WO2014042660A1

WO2014042660A1 - Carbon nanostructure sensor and method for biomolecule sensing

Info

Publication number: WO2014042660A1
Application number: PCT/US2012/060384
Authority: WO
Inventors: Chunhong Li; David J. Ruggieri
Original assignee: Nanoselect Inc
Current assignee: Nanoselect Inc
Priority date: 2012-09-11
Filing date: 2012-10-16
Publication date: 2014-03-20
Anticipated expiration: 2015-03-11
Also published as: WO2014042660A8

Description

CARBON NANOSTRUCTURE SENSOR AND METHOD FOR BIOMOLECULE

SENSING

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is co-pending with and is a continuation-in-part application of International Application Serial No. PCT/US 12/054565 filed on September

11, 2012 which claims the priority benefit of the provisional application entitled "Layer- by-Layer Carbon Nanostructure Surface Functionalization and the Applications of Such Functionalized Nanostructures," Application Serial No. 61/533,310, filed on September

12, 201 1, and claims the priority benefit of the provisional application entitled "A CNT- Based Amperometric pH Sensor," Application Serial No. 61/680,293, filed on August 7, 2012, the entirety of each being incorporated herein by reference. The present

application is co-pending with and related to the non-provisional application entitled, "Layer-by-Layer Carbon Nanostructure Surface Functionalization and Devices,"

International Application Serial No. PCT/US 12/054399, filed on September 10, 2012. The present application is co-pending with and related to the application entitled, "Layer- by-Layer Surface Functionalization of Catalyst-Free Fullerene Nanostructures and the Applications Thereof," International Application Serial No. PCT/US 12/060197, filed on October 15, 2012.

FIELD OF DISCLOSURE

[0002] The disclosed system and method generally relate to a layer-by-layer surface functionalization of carbon nanostructures. More specifically, the disclosed system and method relate to layer-by-layer surface functionalization of carbon

nanostructures and the application of such functionalized carbon nanostructures as sensing elements for measuring, monitoring, analyzing and testing biomolecules in biological fluids. BACKGROUND

[0003] Interest has grown in utilizing carbon nanostmctures including carbon nanotubes (CNT) in sensing applications due to the unique mechanical, electrical and optical properties of CNTs. Pristine CNTs are generally hydrophobic and individual CNTs tend to bundle together due to van der Waals forces. Efforts have been made to covalently graft chemical functions to the surface of CNTs for various applications in attempts to impart new properties to these CNTs. For example, one conventional practice to impart new properties to CNTs is to first oxidize CNT powder in HNO₃ solution or oxygen plasma resulting in -OH and -COOH groups on the CNT surface. Functional groups may then be introduced via amide or ester bond formation. These as- functionalized CNTs may then be used as a powder or in a composite to enhance electrical or mechanical properties in various applications. While a viable approach to introduce -OH and -COOH groups on a CNT surface, an oxidation of the CNT surface may damage the CNT tube structure. Further, some C=C double bonds in the CNT may be altered or broken for any type of subsequent covalent functionalization on the CNT surface thereby leaving pits in the CNT surface structure and modifying both the mechanical and electrical properties of the CNT. Furthermore, such a conventional process does not readily control the degree of functionalization and the density of the introduced functional groups.

[0004] Another conventional practice to impart new properties to CNTs includes non-covalent functionalization of CNTs such as dispersion and solubilization of CNT powders using surfactants and polymers. WO 03/050332 describes a preparation of CNT dispersions in liquid, WO 02/16257 describes a polymer wrapped, single-walled CNT, WO 03/102020 describes a method for obtaining peptides that bind to a CNT and other carbon nanostmctures, WO 02/095099 describes non-covalent sidewall functionalization of CNTs, and WO 07/013872 describes the use of non-covalently functionalized CNTs as a sensing composition. Generally, the non-covalent approach relies upon favorable interactions between adsorbed molecules and CNT sidewalls, namely, van der Waals, π- π, and CH-π interactions. As no covalent bond is formed between the adsorbed molecules and CNT sidewalls, non-covalent functionalization of CNTs using these conventional approaches most likely results in little disturbance to the π system in a CNT and thus minimal alteration to the mechanical, electrical and spectroscopic properties of CNTs. These non-covalent approaches typically perform well in dispersing and solubilizing CNT powders, and such approaches generally include mechanical force processes such as ultrasonication and/or mechanical milling to form such powders.

[0005] An additional conventional approach to impart new properties to harness the superior properties of CNTs is to grow CNTs on a substrate, functionalize these CNTs on the substrate, and then use the resulting carbon nanostmcture on the substrate as an electrode material. For example, a thin film of a metal catalyst such as nickel, cobalt or iron may first be deposited on a silicon substrate with a titanium adhesion or barrier layer. This film may then be annealed at high temperature leading to the formation of small metal particles on the substrate. Feed gases such as acetylene, hydrogen and argon are introduced and contact the surface of each particle of metal catalyst whereby CNTs grow from the particles. The metal catalyst particles may then serve as conducting contacts between the CNTs and the substrate. These grown CNTs, however, are unstable toward strongly oxidizing agents including HNO₃. For example, when these grown CNTs are treated with an HNO₃ solution, CNTs are lost from the substrate as the metal catalyst is oxidized and consumed. Therefore, to maintain the structural integrity of the carbon nanostmcture on a substrate and the electric contact between a CNT and the substrate, the metal catalyst particles must first be protected from strong oxidizing agents and/or the functionalization of CNTs on a respective substrate conducted under mild conditions.

[0006] While conventional non-covalent processes described above may result in the protection and functionalization of carbon nanostmctures, it is unclear what surfactants, polymers or peptides are suitable for various applications. Further, if mechanical force processes such as ultrasonication are required for the non-covalent functionalization, such processes are unsuitable for carbon nanostmctures on a substrate as these stmctures are known to peel off from the substrate upon ultrasonication. Furthermore, when CNTs are dispersed and functionalized with general surfactants and polymers, the control of the deposition thickness of surfactants and/or polymers is difficult. Additionally, most surfactants and polymers used for CNT dispersion and functionalization are non-conducting and introduce an uncontrolled amount of foreign materials (e.g., conducting or non-conducting) to the respective CNT surface which may compromise any superior electrical properties of the CNT. Thus, for sensing

applications, it is important that functionalized carbon nanostructures are free from nonspecific adsorption. For example, serum albumin, an abundant plasma protein in mammal, forms complexes with CNT whereby the binding leads to quenching of the band gap fluorescence of CNT. An uncontrolled thickness of surface deposition of polymers or proteins may effectively block access to or shield the CNT from the environment. Thus, in such instances, the CNT would cease to function as sensing element.

[0007] It is known that exemplary sensing elements such as pH sensors may play an important role in the control and measurement of pH. Such devices may find utility in industries such as, but not limited to, water monitoring, medical diagnostics, agriculture, biology, chemistry, civil engineering, environmental science, food science, forestry, medicine, oceanography, oil production, and other industries. Conventionally, glass electrodes are used for pH measurements as a potentiometric pH sensor; however, potentiometric pH measurements may require the reference electrode to be exceptionally stable and any potential drift from reference electrode may lead to an inconsistent pH measurement. Additionally, the membrane of such glass electrodes may foul easily resulting in a deteriorating performance over time and subsequent cleaning and calibration. Thus, such pH sensors may not be suitable for applications where long-term continuous monitoring of solution pH is required. It follows that due to the fragility of a glass electrode, such a sensor may not be employed in a solution under pressure (e.g., drinking water in pipe).

[0008] Voltammetric pH sensors have also been developed by applying various controlled potential techniques. For example, voltammetric pH sensors conventionally utilize the shift of pH-sensitive peak potential of redox species such as quinone and ferrocene deposited on an electrode surface. One disadvantage in conventional voltammetric pH sensors is the poor long-term stability of redox species. It is also known that electrochemical reactions take place on or near the electrode surface in a

voltammetric pH sensor, and as these reactions consume or generate H⁺ (protons), the local pH near the electrode may be different from the bulk solution pH. Thus, it follows that measured pH may be different from the actual pH in the bulk solution.

[0009] Additionally, while field effect transistor (FET)-based pH sensors have been utilized for pH measurements (e.g., under pressure), such FET pH sensors are also generally unsuitable for continuous long-term monitoring of solution pH. Thus, it is desirable to provide a stable FET-based or other pH sensor using an appropriately functionalized surface to respond to pH-related electric field changes and resist nonspecific deposition of foreign materials on the surfaces (i.e., fouling).

[0010] Generally biosensors are devices that may monitor, detect, identify and/or analyze biological events such as nucleic acid hybridization (e.g., DNA-DNA pairing), protein-protein interaction, antigen-antibody binding, enzyme/substrate interaction and even cell-cell interaction. FET-based biosensors possess several advantages over conventional approaches such as an immunoassay or other biochemical tests. For example, FET biosensors detect biomolecular interactions in a label-free manner through a direct change in electric current or other related electrical property of the device. An immunoassay typically requires plural washing and/or separation steps and may also rely upon the use of analytical agents in association with a detectable label such as a radioactive element and/or fluorescent dye. Additionally, an exemplary FET device may have a small construction and thus, a FET biosensor might be most suitable for use in a portable monitoring system such as a hand-held drug monitoring system.

[0011] Generally, a FET device includes three terminals, source, drain and gate terminals. For a properly functioning FET-based biosensor, the gate terminal surface should be functionalized with probe molecules to interact with target biomolecules in solution. Various probe molecules, such as DNA or RNA strands, DNA or RNA aptamers, proteins, enzymes, haptens, antibodies and cells, may be introduced onto a FET gate surface to construct biosensors such as a DNA FET, an enzyme FET and an Immuno FET. When target biomolecules interact with probe molecules on a FET gate surface, the interaction may elicit changes in gate surface properties such as surface charge

distribution and surface potential subsequently leading to a change in electric current or potential of the device.

[0012] Silicon nanowire and single-walled CNTs (SWCNTs) have been used as sensing elements in FET-based biosensors; however, construction of conventional FET- based biosensors is difficult and laborious due to the fact that the silicon nanowires and SWCNTs must be manipulated to align between the source and drain terminals.

Additionally, for S WCNT-based FET biosensors, sorting and purification of semiconducting CNTs from metallic SWCNTs is also quite laborious and it is difficult to reproducibly establish good electric contact from the source to drain terminals via the semi-conducting nanowires or nanotubes. Further, there exist a limited number and type of functional groups on the nanowire or CNT surface for the covalent attachment of probe molecules. Conventional FET-based biosensors based on conductive diamond utilize the dangling bonds terminated with hydroxyl (-OH), amino (-NH₂) and carboxyl (- COOH). The density of these surface functional groups, however, is relatively low on a flat diamond surface and only a small number of probe molecules can be introduced to the diamond surface. Further, the gate voltage shift is small upon DNA hybridization in such conventional FET-based biosensors.

[0013] Therefore a need exists in the art for a biosensor based on a functionalized multi- walled CNT electrode that may be readily constructed and provide a high sensitivity for biomolecular targets. There also exists a need in the art for a well- controlled surface functionalization of carbon nano structures without altering the superior properties of such carbon nanostructures to provide exemplary nanostructures for biometric and other industry usage. Thus, it is desirable to overcome the limitations of the prior art and provide a carbon nanostructure having functionalized layers and utilize such structures as sensors and the like. SUMMARY

[0014] Embodiments of the present subject matter may protect and functionalize carbon nanostructures using a layer-by-layer approach. For example, various functional groups and functional moieties may be introduced onto the carbon nanostructure surface platform thereby resulting in carbon nanostructures suitable for various applications. Embodiments of the present subject matter may also control the thickness of the functionalization layer thereby resulting in minimal alteration of the intrinsic electrical and optical properties of such carbon nanostructures. Additionally, embodiments of the present subject matter may adjust the density of introduced functional groups and functional moieties and may modulate the degree of surface hydrophilicity of the functionalized carbon nanostructures. Functionalized carbon nanostructures formed according to exemplary embodiments may then be stable and robust in resisting fouling (e.g., mineral deposition and biofouling) when used in aqueous applications. For example, one embodiment of the present subject matter includes a stable CNT

electrochemical sensor which is adaptable to determine free chlorine, bromine, chlorine dioxide and ozone concentrations in flowing tap water.

[0015] Another embodiment of the present subject matter finds applicability as a voltammetric pH sensor when a pH responsive redox mediator moiety is introduced onto a CNT surface. This resulting CNT electrode may then be used in a buffer solution with high ionic strength and/or a non-buffered tap water solution. A further embodiment of the present subject matter provides a functionalized CNT-based potentiometric pH sensor for flowing tap water with low conductivity. Such an exemplary functionalized CNT electrode may also be employed to monitor molecular binding or interaction events on an electrode surface in electrochemical impedance spectroscopy or in a field-effect transistor (FET) device (e.g., ion-selective FET and solution-gate FET). Pristine CNTs may also be dispersed and functionalized using an exemplary layer-by-layer approach for CNT sorting, separation and purification; and, exemplary surface functionalized CNTs according to embodiments of the present subject matter may be utilized as optical sensors by harnessing the unique spectroscopic properties of CNT such as optical absorption, luminescence and Raman scattering.

[0016] One embodiment of the present subject matter provides a layer-by- layer protection and functionalization of a carbon nanostructure by subjecting carbon nanostmctures to a composition comprising an alkyl protective moiety under conditions that permit the formation of an alkyl protective moiety layer adjacent to the carbon nanostructure. Various functional groups and functional moieties may subsequently be introduced to form a second layer above the alkyl protective moiety layer. These introduced functional groups and functional moieties may, in other embodiments, undergo further transformations to incorporate additional layers and/or functionalities to the respective carbon nanostructure surface.

[0017] A further embodiment of the present subject matter provides a method for the protection of carbon nanostmctures. The method may include protecting the surface of such a stmcture, e.g., a carbon and metal catalyst on a substrate, by contacting the nanostmctures with a composition comprising an alkyl protective moiety under conditions that permit the formation of an alkyl protective moiety layer disposed directly adjacent to at least a portion of the metal catalyst, the carbon, or both.

[0018] An additional embodiment of the present subject matter provides a method for subsequent surface functionalization of carbon nanostmctures comprising forming a second layer and/or third layer on nanostmctures that have been protected with an alkyl protective moiety layer. Exemplary functionalization may include, but is not limited to, the introduction of various functional groups such as -OH, -COOH, -NH₂, -NHR, -SH, -S-S-R, -C≡CH, -N₃, -CN, -CHO, -CONH-NH₂, a maleimido group, epoxide, and other functional moieties such as redox mediator stmctures. Of course, these functional groups may be further derivatized to form covalent bonds with other functional moieties including, but not limited to, redox mediator molecules, crown ethers, catalysts, boric acids, carbohydrates, oligonucleotides (DNAs and RNAs), DNA or R A aptamers, peptide aptamers, proteins such as enzymes and antibodies, quantum dotsand

nanoparticles, cells, cell organelles, and other cellular components, either before or after the formation of the second layer on carbon nanostructure surfaces. Thus, such a disclosure should not limit the scope of the claims appended herewith.

[0019] Embodiments of the present subject matter may also control the density of specific functional groups and functional moieties on carbon nanostructure surfaces and may control the degree of hydrophilicity of functionalized carbon nanostructure surfaces. Exemplary methods may be provided to construct a hydrophilic platform on the surface of a CNT and carbon nanostructure. Functional groups and/or moieties such as redox mediator molecules, crown ethers, catalysts, boric acids, carbohydrates, oligonucleotides (DNAs and RNAs), DNA or RNA aptamers, peptide aptamers, proteins such as enzymes and antibodies, quantum dots and nanoparticles, cells, cell organelles, and other cellular components may then be introduced on the hydrophilic surface via covalent bond formation that is free from non-specific adsorption.

[0020] Exemplary devices formed using embodiments of the present subject matter may include, but are not limited to, voltametric pH sensors using a layer-by-layer functionalization of carbon nanostructure surface having, for example, redox mediator molecules that require proton participation for their redox reactions (redox peak potential shift and the solution pH adhere to the Nernst Equation), a surface-functionalized CNT- based potentiometric pH sensor, and an amperometric pH sensor, to name a few.

[0021] One embodiment of the present subject matter may provide an exemplary amperometric pH sensor capable of withstanding solution pressures and suitable for continuous monitoring of solution pH over an extended period of time. Such an exemplary amperometric pH sensor may measure current between two contacts, e.g., source and drain with a sensing electrode (i.e., functionalized carbon nanotube electrode) between the source and drain on the silicon chip. By applying a potential to the sensing electrode, a current may flow through the source and drain whereby the measured current may be proportional to the solution pH. As there are no redox species or acid-base reactive functional groups required on the sensing electrode surface, this type of amperometric pH sensor may offer long-term stability and may be applied for continuous long-term monitoring of solution pH. Furthermore, in an exemplary amperometric pH sensor, the measured species (e.g., H ) is not consumed; thus, there is little or no local pH and bulk solution pH difference in such embodiments.

