Patent Application
20100066346
1. Field of the Invention
The present invention is directed to nanopillar-enhanced microstructures, their methods of use, and processes for developing nanopillar-enhanced electrodes.
2. Background Art
Biosensors are important devices for monitoring biological species in various processes of environmental, fermentation, food and medical concerns. The main challenges biosensors face include low sensitivity, poor specificity and proneness to fouling. The advent of nanotechnology presents promising solutions for alleviating these problems.
In a typical glucose biosensor, an enzyme, such as glucose oxidase, is immobilized onto the electrode surface [1,2]. The performance of such functionalized electrodes can be improved by either adjusting the spatial distribution of the enzyme or by modifying the morphology of the electrode surface. To achieve a high efficiency in immobilizing an enzyme onto the electrode surface, various techniques have been developed, such as the use of self-assembled monolayer [1-4], conducting polymers [5,6] and sol-gels [7]. Among these methods, the self-assembled monolayer (SAM) approach offers a better control for enzyme distribution at the molecular level, a high degree of reproducibility in enzyme immobilization and a short distance between the immobilized enzyme and the electrode surface [1,4]. The SAM approach, however, is limited by the amount of the enzyme that can be immobilized onto the electrode surface, which in turn will affect the sensing performance of the biosensor [8]. To increase the amount of immobilized enzyme various nanostructures such as nanopillars, nanoparticles and nanorods have been explored in order to increase the active surface area of the electrodes.
For example, nanostructures like gold nanopillars [8], carbon nanopillars [5,9] and gold nanoparticles [10] have been incorporated into electrode surfaces and they exhibited better performance than conventional flat electrodes.
Recently Wang et al. [11] used nanostructured platinum electrodes functionalized with glucose oxidase for glucose detection. These electrodes showed a significant (two orders of magnitude) increase in glucose detection sensitivity as compared with a flat electrode, but the response of these electrodes to K4Fe(CN)6 was just 2.3 times that of the flat electrode. They attributed such sensitivity enhancements for glucose detection to the increased enzyme loading and improved retention of hydrogen peroxide near the electrode surface without examining systematically the role of reaction kinetics and mass transport. It is theorized that the electrical current response of these nanostructured electrodes is controlled by reaction kinetics, mass transport and the geometric topography of the nanostructures.
Surface acoustic wave (SAW) sensors are microelectromechanical (MEMS) systems in which the acoustic wave travels along the surface of a piezoelectric substrate. Interdigitated transducers (IDTs) are placed on the surface of a piezoelectric substrate to generate and receive the acoustic waves. The area between the generator and receiver IDTs is very sensitive to surface perturbation like mass loading. In a SAW sensor, this area is generally coated with a chemically selective layer for adsorption of analyte species. SAW based sensors have been widely used for gaseous, chemical and biological species detection. With the advent of nanotechnology, efforts have been made to increase the sensitivity of SAW sensors by integrating nanostructures on the active surface of the sensors [16-19].
The present invention provides nanopillar-enhanced structures, methods for fabricating the same, and methods for using nanopillar-enhanced structures.
In one aspect, the present invention provides nanopillar enhanced electrodes for glucose detection. The electrodes are defined by an active three-dimensional (3D) surface formed by arrays of nanopillars standing on a flat support base. In some embodiments, the outer surface of the nanopillars is further functionalizes with glucose oxidase through either self assembly monolayer (SAM) molecules or polypyrrole polymer. In one embodiment, the pyrrole polymerization is carried out by continuous pumping of an electrolyte containing pyrrole and glucose oxidase.
In another aspect, the present invention provides methods for fabricating nanopillar-enhanced electrodes. In some embodiments, the nanopillar electrodes are fabricated by first coating a silicon wafer with several thin layers of metallic film and anodizing the top layer to form a nanoporous template, followed by electrodeposition of gold nanopillars and removal of the template. Nanopillars prepared by the process described herein are formed via metallic bonds, leading to superior mechanical properties. The resulting smooth nanoscopic surface of the nanopillars aids in the minimization of the surface tension, leading to the resistance of the nanostructures to the capillary interaction forces. Stated otherwise, nanopillars fabricated by electrodeposition are resistant to deformation by capillary forces generated between the vertically aligned nanostructures and liquid medium.
Some embodiments of the present invention provide a process for fabrication of integrated structures of micro- and nano-scale features on a surface. In one embodiments, the nanopillar-enhanced surface can be micropatterned using conventional microfabrication techniques to produce a desired micro-pattern.
Another aspect of the invention provides a micro flow-channel glucose sensor with microscale-interdigitated planar electrodes incorporated with nanopillars. In one embodiment, the nanopillar-enhanced sensor comprises a micro-flow-channel design with an interdigitated arrangement (a working electrode is placed next to a detector electrode in an alternating manner) of micro planar electrodes enhanced by nanopillars. The micro-flow-channel design provides a convective flow for mass transport, whereas the current response of the planar electrodes are further enhanced by the addition of nanopillars and interdigitated arrangement.
In another embodiment, the structures of micro- and nano-scale features fabricated by the process of the present invention are used as integrated elements in surface acoustic wave (SAW) based biosensor. The active surface of a SAW sensor described by the present invention is integrated with standing nanopillars with adjustable diameter and spacing in a process that is microfabrication compatible. Nanopillars formed by the electrochemical anodization and deposition are capable of withstanding capillary forces generated by the nanostructure-liquid interactions, and are ideally suited for sensing applications in aqueous environments. With such a SAW sensor, a multi-fold increase in detection sensitivity is achieved.
