polymer. The polymer can be used to enhance the sensitivity of the sensor or facilitate signal transfer to the CNTs. This polymer can be deposited using electrochemical polymerization (
In another example of the sensor, the sensing element (E) and support polymer are attached directly to the CNTs in solution. This can be facilitated through a chemical reduction of the polymer monomer concurrent in solution (
In another embodiment of the present invention, the CNTs are coated with the sensing element (E) or with a polymer with the sensing element (E) embedded in the polymer. The coated CNTs are deposited in a monolayer on a dielectric gate layer.
In yet another embodiment, the carbon nanotubes are coated onto a working electrode of an electrochemical cell (
An embodiment of the present invention in which a CNT is coated with a polymer that contains an example of an embedded sensing element (E) is shown in
The devices described herein are used for chemical or biological sensing. The sensing element (E) is defined by the type of sensor to be created. If E is a biological enzyme, the sensor will be a nanobiosensor. A glucose sensor would be created if glucose oxidase were immobilized. For example, glucose oxidase will react with glucose to produce hydrogen peroxide, H2O2. The peroxide changes the chemical environment of the CNT through electron transfer. The electron transfer will effectively change the carrier concentration in the CNT film, thus gating ON or OFF the CNT-TFT. Devices can be designed with electron transfer into (donating) or out of (withdrawing) the CNT. Thus, the response is able to be tuned when combined with a p- or n-type transistor.
Devices are tested by measuring current, Id, through the device at a given drain voltage, Vd. When a sensing event is detected, the current will change depending on the type of interaction and the nature of the transistor itself. For example, a p-type transistor will show a decrease in current when an electron withdrawing chemical event occurs, reducing the carrier concentration. The magnitude of current change is directly proportional to the number of analytes encountered by the sensor. The advantage of the TFT architecture is the ability to get orders of magnitude in current change from only a few analytes.
The following example is included to demonstrate particular embodiments of the present invention. It should be appreciated by those of skill in the art that the systems and methods disclosed in the example which follows merely represent exemplary embodiments of the present invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments described and still obtain a like or similar result without departing from the spirit and scope of the present invention.
These examples serve to illustrate certain exemplary embodiments of the present invention that comprise:
Instrumentation: Experiments using electro-polymerization were carried out using a three electrode Gamry (v. 4.02) electrochemical system coupled to a Gamry Potentiostat (PCI4/300). The three electrodes included a working, counter and Ag/AgCl reference. The measurements and analysis were performed and recorded using the Gamry Software (v. 1.02).
Materials: Single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs) may be purchased from any source. In the CNT-TFT, the SWNTs used have diameters that range from 1 to 3 nm and lengths that range from less than one micron to greater than 10 μm. SWNTs have been used “as-produced” (AP) or purified with similar results. This disclosure does not specify how the carbon nanotubes were made, although methods that deliver higher concentrations of semiconducting nanotubes may be used, either as produced or as a post-fabrication process procedure.
In the nanobiosensor, the multi-walled nanotubes had diameters 10-30 nm and lengths 1-10 μm. Single-wall carbon nanotubes (SWNT) used have diameters of 1-2 nm and lengths of 5-20 μm. Organic vehicle and glass frit were obtained from Daejoo Electronic Materials Co., Ltd, and thinner was obtained from Dupont Electronics Microcircuits Industries. Aniline, pyrrole, sulfuric acid, sodium perchlorate, glucose oxidase, hydrogen peroxide, and standard glucose solution were obtained from Aldrich.
Carbon nanotube paste electrodes (0.5 cm2) were prepared by mixing 50% by weight of carbon nanotubes with 43% by weight of organic vehicle and 7% by weight of glass frit in a mortar and pestle for 30 minutes followed by grinding in a three roll mill for 20 minutes to disperse the clusters in the mixture. The composite was then screen printed through a mask of known area onto a silicon substrate, vacuum evaporated with 20 angstroms chromium and 500 angstroms gold. The substrate was then baked at 100° C. for 10 minutes in an oven and cooled at room temperature as illustrated in
Carbon nanotube spray electrodes (0.5 cm2) were prepared by dissolving a known quantity of carbon nanotubes (e.g., 0.1 g) in 20 ml isopropyl alcohol, followed by ultrasonication for 5 minutes and spraying the solution onto the substrate (silicon substrate, vacuum evaporated with 20 angstroms chromium and 500 angstroms gold). The spray electrode was then baked at 100° C. for 10 minutes in an oven and cooled at room temperature.
The carbon nanotubes can also be prepared by a chemical vapor deposition process comprising a catalyst (e.g., nickel, copper, cobalt, iron) and a carbon source (e.g., acetylene, ethylene, methane and other hydrocarbons).