[0022] Another embodiment of the present subject matter provides a method of functionalizing carbon nanostructures. The method may include providing a carbon nanostructure having a first protective layer on a surface of the structure and forming a functional second layer over the first protective layer, where the second layer comprises a bipolar molecule with functional groups or functional moieties. A CNT nanostructure functionalized with the first layer and second layer may be further treated with reagents to form a third layer (e.g., via cross-linking) followed by subsequent transformations to incorporate additional functional groups or functional moieties.

[0023] One embodiment of the present subject matter provides a carbon

nanostructure having a substrate with one or more carbon nanotubes situated on a surface of the substrate. A first protective layer may cover portions of the substrate, and a functional second layer may be situated over the first protective layer. This second layer may comprise a bipolar molecule with functional groups or functional moieties. With judicious selection of chemistry, the functional groups or functional moieties in the second layer can be utilized to form a third layer (e.g., via cross-linking) followed by subsequent transformations to incorporate additional functional groups or functional moieties.

[0024] A further embodiment of the present subject matter provides a method of controlling the density of a functional groups or functional moieties on a surface of a carbon nanostructure. The method may include providing a carbon nanostructure having a first protective layer on a surface of the structure and forming a functional second layer over the first protective layer, the second layer having a controllable density of functional groups or functional moieties. The density may be controlled by applying bipolar molecules having a predetermined ratio of functional groups or functional moieties.

[0025] An additional embodiment of the present subject matter provides a method of modulating hydrophilicity of a carbon nanostructure. The method may include providing a carbon nanostructure having a first protective layer on a surface of the structure, and forming a hydrophilic second layer over the first protective layer using compounds having one or more -OH groups, -NH₂ groups or -NH- groups.

[0026] One embodiment of the present subject matter provides a method for measuring pH in an environment. The method may include providing a pH sensor, the sensor having a reference electrode and a sensing electrode, the sensing electrode disposed between a first contact and a second contact and applying a potential across the reference and sensing electrodes. Current may then be measured resulting from the applied potential, and pH determined in the environment as a function of the measured current.

[0027] Another embodiment of the present subject matter provides a device for measuring pH in a fluid. The device may include a reference electrode in communication with the fluid and a sensing electrode in communication with the fluid and disposed between a first electrical contact and a second electrical contact. The sensing electrode may include one or more carbon nanostructures functionalized with a chemically stable moiety that responds to solution pH changes when a potential is applied across the first and second electrical contacts to thereby provide a current proportional to solution pH.

[0028] A further embodiment of the present subject matter provides a system for monitoring and controlling pH. The system may include a sensor for measuring pH in a fluid. This sensor may have a reference electrode in communication with the fluid and a sensing electrode in communication with the fluid and disposed between a first electrical contact and a second electrical contact. The sensing electrode may also include one or more carbon nanostructures functionalized with a chemically stable moiety that responds to solution pH changes when a potential is applied across the first and second electrical contacts. The system may further have circuitry for measuring a current resulting from the applied potential and for providing an output signal and a transmitter for transmitting the output signal to a location remote from the sensor.

[0029] One embodiment provides a device for measuring a biological target species in a fluid. The device may include a reference electrode in communication with a fluid and a sensing electrode in communication with the fluid and disposed between a first electrical contact and a second electrical contact. The sensing electrode may have one or more carbon nanostructures fimctionalized with a chemically stable moiety that responds to a biological target species in the fluid when a potential is applied across the first and second electrical contacts to provide a current correlating to a concentration of the target species in the fluid.

[0030] Another embodiment provides a method for measuring a biological target species in a fluid. The method may include providing a sensor, the sensor having a reference electrode and a sensing electrode, the sensing electrode disposed between a first contact and a second contact. The method may also include applying a potential across the reference and sensing electrodes and measuring current resulting from the applied potential. A concentration of a biological target species in the fluid may then be determined as a function of the measured current.

[0031 ] In another embodiment, a system for measuring a concentration of a biological target species in a fluid is provided. The system may include a sensor for measuring a concentration of a biological target species in a fluid. The sensor may include a reference electrode in communication with the fluid and a sensing electrode in communication with the fluid and disposed between a first electrical contact and a second electrical contact. The sensing electrode may include one or more carbon nanostructures functionalized with a chemically stable moiety that responds to a biological target species in the fluid when a potential is applied across the first and second electrical contacts. The system may further include circuitry for measuring a current resulting from the applied potential and for providing an output signal, the measured current correlating to the concentration of the biological target species in the fluid. The system may also include a transmitter for transmitting the output signal to a location remote from the sensor.

[0032] These embodiments and many other objects and advantages thereof will be readily apparent to one skilled in the art to which the invention pertains from a perusal of the claims, the appended drawings, and the following detailed description of the embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is a schematic illustration of an exemplary layer-by-layer approach to provide surface functionalization to a CNT and carbon nanostmcture.

[0034] Figure 2 is an illustration of a general structure for a molecule with an attached anthraquinone functional moiety for CNT surface functionalization.

[0035] Figure 3 is an illustration of a hydrophilic CNT nanostmcture surface with controllable density of anthraquinone moieties.

[0036] Figure 4 is a graphical depiction of a square wave voltammogram overlay of CNT nanostmctures functionalized with different ratios of polyoxyethylene alkyl ether anthraquinone 2-carboxylic acid conjugate and C12EG30 for the formation of a second layer, demonstrating the control of functional moiety (anthraquinone) density on functionalized CNT nanostmcture surface.

[0037] Figure 5 is a schematic illustration of controlling the number of -OH groups in a bipolar molecule used for the formation of a second layer on a functionalized CNT surface.

[0038] Figure 6 is a schematic illustration of depositing a polyoxyethylene dialkyl ether on a CNT surface to form a second layer on a functionalized CNT surface.

[0039] Figure 7 is an illustration of an exemplary stmcture of a hydrophilic CNT nanostmcture surface and a covalent functionalization of surface -OH groups with an activated anthraquinone ester.

[0040] Figure 8 is a graphical depiction of a square wave voltammogram overlay of differently functionalized CNT nanostmcture electrodes.

[0041] Figure 9 is a schematic illustration of an exemplary layer-by-layer introduction of various functional groups onto a CNT nanostmcture surface.

[0042] Figure 10 is a graphical depiction of a square wave voltammogram overlay for various embodiments of the present subject matter.

[0043] Figure 11 is a graphical depiction of a square wave voltammogram overlay of a CNT nanostmcture electrode functionalized with anthraquinone in buffer solutions at various pHs. [0044] Figure 12 is a plot of anthraquinone square wave voltammogram redox peak potential versus buffer solution pH for a CNT nanostmcture electrode

functionalized via an embodiment of the present subject matter.

[0045] Figure 13 is a graphical depiction of an open circuit potential of a CNT nanostmcture electrode functionalized using an embodiment of the present subject matter.

[0046] Figure 14 is a plot of open circuit potential versus pH for flowing tap water using an embodiment of the present subject matter.

[0047] Figure 15 is a schematic illustration of a CNT electrode with a cross-linked hydrophilic surface layer.

[0048] Figure 16 is a schematic illustration of constmcting an orderly hydrophilic layer over a cross-linked hydrophilic layer.

[0049] Figure 17 is a schematic illustration of constmcting a functionalized CNT stmcture on conventional ISFET gate oxide as pH sensor.

[0050] Figure 18 is a schematic illustration of functionalizing conventional ISFET gate oxides as a pH sensor.

[0051] Figure 19 is a simplified diagram of a pH sensing device or an exemplary amperometric biosensor.

[0052] Figure 20 is a top view of the sensing electrode depicted in Figure 19.

[0053] Figure 21 is a graphical depiction of a current versus time for a carbon nanostmcture sensing electrode functionalized using an embodiment of the present subject matter.

[0054] Figure 22 is a plot of current versus pH for an exemplary pH sensor.

[0055] Figure 23 is a schematic illustration of an exemplary biosensor according to another embodiment.

DETAILED DESCRIPTION

[0056] With reference to the figures, where like elements have been given like numerical designations to facilitate an understanding of the present subject matter, the various embodiments of a carbon nanostructure sensor and method for biomolecule sensing are described.

[0057] While carbon nanotubes (CNTs) are generally regarded as a superior electrode material, grown CNTs are typically hydrophobic. While reference may be made to specific CNTs herein, the claims appended herewith should not be so limited as it is envisioned that embodiments of the present subject matter are applicable to any type CNT such as, but not limited to, single-walled CNTs (SWCNT), multi-walled CNTs (MWCNT), conductive, semi-conductive, or insulated CNTs, and chiral, achiral, open headed, capped, budded, coated, uncoated, functionalized, anchored, or unanchored CNTs, and the like.

[0058] Hydrophobicity may thus make such CNTs unsuitable for aqueous applications, especially in aqueous solutions with low ionic concentrations.

Embodiments of the present subject matter, however, may chemically modify a CNT surface to impart a certain degree of hydrophilicity. Additionally, the use of CNTs as an electrode material is challenging as good contact between the CNT and a conductive surface or electric lead structure must be established. In this regard, CNTs have been used in a composite format with carbon powder on glassy carbon as an electrode material; however, such a mixture of CNT with carbon powder and a composite binder may result in uncertain electrical properties for the CNT.

[0059] Embodiments of the present subject matter may grow CNTs on a substrate with an established electric contact. For example, a metal catalyst such as, but not limited to, nickel on top of a titanium adhesion/barrier layer may be deposited on a silicon substrate and annealed at a high temperature to form small catalyst particles. Of course, any type of metal catalyst may be employed in embodiments of the present subject matter and the claims appended herewith should not be limited to the example above. Using chemical vapor deposition techniques, CNTs may grow from the catalyst particles and establish electric contact between the grown CNT and substrate. In copending International Application No. PCT/US2010/056350, entitled, "Protection and Surface Modifications of Carbon Nanostructures," having an international filing date of November 11, 2010, the entirety of which is incorporated herein by reference, the use of an alkyl protective moiety forming an alkyl protective moiety layer to protect the metal catalyst particles (i.e., the electric contact between CNT and substrate) is described. This application generally describes a carbon nanostructure employed as an electrode for the determination of free chlorine and total chlorine concentrations in water.

[0060] Figure 1 is a schematic illustration of an exemplary layer-by-layer approach to provide surface functionalization to a CNT and carbon nanostructure. With reference to Figure 1, to exploit the superior electrical and optical properties of CNTs,

embodiments of the present subject matter may covalently attach additional functional groups or functional moieties such as redox mediators and enzyme molecules on top of an exemplary protective layer for the detection of other analytes of interest using a layer- by-layer approach. In step one, grown CNTs 10 on a substrate 12 may be contacted with a composition comprising an alkyl protective moiety under conditions that permit the formation of an alkyl protective layer 15 disposed directly adjacent to at least a portion of the metal catalyst 14 and/or the carbon nanotubes 16. An exemplary alkyl protective moiety may include, but is not limited to, a compound such as an alkane. Non-limiting examples of alkanes include n-octadecane, n-dodecane, eicosane and hexatriacontane. Of course, these examples of alkanes should not limit the scope of the claims appended herewith. The alkyl protective layer 15 may have a thickness in the range of, for example, from about 1 nm to about 500 nm, about 10 nm to about 300 nm, about 50 nm to about 250 nm, or about 50 nm to about 100 nm. At this stage, the CNT surface having the first protective layer 15 may be hydrophobic.

[0061 ] One non-limiting method for the deposition of the first hydrophobic protective layer on an exemplary CNT nanostructure on a substrate may include depositing a solution comprising n-octadecane (10 mM in tetrahydrofuran (THF), 2 x 5μΙ ) onto CNTs on a silicon substrate using standard procedures. Upon drying the solvent in air, the treated sample may be placed in a small vial (I.D. ~2.5 cm) and the vial purged with an Argon stream for 30 seconds and then securely capped. This capped vial may be heated at 120°C for 16-24 h, and the sample then cooled to ambient temperature in the capped vial. The sample may then be removed from the vial with forceps and rinsed with THF before drying in air. At this stage, the CNT may be highly hydrophobic with the alkyl protective layer (first layer) in place. Of course, this exemplary method should not limit the scope of the claims appended herewith and is presented simply for representative purposes only.

[0062] In step two, other functional groups 17 and functional moieties may then be introduced above this first protective, hydrophobic layer 15, leading to the formation of a second layer 18. One non-limiting method for the second layer functionalization of a CNT nanostructure on a substrate may include providing a CNT nanostructure on a silicon substrate with the first alkyl protective layer in place followed by depositing a solution of bipolar molecules or a mixture of bipolar molecules with desired functional groups or functional moieties onto the first layer (e.g., 10 mM in THF, 2 x 5μΙ ). Upon drying the solvent in air, the treated sample may be placed in a small vial (I.D. ~2.5 cm) and the vial purged with an Argon stream for 30 seconds and then securely capped. This capped vial may be heated at 80~120°C for 16-24 h, and the sample cooled to ambient temperature in the capped vial. The sample may then be removed from the vial with forceps and rinsed with a solvent to remove excess deposition before drying in air.

Again, this exemplary method should not limit the scope of the claims appended herewith and is presented for representative purposes only. With the second layer in place, the CNT nanostructure on the substrate may be used as an electrode if no additional functional groups derivatization is required.

[0063] In one embodiment, it may be advantageous to use a bipolar molecule (or a mixture of bipolar molecules) where favorable hydrophobic-hydrophobic interaction assists the anchoring of the bipolar molecule onto the first layer 15 with the polar groups exposed for additional manipulation if necessary. An exemplary bipolar molecule may be represented by a compound having the general formula (I):

(I)

in which:

i represents hydrogen or a Ci_-50 straight or branched alkyl or alkenyl, which is optionally substituted with one or more halogen atoms;

R₂ represents a single bond, an aromatic or alicyclic group, -(OCH₂CH₂)_m-, - (OCH₂CH₂CH₂)_m- or -[OCH₂CH(CH₃)]_m- where m and n are each independently 0 to 500;

X represents hydrogen, halogen, maleimido group, epoxide, -C≡CH, -N₃, -CN, - OH, -OSO₃ ^", -OR, -SH, -SR, -S-S-R, -SO₃H, -SO₃R, -SO₃\ -PO₃H₂, -PO₃H^~, - (PO₃)^2~, -P(=O)(-OR)(OR"), -OPO₃H₂, -OPO₃H^", -O(PO₃)^2", -CHO, -COR, -COOH, - COO^", -COOR, -CONRR", -CONHNH₂, -NH₂, -NRR", -N(COR')R", -N⁺RR"R^m, - N⁺C₅H₅, - (OCH₂CH₂)_m-OR, - (OCH₂CH₂CH₂)_m-OR, - [OCH₂CH(CH₃)]_m-OR, a polyol, a monosaccharide, a disaccharide or a polyethylene oxide derivative thereof;

R may be R_b R_!(CH₂)_nR₂ or -(CH₂)_nR₂X;

R', R", R'" may each be independently hydrogen, alkyl, cycloalkyl, alkyl and cycloalkyl substituted by one or more hydroxyl groups, alkyl and cycloalkyl substituted by one or more carboxylic groups, - (CH₂CH₂₀)_nR, - (CH₂CH₂CH₂O)_nR, or - [CH₂CH(CH₃)O]_nR;

p, q may each be independently an integral number between 0 and 10;

r, s may each be an integral number between 1 and 4, and 1< r+s <= 4; and

V represents a single bond, C, CH, CH₂, Si, N, NH, P, (P=O) or O.

[0064] Any polyol may be selected for use as the X substituent in a compound of the formula (I) above. Polyols are compounds having multiple hydroxyl functional groups and may be, for example, diols, triols, tetrols, pentols, and the like. Non-limiting examples of polyols also include polyethylene glycol, pentaerythritol, ethylene glycol, glycerin pentaerythrityl, polyglycerol, sorbitan, polyethylene oxide derivatives of sorbitan, sugar alcohols, trimethylolethane, and trimethylolpropane, among others.