Another aspect of the present invention provides methods of use of the structures of micro- and nano-scale features described herein. In one embodiment, the micro/nano-structures of the present invention can be used in biosensors. In one embodiment, said biosensors can be used for remote detection of biological warfare agents (i.e., anthrax). In another embodiment, the biosensors with the integrated structures of micro- and nano-scale features fabricated by the process of the present invention can be used for in-vitro and ex-vivo monitoring of bioanalytes.
a illustrates, schematically, a nanopillar-enhanced electrode.
b illustrates, schematically, a nanopillar-enhanced electrode functionalized with glucose-oxidase with the use of anchoring molecule such as SAM or polypyrrole.
a illustrates, schematically, a micro flow-channel glucose sensor with microscale-interdigitated nanopillar-enhanced planar electrodes.
b illustrates, schematically, a micro flow-channel glucose sensor equipped with a pump.
a illustrate, schematically, a conventional design for the microflow channel biosensor.
b illustrate, schematically, a microinterdigitated design for the microflow channel biosensor.
a illustrates amperometric current responses obtained for the bare NAEs and flat electrode when incremental drops of K4Fe(CN)6 were added to the solution.
b provides calibration curves obtained based on a linear regression analysis for the relationship between the steady-state current and K4Fe(CN)6 concentration.
a illustrates amperometric current responses obtained for the functionalized NAEs and flat electrode when incremental drops of glucose were added to the solution.
b provides calibration curves obtained based on a linear regression analysis for the relationship between the steady-state current and glucose concentration (from 2.5 mM to 15 mM).
a illustrates simulated currents responses for a functionalized NAEs electrode and a flat electrode at a reaction constant of 5×10−5 m/s and 5×10−7 m/s.
b provides a contour plot for glucose concentration near the electrode at a reaction constant of 5×10−7 m/s.
c provides a contour plot for K4Fe(CN)6 concentration near the electrode at a reaction rate constant of 5×10−4 m/s.
a illustrates a collection efficiency obtained at different electrode designs.
b illustrates a conversion efficiency obtained at different electrode designs.
c illustrates an amperometric current obtained for different electrode designs.
a illustrates a current response for 2.5 mM glucose by different polypyrrole deposition procedures in nanopillar electrodes.
b illustrates current response for 2.5 mM glucose by different polypyrrole deposition procedures in flat gold electrodes.
a illustrates amperometric current responses at various glucose concentrations for the flat electrodes.
b illustrates a calibration plot for the flat electrodes.
a illustrates amperometric current responses at various glucose concentration for the nanopillar electrodes.
b illustrates a calibration curve for nanopillar electrodes.
It is understood that the illustrations and figures of the present application are not necessarily drawn to scale and that these figures and illustrations merely illustrate, but do not limit, the present invention.
In the following description, for purposes of explanation, specific numbers, materials and configurations are set forth in order to provide a thorough understanding of the invention. It will be apparent, however, to one having ordinary skill in the art that the invention can be practiced without these specific details. In some instances, well-known features can be omitted or simplified so as not to obscure the present invention.
The embodiment(s) described, and references in the specification to “one embodiment”, “an embodiment”, “an example embodiment”, etc., indicate that the embodiment(s) described can include a particular feature, structure, or characteristic, but every embodiment may not necessarily include the particular feature, structure, or characteristic. Moreover, such phrases are not necessarily referring to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is understood that it is within the knowledge of one skilled in the art to effect such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described.
As used herein, the term “biosensor” refers to a device for the detection of an analyte that combines a biological component with a physicochemical detector component. The term “analyte” refers to a naturally occurring and/or synthetic compound, which is a marker for a condition (i.e., drug abuse), disease state (i.e., infection disease), disorder (i.e., neurological disorder), or a normal or pathologic process that occurs in a patient (i.e., drug metabolism). The term “analyte”, as used herein, can refer to any substance, including chemical and/or biological agents, that can be measured in an analytical procedure. Biosensors have potential use as a method of detection in many areas, including environmental, fermentation, food and medical areas. Biosensors could be used for in vivo or in vitro sensing in humans or animals. Currently, biosensors have a tendency to have low sensitivity, poor specificity and are prone to fouling. The biosensors of the present invention alleviate these problems.
One aspect of the present invention, as illustrated in
In one embodiment, the nanopillar-enhanced electrode comprises a 3D surface 100 formed by arrays of nanopillars 110 standing on a solid flat support base 120. Said 3D surface is used as the active surface for the electrochemical reactions. The 3D surface of the present invention can be made of any suitable metal, reduced or oxidized form of a metal, or metal alloys. In some embodiments, a suitable metal of the present invention comprises metals, metal oxides, and metal alloys such as, but not limited to, gold, silver, platinum, aluminum, aluminum oxide, copper, palladium, or combinations thereof.
Base 120 can be any substantially flat or planar material. In certain embodiments, base 120 is a glass disk or a glass plate. In other embodiments, base 120 is a silicon chip or wafer. In other embodiments, base 120 can be a ceramic or concrete plate that has been manufactured to be substantially flat. Although reference is made above to disks, plates, or chips, it is understood that the nano-enhanced electrodes of the present invention can be formed onto any shaped base 120, so long as that base 120 is substantially flat or planar. The term “substantially flat or planar” as used herein means an active surface that is uniformly flat or planar. It is understood that under today's conventional manufacturing techniques no surface is perfectly flat or planar. Some irregularities on the surface is acceptable.
The term “electrochemical reaction”, as used herein, refers to any chemical reaction that takes place in a solution at the interface of an electron conductor (a metal or a semiconductor) and an ionic conductor (the electrolyte), and that elicits chemical potential by the means of electron transfer between the electrode and the electrolyte or species in solution.