Electrochemical polymerization onto the carbon nanotube electrodes (applicable to all the electrodes discussed above) was carried out by the oxidation of aniline (0.1 M) in a solution containing 1 M H2SO4 in a pH 7.0 buffer solution. A potential window of −1.04 V to 0.96 V was employed for the electropolymerization with a scan rate of 50 mV/s for 10 cycles. Electropolymerization to form the polypyrrole was carried out by the oxidation of pyrrole (0.1 M) in a solution containing 0.1 M NaClO4 in a pH 7.0 buffer solution under the same electrochemical conditions. The electrodes were washed with water and dried in air.
A biological enzyme, glucose oxidase (e.g., 5 mg/ml) was mixed with aniline (0.1 M) in a solution containing 1 M H2SO4 in a pH 7.0 buffer solution. A potential window of −1.04 V to 0.96 V was employed for the immobilization of the enzyme in situ with the conducting polymer onto the carbon nanotubes with a scan rate of 50 mV/s for 10 cycles. The electrode was washed with water and dried in air. The electrode is comprised of an enzyme embedded in a polymer matrix which in turn is interdispersed with the carbon nanotubes.
Preparation of CNT-TFTs: Solutions of CNTs were prepared by suspending CNTs in a liquid matrix. This liquid matrix may contain the solvent itself, either aqueous or organic, and/or agents to help suspension and stability, including surfactants and/or polymers. CNTs were suspended in a dilute aqueous solution containing sodium-dodecyl sulfate. Suspension was aided by the use of sonication and then centrifuged to remove non-suspended material as has been reported in Haddon et al., U.S. Pat. No. 6,641,793; Lei Zhang, et al., Chem. Mater. 2004, Vol. 16, pp. 2005-2061; Jiang Zhu et al., Nano Letters, 2003; Vol. 3, No. 8, pp. 1107-1113; Furtado, et al., Journal of the American Chemical Society, 2004, Vol. 126, No. 19, pp. 6095-6105; and PCT/US03/01159.
Gum Arabic and other polymers have also been used in place sodium-dodecyl sulfate as a suspension agent for aqueous solutions. (Nativ-Roth et al., Journal of Polymer Engineering, Vol. 22, No. 5, pp. 353-368, 2002; and Nativ-Roth et al, Nano Letters, Vol. 2, No. 1, pp. 25-28, 2002; and U.S. Patent Publication 20040131859).
Substrates were prepared and CNT films were deposited using similar methods to those published in Lay et al., Nano Lett. 2004 (4) 603 and Novak et al., Solid State Elec, 2004, in press. Surface modifications comprising the polymers and sensing elements were added to the substrates and CNT films.
Electrochemical polymerization of conducting polymers was carried out by the oxidation of the polymer monomer unit. For example, aniline (0.1 M in 1 M H2SO4) can be oxidized to polyanailine (PANI) using a cyclic voltammetry window between −1 and +1 V (vs. Ag/AgCl reference electrode). It is foreseen that other polymers could be used in the present invention. This electrochemical polymerization can be performed with other monomers, conditions and counter ions in solution. Varying the sweep rate and the number of cycles will vary the thickness of the polymer formed. The substrate can be used as the working electrode where the polymerization takes place. Chemical polymerizations can also be performed using the correct reducing agent mixed in solution with the polymer monomer. For example, KFeCN can be used to polymerize pyrrole. Additional agents can be added and co-polymerized with the conducting polymer. Additional agents include but are not limited to specific biological enzymes that can be mixed with the aniline monomer during polymerization. This creates a polymer film with an embedded enzyme. The enzyme does not loose any function or reactivity. This enzyme or the enzyme imbedded in the polymer can be used as a sensing element (E).
All patents and publications referenced herein are hereby incorporated by reference. It will be understood that certain of the above-described structures, functions, and operations of the above-described embodiments are not necessary to practice the present invention and are included in the description simply for completeness of an exemplary embodiment or embodiments. In addition, it will be understood that specific structures, functions, and operations set forth in the above-described referenced patents and publications can be practiced in conjunction with the present invention, but they are not essential to its practice. It is therefore to be understood that the invention may be practiced otherwise than as specifically described without actually departing from the spirit and scope of the present invention as defined by the appended claims.
The present application claims priority to U.S. Provisional Application Ser. No. 60/507,436 filed Sep. 30, 2003 and U.S. Provisional Application Ser. No. 60/598,293 filed Aug. 3, 2004.
| Number | Date | Country | |
|---|---|---|---|
| 60507436 | Sep 2003 | US | |
| 60598293 | Aug 2004 | US |