[0065] In another embodiment, the bipolar molecule may be represented by a compound having the eneral formula (II) with two sub-units connected by a linker:

in which:

X represents hydrogen, halogen, maleimido group, epoxide, -C≡CH, -N₃, -CN, - OH, -OSO₃ ^", -OR, -SH, -SR, -S-S-R, -SO₃H, -SO₃R, -S0₃ ^", -PO₃H₂, -PO₃H^~, - (PO₃)^2~, -P(=O)(-OR)(OR"), -OPO₃H₂, -OPO₃H^", -O(PO₃)^2", -CHO, -COR, -COOH, - COO^", -COOR, -CONRR", -CONHNH₂, -NH₂, -NRR", -N(COR')R", -N⁺RR"R^m, - N⁺C₅H₅, - (OCH₂CH₂)_m-OR, - (OCH₂CH₂CH₂)_m-OR, - [OCH₂CH(CH₃)]_m-OR, a polyol, a monosaccharide, a disaccharide or a polyethylene oxide derivative thereof;

R may be R_b R^CH^R, or -(CH₂)_nR₂X;

R, R", R'" may each be independently hydrogen, alkyl, cycloalkyl, alkyl and cycloalkyl substituted by one or more hydroxyl groups, alkyl and cycloalkyl substituted by one or more carboxylic groups, - (CH₂CH₂₀)_nR, - (CH₂CH₂CH₂O)_nR, or - [CH₂CH(CH₃)O]_nR; , q may each be independently an integral number between 0 and 10; t, v may each be an integral number between 1 and 3,u and w may each be an integral number between 0 and 2, and 1 <= t+u <= 3, and 1 <= v+w <= 3;

Wi, W₂ may be independently C, CH, CH₂, Si, N, NH, P, (P=O) or O;

Y represents a single bond or a divalent linker that comprises: Ci_-50 alkyl, alkenyl or aromatic group which is optionally substituted with one or more X; -(OCH₂CH₂)_m-, - (OCH₂CH₂CH₂)_m-, or - [OCH₂CH(CH₃)]_m- where m and n may each be independently 0 to 500.

[0066] Any polyol may be selected for use as the X substituent in a compound of the formula (II) above. Exemplary polyols may be, but are not limited to, diols, triols, tetrols, pentols, polyethylene glycol, pentaerythritol, ethylene glycol, glycerin

pentaerythrityl, polyglycerol, sorbitan, polyethylene oxide derivatives of sorbitan, sugar alcohols, trimethylolethane, and trimethylolpropane, and the like.

[0067] In a further embodiment, the bipolar molecule may be a compound similar to the compound represented by formula (II) above but may include more than two sub- units connected with multiple linker groups. For example, a bipolar molecule having three sub-units connected with two linker groups in a linear manner may be utilized. It should be appreciated by those skilled in the art that a bipolar molecule with three or more sub-units may be connected with three or more linker groups to form a macro-ring structure as well and such examples should not limit the scope of the claims appended herewith.

[0068] Through judicious selection of an exemplary chemical structure of the bipolar molecule, embodiments may introduce an array of functional groups onto a CNT surface above the hydrophobic alkyl protective layer 15. For the CNT nanostructure to be a useful electrode material with long-term stability in aqueous applications, the functionalized CNT surface should, however, be resistant to non-specific adsorption. Additionally, for many surface electrochemical reactions that require participation of H⁺, OH^" or H₂O, the functionalized CNT surface should also be highly hydrophilic. [0069] Polyethylene glycol may generally resist non-specific adsorption when deposited on a surface and may render a respective surface hydrophilic to a certain degree. Polyoxyethylene alkyl ethers may also be suitable to be deposited above the hydrophobic alkyl protective layer or first layer 15 on an exemplary CNT to form a hydrophilic polyethylene glycol layer or second layer 18. For example, polyoxyethylene alkyl ethers possess the general formula:

R₃-(OCH₂CH₂)_m-OH (III)

in which:

R₃ represents an optionally substituted, linear or branched, saturated or

unsaturated, carbo- or heteroalkyl chain bearing 4 to 50 carbon atoms; and

m represents an integer of 1 to 500, and preferably 4 to 200.

[0070] Exemplary polyoxyethylene alkyl ethers include, but are not limited to, tetraethyleneglycol monooctyl ether (designated as C8EG4), hexaethyleneglycol monododecyl ether (C12EG6), heptaethyleneglycol monohexadecyl ether (C16EG7) and commercially available detergents, identified by the trade names Brij®30 (C12EG4), Brij®52 (C16EG2), or Brij®56 (C16EG10), Brij®58 (C16EG20), Brij®35 (C 12EG30), Brij®78 (C18EG20), Brij®S 100 (C18EG100), Brij®S 200 (C 18EG200) (Croda

International PLC, East Yorkshire, England).

[0071] Embodiments of the present subject matter may employ a myriad of processes to synthesize exemplary bipolar molecules having various functional groups and functional moieties. It should be noted, however, that the subsequent processes detailed below are exemplary only and should not limit the scope of the claims appended herewith. For example, a first process may be used to synthesize N-( 6-hydroxy-n-hexyl)7-decylbenzamide represented by the general formula:

[0072] The process may include adding N, N'-dicyclohexylcarbodiimide (DCC,

2.471 g, 1 1.98 mmol) to a CH₂C1₂ solution (30 mL) ofp-decylbenzoic acid (3.143 g, 1 1.98 mmol), N-hydroxysuccinimide (NHS, 1.406 g, 12.218 mmol) and triethylamime (Et₃N, 1.67 mL, 1 1.98 mmol) resulting in a white slurry. After approximately 16 hours at room temperature, the slurry may be filtered using a Buchner filter funnel and rinsed with additional CH₂C1₂ (30 mL). The filtrate ( -decylbenzoic acid NHS ester) may then be combined and used for subsequent reaction without further purification. A portion of the this filtrate (0.71 1 mmol) may be mixed with 6-aminohexan- 1 -ol (0.1755 g, 1.5 mmol) upon stirring. After approximately 2 hours, the reaction mixture may be loaded onto a SiO₂ column and eluted with 5% MeOH in CH₂C1₂. The fractions containing the desired product may be combined and concentrated to yield a white solid (0.239 g, 93%).

[0073] A second process may be used to synthesize N, N'-[2,2'-(ethylenedioxy) bis(ethyl)] di(/?-decylbenzamide) represented by the general formula:

(0.104 mL, 0.71 1 mmol) to a CH₂C1₂ solution (8mL) of p-decylbenzoic acid NHS ester (1.422 mmol) and Et₃N (0.198 mL, 1.422 mmol). The mixture may be stirred at room temperature for approximately 16 hours before loading onto an SiO₂ column and eluted with 3% MeOH in CH₂C1₂ and then 5% MeOH in CH₂C1₂. The fractions containing the desired product may be combined and concentrated to yield a white solid (0.326 g, 72%) [0075] A third process may be used to synthesize N-(l l-hydroxy-3,6,9- trioxaundecyl) /?-decylbenzamide represented by the general formula:

[0076] The process may include adding 1 l-amino-3,6,9-trioxaundecan-l-ol (0.2 g, 1.0 mmol) to a CH₂C1₂ solution (7 mL) ofp-decylbenzoic acid NHS ester (1.0 mmol) and Et₃N (0.14 mL, 1.0 mmol). The mixture may be stirred at room temperature for approximately 1 hour before loading onto an SiO₂ column and eluted with 3% MeOH in CH₂C1₂. The fractions containing the desired product may be combined and concentrated to yield a solid with a low melting point (0.31 g, 71%).

[0077] A fourth process may be used to synthesize N-(l 1 -hydro xy-3, 6,9- trioxaundecyl) octadecanamide represented by the general formula:

[0078] The process may include adding DCC (2.512 g, 12.173 mmol) to a CH₂C1₂ solution (30 mL) of stearic acid (3.463 g, 12.173 mmol), NHS (1.429 g, 12.416 mmol) and Et₃N (1.7 mL, 12.173 mmol) resulting in an opaque solution, which may slowly turn into a white slurry. After approximately 16 hours at room temperature, a white solid may be filtered using a Buchner filter funnel and rinsed with additional CH₂C1₂ (30 mL) whereby the filtrate (stearic acid NHS ester) may be combined and used for subsequent reaction without further purification. A portion of the filtrate (2.815 mmol) may be mixed with 1 l-amino-3,6,9-trioxaundecan-l-ol (0.483 g, 2.5 mmol) and Et₃N (0.39 mL, 2.82 mmol) upon stirring. The mixture may then be concentrated to ~5 mL and then loaded onto an SiO₂ column and eluted with 3% MeOH in CH₂C1₂. The fractions containing the desired product may be combined and concentrated to yield a white solid (0.772 g, 72%).

[0079] A fifth process may be used to synthesize C16EG10CH₃ represented by the general formula:

[0080] The process may include dissolving waxy solid Brij®56 (a mixture designated as C16EG10) (1.808 g, 2.65 mmol) in anhydrous DMF (6 mL). Upon the addition of NaH (57% oil dispersion, 0.223 g, 5.29 mmol), the mixture may turn slightly foamy with gas evolution. After introduction of CH₃I (0.66 mL, 10.6 mmol), the reaction may become warm and gas evolution subside. After approximately 5 hours, the solvent may be removed in vacuo and the resultant white residue suspended in CH₂C1₂ (2 mL) and then loaded onto an SiO₂ column and eluted with 3% MeOH in CH₂C1₂ and then 5% MeOH in CH₂C1₂. After removal of the solvent, the desired product may be obtained as a white waxy solid (0.963 g, 53% yield).

[0081] A sixth process may be used to synthesize C16EG10C6 represented by the eneral formula:

g, 5.17 mmol) in anhydrous DMF (10 mL), followed by adding NaH (57% oil dispersion, 1.088 g, 25.84 mmol). 1-bromohexane (4.354 mL, 31.02 mmol) may then be introduced to the mixture resulting in a slurry which may be stirred at room temperature in a sealed flask after gas evolution subsides. After approximately 16 hours, the solvent may be removed in vacuo and the resultant white residue mixed with an ethyl acetate/hexanes mixture solvent (EtO Ac/hex, 1 :1 v/v, ~5 mL). This mixture may then be loaded onto an SiO₂ column and eluted with EtOAc/hex (1 : 1 v/v) and then 3% MeOH in CH₂C1₂. The fractions containing the product may be combined to yield a waxy solid (-3.15 g) and may then be subjected to a second SiO₂ column and eluted with 3% MeOH in CH₂C1₂ to yield a white waxy solid (2.872 g, 72% yield).

[0083] A seventh process may be used to synthesize heptaethylene glycol dihexadecyl ether (C16EG7C16) represented by the general formula:

[0084]

monohexadecyl ether (pure compound from Sigma- Aldrich, designated as C16EG7) (0.1 1 g, 0.2 mmol) in anhydrous DMF with NaH (57% oil dispersion, 42 mg, 1.0 mmol), followed by the addition of 1-bromohexadecane (0.366 g, 1.2 mmol). The resulting mixture may be stirred at room temperature for approximately 16 hours in a sealed flask before removal of the solvent in vacuo and suspension of the resultant white residue in an ethyl acetate/hexanes mixture solvent (EtO Ac/hex, 1 : 1 v/v, ~2 mL). The suspension may then be loaded onto an SiO₂ column and eluted with EtO Ac/hex (1 : 1 v/v) and then 3% MeOH in CH₂C1₂ to afford the desired product as a waxy film (0.147 g, 95% yield).

[0085] An eighth process may be used to synthesize C 18EG20C 16 represented by the general formula:

(XI)

[0086] The process may include mixing waxy solid Brij®78 (a mixture designated as C18EG20) (2.302 g, 2.0 mmol) with anhydrous DMF (10 mL), followed by the addition of NaH (57% oil dispersion, 0.21 g, 5.0 mmol). The mixture may be heated whereby gas evolution may commence and the mixture may become free-flowing. After approximately 15 minutes, 1-bromohexadecane (1.832 g, 6.0 mmol) may be introduced and the resulting slurry stirred at room temperature in a sealed flask. After approximately 16 hours, the solvent may be removed in vacuo and the white residue mixed with 3% MeOH in CH₂C1₂ (5 mL) and SiO₂ (2 g). The slurry may then be loaded onto an SiO₂ column, and eluted with 3% MeOH in CH₂C1₂ and then 5% MeOH in CH₂C1₂. Less polar fractions of the product may be discarded, and the more polar fractions of product may be combined and concentrated in vacuo to afford a white waxy solid (1.619 g, 59%). [0087] A ninth process may be used to synthesize C12EG30C12 represented by the general formula:

Χ^Χ^ ^Χ^· ^· ^ O (XII)

[0088] The process may include mixing white solid Brij®35 (a mixture designated as C12EG30) (2.624 g, 1.741 mmol) with anhydrous DMF (10 rnL), followed by adding NaH (57% oil dispersion, 0.183 g, 4.35 mmol). The mixture may be heated whereby gas evolution may commence and the mixture may become free-flowing. After

approximately 15 minutes, 1 -bromododecane (1.252 mL, 5.223 mmol) may be introduced and the resulting slurry stirred at room temperature in a sealed flask. After approximately 16 hours, the solvent may be removed in vacuo and the white residue was mixed with 3% MeOH in CH₂C1₂ (5 mL) and SiO₂ (2 g). The slurry may then be loaded onto an SiO₂ column, and eluted with 1 % MeOH in CH₂C1₂, 5% MeOH in CH₂C1₂ and then 8% MeOH in CH₂C1₂. The fractions containing the desired product may be combined and concentrated in vacuo to afford a white solid (2.91 g, 100%).

[0089] A tenth process may be used to synthesize C16EG9CH₂CH₂N₃ represented by the general formu

[0090] The process may include dissolving waxy solid Brij®56 (C16EG10) (6.20 g, 9.078 mmol) in anhydrous THF (30 rnL), followed by adding Et₃N (1.9 mL, 13.62 mmol) thereto. Toluenesulfonyl chloride (1.904 g, 10.0 mmol) may be introduced to the mixture resulting in a slurry which may be stirred at room temperature in a sealed flask. After approximately 3 days, a solid may be filtered using a Buchner filter funnel and the filtrate concentrated to afford a milky liquid (8.16 g). Anhydrous DMF (10 mL) and NaN₃ (0.649 g, 10.0 mmol) may be mixed with the milky liquid and then stirred at 80°C in a sealed flask for approximately 24 hours. The solvent may then be removed in vacuo and the residue mixed with EtO Ac/hex (1 : 1 v/v) (~10 mL) and SiO₂ (5 g). The resulting slurry may be loaded onto an SiO₂ column and eluted with EtO Ac/hex (1 : 1 v/v), 3% MeOH in CH2CI2 and then 5% MeOH in CH₂C1 . Fractions containing the desired product may be combined to afford a light yellow waxy solid (5.3 g, 82%).

[0091 ] An eleventh process may be used to synthesize C 16EG9CH₂CH₂NH₂ represented by the general formula:

[0092] The process may include dissolving a light yellow waxy solid (a mixture designated as C16EG9CH₂CH₂N₃) (1.87 g, -2.63 mmol) in THF (20 mL), followed by adding a Raney Ni suspension (50% slurry in H₂O, ~1 mL). Upon gas evolution subsiding, the solid may be filtered using glass wool in a pipette and rinsed with THF. The filtrate may then be concentrated and loaded onto an SiO₂ column and eluted with 5% MeOH in CH₂C1₂, then 10% MeOH in CH₂C1₂ and then MeOH/CH₂Cl₂/saturated NH₃ aqueous solution (1 :5:0.1 v/v/v). The desired product may be obtained as a white solid (1.006 g, 56%).

[0093] A twelfth process may be used to synthesize (C 16EG9CH₂CH₂S)₂ represented by the general formula:

[0094] The process may include dissolving waxy solid Brij®56 (C16EG10) (1.282 g, 1.877 mmol) in anhydrous CH₂C1₂ (10 mL), followed by adding Et₃N (0.53 mL, 3.75 mmol) thereto. Methanesulfonyl chloride (0.22 mL, 2.82 mmol) may be introduced to the mixture at 0°C resulting in a suspension which may be stirred at room temperature in a sealed flask for approximately 30 min. This reaction mixture may then be loaded onto an SiO₂ column and eluted with 3% MeOH in CH₂C1₂ and then 10% MeOH in CH₂C1₂. The fractions may be combined and concentrated to afford a waxy solid (1.27 g), which may be mixed with anhydrous DMF (5 mL) and KSAc (0.381 g, 3.338 mmol). This mixture may be stirred at 80°C in a sealed flask for approximately 24 hours resulting in a gel-like suspension. The suspension may be cooled to room temperature and mixed with 5% MeOH in CH₂C1₂ (5 mL) and SiO₂ (5 g) and then loaded onto an SiO₂ column and eluted with 5% MeOH in CH₂C1₂ and then 10% MeOH in CH₂C1₂. Fractions containing the product may be combined and concentrated into a red oil, and the red oil subjected to a second SiO₂ column and eluted with 5% MeOH in CH₂C1₂ and then 10% MeOH in CH₂C1₂ to afford a pale yellow waxy solid (1.186 g). This solid may then be dissolved in MeOH (5 mL) and then treated with NaOH (0.134 g, 3.34 mmol), stirred at room temperature in a sealed flask for approximately 16 hours, and then stirred in open air for approximately 16 hours to oxidize any free thiol -SH to its corresponding disulfide. The resulting solid may then be mixed with 3% MeOH in CH₂C1₂ (3 mL) and SiO₂ (2 g) resulting in a slurry. This slurry may be loaded onto an SiO₂ column and eluted with 3% MeOH in CH₂C1₂ and then 10% MeOH in CH₂C1₂. The product may be obtained as a pale yellow solid (1.04 g, 79% yield).