In another embodiment, a preferred feature of the invention is to functionalize the outer surface of at least one of the nanopillar-enhanced electrodes. In one embodiment, the electrodes are functionalized with macromolecules 130 on the surface. Examples of the macromolecules include, but are not limited to, any biomolecule capable of accelerating a reduction/oxidation chemical transformation utilizing any known redox co-factor. One example of a macromolecule suitable for use in the present invention is glucose oxidase.
An increase in a detection sensitivity obtained with the electrodes of the present invention will be immediately appreciated by people skilled in the art. In some embodiments, the nanopillar-enhanced electrodes are characterized by an increased sensitivity in an analyte detection of at least 2-fold from that of a flat electrode. In one embodiment, the nanopillar-enhanced electrodes are characterized by an increased sensitivity in an analyte detection of at least 10-fold from that of a flat electrode. In yet another embodiment, the detection sensitivity of the nanopillar-enhanced is 100-fold higher than that of a flat electrode. As used herein, the term “flat electrode”, “planar electrode”, or “microplanar electrode” are used interchangeably to refer to an electrode that has not been enhanced by any nanostructures.
Previously, Delvaux et al. reported a sensitivity of a gold nanostructure-enhanced electrode for glucose detection to be 0.4 μA·mM−1·cm−2 [8]. In some embodiments, the sensitivity of the nanopillar-enhanced electrodes of the present invention in glucose detection is at least 2-fold higher than that of the gold nanostructure-enhanced electrode reported by Delvaux. In one embodiment, the sensitivity of the nanopillar-enhanced electrodes of the present invention in a glucose detection is at least 10-fold higher than the gold nanostructure-enhanced electrode reported by Delvaux. In yet another embodiment, the sensitivity of the nanopillar-enhanced electrodes of the present invention in glucose detection is at least 100-fold higher than that of the gold nanostructure-enhanced electrode reported by Delvaux.
Another aspect of the present invention describes a process for fabricating the nanopillar-enhanced electrodes, which comprises:
1) developing a nanoporous template by anodizing an aluminum sheet,
2) electrodepositing gold nanopillars, and
3) removing the template.
To date, a number of techniques have been described for fabricating various nanostructures. Among them, chemical vapor deposition technique (CVD), physical vapor deposition technique (PVD), and template-based electrodeposition technique are the most commonly used methods. One of the major disadvantages of nanostructures prepared by CVD or PVD is their inability to sustain the capillary forces generated by the nanostructure-liquid interactions. When vertically aligned nanostructures are exposed to a liquid environment, capillary forces are generated between the vertically aligned nanostructures and the liquid medium. Often, the nanostructures are unable to sustain these forces, leading to their deformation or bunching. As a consequence of such deformations, the magnitude of increase in the surface area is drastically reduced, posing a serious problem for utilization of such nanostructures in aqueous-based biosensors. Nanopillars prepared by the process described herein possess sufficient mechanical stability to resist the capillary interaction forces.
As used herein, vertically standing nanopillars refers to nanopillars that are substantially vertical in orientation to the support substrate. In certain embodiments, the vertically standing nanopillars are essentially at a 90 degree angle to the support substrate.
It is understood that the above description is but one embodiment for fabricating the nanopillar-enhanced electrodes. For purposes of the present invention, the term “anodization” refers to a process whereby the valve metal in question (Al, Ti, Cr, Ta, etc.) is converted to its anodically generated oxide in aqueous acidic solution, typically a diprotic acid such as H2SO4, oxalic, phosphoric, etc. For the purposes of the present invention, the term “valve metal” refers to a metal that produces a stable oxide layer, such as titanium, tantalum, zirconium, niobium, chromium, etc. When a valve metal is anodized in an appropriate acidic electrolyte under controlled conditions, it oxidizes to form a hydrated metal oxide containing a two dimensional organized hexagonal array of cylindrical pores. The pore diameter and the interpore spacing depend primarily on the applied electrical potential and in a secondary fashion on electrolyte pH, temperature, and metal microstructure (grain size).
In this embodiment, a nanoporous template 240 is prepared from any suitable metal known to those skilled in the art and guided by the teachings herein provided. In some embodiments, a suitable metal of the present invention comprises metals and alloys such as, but not limited to, aluminum, titanium, zinc, magnesium, niobium, or combinations thereof. In one embodiment, the metal used for formation of the nanoporous template is aluminum.
In one embodiment, the template (e.g., Al, Ti, Cr, Ta, etc.) 240 can be created by first coating a flat surface 210 with several thin layers of metal. In one embodiment, the flat surface can be coated with at least two layers of metal. In one embodiment, the template (e.g., Al, Ti, Cr, Ta, etc.) is created by first coating the flat surface with a thin layer of metal (about 5 to about 20 nm) 220, followed by another layer of metal (about 10 to about 150 nm) 230. Examples of said surface suitable for the purposes of the present invention are those of a silicon wafer or a glass substrate. Examples of the metal suitable for the present invention include, but are not limited to, gold, silver, titanium, platinum, copper, palladium, or combinations thereof, and oxides or alloys of above-mentioned metals. In one embodiment, the metal used for formation of the first layer is titanium, and the second layer is gold.
