Patent Application
20090017197
1. Field of the Invention
This invention generally relates to integrated circuit (IC) fabrication and, more particularly, to a fine resolution protein sensor fabricated with iridium oxide nanowire electrodes.
2. Description of the Related Art
The current industry standard for protein detection is fluorescent-based detection. Other detection means include: (1) Amperometry, (2) Potentiometry, and (3) Conductance. Table 1 highlights the advantages and shortcomings of these techniques for protein sensing.
From the above comparison, it can be seen that there are advantages to choosing the capacitance method of detection. At the nano-scale, the amount of surface area available for adherence is very high and the distance of separation between electrodes is reduced. Both these factors suggest to possibility of improved sensitivity, as capacitance is directly proportional to the surface area. To induce a change in capacitance, no additional reactions, such as reduction-oxidation reactions, need be instigated. Among electrical parameters, capacitance is the least affected by inherent background currents, making it a highly stable parameter for biomolecule detection.
Proteomics research has resulted in the identification of a large range of biomarkers that have the potential of greatly improving disease diagnosis. The availability of multiple protein markers is believed to be especially important in the diagnosis of complex diseases ranging from cancer identification to cardiovascular diseases. For these complex diseases, the heterogeneity of the disease makes tests of single protein markers inadequate. Patterns of multiple protein markers might, however, provide the information necessary for the robust diagnosis of disease in any person within a population. Moreover, the detection of markers associated with different stages of disease pathogenesis could facilitate early detection.
Widespread use of protein markers in healthcare will depend upon the development of techniques that will enable the rapid and selective detection of multiple markers. This goal has not yet been achieved by any of the existing detection methods that include the use of micro cantilevers, surface plasmon resonance, enzyme linked immunosorbant assays (ELISA). and carbon nanotube based sensors.
Nanomaterials have been used to improve sensitivity in the nanogram sensitivity regime using silicon nanowire and carbon nanotube based devices. However, these materials are not suitable for multiplexed detection due to the complexity associated with the device fabrication and issues with repeatability.
Another limitation associated with micromachining technology is size, as the individually machined electrodes cannot be made to a nano-size resolution. Even if a template of nano-sized structures could be micro-machined, plating an array of nanostructures, with a noble metal for example, in a sufficiently high aspect ratio is a big challenge. Micro-machined electrodes are normally formed from a thick film that is deposited using a physical vapor deposition (PVD) process or electrode plating. In either case, the resultant film, and micro-machined electrode post are typically a polycrystalline material.
IrOx nanowire-based electrodes have a better surface-to-volume ratio, as compared to carbon nanotubes (CNTs) for example, as well as a high resolution stimulation, biocompatibility, and ability to grow on transparent conducting electrodes such as ITO, SnO2, ZnO and TiO2 with or without any doping. Single-crystal IrO2 nanowires/rods/tips have a much longer life than polycrystalline IrO2, due to their higher chemical reaction resistance. Single-crystal IrOx nanostructures also have a higher conductance than polycrystalline IrO2, so they can pass through current more efficiently. However, it is difficult to form single-crystal IrO2 films using conventional PVD or electrode plating methods. IrO2 nanostructures can be formed using a solution method, but these structures have a low mechanical strength and poor crystal quality. Vapor phase transport methods can also be used to form IrO2 nanostructures, but this process requires high substrate temperature, and it is not suitable for use with glass and polyimide substrates.
It would be advantageous if a sensor could be fabricated using IrOx nanowire electrodes for capacitively measuring the detection of proteins.
This disclosure presents integrated nanowire arrays in which distinct nanowire surfaces can be integrated with distinct receptors/antibodies, to function as individual components of device elements. The detection technique is such that the variation in the electrical conductivity associated with the binding of specific proteins onto selectively functionalized nanowire arrays can be measured. The result is an electrochemical signature that is unique to a specific antibody or protein/antigen pair. Non-specific bindings can be eliminated by such a technique, thus reducing background noise effects considerably and improving the signal to noise ratio. The individual device elements, due to their specific functionalization, are able to detect specific proteins. Hence, a large array of proteins can be detected in a matrix format within a few minutes i.e., in near real time as opposed to conventional detection methods that range from a few hours to a few days. The quantity of test sample required for such detection process is in the order of microliters, as compared to milliliters in the conventional methods.
Iridium oxide nanowires are used as the active elements in the detection process. These nanowires are biocompatible, and amenable to the addition of multifunctionality suitable for multiplexed detection. The large surface area afforded by these nanowires enables a reduction in the device footprint by increasing the active area for detection. Comparing to a planar IrO2 electrode, IrO2 nanowires have an improved surface-to-volume ratio, resulting in a high selectivity, high sensitivity larger linear dynamic range of detection, and rapid response time.