[0095] A thirteenth process may be used to synthesize C 16EG10SO₃ ^~ represented by the general formula:

[0096] The process may include mixing solid Brij®56 (C16EG10) (0.8829 g,

1.293 mmol) with a solid sulfur trioxide trimethylamine complex (SO₃.NMe₃, 0.201 g, 1.44 mmol) in a sealed flask under Argon. The mixture may then be warmed at 90°C for approximately 16 hours resulting in a white slurry which slowly turns into a clear oil, indicating the consumption of SO₃.NMe₃. The oil may then turn into a white solid upon cooling. As the solid is not soluble in THF, this indicates conversion of C16EG10 to its sulfate C16EG10SO₃ ^~. The resultant solid is soluble in a THF/MeOH (1 : 1 v/v) mixture solvent and may be employed for CNT nanostructure surface functionalization without further purification.

[0097] A fourteenth process may be used to synthesize C 18EG20SO₃ ^" represented by the general fo

(XVII) [0098] The process may include mixing Brij®78 (C18EG20) (1.331 g, 1.157 mmol) with a solid sulfur trioxide trimethylamine complex (SOs. Me , 0.177 g, 1.272 mmol) in a sealed flask under Argon. The mixture may then be warmed at 90 °C for approximately 16 hours turning into a clear oil which indicates the consumption of SO₃ ^'NMe₃. The oil may then turn into a white solid upon cooling which is soluble in a THF/MeOH (1 : 1 v/v) mixture solvent for CNT nanostmcture surface functionalization.

[0099] A fifteenth process may be used to synthesize C12EG30SO₃ ^~ represented by the general formula:

(XVIII)

[00100] The process may include mixing Brij®35 (C12EG30) (1.574 g, 1.044 mmol) with a solid sulfur trioxide trimethylamine complex (SO₃.NMe₃, 0.16 g, 1.149 mmol) in a sealed flask under Argon. The mixture may then be warmed at 90 °C for approximately 16 hours turning into a clear oil which indicates the consumption of SO₃ ^'NMe₃. The oil may then turn into a white waxy solid upon cooling which is soluble in a THF/MeOH (1 : 1 v/v) mixture solvent for CNT nanostmcture surface

functionalization.

[00101] A sixteenth process may be used to synthesize C 16EG7SO₃ ^~ represented by the general formula:

[00102] The process may include mixing solid heptaethylene glycol monohexadecyl ether (C16EG7, 0.712 g, 1.293 mmol) with a solid sulfur trioxide trimethylamine complex (SO₃NMe₃, 0.198 g, 1.42 mmol) in a sealed flask under Argon. The mixture may then be warmed at 90°C for approximately 16 hours turning into a clear oil which indicates the consumption of SO₃ NMe₃. The oil may then turn into a white waxy solid upon cooling which is soluble in a THF/MeOH (1 : 1 v/v) mixture solvent for CNT nanostmcture surface functionalization. [00103] A seventeenth process may be used to synthesize C8EG3CH₂CH₂N₃ represented by the general formula:

mmol) to a THF solution (8 mL) of tetraethylene glycol monooctyl ether (pure compound from Sigma-Aldrich designated as C8EG4) (0.646 g, 2.1 1 mmol) and Et₃N (0.593 mL, 4.22 mmol) resulting in a slurry. This slurry may be stirred in a sealed flask at room temperature for approximately 24 hours whereby an additional 0.2 eq of toluenesulfonyl chloride may be introduced followed by additional stirring at 40°C for approximately 16 hours. The solvent may then be removed, and the residue loaded onto an SiO₂ column and eluted with EtO Ac/hex 1 :2 then 1 : 1 to yield an oil (0.865 g, 89%). This oil

(tetraethylene glycol monooctyl ether tosylate) (0.216 g, 0.469 mmol) may be mixed with anhydrous DMF (5 mL) and NaN₃ (46 mg, 0.704 mmol) and then stirred at 85 °C in a sealed flask for approximately 24 hours. The solvent may be removed in vacuo and the residue mixed with EtO Ac/hex (1 :2 v/v) (~2 mL) resulting in a slurry. This slurry may be loaded onto an SiO₂ column and eluted with EtO Ac/hex (1 : 1 v/v) to afford the desired product as a clear oil (0.156 g, 100%).

[00105] An eighteenth process may be used to synthesize C8EG3CH₂CH₂NH₂ represented by the general formula:

[00106] The process may include mixing an oil C8EG3CH₂CH₂N₃ (0.156 g, 0.469 mmol) with THF (3 mL), H₂O (20 μΕ) and triphenyl phosphine (0.185 g, 0.704 mmol). The resulting mixture may then be stirred at room temperature under Argon in a sealed flask for approximately 16 hours. The solvent may be removed and residue loaded onto an SiO₂ column and eluted with 10% MeOH in CH₂C1₂ and then 10% MeOH in CH₂C1₂ with a 1 % saturated NH₃ aqueous solution to afford the desired product as a clear film (0.1 1 g, 77% yield). [00107] A nineteenth process may be used to synthesize 12-(n-octyl)-12-aza-3,6,9- trioxa- 1 -eicosanol represented b the general formula:

[00108] The process may include mixing dioctylamine (0.71 mL, 2.37 mmol) with tetraethylene glycol monotosylate (0.412 g, 1.18 mmol) in a sealed flask. The mixture may be stirred and warmed at 80°C for approximately 16 hours resulting in a slurry. Upon cooling, the slurry may be suspended in CH₂C1₂ (5 mL), followed by adding Et₃N (0.164 mL, 1.18 mmol) and acetic anhydride (0.1 12 mL, 1.18 mmol) at 0°C. After approximately 30 min, the reaction mixture may be diluted with MeOH (1.0 mL) and then concentrated whereby the residue may be loaded onto an SiO₂ column and eluted with 5% MeOH in CH₂C1₂ and then 10% MeOH in CH₂C1₂ to yield the desired product as a clear oil (0.402 g, 82% yield).

[00109] A twentieth process may be used to synthesize N, N-di-(n-octyl)-N'-( 1 1 - hydroxy-3,6,9-trioxaundec l) succinamide represented by the general formula:

(XXIII)

[001 10] The process may include mixing dioctylamine (0.302 mL, 1.0 mmol) with succinic anhydride (0.1 1 g, 1.1 mmol) and diisopropylethylamine (DIPEA, 0.348 mL, 2.0 mmol) in CH₂C1₂ (2 mL). After approximately 16 hours, the solution may be treated with 1 l-amino-3,6,9-trioxaundecan-l-ol (0.193 g, 1.0 mmol) and 3-diethoxyphosphoryloxy- l ,2,3-benzotriazin-4(3H)-one (0.329 g, 1.1 mmol) resulting in a light yellow solution. This solution may be stirred at room temperature for approximately 4 hours before quenching with ethylene diamine (0.1 mL) to form a yellow suspension. The suspension may be loaded onto an SiO₂ column and eluted with 5% MeOH in CH₂C1₂ to afford the desired product as a clear oil (0.306 g, 59% yield over two steps).

[001 1 1] A twenty first process may be used to synthesize N, N-di-(«-octadecyl)-N- (1 l-hydroxy-3,6,9-trioxaundecyl) succinamide represented by the general formula:

[001 12] The process may include mixing dioctadecylamine (0.261 g, 0.5 mmol) with succinic anhydride (0.055 g, 0.55 mmol) and diisopropylethylamme (DIPEA, 0.174 mL, 1.0 mmol) in CH₂CI₂ (1 mL). After approximately 16 hours, the solution may be treated with 1 l-amino-3,6,9-trioxaundecan-l-ol (0.0966 g, 0.5 mmol) and 3- diethoxyphosphoryloxy-l,2,3-benzotriazin-4(3H)-one (0.165 g, 0.55 mmol) resulting in a light yellow solution. This solution may be stirred at room temperature for

approximately 4 hours before quenching with ethylene diamine (0.15 mL) to form a yellow suspension. The suspension may be loaded onto an SiO₂ column and eluted with 3% MeOH in CH₂C1₂ to afford the desired product as a clear oil (0.355 g, 89% yield over two steps).

[001 13] A twenty second process may be used to synthesize N, N-di-(n-octadecyl)- N'-(6-hydro

[001 14]

with succinic anhydride (0.055 g, 0.55 mmol) and diisopropylethylamme (DIPEA, 0.174 mL, 1.0 mmol) in CH₂C1₂ (1 mL). After approximately 16 hours, the solution may be treated with 6-amino-hexan-l-ol (0.0585 g, 0.5 mmol) and 3-diethoxyphosphoryloxy- l,2,3-benzotriazin-4(3H)-one (0.165 g, 0.55 mmol) resulting in a light yellow solution. This solution may be stirred at room temperature for 16 hours before quenching with ethylene diamine (0.15 mL) to form a yellow suspension. The suspension may be loaded onto an SiO₂ column and eluted with 3% MeOH in CH₂C1₂ to afford the desired product as a clear oil (0.305 g, 86% yield over two steps).

[001 15] A twenty third process may be used to synthesize 12-(n-octadecyl)-12-aza- 3,6,9-trioxa-l-triacontanol represented by the general formula:

[001 16] The process may include mixing solid dioctadecylamine (0.367 g, 0.703 mmol) with tetraethylene glycol monotosylate (0.223 g, 0.639 mmol) in a sealed flask. The mixture may then be stirred at 90°C for approximately 16 hours to form an amber oil. Upon cooling, the resultant yellow solid may be suspended in 3% MeOH in CH₂C1₂ (5 mL) followed by adding Et₃N (0.21 mL, 1.5 mmol) and acetic anhydride (0.0354 mL, 0.375 mmol) resulting in a clearly slurry. After approximately 30 min, the reaction mixture may be quenched with ethylenediamine (0.15 mL) and loaded onto an SiO₂ column and eluted with 3% MeOH in CH₂C1₂, 5% MeOH in CH₂C1₂ and then 10% MeOH in CH₂C1₂ to yield the desired product as a waxy solid (0.212 g, 48% yield).

[001 17] A twenty fourth process may be used to synthesize 3a, 7a, 12a-trihydroxy- 5 -cholan-2 -oic acid N, N-di-(n-octadecyl) amide represented by the general formula:

[001 18] The process may include introducing diisopropylethylamine (0.082 mL,

0.472 mmol) and 3-diethoxyphosphoryloxy-l,2,3-benzotriazin-4(3H)-one (77.7 mg, 0.26 mmol) to a slurry of 3a, 7a, 12a-trihydroxy-5 -cholan-24-oic acid (96.5 mg, 0.236 mmol) and dioctadecylamine (123.3 mg, 0.236 mmol) in CH₂C1₂ (5 mL) turning the slurry into a clear yellow solution after approximately 16 hours of stirring.

Ethylenediamine (0.025 mL) may then be added to the slurry and mixed with SiO₂ (1 g) and then loaded onto an SiO₂ column and eluted with 5% MeOH in CH₂C1₂ to yield the desired product as a white solid (0.186 g, 86% yield).

[001 19] A twenty fifth process may be used to synthesize (2,2,2- trimethylol) azidoethane tri(3 ,6,9, 12-tetraoxaeicosanyl) ether represented by the general formula:

(XXVIII)

[00120] The process may include stirring (2-bromomethyl)-(2-hydroxymethyl)-l,3- propanediol (2.5747 g, 12.93 mmol) with NaN₃ (1.163 g, 17.9 mmol) in anhydrous DMF (10 mL) in a sealed flask at 85°C for approximately three days. The solvent may be removed in vacuo, and the resulting white slurry purified by loading onto an SiO₂ column and eluted with 10% MeOH in CH₂C1₂ and then 20% MeOH in CH₂C1₂ to yield the desired product (2-azidomethyl)-(2-hydroxymethyl)- 1,3 -propanediol as a white soft solid upon standing (2.059 g, 99% yield). (2-azidomethyl)-(2-hydroxymethyl)-l,3-propanediol (57.4 mg, 0.357 mmol) may then be mixed with NaH (57% oil dispersion, 68 mg, 1.61 mmol) in anhydrous DMF (5 mL). Tetraethylene glycol monooctyl ether tosylate (0.525 g, 1.14 mmol) may then be introduced into the mixture resulting in a slurry. The slurry may then be stirred in a sealed flask at room temperature for approximately 24 hours, and additional NaH (57% oil dispersion, 42 mg) introduced followed by the addition of tetraethylene glycol monooctyl ether tosylate (0.10 g). This reaction mixture may then be stirred at room temperature for approximately three days whereupon the solvent may be removed and residue loaded onto an SiO₂ column and eluted with EtO Ac/hex (1 :2 v/v) then 5% MeOH in CH₂C1₂ to yield the desired product (2,2,2-trimethylol) azidoethane tri(3,6,9,12 tetraoxaeicosanyl) ether as a clear oil (0.37 g).

[00121] A twenty sixth process may be used to synthesize (2,2,2-trimethylol) ethylamine tri(3,6 9,12-tetraoxaeicosanyl) ether represented by the general formula:

[00122] The process may include subjecting the clear oil (2,2,2-trimethylol) azidoethane tri(3,6,9, 12-tetraoxaeicosanyl) ether (0.37 g, -0.357 mmol) to reduction with triphenyl phosphine (0.14 g, 0.536 mmol) in THF (3 mL) with H₂O (10 mg) in a sealed flask under Argon upon stirring for approximately 24 hours. The solvent may then be removed and the residue mixed with CH₂C1₂ (1 mL) and then loaded onto an SiO₂ column and eluted with 5% MeOH in CH₂C1₂, and then a mixture solvent of

MeOH/CH₂Cl₂/saturated aqueous ammonia (10:90: 1 v/v/v) to afford the desired product (2,2,2-trimethylol) ethylamine tri(3,6,9,12-tetraoxaeicosanyl) ether as a clear oil (0.26 g, 73% yield).

[00123] A twenty seventh process may be used to synthesize

C12EG29CH₂CH₂NHCH₂CH₂OH re resented by the general formula:

[00124] The process may include mixing pellets of Brij®35 (C12EG30) (9.042 g, 6.0 mmol) with Et₃N (1.254 mL, 9.0 mmol) in THF (4 mL). The mixture may be heated to a clear solution and toluenesulfonyl chloride (1.258 g, 6.6 mmol) introduced thereto resulting in a milky slurry. The slurry may be stirred at room temperature for

approximately three days and a solid filtered using glass wool in a glass pipette. The solid may then be rinsed with THF (-1 0 mL) and the filtrate concentrated to a viscous oil in vacuo which may then be mixed with ethanolamine (3.62 mL, 60 mmol) in a sealed flask upon stirring at 90°C for approximately 16 hours resulting in a slightly yellow reaction mixture. Upon cooling the mixture may become a waxy solid whereupon the solid may be dissolved in 5% MeOH in CH₂C1₂, loaded onto an SiO₂ column and eluted with 5% MeOH in CH₂C1₂, 10% MeOH in CH₂C1₂, and then a mixture solvent of MeOH/CH₂Cl₂/saturated aqueous ammonia (10:90: 1 followed by 20:80:2 v/v/v) to yield the desired product as a slightly yellow waxy solid (6.408 g, 69%).

[00125] A twenty eighth process may be used to synthesize N-(C 16EG9CH₂CH₂) (±)-a-lipoic

(XXXI)

[00126] The process may include introducing C 16EG9CH₂CH₂NH₂ (0.1076 g, 0.1577 mmol) to a solution of (±)-a-lipoic acid (39 mg, 0.189 mmol) and

diisopropylethylamine (55 μΕ, 0.315 mmol) in CH₂C1₂ (2 mL), followed by adding 3- diethoxyphosphoryloxy-l ,2,3-benzotriazin-4(3H)-one (56.6 mg, 0.189 mmol) resulting in a yellow solution. After approximately 16 hours, ethylenediamme (15 μΐ.) may be added to the solution resulting in a slurry which may be loaded onto an SiO₂ column and eluted with 3% MeOH in CH₂C1₂, 5% MeOH in CH₂C1₂ and then 10% MeOH in CH₂C1₂ to afford the desired product as a light yellow waxy solid (0.103 g, 75% yield).