A film of the valve metal with a thickness in a range of 10 nm-50 μm can be subsequently deposited onto the gold layer using any physical vapor deposition techniques known to the skilled artisans (i.e., an electron beam evaporation), followed by an electropolishing in a 9:1 ethanol to water solution. The metal template 240 will be made porous by anodization. An anodization of the metal film (e.g., Al, Ti, Cr, Ta, etc.) can be performed under a variety of anodization conditions. In one embodiment, a one-step anodization is carried out with the metallized wafer serving as the working electrode, and a piece of aluminum foil as the counter electrode. As used herein, “working electrode” refers to an electrode on which a reduction or oxidation reaction occurs. In an alternative embodiment, a two-step anodization is performed, wherein the formed oxide layer is removed before anodization is continued to the gold layer. The anodization conditions can be chosen, for example, to be constant potential at 40 V for 25 minutes in 0.3 M oxalic acid electrolyte at 3° C. The anodization potential can be kept constant at a value of from about 5 V to about 300 V. In one embodiment of the present invention, the barrier layer at the bottom of the metal (e.g., Al, Ti, Cr, Ta, etc.) layer is removed. In one embodiment, the barrier layer at the bottom of the metal (e.g., Al, Ti, Cr, Ta, etc.) layer is removed by immersing the wafer in 5 wt % phosphoric acid solution for 25 minutes, leaving a wafer with the anodized porous template 240 sitting on top of the film (e.g., Au, Pt, Pd, Ti, Ag, etc.) 230. In one embodiment, the template 240 is the anodized aluminum oxide (AAO) template.
Nanopillars 250 can be formed through the open pores of the porous template (e.g., porous anodic alumina (PAA) template) from any suitable material and by any of the suitable plating techniques known to the persons skilled in the art and guided by the teachings herein provided. Examples of material suitable for forming nanopillars include, but are not limited to, any metal resistant to corrosion or oxidation, or any alloy of such metal. In some embodiments, suitable metal comprise metals such as gold, silver, platinum, copper, palladium, or combinations thereof. In one embodiment, the metal used for formation of nanopillars is gold.
In some embodiments, the nanopillars of a desired height are electrodeposited onto the porous template 240. The conditions for electrodeposition can be chosen, as a way of an example, to be 5 mA/cm2 electrical current applied to the PAA at 65° C. in a gold potassium cyanide bath. It will be appreciated by the skilled artisans that the height of the nanopillars can be controlled by varying the electrodeposition time. In some embodiment of the present invention, electrodeposition time is varied between about 1 and about 15 minutes.
The nanopillars developed by electrodeposition are mechanically strong enough to sustain the hydrodynamic interactions produced during the electrochemical processes. Stated otherwise, nanopillars fabricated by this technique are resistant to deformation by capillary forces generated between the vertically aligned nanostructures and liquid medium.
In some embodiments, the porous template can be removed following nanopillar formation. It is apparent to those skilled in the art and guided by the teachings herein that any suitable condition can be used for removal of the porous template. By way of example, the porous template can be removed by immersing the wafer in 1M NaOH solution for 25 minutes. In one embodiment, the porous template can be removed completely. In another embodiment, the porous template can be partially removed to expose the tips of nanopillars. The term “partially”, as used herein, refers to removal of about 2 to about 98% of the template.
Microfabrication procedure has a strong impact in most of the areas of contemporary science and technology and the knowledge and experimental procedures for miniaturisation were transferred from electronics also to chemistry and biochemistry for creating sensors with better performances. The ability to generate patterns of biomolecules on different material surfaces is important for biosensor technology, tissue engineering, and fundamental studies in cell biology. There are several well established ways to pattern biomolecules onto substrates, such as photolithography, soft lithography, nano-pen lithography, and spotting techniques.
Photolithography, or patterning materials using photoresists and etching, is a technology known in the art, which has been advanced by progress in microelectronics where structures on the order of microns and submicrons are used. One of the major disadvantages of this technique, as it is known to date, is its inability to form structures with micro and nano (<100 nm) features on common wafers such as glass or silicon without causing severe deformation in the nanostructures due to its wet-process nature. Since the techniques of photolithography and microfabrication are widely accessible and commonly used at research labs and manufacturing facilities, any new process that is compatible with these techniques will bring widespread applications. Moreover, such a compatibility is also vital for a large-scale production of the said structures and electrodes, thus lowering the cost of production
One aspect of the present invention provides a process for producing integrated structures of micro- and nano-scale features on glass or silicon substrates. In some embodiments, such structures can be produced by micropatterning. In one embodiment, micropatterning can be achieved by coating the wafer enhanced with nanopillars fabricated by the process described above with a positive photoresist 260 (i.e., Photoresist 1818). The term “photoresist”, as used herein, refers to light-sensitive materials used to form a patterned coating on a surface (i.e., polyhydroxystyrene-based polymers). In one embodiment, photoresist can be applied to the wafer prior to the PAA template removal. In another embodiment, the PAA template will be removed prior to photoresist deposition. Photoresist can be deposited on the wafer using any of the variety of deposition techniques known to people skilled in the art. Examples of the suitable deposition techniques include, but are not limited to, spin-coating and electrodeposition. In some embodiments, photoresist will be spin-coated onto the wafer, followed by an exposure to a UV light through a micropattern mask 270. In some embodiments, the micropattern will be developed using a suitable developer solution (i.e., Microposit M 319). Following the development of the micropattern, the unmasked titanium and gold layers can be removed. A person skilled in the art will be familiar with a plethora of techniques available for metal removal. In some embodiments, the unmasked titanium and gold layers will be chemically etched, and photoresist stripped. Chemical etching can be done using any suitable material capable of dissolving metal (i.e., acid or base). In one embodiment, photoresist will be stripped from the wafer using any suitable photoresist strippers (i.e., hydroxylamine). Following micropatterning, PAA template can be removed by, for example, immersing the wafer in 1M NaOH solution for 25 minutes.
Another aspect of the present invention provides a microflow channel biosensor with planar electrodes incorporated with nanopillars. As used herein, the term “microflow-channel” refers to an apparatus of micro-scale dimensions designed for driving a microflow, or a fluid, in microliter amounts. It has been theorized that electrical current response of nanostructured electrodes depends on, among other factors, transport of analyte molecules to the active surface of the nanostructures, a process known as mass transport. Higher current response is achieved in instances wherein an analyte is able to diffuse into the deep spaces between the nanopillars to get oxidized. The detection sensitivity of the nanostructured devices described thus far has been limited by the diffusion rates of the analytes. A nanopillar-enhanced microflow channel biosensor described herein allows for bypassing a diffusion-limited sensor response by providing a convective transport of analyte molecules within said biosensor.
An example of a conventional arrangement of electrodes in the microflow channel biosensors is represented schematically in
In some embodiments of the present invention, a microflow channel 450 comprises a micro-interdigitated array 400 of working electrodes 410 and detector electrodes 420.
Although microflow channel shown in
In some embodiment, the surface of the working electrode can be functionalized with a macromolecule. Example of the macromolecules includes, but is not limited to, any biomolecule capable of accelerating a reduction/oxidation chemical transformation utilizing any known redox co-factor (i.e., FAD). In one embodiment, the macromolecule in the present invention is glucose oxidase. It will be apparent to the skilled artisan that glucose oxidase can be obtained by standard enzyme manufacturing techniques, such as microbial fermentation using traditional techniques or genetic recombination techniques. Alternatively, glucose oxidase can be purchased from industrial makers of enzymes, such as Amano Enzyme, Inc.
In certain embodiments, the surface of the working electrode is functionalized with macromolecules using a self-assembly monolayer (SAM) such as, for example, alkyl thiol. In an alternative embodiment, the surface is functionalized by entrapping the macromolecules in a film of conducting polymer that coats the electrode. As used herein, the term “conducting polymer” refers to an organic polymer capable of conducting electricity or serving as an electrical semiconductor. Examples of conducting polymers suitable for the present invention include, but are not limited to, polyacetylene, polyaniline, and polypyrrole, and combinations thereof. In one embodiment, the conducting polymer used to functionalize the electrodes of the present invention is polypyrrole. Optionally, materials such as sol gel and/or carbon paste can be used to modify the surface (as a replacement for SAM or polypyrrole polymer, or in combination with either).
Glucose oxidase can be entrapped in the conducting polymers by subjecting a mixture of glucose oxidase and polymerizable monomers (e.g., acetylene, aniline, pyrrole, etc.) to at least one of the following conditions, such as low pH (4 and below), temperatures of at least 85° C., actinic radiation of sufficient energy to bring about polymerization, and electrical current having a constant density of about 10 μA/cm2 to about 150 μA/cm2 (galvanostatic polymersization). Various combinations of these steps can also be used to bring about polymerization. In certain embodiments, a film of the conducting polymer containing entrapped glucose oxidase is formed by galvanostatic polymerization of polymerizable monomers (e.g., acetylene, aniline, pyrrole, etc.). In one embodiment, a film of a polypyrrole polymer containing entrapped glucose oxidase is formed by galvanostatic polymerization. In certain embodiments, the polymerization can be carried out by a continuous pumping of the polymerizable monomers (e.g., acetylene, aniline, pyrrole, etc.)/glucose oxidase mixture through the microflow channel. The rate of pumping can be varied between about 1 μL/min to about 50 μL/min. Pumping can be done by any device capable of moving fluids, such as gases, liquids, or slurries. Examples of pumps suitable for the present invention include, but are not limited to, vacuum pumps, heating pumps, circulator pumps, centrifugal pumps, peristaltic pumps, and cyclic pumps. Conditions for polymerization can be chosen, as a way of an example, to be galvanostatic polymerization in 0.1M KCl containing 0.05M pyrrole and 0.5 mg/L of glucose oxidase, wherein the polymerization is carried out at a current density of 50 μA/cm2 for about 45 minutes. In the embodiment illustrated in
Another aspect of the present invention provides MEMS devices integrated with micro/nano structures disclosed by the present invention. A person skilled in the will be familiar with a variety of conventional microfabrication techniques used for incorporating micro/nano structures into MEMS devices. Examples of the suitable microfabrication techniques include, but are not limited to, laser technology, microlithography, micromechatronics, micromachining and microfinishing (nanofinishing).
In some embodiments, micro/nano structures of the present invention will be incorporated into a surface acoustic wave (SAW)-based biosensor. Although
It will be appreciated by people skilled in the art that the following description is but one embodiment of a surface acoustic wave-based biosensor integrated with nanopillar-enhanced electrodes.
In one embodiment, depicted in
People skilled in the art should know the requirement for a piezoelectric substrate in SAW sensors. The term “piezoelectric”, as used herein, refers to any material capable of generating an electric potential in response to an applied mechanical stress. Examples of the piezoelectric substrates suitable for use in the present invention include, but are not limited to, lithium niobate, potasium niobate, lithium tantalate, sodium tungstate, polyvinylidene fluoride, quartz, cane sugar, topas, Rochelle salt, berlinite, and the like, and the combinations thereof. In one embodiment of the present invention, lithium niobate is used as the piezoelectric substrate 650.
In one embodiment, the chemically active layer will be applied on the propagation path of an acoustic wave. As used herein, the term “active layer” refers to any material capable of adsorbing an analyte. Examples of a suitable material that can be used as an active layer in the SAW biosensors of the present invention include, but are not limited to, gold, and piezoelectric materials. It will be easily recognized by the skilled artisan that the dimensions of the active layer will depend on the dimensions of the biosensor, and the distance between the IDTs. In the embodiment depicted in
Another aspect of the present invention provides methods of using the micro/nano-structures described herein. The vast number of potential applications of the micro/nano-structures described herein will be immediately apparent to persons skilled in the art. Below are but a few embodiments describing a potential utility of such structures.