[00127] A twenty ninth process may be used to synthesize N-(C 16EG9CH₂CH₂) anthraquinone-2-carboxylic acid amide represented by the general formula:

(XXXII)

[00128] The process may include introducing C 16EG9CH₂CH₂NH₂ (0.101 g, 0.149 mmol) to a solution of anthraquinone-2-carboxylic acid (45 mg, 0.178 mmol) and diisopropylethylamine (52 μΐ_^, 0.297 mmol) in CH₂C1₂ (2 mL), followed by adding 3- diethoxyphosphoryloxy-l ,2,3-benzotriazin-4(3H)-one (53.4 mg, 0.178 mmol) resulting in a yellow solution. After approximately 3 hours, ethylenediamme (10 uL) may be added to the solution resulting in a slurry which may be loaded onto an SiO₂ column and eluted with 3% MeOH in CH₂C1₂, 5% MeOH in CH₂C1₂ and then 10% MeOH in CH₂C1₂ to afford the desired product as a light yellow waxy solid (93 mg, 68% yield).

[00129] A thirtieth process may be used to synthesize N-(C 12EG29CH₂CH₂)-N-(2- hydroxyethyl) anthraquinone-2-carboxylic acid amide represented by the general formula:

(XXXIII)

[00130] The process may include introducing C12EG29CH₂CH₂NHCH₂CH₂OH (0.203 g, 0.131 mmol) to a solution of anthraquinone-2-carboxylic acid (33 mg, 0.131 mmol) and diisopropylethylamine (46 μΕ, 0.262 mmol) in CH₂C1₂ (1 mL), followed by adding 3-diethoxyphosphoryloxy-l ,2,3-benzotriazin-4(3H)-one (39.2 mg, 0.131 mmol) resulting in a yellow solution. After approximately 16 hours, ethylenediamine (10 μί_^) may be added to the solution resulting in a slurry which may be loaded onto an SiO₂ column and eluted with 5% MeOH in CH₂C1₂ and then 10% MeOH in CH₂C1₂ to afford the desired product as a light yellow waxy solid (172 mg, 75% yield).

[00131] A thirty first process may be used to synthesize N-(C 12EG29CH₂CH₂)-N- (2-hydroxyethyl) 3-(2,5-dimethoxyphenyl)propionic acid amide represented by the general formula:

(XXXIV)

[00132] The process may include introducing C 12EG29CH₂CH₂NHCH₂CH₂OH (0.576 g, 0.371 mmol) to a solution of 3-(2,5dimethoxyphenyl) propionic acid (78.08 mg, 0.371 mmol) and diisopropylethylamine (129 μL·, 0.742 mmol) in CH₂C1₂ (4 mL), followed by adding 3-diethoxyphosphoryloxy-l ,2,3-benzotriazin-4(3H)-one (1 1 1 mg, 0.371 mmol) resulting in a yellow solution. After approximately 16 hours,

ethylenediamine (30 μΐ_^) may be added to the solution resulting in a slurry which may be loaded onto an SiO₂ column and eluted with 5% MeOH in CH₂C1₂ and then 10% MeOH in CH₂C1₂ to afford the desired product as a light yellow waxy solid (0.42 g, 65% yield).

[00133] With continued reference to Figure 1 and the aforementioned examples, an exemplary CNT nanostructure functionalized using a layer-by-layer approach, i.e., forming a first protective (e.g., alkyl) layer followed by a second layer of

polyoxyethylene alkyl ether or other layer. These embodiments may be employed as an electrode for free chlorine concentration determination in tap water, as a potentiometric pH sensor, an amperometric pH sensor, a biometric sensor or electrode, or a voltammetric pH sensor or other electrochemical sensor or electrode.

[00134] It should be noted that polyoxyethylene alkyl ethers may be derivatized to form bipolar molecules with additional functionalities. For example, the -OH group in polyoxyethylene alkyl ethers may react with SO₃ or P₂O₅ to create a bipolar molecule which can be used to introduce -OSO₃ ^~ or -OPO₃H₂ groups onto an exemplary CNT electrode surface. It should also be noted that an -OH group can form esters with various carboxylic acids and may undergo a variety of transformations to be replaced by groups such as, but not limited to, -N₃, -NH₂ and -SH or -S-S-, to name a few.

[00135] Additionally, in one embodiment polyoxyethylene alkyl ether may be converted to a respective mesylate or tosylate, which could then be substituted with nucleophilic groups including, but not limited to, halide, azide, sulfide or masked thiol such as thioacetate, NH₃, primary amine, secondary amine and tertiary amine. In yet another embodiment, polyoxyethylene alkyl ether may react in the presence of NaH with activated acetate such as tert-butyl bromoacetate followed by deprotection of tert-butyl ester to yield polyoxyethylene alkyl ether with a terminal -COOH group. Another embodiment may employ polyoxyethylene alkyl ether with a terminal -NH₂ group to react with succinic anhydride to introduce a terminal -COOH group.

[00136] A terminal -NH₂ or -NH- group in derivatized polyoxyethylene alkyl ether may react with various carboxylic acids via an amide bond formation. Thus, a range of exemplary redox mediator moieties may be covalently linked to polyoxyethylene alkyl ether. Exemplary, non-limiting mediators include anthraquinone 2-carboxylic acid, 3- (2,5-dimethoxyphenyl) propionic acid and (±)-a-lipoic acid. In the case of 3-(2,5- dimethoxyphenyl) propionic acid, the hydroquinone moiety may be protected with methyl groups and hence the hydroquinone/benzoquinone redox pair would not be present after the second layer deposition. After a few scans of cyclic voltammetry, the 2,5-dimethoxyphenyl moiety may then be oxidized to generate a desired

hydroquinone/benzoquinone redox pair for electrochemical sensing of solution pH. It should be noted that many other masked/protected functional groups or functional moieties may be unmasked/deprotected electrochemically once they are introduced onto an exemplary CNT electrode surface, thus, such examples should not limit the scope of the claims appended herewith.

[00137] Figure 2 is an illustration of a general structure for a molecule with an attached anthraquinone functional moiety for CNT surface functionalization. With reference to Figure 2, a molecule 20 is provided having a polyoxyethylene alkyl ether covalently attached with redox mediators [e.g., anthraquinone (AQ)] or another functional group (e.g., polyoxyethylene alkyl ether conjugate) to form an exemplary second layer 18 above the first protective layer 15 on a CNT structure (see Figure 1). Peak potential of the respective redox mediators may be used to determine solution pH according to the Nernst Equation.

[00138] Figure 3 is an illustration of a hydrophilic CNT nanostructure surface with controllable density of anthraquinone moieties. With reference to Figure 3, the density of exemplary functional groups may be, in one embodiment, controlled by mixing polyoxyethylene alkyl ether containing a first functional group with polyoxyethylene alkyl ether containing a second functional group. For example, polyoxyethylene alkyl ether derivatized with a terminal -NH₂ or -NH- group can be mixed with a non- derivatized polyoxyethylene alkyl ether to control the density of the surface -NH₂ or - NH- group. Similarly, polyoxyethylene alkyl ether anthraquinone 2-carboxylic acid conjugate may be diluted with non-derivatized polyoxyethylene alkyl ether to control the density of anthraquinone functional moieties on a CNT surface as illustrated in Figure 3.

[00139] Figure 4 is a graphical depiction of a square wave voltammogram overlay of CNT nanostructures functionalized with different ratios of polyoxyethylene alkyl ether anthraquinone 2-carboxylic acid conjugate and C12EG30 for the formation of a second layer. With reference to Figure 4, when N-(C 12EG29CH₂CH₂)-N-(2-hydroxyethyl) anthraquinone-2-carboxylic acid amide and C12EG30 were mixed in different ratios (1 :0, 1 :4, 1 :9 and 1 : 19) for the formation of a second layer on a CNT, it was discovered that the redox signal amplitude in square wave voltammetry (SWV) could be modulated. It should be noted that while the mixtures of only two compounds (N-(C12EG29CH₂CH₂)- N-(2-hydroxyethyl) anthraquinone-2-carboxylic acid amide and C12EG30) are

demonstrated in Figure 4, this should not limit the scope of the claims appended herewith as it is envisioned that mixtures of three or more compounds, including but not limited to the aforementioned compounds, may also be employed to introduce three or more functional groups or functional moieties with controlled density of each functional group or functional moiety.

[00140] Surface hydrophilicity of exemplary functionalized CNT nanostructures is important for such nanostructures to be used as electrodes since many electrochemical reactions in aqueous solutions require the participation of H⁺ or OH^". It follows that one may then control the degree of surface hydrophilicity at the molecular level. Thus, by increasing the number of terminal -OH groups in the polyoxyethylene alkyl ether chain, the degree of hydrophilicity of the subsequently functionalized CNT surface may be increased. For example, the tosylate of polyoxyethylene alkyl ether may be treated with ethanolamine, 2-amino-l,3-propandiol, 3-amino-l,2-propandiol and tris(hydroxymethyl) amino methane to introduce 1 , 2 and 3 terminal -OH groups onto the polyoxyethylene alkyl ether chain. Figure 5 is a schematic illustration of controlling the number of -OH groups in a bipolar molecule used for the formation of a second layer on a functionalized CNT surface. Of course, many other aminopolyols may be used in embodiments of the present subject matter including, but not limited to, amino saccharides that can be covalently linked to polyoxyethylene alkyl ether chain in similar fashion and such an example should not limit the scope of the claims appended herewith. When used to form the second layer on a CNT surface, these derivatized polyoxyethylene alkyl ethers may lead to a surface having various degrees of hydrophilicity due to the presence of different numbers of terminal -OH groups. Further, the use of primary aminoalcohols may also provide for subsequent derivatization of a resulting secondary amino group with various carboxylic acids including anthraquinone 2-carboxylic acid, 3-(2,5-dimethoxyphenyl) propionic acid and (±)-a-lipoic acid via amide bond formation. It should be noted that other functional groups and functional moieties such as, but not limited to, redox mediator molecules, crown ethers, catalysts, boric acids, carbohydrates, oligonucleotides (DNAs and RNAs), DNA or RNA aptamers, peptide aptamers, proteins such as enzymes and antibodies, quantum dots and nanoparticles, cells, cell organelles, and other cellular components, may be covalently attached to the hydrophilic surface via ester or amide bond formation. [00141] With continued reference to Figure 5, when surface hydrophilicity is of concern, the tosylate of polyoxyethylene alkyl ether may react with various alcohols in the presence of NaH to afford polyoxyethylene alkyl ether with 0, 1, 2, 3, 4 or more -OH groups per polyoxyethylene alkyl ether chain. Exemplary, non-limiting alcohols may be any monoalkyl alcohol, ethylene glycol, glycerol, erythritol, threitol, pentaerythritol, inositol, xylitol, mannitol and other sugar alcohols. Certain embodiments may also glycosylate the terminal -OH group in polyoxyethylene alkyl ether to covalently link various sugar alcohols or polyols to the polyoxyethylene alkyl ether chain. When such exemplary bipolar molecules are used in the second layer of a CNT nanostructure according to embodiments of the present subject matter, the density of -OH groups may be controlled and the surface hydrophilicity modulated as desired.

[00142] One embodiment may modulate and/or control the density of various surface functional groups and functional moieties by mixing a bipolar compound containing the functional groups and/or functional moieties described herein with a similar bipolar compound containing no such functional groups and/or functional moieties according to a specific ratio (e.g., 1 : 1, 1 :2, etc.) in a solution used for the second layer functionalization of an exemplary CNT surface. Further, more than two compounds may also be utilized to simultaneously introduce functional groups with desired density.

[00143] Due to the size and/or polarity of a respective functional group, it may be difficult to construct a second layer structure having certain functional groups attached to the polyoxyethylene alkyl ether chain and/or it may be difficult to ensure that certain functional groups are exposed on the outer surface of the second layer. For example, when the functional moiety of a prospective functional group is an enzyme molecule, it may be difficult to eliminate a hydrophobic interaction between the alkyl chain in the polyoxyethylene alkyl ether and the enzyme molecule if the enzyme molecule is first covalently attached to the polyoxyethylene alkyl ether simply due to the sheer size of the enzyme molecule. Further, if the polarity of a certain functional group is similar to the alkyl chain in a polyoxyethylene alkyl ether, when forming the second layer above the protective first layer on an exemplary CNT surface the hydrophobic functional group may not necessarily separate from the alkyl chain and as a result may be buried underneath the hydrophilic polyoxyethylene structure. Figure 6 is a schematic illustration of depositing a polyoxyethylene dialkyl ether on a CNT surface to form a second layer on a functionalized CNT surface. With reference to Figure 6, the surface of an exemplary functionalized CNT nanostructure electrode 60, having a protective first layer 62 and a polyoxyethylene dialkyl ether C18EG20C16 in the second layer 64, may be hydrophilic thereby providing an indication that the polyoxyethylene portion thereof is exposed on the outermost surface. In other instances, when the prospective functional group is relatively unstable under the conditions for the formation of the second layer, it may be desirable that the functional group be covalently attached after the second layer structure is established on the CNT surface.

[00144] Figure 7 is an illustration of an exemplary structure of a hydrophilic CNT nanostructure surface and a covalent functionalization of surface -OH groups with an activated anthraquinone ester. With reference to Figure 7, an exemplary method may establish a hydrophilic platform on a CNT nanostructure 10 amenable for subsequent covalent attachment of various functional groups regardless of their size, polarity, hydrophobicity/hydrophilicity, and/or stability under elevated temperatures. In one embodiment, an exemplary CNT nanostructure electrode 10 may be protected with n- octadecane to form the first protective layer, followed by the deposition of molecules such as C12EG30, (2,2,2-trimethylol) ethylamine tri(3,6,9,12-tetraoxaicosanyl) ether or dioctadecylamine [(n-Ci₈H₃₈)₂NH] to form a second layer with -OH groups (see Figure 7) or -NH₂ or -NH- groups. Of course, such exemplary groups may be useful for covalent attachment of other functional groups or functional moieties. For example, various carboxylic acids may be introduced to the surface via ester and amide bond formation, and other functional groups and functional moieties including, but not limited to, redox mediator molecules, crown ethers, catalysts, boric acids, carbohydrates, oligonucleotides (DNAs and RNAs), DNA or RNA aptamers, peptide aptamers, peptides and proteins such as enzymes and antibodies, quantum dots and nanoparticles, cells, cell organelles, and other cellular components, may also be covalently linked to the amino and/or hydroxyl groups on the hydrophilic surface.

[00145] Figure 8 is a graphical depiction of a square wave voltammogram overlay of differently functionalized CNT nanostructure electrodes. With reference to Figure 8 and continued reference to Figure 7 to illustrate certain principles underlying

embodiments of the present subject matter, a series of CNT nanostmctures 10 were used to demonstrate the covalent nature of an exemplary functionalization process with functional groups such as -OH and -NH₂ in the second layer. These CNT nanostmctures 10 were first protected with n-octadecane in the first layer 62 and then deposited with C12EG30 to form the second layer 64 with -OH groups on the surface thereof. When the surface was treated with an activated AQ ester, the CNT nanostmcture electrode 10 yielded a strong redox signal for the AQ as graphically illustrated by a first trace 82. When a similar surface was treated with a solution with anthraquinone 2-carboxylic acid methyl ester (AQ methyl ester) and a trace of the activated AQ ester, the CNT

nanostmcture electrode 10 subsequently yielded a considerably smaller redox signal for the AQ as graphically illustrated by a second trace 84. When a CNT nanostmcture 10 was only protected with n-octadecane and no deposition of C12EG30 occurred for the second layer (hence no -OH groups on the nanostmcture surface), subsequent treatment of the electrode with activated AQ ester under identical condition resulted in no redox signal for the AQ as illustrated by a third trace 86. Finally, another CNT nanostmcture 10 having n-octadecane and then C12EG30 deposition was treated with only

anthraquinone 2-carboxylic acid methyl ester, and the resulting CNT nanostmcture electrode 10 subsequently did not produce a redox signal for the AQ as graphically illustrated by a fourth trace 88 thereby indicating no non-specific adsorption of anthraquinone 2-carboxylic acid methyl ester onto the hydrophilic surface. These experiments indicate the covalent nature of the hydrophilic surface functionalization of certain embodiments of the present subject matter and provide evidence that exemplary CNT nanostmctures according to embodiments of the present subject matter having poly(ethylene glycol) functionality on the surface thereof following a layer-by-layer process may be resistant to non-specific adsorption.