In some embodiments, the micro/nano-patterned structures fabricated by the process of the present invention can be used in biosensors. One embodiment provides a method for use of the biosensors integrated with the nanopillar-enhanced electrodes fabricated by the process of the present invention for monitoring a target analyte level, comprising:
In some embodiments, biosensors integrated with nanostructures described herein can be used for a detection of a target analyte level in biological fluids. Examples of the target analytes include, but are not limited to, endogenously found molecules (i.e., glucose or lactose), exogenously consumed species (i.e., drugs or alcohol), toxic metabolites (mycotoxins), and pathogens (i.e., E. coli or Salmonella). Examples of the biological fluids include, but are not limited to blood, urine, serum, saliva, cerebra-spinal fluid, and semen. In other embodiments, biosensors integrated with nanostructures described herein are useful for environmental applications, such as detection of pesticides and river water contaminations. In some embodiments, biosensors integrated with nanostructures described herein can be used for a remote detection of biological warfare agents. Examples of the biological warfare agents include but not limited to: anthrax, ebola virus, ebola, Marburg virus, plague, cholera, tularemia, brucellosis, Q fever, machupo, Coccidioides mycosis, Glanders, Melioidosis, Shigella, Rocky Mountain spotted fever, typhus, Psittacosis, yellow fever, Japanese B encephalitis, Rift Valley fever, and smallpox. Naturally-occurring toxins that can be used as weapons include ricin (WA), SEB (UC), botulism toxin (XR), saxitoxin (TZ), and many mycotoxins.
In some embodiments, biosensors integrated with nanostructures described herein can be used for determining levels of toxic substances before and after bioremediation. In other embodiments, the biosensors integrated with nanostructures described herein find their application in drug discovery and evaluation of biological activity of new compounds. In yet another embodiment, the biosensors described herein are useful in determination of drug residues in food, such as antibiotics and growth promoters.
In some embodiment, the micro/nano-patterned structures of the present invention can be used for tissue engineering. For example, cell growth (i.e., neuronal) can be directed into intricate micro/nano-patterns of the present invention in a controlled way. In such embodiments, a micro/nano-pattern is prepared on a suitable substrate, i.e., glass plate or silicon wafer, cells are plated on the micropatterned substrate, and the cells are permitted to grow in a suitable nutrient medium. As the cells are permitted to grow, their growth along the micropattern along with exposure to nanoscale topographic environment can be stimulated by the signaling of the nanoelectrodes. In other words, the transmission of electrical signals along the micropatterned nanoelectrode stimulates cell growth along the path of the micropattern. Certain cell types can be stimulated by the use of appropriate hormones or cell active agents, such as cytokines or the like. In at least certain embodiments, the cells plated in the micropattern are stem cells.
The invention will be further appreciated with respect to the following non-limiting examples. Other variations or embodiments of the invention will also be apparent to one of ordinary skill in the art from the above descriptions and examples. Thus, the forgoing embodiments are not to be construed as limiting the scope of this invention.
Nanopillar array electrodes (NAEs) with three different pillar heights tested herein were fabricated using a template method [12]. It will be apparent to the skilled artisan that similar results will be obtained with the nanopillar-enhanced electrodes prepared by the process detailed in this invention.
In fabricating these electrodes, a layer of gold film about 150 nm thick was first sputter-coated onto one side of a porous anodic alumina (PAA) circular disc (d=25 mm; Whatman Inc, Maidstone, England) having an average pore diameter of 150 nM using a SPI sputter coater (Structure probe Inc, West Chester, Pa.). Then, a thicker gold layer was electrodeposited on top of the sputtered gold film to form a strong supporting base in an Orotemp24 gold plating solution (Technic Inc, Cranston, R.I.) with a current density of 5 mA/cm2 for two minutes. This supporting base was masked with Microstop solution (Pyramid plastics Inc., Hope, Ark.) for insulation. After that, gold nanopillars were electrodeposited through the open pores of the PAA disc from the uncoated side under an electrical current density of 5 mA/cm2 at 65° C. The deposition time was varied for achieving nanopillars of different heights. Specimens with three different nanopillar heights were prepared with the electrodeposition time controlled at 1, 7 and 15 minutes. After nanopillar deposition, the PAA disc was dissolved in 2.0 M NaOH resulting in a thin gold sheet with arrays of vertically standing gold nanopillars.
All resulting nanopillars had a diameter of about 150 nm, and varying heights of 1 μm, 2.5 μm and 6 μm for specimen A, B and C, respectively. The insets in
The electrochemical characteristics of the developed nanopillar array electrodes (NAE's) were evaluated in a three-electrode electrochemical system with nano-structured electrode used as a working electrode. Cyclic voltammetry (CV) was performed on the NAE's, using a flat gold electrode having the same geometrical area (about 16 mm2) as a control. The flat gold electrode was prepared by depositing a thin film (300 nm) of gold on titanium-coated glass plate using a thermal evaporator (built in-house). CV was performed in 0.5 M Na2SO4 supplemented with 4 mM K4Fe(CN)6 (JT Baker Inc., Phillipsburg, N.J., USA) at various scan rates (50 mV/s, 100 mV/s, 150 mV/s, and 200 mV/s). All runs were conducted in an unstirred solution using high purity deionized water.
The sensitivity of the nano-structured electrode as compared to the flat electrode was assess by evaluating their amperometric current responses in 0.5 M Na2SO4 solution supplemented with either 6 different concentrations (4 mM, 8 mM, 12 mM, 16 mM, 20 mM, and 24 mM) of K4Fe(CN)6. The transient current was measured at a constant potential of 350 mV versus Ag/AgCl, and the change in the current response upon the change in K4Fe(CN)6 concentration for both the NEAs and flat electrode was determined. The solution was stirred constantly for the duration of the experiments using a magnetic stirrer.
a shows the amperometric current response for the NAEs and flat electrodes at various K4Fe(CN)6 concentrations. In general, all the NAEs exhibited a higher current than the flat electrode at each K4Fe(CN)6 concentration. To further quantify the sensing performance of these electrodes, the relationship between the current response and K4Fe(CN)6 concentration was analyzed by a linear regression analysis.