[00146] Figure 9 is a schematic illustration of an exemplary layer-by-layer introduction of various functional groups onto a CNT nanostructure surface. With reference to Figure 9, ring opening reactions of epoxide may be advantageously employed using a CNT surface having a second layer with -OH groups or -NH₂ or -NH- groups that readily react with glycidyl ethers such as polyetheneglycol diglycidyl ether and trimethylolpropane triglycidyl ether. Other epoxide-containing molecules can also be used including glycidol, trimethylolethane triglycigyl ether, glycerol diglycidyl ether, glycerol triglycidyl ether, glycerol polyglycidyl ether, diglycerol polyglycidyl ether, polyglycerol polyglycidyl ether, pentaerythritol glycidyl ether, pentaerythritol

polyglycidyl ether, sorbitol polyglycidyl ether and so on. Exemplary crosslinking 92 may result in a new polymeric layer (e.g., third layer) 94 with excess epoxide groups. When treated with amino alcohols such as ethanolamine, 2-amino-l,3-propandiol, 3-amino-l,2- propandiol and tris(hydroxymethyl)aminomethane, secondary amine -NH- and 1, 2 or 3 terminal -OH groups may be introduced onto the CNT surface. Of course, many other aminopolyols including, but not limited to, aminosaccharides may be employed in similar fashion and such a disclosure should not limit the scope of the claims appended herewith. The resulting amino and/or hydroxyl groups may also be derivatized with various carboxylic acids including 3-(anthracen-9-yl) propionic acid, anthraquinone 2-carboxylic acid, 3-(2,5-dimethoxyphenyl) propionic acid and (±)-a-lipoic acid. Similarly, other functional groups and functional moieties including, but not limited to, redox mediator molecules, crown ethers, catalysts, boric acids, carbohydrates, oligonucleotides (DNAs and RNAs), DNA or RNA aptamers, peptide aptamers, peptides and proteins such as enzymes and antibodies, quantum dots and nanoparticles, cells, cell organelles, and other cellular components, may also be covalently linked to the amino and/or hydroxyl groups on the hydrophilic surface layer 96.

[00147] In embodiments of the present subject matter where it may be necessary to further derivatize functional groups or functional moieties in the second layer on a CNT surface, the CNT nanostmcture may be treated with reagents in an appropriate solvent, e.g., activated AQ ester in CH₂C1₂ for a predetermined period as described in examples above. Upon completion of this further reaction, the CNT nanostmcture on the respective substrate may be rinsed with a solvent (e.g., THF), dried in air, and then wire- bonded and assembled for testing. In an alternative embodiment, a CNT nanostmcture functionalized with the first layer and second layer may be treated with reagents to form a third layer (e.g., via cross-linking) followed by subsequent transformations to incorporate additional functional groups or functional moieties. For example, after the formation of a C12EG30 layer on top of a first n-octadecane layer, a CNT nanostmcture on a substrate may be treated with a mixture solution of polyethylene glycol diglycidyl ether

(PEGDGE) and trimethylolpropane triglycidyl ether (TMPTGE) in THF (25mM/25mM, 2 x 5 μΕ), dried in air, and then warmed at 120°C in a tightly capped vial under Argon for approximately five hours. The CNT nanostmcture on the substrate may then be cooled to room temperature, rinsed with THF to remove excess PEGDGE and TMPTGE on the substrate. The CNT nanostmcture may then be dried in air and placed in a tightly capped vial with a mixture of Tris (121 mg) in DMF (1 mL) under Argon atmosphere and warmed at 80°C for approximately 24 hours before removal from the DMF solution. This nanostmcture may then be rinsed with MeOH and THF and dried in air. In a separate vial, anthraquinone 2-carboxylic acid (9 mg, 0.0356 mmol) may be mixed with diisopropylethylamine (10.4 μΕ, 0.071 mmol) and 3-diethoxyphosphoryloxy- 1,2,3- benzotriazin-4(3H)-one (10.6 mg, 0.0356 mmol) resulting in a yellow solution. The CNT nanostmcture may then be placed in the yellow solution for approximately 16 hours and rinsed with THF and dried. An exemplary CNT nanostmcture functionalized in this manner may then, for example, be used as voltammetric pH sensor with long-term stability. Of course, such a process is exemplary only and should not limit the scope of the claims appended herewith.

[00148] Figure 10 is a graphical depiction of a square wave voltammogram overlay for various embodiments of the present subject matter. With reference to Figure 10 and continued reference to Figure 9, in another embodiment a series of bipolar molecules having -OH, -NH₂ and secondary amine groups may be used for the deposition of the second layer on an exemplary CNT surface. For example, (2,2,2-trimethylol) ethylamine tri(3,6,9,12-tetraoxaeicosanyl) ether was employed for this purpose as schematically illustrated by the generic process depicted in Figure 9. Dialkyl amine including dioctadecylamine may also be applied to achieve a similar level of layer-by-layer surface functionalization. Polyoxyethylene alkyl ethers such as, but not limited to, C12EG30 and C12EG30NHCH₂CH₂OH may also be used as the second layer material for crosslinking to construct an exemplary third layer on a CNT surface. Upon subsequent epoxide ring opening and covalent bond formation with an activated AQ ester, a strong redox signal may be observed by a first and second trace 1010, 1012 in Figure 10. When no cross- linking step was employed as illustrated by a third trace 1014, subsequent treatment with tris(hydroxymethyl)aminomethane (Tris) in dimethylformamide (DMF) at an elevated temperature (80°C for 16 h) resulted in the removal of uncrosslinked C12EG30 and thus little or no -OH groups remained on the CNT surface for AQ functional moiety attachment which appears to explain the small redox signal for the AQ observed in the third trace 1014. These results provide an indication of the formation of a third layer by cross-linking on the CNT surface. Additionally, a cross-linked third layer may also stabilize the anchoring of functional groups and functional moieties on an exemplary CNT surface and may maintain the structural integrity of the surface layers thereby rendering the surface less prone to non-specific adsorption. Exemplary CNT surfaces functionalized in this fashion may provide excellent long-term stability and are suitable for subsequent introduction of proteins and enzymes. Of course, other cross-linking and polymerization approaches may be employed by embodiments of the present subject matter for the construction of a third layer and such an example should not limit the scope of the claims appended herewith. For example, it is envisioned that polymer brushes may be grown on top of the second or third layer of an exemplary CNT surface following the layer-by-layer approach and, with the appropriate chemistries, construction of a multilayer, organized structure possessing a controlled layer thickness may be performed. [00149] Figure 11 is a graphical depiction of a square wave voltammogram overlay of a CNT nanostmcture electrode functionalized with anthraquinone in buffer solutions at various pHs. Figure 12 is a plot of an anthraquinone square wave voltammogram redox peak potential versus buffer solution pH for a CNT nanostmcture electrode

functionalized via an embodiment of the present subject matter. With reference to Figures 11 and 12, exemplary functionalized carbon nanostmctures may be used as sensing elements in various applications. For example, a voltammetric pH sensor was fabricated using a CNT nanostmcture on a silicon substrate functionalized using an exemplary layer-by-layer approach with anthraquinone 2-carboxylic acid. When subjected to square-wave voltammetry (SWV) in a 0.05 M phosphate buffer solution with 0.1 M NaClO₄ as a supporting electrolyte at various pHs (2.0, 4.36, 7.0, 10.0 and 11.88), the sensor electrode generated symmetrical redox peaks for the respective pHs. In this embodiment of a surface functionalized CNT nanostmcture as a voltammetric pH sensor, SWV measurements were recorded using a Reference 600 potentiostat 5.61 with a standard three-electrode configuration, consisting of a Ag/AgCl reference electrode, a carbon rod auxiliary counter electrode, and a CNT nanostmcture on Si substrate as working electrode in a specially designed electrochemical cell. The exemplary CNT nanostmcture was functionalized with redox mediator molecules using an exemplary layer-by-layer approach, and then exposed to different pH solutions (about 5 mL) in an electrochemical cell. Different pH solutions were then prepared in deionized water as follows: pH 2.0, 0.05 M H₃PO₄ adjusted with 10% NaOH solution; pH 4.36, 0.05 M NaH₂PO₄; pH 7.0, 0.05 M Na₂HPO₄ adjusted with 0.05 M NaH₂PO₄; pH 10.0, 0.05 M Na₂HPO₄ adjusted with 10% NaOH solution; pH 11.88, 0.05 M Na₂HPO₄ adjusted with 10% NaOH solution. NaClO₄ may be added into these solutions as a supporting electrolyte to a concentration of 0.1 M. The pH values of these solutions may be obtained using a pH meter. SWV were performed with the following parameters:

frequency 10 Hz, step potential 2 mV, amplitude 25 mV within the potential range of -1.0 V and 0.5 V. Square wave (SW) voltammograms recorded using an AQ functionalized CNT nanostmcture electrode were overlaid and are illustrated in Figure 1 1. These voltammograms indicate that as pH increases from pH 2 to pH 11.88, the AQ redox peak shifts to more negative potential.

[00150] A plot of redox peak potential against pH illustrated in Figure 12 provides a linear, Nernstian response having a slope of -55.8 mV/pH and linearity R of 0.9993, substantially close to the theoretical slope of -59.1 mV/pH. Figure 12 thus reflects the plot of the AQ redox peak potential versus solution pH demonstrating a linear response from pH 2 to pH 1 1.88 with a slope of -55.788 mV per pH unit. It is apparent that an exemplary CNT nanostmcture electrode functionalized using a layer-by-layer approach with redox mediator molecules such as, but not limited to, AQ may be advantageously utilized as pH sensors for aqueous solutions.

[00151] Figure 13 is a graphical depiction of an open circuit potential of a CNT nanostmcture electrode functionalized using an embodiment of the present subject matter. Figure 14 is a plot of open circuit potential versus pH for flowing tap water using an embodiment of the present subject matter. With reference to Figures 13 and 14, a potentiometric pH sensor was fabricated using a CNT nanostmcture on a silicon substrate functionalized using an exemplary layer-by-layer approach with n-octadecane for the first layer and then polyoxyethylene alkyl ether Brij®35 (C12EG30) for the second layer. The stmcture was then placed in a polydimethylsiloxane (PDMS) flow cell and used as a potentiometric pH sensor for flowing tap water with low conductivity. As shown in Figure 13, open circuit potential (OCP) was measured against an Ag/AgCl reference electrode (RE-6, BASi Analytical Instmments) placed 3 mm above the CNT

nanostmcture electrode. To generate the results depicted in Figure 13, OCP

measurements were recorded using a Reference 600 potentiostat 5.61 with a two- electrode configuration having an Ag/AgCl reference electrode and a CNT nanostmcture on a silicon substrate as working electrode in a specially designed (PDMS) flow cell. The CNT nanostmcture may be functionalized using an exemplary layer-by- layer approach and then exposed to flowing tap water (after filtration over activated carbon to remove free chlorine in tap water) at different pH. The pH of the flowing tap water may be monitored using a glass pH meter and adjusted in a reservoir with dilute HCl or NaOH solutions upon constant stirring. Thus, Figure 13 illustrates the OCP change with different water pH whereby at a given pH, the CNT nanostructure electrode possessed a definite potential. It is shown that the potential shifted to more negative as the solution pH increased.

[00152] As illustrated in Figure 14, it was discovered that the OCP of the CNT electrode provided a linear response to pH in flowing tap water. This plot of the OCP against flowing tap water pH produced a linear response (R = 0.9928) with a gradient of -45.2 mV per pH unit between pH 3 and 12. Thus, the use of an exemplary CNT nanostructure electrode as a potentiometric pH sensor is evident, and such an exemplary potentiometric pH sensor may find utility in water or other aqueous solutions with low ionic concentrations as well as under pressure.

[00153] Nevertheless, functionalized CNT-based potentiometric pH sensors are generally responding to redox species present in the respective solution. To overcome such interferences, one embodiment of the present subject matter may provide an exemplary electrochemical pH sensor using a surface functionalized CNT electrode on a field effect transistor (FET) structure. Long-term stability of conventional FET-based pH sensors may be challenging as the sensing element surfaces are not appropriately functionalized to respond to pH-related electric field changes and cannot resist nonspecific adsorption of foreign materials on the surfaces (e.g., fouling) at the same time. As a result of exemplary functionalized CNT electrodes, the electrode surface could be both highly hydrophilic and resistant to fouling. As the pH sensing mechanism generally results from the structured hydrophilic layer on the respective CNT electrode surface, this type of pH sensor might have long-term stability as well as be interference-free. Another embodiment may provide an exemplary amperometric pH sensor having a reference electrode and a sensing electrode with a carbon nanostructure functionalized with a chemically stable moiety that responds to solution pH changes and may provide a stable current between a respective source and drain at a given solution pH when a fixed potential is applied to the sensing electrode. [00154] One exemplary method of fabrication of a pH sensor according to an embodiment of the present subject matter includes providing or fabricating the

underlying silicon chip, growing or depositing appropriate carbon nanostructures such as CNTs, and functionalizing the surface of such nanostructures. For example, one exemplary method of silicon chip fabrication is described in International Application No. PCT/US07/02104, the entirety of which is incorporated herein by reference. In this method, an insulating layer (e.g., SiO₂ or the like) may be deposited on top of a silicon substrate. A conductive layer having a defined geometry may be deposited and may be situated between two terminals, one serving as a source and the other as a drain. Thus, an exemplary conductive layer may act as an interconnect for CNT nodes and/or may act as an electric conduit between the source and drain. A barrier layer (e.g., Ti or the like) may be deposited on the conductive layer area to prevent segregation of subsequent catalyst material from the conductive layer. A thin catalyst layer (e.g., Ni, Fe or Co, etc.) may then be deposited and patterned by conventional lithography to form nodes of catalyst in a defined geometric shape (e.g., circle, rectangle, strips, etc.) with appropriate insulating layers (SiO₂, Si₃N₄, etc.) surrounding the nodes of catalyst. The insulating layers may be used to ensure the conductive layer is not exposed to solution in the pH sensing electrode.

[00155] Exemplary CNTs as described herein may then be grown on the underlying substrate by any number of methods including, but not limited to, an exemplary chemical vapor deposition (CVD) process described in PCT/US07/02104 and may be, in one embodiment, undoped aligned CNTs assemblies. Other methods may include an exemplary arc discharge process, laser-ablation process, natural, incidental and/or controlled flame environments, plasma enhanced chemical vapor deposition, a

capacitively coupled microwave plasma process, a capacitively coupled electron cyclotron resonance process, a capacitively coupled radio frequency process, an

inductively coupled plasma process, a dc plasma assisted hot filament process, template synthesis, carbo thermal carbide conversion, or combinations thereof, to name a few.

[00156] For example, the CNTs may include an electrically conductive layer covering a portion or all of a substrate and may include an assembly of undoped CNT antennae vertically oriented with respect to the electrically conductive layer. Any or each of the undoped CNT antennae may include a base end attached to the electrically conductive layer, a mid-section having an outer surface surrounding a cavity or channel therein (i.e., lumen), and a top end disposed opposite the base end. In one embodiment, the outer surface of the mid-section may be in fluidic contact with an environment (e.g., a liquid solution) that is in contact with the CNT antennae.

[00157] The CNTs may then be functionalized as described herein and in copending Application No. _/ and International Application No.

PCT/US2010/056350, the entirety of both incorporated herein by reference. Such exemplary CNT surface functionalization process may provide chemical and structural stability for the assembly electrodes and surface hydrophilicity. These functionalized CNT electrodes may then be assembled into a pH sensing device and used to measure the pH of aqueous solutions. Thus, when both reference electrode and the functionalized CNT electrode are in contact with an aqueous solution and a given potential is applied to the CNT electrode, a measured current between the source and drain may be proportional to the solution pH.

[00158] Figure 15 is a schematic illustration of a CNT electrode with a cross-linked hydrophilic surface layer. With reference to Figure 15, one embodiment may (or may not) circumvent a cumbersome conventional CNT FET fabrication process by fabricating a pH sensor using an exemplary layer-by-layer surface modification of a CNT electrode grown on a substrate. For example, a substrate 12 may include an insulating layer, a conducting layer and a catalyst layer 14 deposited thereon. The CNT 10 grown on the catalyst layer 14 may then be functionalized with a cross-linked, highly hydrophilic surface layer 150. The cross-linking level and the thickness of the hydrophilic layer 150 should be sufficient such that the layer 150 may be impermeable to free chlorine and other redox species and also such that the underlying CNT electrode may sense the electric field effect related to solution pH change.

[00159] One embodiment of the present subject matter may provide an exemplary pH sensor having a sensing and a reference electrode where the sensing electrode may include one or more carbon nanotubes functionalized with a chemically stable moiety described above. These nanotubes may respond to solution pH changes and provide a stable current between a source and drain at a given solution pH when a fixed potential is applied to the sensing electrode. In one embodiment, the carbon nanotube sensing electrode may be flanked by the source and drain on a silicon chip. Two exemplary contacts may be established from the conducting layer and used as a source and drain, respectively, whereby an exemplary CNT structure is placed in an aqueous solution with a reference electrode approximately 3 mm above the CNT surface to complete the circuit. Through the application of a given voltage (e.g., 10 mV, 1000 mV, -2000 mV, etc.) across the source and drain, a current may move across the CNT electrode whereby the current level or value generally responds to solution pH changes.

[00160] In another embodiment, the surface layer structure may be modified to ensure a stable hydrate layer on the CNT electrode surface. Figure 16 is a schematic illustration of constructing an orderly hydrophilic layer over a cross-linked hydrophilic layer. With reference to Figure 16, one embodiment of the present subject matter may provide a more orderly surface structure which results in a more stable pH response for an exemplary amperometric pH meter. In one embodiment, the method may include introducing additional glycidyl groups using trimethylolpropane triglycidyl ether

(TMPTGE) 155, and cross-linking and reacting glycidyl groups 157 with free -OH groups in C16EG10 to render a hydrophobic C16 chain on top of the cross-linking layer 150. The method may also rely on the hydrophobic-hydrophobic interaction between C16 and C18 chains to establish a C18EG227C18 layer, which may then render the electrode surface highly hydrophilic. These additional cross-linking and thicker layers may result in a barrier 160 impermeable to free chlorine and other redox species and yet, at the same time, responsive to solution pH changes.

[00161] Figure 17 is a schematic illustration of constructing a functionalized CNT structure on a conventional ion-sensitive FET (ISFET) gate oxide as a pH sensor. With reference to Figure 17, another embodiment may grow CNTs 185 on a gate oxide 181 of conventional FET device 180, followed by the functionalization of the CNTs 185 as described above. One issue with an oxide-based ISFET device is its general instability over time due to non-specific interactions with ionic species in solution. For example, iron oxide readily deposits on a glass surface, and a SiO₂ surface may also interact with alkaline earth metal ions such as Ca , Mg , etc. Exemplary CNTs functionalized with poly(ethylene glycol) alkyl ethers described above may resist non-specific adsorption and deposition (e.g., fouling). Thus, by growing CNTs on the surface of a conventional ISFET gate oxide and then functionalizing the CNTs with poly( ethylene glycol) alkyl ethers, the gate oxide may be utilized as the barrier for free chlorine and other redox species while the functionalized CNT would function as a pH sensing structure.

[00162] Figure 18 is a schematic illustration of functionalizing conventional ISFET gate oxides as a pH sensor. With reference to Figure 18, an exemplary surface structure 190 for a gate-oxide SiO₂ surface 192 may have deposited thereon a monolayer of octadecyl phosphonic acid 194. The resulting hydrophobic surface may then interact with poly(ethylene glycol) alkyl ethers to form an orderly surface structure 190. The non-specific adsorption and fouling problems associated with ISFET gate oxides may thus be eliminated by direct functionalization of the gate oxide. Of course, other approaches may be implemented to functionalize gate oxide surfaces and such an example should not limit the scope of the claims appended herewith. For example, SiO₂ may be functionalized with (3-aminopropyl) triethoxysilane. Further, subsequent amide formation with octadecanoic acid may result in a highly hydrophobic surface suitable for the non-covalent functionalization with poly(ethylene glycol) alkyl ethers as described above. The resulting hydrophilic surface may then be responsive to solution pH changes while maintaining superior fouling-resistant properties. Thus, such exemplary FET- based approaches may be employed to provide pH sensors free from redox species interferences and provide long-term stability therefore due to the presence of a highly ordered hydrophilic poly(ethylene glycol) surface.

[00163] Figure 19 is a simplified diagram of a pH sensing device or an exemplary amperometric biosensor. Figure 20 is a top view of the sensing electrode depicted in Figure 19. With reference to Figures 19 and 20, a biosensor or pH sensing device 200 is illustrated having a conductive layer 202 covering a portion(s) of a substrate (e.g., silicon or otherwise) 204. The biosensor 200 may include ohmic contacts 206 for the source and drain and may include a catalyst thin layer 208 as a node in a specific geometric shape on top of the conductive layer 202. The biosensor 200 may include any number of sensing electrodes 210 having functionalized carbon nanostructures thereon. For example, the sensing electrode 210 may have in one embodiment, one or more CNTs functionalized with a probe molecule(s), which responds to a target molecule(s) in solution. Exemplary probe molecules include, but are not limited to, DNA or RNA strands, DNA or RNA aptamers, proteins, enzymes, haptens, antibodies, cells and the like. The biosensor 200 may include a reference electrode 212 whereby the reference and sensing electrodes 212, 210 are in fluid contact with a surrounding environment (e.g., aqueous solution). The biosensor 200 may also include applicable hardware 214 such as, but not limited to, a potentiostat (e.g., polypotentiostat, etc.) to control the device and electrodes therein. The biosensor 200 may provide a stable current between the source and drain 206 at a given solution condition when a fixed potential is applied to the sensing electrode 210 flanked by the source and drain 206 on a silicon chip. When both the sensing and reference electrodes 210, 212 are in contact with an aqueous solution or fluid, the measured current between the source and drain 206 may be related to the concentration and/or identity of the target molecule(s). Probe molecules attached to the functionalized layer on an exemplary CNT electrode surface may thus provide sensitivity to target molecules in biological solution. The dashed lines 220 in Figure 20 indicate a conductive layer beneath the chip surface to complete an electric conduit between the source and drain contacts 206. The source and drain contacts 206 may be aligned on the same side of the chip or may be positioned on opposite sides thereof so long as the sensing electrodes 210 are between the source and drain contacts 206 in the electric circuit. An exemplary biosensor 200 may be, but is not limited to, a FET-based biosensor (e.g., a DNA FET, an enzyme FET, an Immuno FET, and the like).

[00164] Figure 21 is a graphical depiction of a current versus time for a carbon nanostructure sensing electrode functionalized using an embodiment of the present subject matter. With reference to Figure 21, current between exemplary source and drain contacts was measured and is shown changing over time with different solution pH after a stabilization period. To provide the results shown, solution pH (from 3 to 12) was adjusted in a reservoir with constant stirring. A pH sensing electrode and reference electrode were then exposed to a flow of solution from the reservoir whereby solution pH was monitored with a commercial glass pH meter to provide the results in Figure 21.

[00165] Figure 22 is a plot of current versus pH for an exemplary amperometric pH sensor. Figure 22 also is illustrative of the principle that an exemplary functionalized CNT electrode according to embodiments of the present subject matter may respond to solution pH change. Thus, For example, when both the sensing and reference electrodes are in contact with an aqueous solution, measured current between the source and drain may be substantially proportional to solution pH (see Figure 22). An exemplary functionalization layer on a CNT electrode surface may thus provide sensitivity to pH due, in part, to a self-aligned layered structure on the CNT surface and the hydrophilicity of the functionalization layer which facilitate the formation of a hydrated layer on the functionalized CNT electrode surface.

[00166] In one embodiment a pH sensing electrode is provided having an assembly of electrodes. This pH sensing electrode may include an electrically conductive layer covering a portion of a substrate and an assembly of functionalized carbon nanostmctures vertically oriented with respect to the electrically conductive layer, wherein each of the functionalized carbon nanostmctures may be functionalized CNTs. Exemplary functionalized CNTs may include a base end attached to an electrically conductive layer, a mid-section, and a top end disposed opposite the base end. An exemplary

functionalization layer may be attached to or contained within the outer surface of the mid-section and/or the top end. In one embodiment, the pH sensing electrode may be flanked by two electric contacts as a source and drain on a substrate. An exemplary electric resistance between the source and drain may be, but is not limited to, between 10 Ω and 2000 Ω. Through control of the electric resistance between the source and drain, the H response slope and thus the pH sensitivity of an exemplary pH sensor may be adjusted.

[00167] Figure 23 is a schematic illustration of an exemplary biosensor according to another embodiment. With reference to Figure 23, an exemplary biosensor 200 is illustrated having a conductive layer 202 covering a portion(s) of a substrate (e.g., silicon or otherwise) 204. The biosensor 200 may include ohmic contacts 206 for the source and drain and may include a catalyst thin layer 208 as a node in a specific geometric shape on top of the conductive layer 202. The biosensor 200 may include any number of sensing electrodes 210 having functionalized carbon nanostructures thereon. For example, the sensing electrode 210 may have in one embodiment, one or more CNTs functionalized with a probe molecule, which responds to a target molecule(s) in solution. The biosensor 200 may include a reference electrode (not shown) whereby the reference electrode and sensing electrode 210 are in fluid contact with a surrounding environment (e.g., aqueous solution). The biosensor 200 may also include applicable hardware to control the device and electrodes therein. The biosensor 200 may provide a stable current between the source and drain 206 at a given solution condition when a fixed potential is applied to the sensing electrode 210 flanked by the source and drain 206 on the substrate 204. When both the sensing electrode 210 and reference electrodes are in contact with an aqueous solution, the measured current between the source and drain 206 may be related to the concentration and/or identity of the target molecule(s). Probe molecules 201 attached to the functionalization layer on an exemplary CNT electrode surface may thus provide sensitivity to target molecules 203 in biological solution.

[00168] With reference to Figures 19, 20 and 23, an exemplary sensing electrode 210 may also include an assembly of electrodes each having an electrically conductive layer 202 at least partially surmounting a substrate 204 and an assembly of functionalized carbon nanostructures vertically oriented with respect to the electrically conductive layer 202. In one embodment, the nanostructures include functionalized CNTs having a base end attached to the electrically conductive layer 202, a mid-section, and a top end distal the base end. For example, the CNTs may include an electrically conductive layer covering a portion or all of a substrate and may include an assembly of undoped CNT antennae vertically oriented with respect to the electrically conductive layer. Any or each of the undoped CNT antennae may include a base end attached to the electrically conductive layer, a mid-section having an outer surface surrounding a cavity or channel therein (i.e., lumen), and a top end disposed opposite the base end. In one embodiment, the outer surface of the mid-section may be in fluidic contact with an environment (e.g., a liquid solution) that is in contact with the CNT antennae.

[00169] As illustrated in Figure 23, an exemplary electrode 210 may be

functionalized with probe molecules 201 whereby a current 211 passes through the conductive layer 202 when a potential is applied to the electrode 210. Exemplary probe molecules include but are not limited to, DNA or RNA strands, DNA or RNA aptamers, proteins, enzymes, haptens, antibodies, cells, and the like. When target molecules 203 (e.g., DNA) in solution interact with the probe molecules 201, the interaction (e.g., binding) therebetween may cause the surface of the electrode 210 surface to change its surface properties such as, for example, a change in charge distribution, surface hydrophilicity, and the like, which in turn may lead to a change in interfacial potential. Consequently, the current 213 between the source and drain 206 may also change.

[00170] One embodiment of the present subject matter is directed to fabricating an exemplary sensor (e.g., pH sensor, biosensor, FET-based biosensor, etc.) including fabricating the underlying silicon chip, growing or depositing appropriate carbon nanostructures such as CNTs, and functionalizing the surface of such nanostructures. In this embodiment, the sensor fabrication technique includes fabricating a substrate (e.g., silicon chip) as described in International Application No. PCT/US07/02104, the entirety of which is incorporated herein by reference. In this method, an insulating layer (e.g., SiO₂ or the like) may be deposited on top of a silicon substrate. A conductive layer having a defined geometry may be deposited and may be situated between two terminals, one serving as a source and the other as a drain. Thus, an exemplary conductive layer may act as an interconnect for CNT nodes and/or may act as an electric conduit between the source and drain. A barrier layer (e.g., Ti or the like) may be deposited on the conductive layer area to prevent segregation of subsequent catalyst material from the conductive layer. A thin catalyst layer (e.g., Ni, Fe or Co, etc.) may then be deposited and patterned by conventional lithography to form nodes of catalyst in a defined geometric shape (e.g., circle, rectangle, strips, etc.) with appropriate insulating layers (SiO₂, Si₃N₄, etc.) surrounding the nodes of catalyst. The insulating layers may be used to ensure the conductive layer is not exposed to solution in the pH sensing electrode.

[00171] Exemplary nanostructures (e.g., CNTs) as described herein may then be grown on the underlying substrate by any number of methods including, but not limited to, an exemplary CVD process described in PCT/US07/02104 and may be, in one embodiment, undoped aligned CNTs assemblies. Other methods may include an exemplary arc discharge process, laser-ablation process, natural, incidental and/or controlled flame environments, plasma enhanced chemical vapor deposition, a capacitively coupled microwave plasma process, a capacitively coupled electron cyclotron resonance process, a capacitively coupled radio frequency process, an inductively coupled plasma process, a dc plasma assisted hot filament process, template synthesis, carbo thermal carbide conversion, or combinations thereof, to name a few.

[00172] The CNTs may then be functionalized as described herein and in copending Application No. _/ and International Application No.

PCT/US2010/056350, the entirety of both incorporated herein by reference. Such exemplary CNT surface functionalization process may provide chemical and structural stability for the assembly electrodes and surface hydrophilicity and biocompatibility which is necessary for pH and biosensor applications. These functionalized CNT electrodes may then be assembled into a pH sensing device and used to measure the pH of aqueous solutions. Thus, when both reference electrode and the functionalized CNT electrode are in contact with an aqueous solution and a given potential is applied to the CNT electrode, a measured current between the source and drain may be proportional to the solution pH or concentration and/or identity of a target molecule(s).

[00173] Functionalization also provides ample functional groups for subsequent attachment of probe molecules onto an exemplary electrode surface. Exemplary functionalization may include, but is not limited to, the introduction of various functional groups such as -OH, -COOH, -NH₂, -NHR, -SH, -S-S-R, -C≡CH, -N₃, -CN, -CHO, -CONH-NH₂, a maleimido group, and other functional moieties such as redox mediator structures. These functional groups may also be further derivatized to form covalent bonds with other functional moieties (probe molecules) including, but not limited to, redox mediator molecules, crown ethers, catalysts, boric acids, carbohydrates,

oligonucleotides (DNAs and RNAs), DNA or R A aptamers, peptide aptamers, proteins such as enzymes and antibodies, quantum dots, and nanoparticles to name a few. The functionalized CNT electrode may then be assembled into a sensing device to sense biomolecules in an aqueous solution. In one embodiment, attachment of probe molecules onto an exemplary CNT electrode surface may be accomplished after the sensing device assembly is completed. In this embodiment, when the probe molecule is in place on CNT electrode surface and both reference electrode and the functionalized sensing electrode are in contact with an aqueous solution, a given potential may be applied to the sensing electrode. The measured current between the source and the drain may then correspond to the presence and concentration of target biomolecules in solution. While reference has been made herein to fluids and/or aqueous solutions, such terminology should not limit the scope of the claims appended herewith as these terms may refer to any type of a substance that deforms (flows) under any amount of applied shear stress.

[00174] In another embodiment, a method is provided for generating an assembly of electrodes. The method may include depositing an electrically conductive layer onto a substrate, and providing or growing an assembly of functionalized carbon nano structures on the electrically conductive layer. These nanostructures may be vertically oriented with respect to the electrically conductive layer and may be, in one embodiment, CNTs having a base end attached to the electrically conductive layer and a mid-section comprising an outer surface surrounding a lumen, where at least a portion of the outer surface of the mid-section may be in fluidic contact with an environment (e.g., a liquid solution). The nanostructure may also include a top end disposed opposite the base end. A portion of the CNT may be treated with functionalization layers, a covalent bond linkage, a functional dopant molecule, a fill material, or any combination thereof.

[00175] Exemplary sensing electrodes according to embodiments of the present subject matter may be, but are not limited to, any carbon- forming electrode made of carbon nanotubes, single walled or multi-walled nanotubes, carbon nanotube pastes, glassy carbon or highly ordered basal plane pyrolytic graphite, highly ordered edge plane pyrolytic graphite, graphene or fullerene nanostructure, conductive diamond formed via thermal chemical vapor deposition, arc discharge process, laser-ablation process, natural, incidental and controlled flame environments, plasma enhanced chemical vapor deposition, a capacitively coupled microwave plasma process, a capacitively coupled electron cyclotron resonance process, a capacitively coupled radiofrequency process, an inductively coupled plasma process, a dc plasma assisted hot filament process, template synthesis, carbo thermal carbide conversion, and/or any combination thereof.

[00176] An exemplary CNT sensing electrode may include one or more nodes of a CNT or an ensemble of CNTs connected to the conductive layer on the substrate. Each node may be in various dimensions ranging from, for example, 1 nm to an ensemble of CNTs several cm in any geometric shape (e.g., bands, circles, grids, loops, meshes, rectangles, squares, stripes, or their combinations, etc.) between the source and drain. Of course, the length of CNTs may vary from tens of microns to sub-microns. The CNT sensing electrode may also include an array of nodes that vary from a few nodes to as many as hundreds of thousands of nodes with or without a pitch (i.e., distance between the center of neighboring nodes) ranging from sub-microns to several thousands of microns. Of course, such an array of nodes may be in any pattern (e.g., bands, circles, grids, loops, meshes, rectangles, squares, stripes, or their combinations, etc.) between the source and drain.