The sensitivity of the bare NAEs did not increase with the increase of the roughness ratio. This is explained by the fact that only the top part of the nanopillars is contributing to the increase of active electrode surface for electron transfer, and the electroactive species K4Fe(CN)6 may encounter certain difficulties in its transport to the small spaces between the bare nanopillars as the result of either a low diffusivity or a fast electron transfer rate constant. With a low diffusivity, it is difficult for K4Fe(CN)6 to diffuse deep into the small spaces between the nanopillars, while with a fast electron transfer rate constant, most of the species K4Fe(CN)6 get oxidized near the top ends of the nanopillars before it diffuses down the gaps.
To functionalize the electrodes, their surfaces were first modified with a SAM layer by placing them in a 75% ethanol solution containing 10 mM 3-mercaptopropionic acid. Then the SAM modified electrodes were rinsed in 75% ethanol and immersed in a 0.1 M 2-(Nmorpholino)ethanesulfonic buffer solution (pH of 3.5) containing 2 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 5 mM N-hydroxysuccinimide for activation for two hours. After washing in phosphate buffer solution (PBS), the activated NAEs were placed in PBS solution at pH 7.4 containing 1 mg/ml of glucose oxidase for two hours under constant stirring. The reason for setting the immobilization time to two hours is that according to literature [13], enzyme loading reaches its maximum in about 2 hours and it saturates afterwards. From the electrochemical experiments, the amperometric current responses of both bare and functionalized NAEs along with flat controls were measured using a conventional three-electrode cell with an Ag/AgCl reference electrode and a platinum counter electrode with the Multistat 1480 (Solartron Analytical, Houston Tex., USA) electrochemical system.
For the functionalized NAEs, the amperometric current responses to each incremental addition of 50 μl of 1 M glucose to a 20 ml PBS solution (equivalent to a 2.5 mM increase in glucose concentration) containing 3 mM p-benzoquinone as a mediator were measured at a constant potential of 350 mV (vs. Ag/AgCl). In all experiments, the background current of all electrodes was allowed to stabilize before drops of target species were added. Prior to these experiments the electrolyte solution was de-aerated with nitrogen and during experiments the solution was blanketed with nitrogen and stirred constantly at 600 rpm.
a shows the amperometric currents for the functionalized NAEs and flat electrode at various glucose concentrations. All the NAEs exhibited a higher current response than the flat electrode at each glucose concentration. In each incremental step, the current response of Nano C is still rising indicating that it has not reached its steady state. This phenomenon may be due to the increased response times for electrodes with taller nanopillars. However, for a quick comparison between these nano electrodes, a more conservative approach was taken to get the current readings for Nano C at the same time as for Nano B and Nano C.
b shows the variations of the steady-state amperometric current with glucose concentration (from 2.5 mM to 15 mM) along with the corresponding linear regression lines. By taking the slope of the regression lines and normalizing it with respect to the geometric area of the electrode in each case, the sensitivity measurement were obtained for the functionalized electrodes (NAEs and flat). From the obtained sensitivity values listed in Table 2, it was observed that unlike in the bare electrode cases, the sensitivity of NAEs increases as the roughness ratio increases. The highest sensitivity value (Nano C) is about 3.13 μA·mM−1·cm−2 (about 12 times higher than that for a flat electrode) which is significantly higher than the value reported for a gold nanopillar electrode (0.4 μA·mM−1·cm−2) [8]. So for the functionalized NAEs, increasing the surface roughness of the NAEs does contribute to an increase in detection sensitivity.
In comparing the bare with the functionalized electrodes, it was observed that the highest nanostructure-induced sensitivity increase for the functionalized electrodes (12 times) is higher than that for the bare electrodes (2 times). This could be due to the difference in electrochemical species involved (i.e., glucose versus K4Fe(CN)6). These two electroactive species, however, have a similar diffusivity value (8×10−10m2/s for K4Fe(CN)6 and 7.6×10−10m2/s for glucose) [14]. This fact suggests that the difference in the reaction rate constant at the bare and functionalized electrodes can play a more dominate role in affecting the current response. It is also possible that such an increase in the sensitivity of functionalized NAEs is the result of heightened retention of the mediator during glucose detection [11].
An electrochemical process was simulated using a finite element analysis method with commercial software COMSOL Multiphysics (COMSOL Multiphysics, Burlington, Mass.). To simplify the situation, two-dimensional situations were considered. As shown schematically in
For the electrode reaction at the functionalized NAEs, it was assumed that glucose was consumed at a flux of Jg at the electrode surface to produce the mediator in its reduced form at a flux of JM. Here Jg and JM can be described by the following equations:
Jglucose=kcG (5)
J
M
=kc
G
−k
0
c
M exp(−αF(E−Estd)/RT) (6)
where k represents the rate constant for Eq. 5, cG the concentration of glucose, cM the concentration of mediator, k0 the standard rate constant, α the charge transfer coefficient, F the Faraday constant, E the electrode potential, and Estd the standard potential of the mediator. To simulate the actual event, the electrode was held at a constant overpotential of 350 mV. Under this condition, the reduced-form mediator was oxidized at the electrode surface to generate a current flux of Jc:
J
C=−2k0cM exp(−αF(E−Estd)/RT) (7)
With these considerations, the amperometric current response of the electrodes in response to a drop of glucose was determined while the electrolyte solution was constantly stirred by a swirling vortex force applied at the center of the cell.
For the electrode reaction at the bare-electrode, it was considered the redox of K4Fe(CN)6 with the reduction flux of K4Fe(CN)6 governed by:
JF=−k
0
FcF1 exp(−αF(E−Estd′)/RT)+k0FcF2 exp(−αF(E−Estd′)/RT) (8)
where k0F is the electron transfer rate for both ferrocyanide and ferricyanide (assumed to be the same), cF1 the concentration of ferrocyanide, cF2 the concentration of ferricyanide, E the electrode potential, and Estd′ the standard potential of ferro- and ferri-cyanide.
Besides the reaction kinetics discussed above, the mass transport in these electrochemical processes was mainly governed by diffusion and convection for the mobile species such as glucose and K4Fe(CN)6. The electromigration was ignored because of the presence of the supporting electrolyte in a high concentration.
After these considerations, the diffusion/convection-controlled electrochemical reaction problems upon a step potential excitation (350 mV) at the electrode were solved using the combined Electrokinetic-Flow and Navier-Stokes applications in COMSOL Multiphysics. In the simulation process, two initial analyses were performed. First, a stationary nonlinear analysis in Navier-Stokes mode was performed for reaching a fully developed vortex flow inside the center inner circle (
where A represents the solute (e.g., glucose or the mediator) and B the solvent (e.g., water), εB the association factor of the solvent, MB the molecular weight of the solvent, μ the viscosity of solution, VA the molar volume of solute glucose, and T the absolute temperature.
The influence of the reaction rate constant on the current response of the NAEs, can be seen from the simulation results.
Two-dimensional simulations were performed by solving the steady-state Navier-Stokes equation and electrokinetic equations.
Flow of solution inside the channel is defined by steady state navier-stokes equations and the mass transport of species is defined by convection and diffusion equation. The steady state Navier-stokes equation for Newtonian incompressible fluid can be written as:
∇.η(∇u+(∇u)T)+ρ(u.∇)u+∇p=F (10)
∇.u=0 (11)
Where ρ denotes the density of the fluid, η the dynamic viscosity, u the velocity vector, p the pressure and F is a body term. Using the above assumptions, the mass balance equation of glucose and hydrogen peroxide can be given by equation 12:
Where Di denotes the diffusion coefficient of the species i, Ni denotes the flux of the species i, Ci denotes the concentration of the species i, u denotes velocity, R denotes the reaction term, Km denotes the Michaelis-Menten constant, [S] the substrate concentration and Vmax the maximum reaction rate. The reaction rate of glucose to hydrogen peroxide was assumed to obey the Michaelis-Menten kinetics which is given by equation 13.
At the walls of the channel no-slip boundary condition (velocity vector, u=0) is applied. A normal flow/pressure boundary condition is imposed at the outlet, i.e. u.t=0, p=0, where t and p denotes the tangential velocity to the boundary and pressure, respectively. At the inlet of the channel a fully developed flow is assumed to be entering which is defined by equation 5:
u=u
max4s(1−s) (14)
Where umax denotes the maximum velocity in the parabolic expression and s the boundary variable that varies from 0 to 1 along the boundary.
The species such as glucose and hydrogen peroxide entering the channel has an initial concentration of 6 mM and 0 mM, respectively. At the enzyme layer, it was assumed that the flux of glucose reacting is equal to the flux of hydrogen peroxide produced. It is reported that the electrochemical oxidation of hydrogen peroxide at the electrode is mass transport controlled. Therefore, the concentration of hydrogen peroxide at the detector electrode is considered to be zero. At the outlet a convective flux boundary condition is applied, which assumes that the mass flux due to diffusion and migration across this boundary is zero.
To evaluate the sensing performances of the fluidic glucose sensor, 0.01M phosphate buffer solution (PBS) containing 3 mM p-benzoquinone as a mediator was continuously fed into the fluidic channel at a constant rate of 5 μL/min. A potential of 0.305V (vs. saturated calomel electrode (SCE)) was applied at the working electrode to detect the amperometric current response caused by oxidation of the reduced mediator species. After the current response stabilized in the PBS solution, a known concentration of glucose in PBS solution was introduced into the channel. For comparison, the same experiment for a fluidic sensor with flat electrodes was performed.
For the nanopillar case, the roughness factor of nanopillar electrodes was determined to be around 18 to 20 when gold was deposited for 6 minutes at 0.6 mA/cm2. The increase in area can be seen in the CV graph plotted in
From the measured amperometric current responses (
Interdigitated transducer pattern 1520 having at least two pairs of fingers with a width of 10 μm and spacing of 10 μm was lithographed onto a piece of a lithium niobate substrate 1510 with the dimensions of 80 μm×200 μm×40 μm coated with aluminum by evaporation. Ti, Au, and Al were shadow mask deposited in an area of 20 μm×20 μm×1 μm between the two IDTs to represent the active gap region 1530, followed by anodization of Al to form nanoporous alumina template 1540. Gold nanopillars with a diameter of 100 nm and a height of 1 μm 1550 were formed by the electrochemical deposition of gold onto the nanoporous alumina template 1540, followed by the removal of the template. The surface of the nanopillar-enhanced active area was then functionalized using the procedure described herein.
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The above references are herein incorporated by reference in their entirety.
This application claims the benefit of U.S. provisional application Ser. No. 61/039,338, filed Mar. 25, 2008, which is incorporated herein by reference in its entirety. It is also a continuation-in-part of U.S. patent application Ser. No. 12/232,152, filed Sep. 11, 2008, which is incorporated herein by reference in its entirety.
Part of the work performed during development of this invention utilized U.S. Government funds under ECS-0304340 awarded by National Science Foundation. Therefore, the U.S. Government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 61039338 | Mar 2008 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12232152 | Sep 2008 | US |
| Child | 12382860 | US |