[00177] One exemplary CNT-based amperometric sensor may be employed to continuously monitor solution pH in a fluid or other environment. Such a system may include a processing unit wirelessly (or via wire-line) coupled to the pH sensor and at least one communication unit being configured to operate in conjunction with the pH sensor to monitor the fluid. Of course, the communication unit may be configured to report pH sensor measurements and other data to a remote communication device, which may transmit this information to a user, server, processor, etc. Thus, embodiments of the present subject matter including any type of sensor or combinations thereof may include some form of real-time remote monitoring and reporting of pH in an environment.

[00178] An additional embodiment of the present subject matter may have utility in a pH monitoring and control system. Such a system may include one or more CNT-based pH sensors (voltammetric, potentiometric, amperometric, etc.) located within a water treatment system or within a part of a water treatment device being monitored. The sensor may include appropriate measurement circuitry (ammeter, voltmeter, etc.) to measure current between a source and drain, conversion circuitry (if necessary) to convert analog measurement signals into digital signals, a transceiver or transmitter to wirelessly (or via wire-line) provide these digital signals to a remote location, device, processor, etc. for a real-time or delayed analysis of the water treatment system. An exemplary system may also include control circuitry for controlling the pH in the respective water treatment system based on such data analysis from the centralized unit to maintain the proper pH in the water treatment system and/or to determine whether the applicable dosing units are functioning properly.

[00179] As exemplary pH sensors according to embodiments of the present subject matter are suitable for long-term continuous monitoring of solution pH while requiring no routine calibration and maintenance, water quality measurements may be gathered in real time. Such real-time data, whether in the form of raw data or analyzed results, of water quality in a respective water distribution system may improve system performance and reduce costs. In municipal, industrial, commercial, and residential applications, the need to remotely monitor water treatment systems and devices has also increased dramatically to ensure water treatment systems or device are operating properly and providing water of a certain quality. Therefore, it is an aspect of embodiments of the present subject matter to provide a monitoring, feedback and/or control system having one or more CNT- based pH sensors located within a water treatment system or portion thereof. Through the data measured and provided by such sensors, appropriate circuitry may be employed to control and monitor the pH of the respective system to assure compliance with water quality standards.

[00180] While embodiments have been heretofore described in connection with amperometric pH sensors, potentiometric pH sensors, voltammetric pH sensors, electrodes and other sensors, the scope of the claims appended herewith should not be so limited. For example, it is envisioned that embodiments of the present subject matter may find utility in the biometric industry and as intracorporeal electrodes and sensors.

[00181] Additionally, data, commands and other information or messages may be sent or received, wirelessly or via wire-line depending upon the application, from or to various electrodes and/or sensors utilizing an exemplary system. For example, an exemplary monitoring system may collect information from a sensor monitoring the pH of a remote or local fluid system and may provide such information to a user or to a database for real-time or stored use. Further, an exemplary monitoring system may collect information transmitted wirelessly from an intracorporeal sensor or matrix of sensors or electrodes. Such provision (i.e., transmission) of information may be via any known mode of transmission (e.g., wireless or wire-line, as applicable). Such

information may also be provided directly to a user or may be provided to a user via a processor for manipulation and/or storage thereof. Of course, the processor and supporting systems may also be employed to provide messages and/or commands to the remote or local sensor or electrode as the need arises. Thus, it is envisioned that embodiments may be implemented using a general purpose computer programmed in accordance with the principals discussed herein. It is also envisioned that embodiments of the subject matter and the functional operations described in this specification may be implemented in or utilize digital electronic circuitry, or in computer software, firmware, or hardware, including the structures disclosed in this specification and their structural equivalents, or in combinations of one or more of them. Thus, embodiments of the subject matter described in this specification can be implemented in or utilize one or more computer program products, i.e., one or more modules of computer program instructions encoded on a tangible program carrier for execution by, or to control the operation of, data processing apparatus. The tangible program carrier can be a computer readable medium. The computer readable medium can be a machine-readable storage device, a machine-readable storage substrate, a memory device, or a combination of one or more of them.

[00182] To note, the term "processor" encompasses all apparatus, devices, and machines for processing data, including by way of example a programmable processor, a computer, or multiple processors or computers. The processor can include, in addition to hardware, code that creates an execution environment for the computer program in question, e.g., code that constitutes processor firmware, a protocol stack, a database management system, an operating system, or a combination of one or more of them.

[00183] A computer program (also known as a program, software, software application, script, or code) can be written in any form of programming language, including compiled or interpreted languages, or declarative or procedural languages, and it can be deployed in any form, including as a standalone program or as a module, component, subroutine, or other unit suitable for use in a computing environment. A computer program does not necessarily correspond to a file in a file system. A program can be stored in a portion of a file that holds other programs or data (e.g., one or more scripts stored in a markup language document), in a single file dedicated to the program in question, or in multiple coordinated files (e.g., files that store one or more modules, sub programs, or portions of code). A computer program can be deployed to be executed on one computer or on multiple computers that are located at one site or distributed across multiple sites and interconnected by a communication network.

[00184] Of course, the general processes described by monitoring systems herein may be performed by one or more programmable processors executing one or more computer programs to perform functions by operating on input data and generating output. These processes may also be performed by special purpose logic circuitry, e.g., a field programmable gate array (FPGA) or an application specific integrated circuit (ASIC). Processors suitable for the execution of an exemplary computer program include, by way of example, both general and special purpose microprocessors, and any one or more processors of any kind of digital computer. Generally, a processor will receive instructions and data from a read only memory or a random access memory or both. The essential elements of a computer are a processor for performing instructions and one or more data memory devices for storing instructions and data. Generally, a computer will also include, or be operatively coupled to receive data from or transfer data to, or both, one or more mass storage devices for storing data, e.g., magnetic, magneto optical disks, or optical disks. However, a computer need not have such devices.

Moreover, a computer can be embedded in another device, e.g., a mobile telephone, a personal digital assistant (PDA), a mobile audio or video player, a game console, a Global Positioning System (GPS) receiver, to name just a few.

[00185] Computer readable media suitable for storing computer program

instructions and data include all forms of data memory including no n- volatile memory, media and memory devices, including by way of example semiconductor memory devices, e.g., EPROM, EEPROM, and flash memory devices; magnetic disks, e.g., internal hard disks or removable disks; magneto optical disks; and CD ROM and DVD- ROM disks. The processor and the memory can be supplemented by, or incorporated in, special purpose logic circuitry.

[00186] To provide for interaction with a user, exemplary systems according to embodiments of the subject matter may be implemented on a computer having a display device, e.g., a cathode ray tube (CRT) or liquid crystal display (LCD) monitor, for displaying information to the user and a keyboard and a pointing device, e.g., a mouse or a trackball, by which the user can provide input to the computer. Other kinds of devices can be used to provide for interaction with a user as well; for example, input from the user can be received in any form, including acoustic, speech, or tactile input.

[00187] Embodiments of the subject matter described in this specification may also be implemented in a computing system that includes a back end component, e.g., as a data server, or that includes a middleware component, e.g., an application server, or that includes a front end component, e.g., a client computer having a graphical user interface or a Web browser through which a user can interact with an implementation of the subject matter described is this specification, or any combination of one or more such back end, middleware, or front end components. The components of the system may be interconnected by any form or medium of digital data communication, e.g., a

communication network. Examples of communication networks include a local area network (LAN) and a wide area network (WAN), e.g., the Internet. The computing system may also include clients and servers as the need arises. A client and server are generally remote from each other and typically interact through a communication network. The relationship of client and server arises by virtue of computer programs running on the respective computers and having a client-server relationship to each other.

[00188] It may be emphasized that the above-described embodiments, particularly any "preferred" embodiments, are merely possible examples of implementations, merely set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiments of the disclosure without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and the present disclosure and protected by the following claims.

[00189] While this specification contains many specifics, these should not be construed as limitations on the scope of the claimed subject matter, but rather as descriptions of features that may be specific to particular embodiments. Certain features that are described in this specification in the context of separate embodiments can also be implemented in combination in a single embodiment. Conversely, various features that are described in the context of a single embodiment can also be implemented in multiple embodiments separately or in any suitable sub-combination. Moreover, although features may be described above as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can in some cases be excised from the combination, and the claimed combination may be directed to a subcombination or variation of a sub-combination. [00190] As shown by the various configurations and embodiments illustrated in Figures 1-23, a carbon nanostructure sensor and method for biomolecule sensing have been described.

[00191] While preferred embodiments of the present subj ect matter have been described, it is to be understood that the embodiments described are illustrative only and that the scope of the invention is to be defined solely by the appended claims when accorded a full range of equivalence, many variations and modifications naturally occurring to those of skill in the art from a perusal hereof.

Claims

What is claimed is:

1. A device for measuring a biological target species in a fluid comprising: a reference electrode in communication with a fluid;

a sensing electrode in communication with the fluid and disposed between a first electrical contact and a second electrical contact;

wherein the sensing electrode includes one or more carbon nanostmctures functionalized with a chemically stable moiety that responds to a biological target species in the fluid when a potential is applied across the first and second electrical contacts to provide a current correlating to a concentration of the target species in the fluid.

2. The device of Claim 1 wherein the sensing electrode further comprises an aligned or non-aligned carbon nanotube assembly including:

an electrically conductive layer covering a portion of a substrate; and

an assembly of functionalized carbon nanotubes substantially orthogonal to a plane formed by the electrically conductive layer, wherein each of the functionalized carbon nanotubes includes:

a proximate base end attached to the electrically conductive layer, a mid-section having an outer surface in communication with the fluid, and a distal top end opposite the base end,

wherein the outer surface and top and base ends form a lumen.

3. The device of Claim 1 wherein the chemically stable moiety is an alkyl protective moiety selected from the group consisting of linear alkanes, branched alkanes, alkenes, alkenes containing 10 to 50 carbon atoms, alkenes substituted with one or more halogen atoms, n-octadecane, n-dodecane, eicosane and hexatriacontane, and

combinations thereof.

4. The device of Claim 1 wherein the sensing electrode includes one or more carbon nanostmctures functionalized with a bipolar molecule having functional groups or functional moieties.

5. The device of Claim 1 wherein the chemically stable moiety is selected from the group consisting of redox mediator molecules, crown ethers, catalysts, boric acids, carbohydrates, oligonucleotides, DNA apatmers, RNA aptamers, peptide aptamers, proteins, enzymes, antibodies, quantum dots, nanoparticles, cells, cell organelles, or other cellular components, and combinations thereof.

6. The device of Claim 1, wherein the sensing electrode further comprises carbon nanotubes grown on a metal catalyst.

7. The device of Claim 6, wherein the metal catalyst includes an element selected from the group consisting of Ni, Fe, Co, or any combination thereof.

8. The device of Claim 1, wherein the carbon nanostructures are selected from the group consisting of single-walled carbon nanotubes, multi-walled carbon nanotubes, conductive, semi-conductive, or insulated carbon nanotubes, chiral, achiral, open headed, capped, budded, coated, uncoated, functionalized, anchored, or unanchored carbon nanotubes, amorphous carbon, graphene, edge plane highly oriented pyroptic graphite, basal plane highly oriented pyroptic graphite, or conductive diamond nanotubes, and combinations thereof.

9. The device of Claim 1, wherein the sensing electrode further comprises one or more nodes, each node having a carbon nanotube or an ensemble of carbon nanotubes.

10. The device of Claim 9 wherein the one or more nodes are arranged in bands, circles, grids, loops, meshes, rectangles, squares, stripes, etc, or any combination thereof.

11. The device of Claim 1 wherein the carbon nanostructure includes one or more cross-linking layers.

12. A method for measuring a biological target species in a fluid comprising the steps of:

providing a sensor, the sensor having a reference electrode and a sensing electrode, the sensing electrode disposed between a first contact and a second contact; applying a potential across the reference and sensing electrodes;

measuring current resulting from the applied potential; and

determining a concentration of a biological target species in the fluid as a function of the measured current.

13. The method of Claim 12 wherein the sensor is a field effect transistor (FET) biosensor.

14. The method of Claim 12 wherein the sensing electrode comprises a carbon nanotube assembly including an electrically conductive layer and an assembly of functionalized antennae vertically oriented with respect to the electrically conductive layer.

15. The method of Claim 14 wherein the carbon nanotube assembly includes nanotubes selected from the group consisting of single-walled carbon nanotubes, multi- walled carbon nanotubes, conductive, semi-conductive, or insulated carbon nanotubes, chiral, achiral, open headed, capped, budded, coated, uncoated, functionalized, anchored, or unanchored carbon nanotubes, amorphous carbon, graphene, edge plane highly oriented pyroptic graphite, basal plane highly oriented pyroptic graphite, or conductive diamond nanotubes, and combinations thereof.

16. The method of Claim 14 wherein the carbon nanotube assembly further comprises a first layer having an alkyl protective moiety selected from the group consisting of linear alkanes, branched alkanes, alkenes, alkenes containing 10 to 50 carbon atoms, alkenes substituted with one or more halogen atoms, n-octadecane, n- dodecane, eicosane and hexatriacontane, and combinations thereof.

17. The method of Claim 14 wherein the carbon nanotube assembly further comprises a second layer having a bipolar molecule with functional groups or functional moieties.

18. The method of Claim 14 wherein the carbon nanotube assembly further comprises functional groups or functional moieties selected from the group consisting of redox mediator molecules, crown ethers, catalysts, boric acids, carbohydrates,

oligonucleotides, DNA apatmers, RNA aptamers, peptide aptamers, proteins, enzymes, antibodies, quantum dots, nanoparticles, cells, cell organelles, or other cellular components, and combinations thereof.

19. The method of Claim 12 wherein the step of providing a sensor further comprises the step of growing a carbon nanostmcture on a substrate by a process selected from the group consisting of chemical vapor deposition, arc discharge process, laser- ablation process, natural flame environment, incidental flame environment, controlled flame environments, plasma enhanced chemical vapor deposition, capacitively coupled microwave plasma process, capacitively coupled electron cyclotron resonance process, capacitively coupled radiofrequency process, inductively coupled plasma process, dc plasma assisted hot filament process, template synthesis, carbo thermal carbide conversion, and combinations thereof.

20. The method of Claim 12 further comprising the step of controlling electric resistance between the first and second contacts to adjust sensitivity of the sensor.

21. A system for measuring a concentration of a biological target species in a fluid comprising:

a sensor for measuring a concentration of a biological target species in a fluid having:

a reference electrode in communication with the fluid, and

a sensing electrode in communication with the fluid and disposed between a first electrical contact and a second electrical contact, wherein the sensing electrode includes one or more carbon nanostructures functionalized with a chemically stable moiety that responds to a biological target species in the fluid when a potential is applied across the first and second electrical contacts;

circuitry for measuring a current resulting from the applied potential and for providing an output signal, the measured current correlating to the concentration of the biological target species in the fluid; and

a transmitter for transmitting the output signal to a location remote from the sensor.

22. The system of Claim 21 wherein the transmitter is a wireless or wire-line transmitter.

23. The system of Claim 21 further comprising a converter for converting the output signal into a digital signal.

24. The system of Claim 21 wherein the sensing electrode comprises a carbon nanotube assembly including an electrically conductive layer and an assembly of functionalized antennae vertically oriented with respect to the electrically conductive layer.

25. The system of Claim 21 wherein the carbon nanostmctures are selected from the group consisting of single-walled carbon nanotubes, multi-walled carbon nanotubes, conductive, semi-conductive, or insulated carbon nanotubes, chiral, achiral, open headed, capped, budded, coated, uncoated, functionalized, anchored, or unanchored carbon nanotubes, amorphous carbon, graphene, edge plane highly oriented pyroptic graphite, basal plane highly oriented pyroptic graphite, or conductive diamond nanotubes, and combinations thereof.

26. The system of Claim 21 wherein the chemically stable moiety is an alkyl protective moiety selected from the group consisting of linear alkanes, branched alkanes, alkenes, alkenes containing 10 to 50 carbon atoms, alkenes substituted with one or more halogen atoms, n-octadecane, n-dodecane, eicosane and hexatriacontane, and

combinations thereof.

27. The system of Claim 21 wherein the sensing electrode includes one or more carbon nanostmctures functionalized with a bipolar molecule having functional groups or functional moieties.

28. The system of Claim 21 wherein the chemically stable moiety is selected from the group consisting of redox mediator molecules, crown ethers, catalysts, boric acids, carbohydrates, oligonucleotides, DNA apatmers, RNA aptamers, peptide aptamers, proteins, enzymes, antibodies, quantum dots, nanoparticles, cells, cell organelles, or other cellular components, and combinations thereof.