As noted above, iridium oxide has very good conductivity and charge storing capacity. As such, it can be used to detect even a very small change in surface charge. High selectivity can be achieved by incorporating protein receptors (antibodies) on the nanowires, which bind only to specific proteins. This binding induces a change in surface charge, on the nanowire surface. This change in surface charge is due to the modification of the surface charge of the proteins as a result of the binding, which can be efficiently detected. This technique is extremely sensitive to these surface charge variations, enabling the detection of very small concentrations of proteins.
Accordingly, a method is provided for forming an iridium oxide (IrOx) nanowire protein sensor. The method provides a substrate and forms overlying working and counter electrodes. A dielectric layer is deposited over the working and counter electrodes, and contact holes are formed in the dielectric layer, exposing regions of the working and counter electrodes. IrOx nanowires (where 0<X≦2) are grown from exposed regions of the working electrode. In one aspect, the IrOx nanowires are additionally grown on the dielectric, and subsequently etched from the dielectric. In another aspect, IrOx nanowires are grown from exposed regions of the counter electrode.
The working and counter electrodes may be a material such as ITO, SnO2, ZnO, TiO2, doped ITO, doped SnO2, doped ZnO, doped TiO2, TiN, TaN, Au, Pt, or Ir. The substrate may be Si, SiO2, quartz, glass, or polyimide. The dielectric layer may be made from a material such as SiO2 or SiN.
The contact holes have openings about equal to, and aligned with top surfaces of the underlying working and counter electrodes. The counter electrode has a top surface area in a range of 1 square micron to 1 square millimeter (mm2), and the working electrode has a top surface area in a range of about 1 to 1000 times smaller than the counter electrode top surface area. Both electrodes come in a variety of shapes, and are typically separated by a distance in a range between 0.1 and 10 microns.
Additional details of the above-described method, a method for capacitively detecting the presence of proteins, and an IrOx nanowire protein sensor array are presented below.
A nanowire may alternately be known as a nanostructure, nanorod, nanotip, or nanotube. The average IrOx nanowire has an aspect ratio in a range of about 1:1 to about 1000:1. As used herein, aspect ratio is defined as the ratio of the nanowire height, to the nanowire diameter or width at the base, where it is attached to the electrode. The IrOx nanowires have an average height in the range of about 10 nanometers (nm) to about 10 micrometers (am). The IrOx nanowires have an average base end diameter in the range of about 1 nm to about 1 μm.
The working and counter electrodes 306/308 are a material such as ITO, SnO2, ZnO, TiO2, doped ITO, doped SnO2, doped ZnO, doped TiO2, TiN, TaN, Au, Pt, or Ir. The substrate 302 may be a material such as Si, SiO2, quartz, glass, or polyimide. The dielectric layer 310 may be a material such as SiO2 or SiN. However, it should be noted that the list of above-mentioned materials are examples of materials that are already conventionally used in many IC fabrication processes, and that the sensor device 300 may be enabled with other materials that would be well known in the art.
As seen in
The counter electrode 308 has a top surface 330 area in a range of 1 square micron to 1 square millimeter (mm2). The working electrode 306 has a top surface 328 area in a range of about 1 to 1000 times smaller than the counter electrode top surface area. Both the working and counter electrodes 306/308 come in a variety of shapes and orientations, depending upon the specific function of the sensor. As shown, both electrodes 306/308 have a substantially rectangular shape, and the counter electrode substantially surrounds the working electrode. In other aspects not shown, the traces are carried in layers either overlying or underlying the electrodes, and connected to the electrodes using vias. Typically, the working electrode 306 is separated from its corresponding counter electrode 308 by a (minimum) distance 332 in the range between 0.1 and 10 microns. However, the distance 332 need not necessarily be uniform.
One limitation to the capacitance measuring technique is its dependence upon the protein receptor specificity. However, covalent linker chemistry can be incorporated to promote greater specificity. To characterize protein detection based on capacitance measurements, the following measurement technique may be employed: (1) Baseline and Control Measurements, (2) Protein Receptor (Antibody) Saturation Measurements, and (3) Protein (Antigen) binding measurements.
The thickness of the diffuse double layer at room temperature is derived as,
λdouble=3.3×106∈r/(zc1/2) (1)
Where, ∈r is the relative permittivity of the medium
From this equation it can be seen that the double layer thickness decreases with increasing valence and concentration. It can also be seen that the properties of the electrical double layer depend on the surface charge on the electrode, the DC bias voltage applied, the concentration of ions in the solution, and the charge of the individual ions.
Capacitance is inversely proportional to the double layer thickness:
Cα1/λdouble
Hence the capacitance induced can be sufficiently enhanced by using a high concentration ionic solution. Typically, the thickness of the double layer can be changed by varying the bias voltage and sweeping it over a finite frequency range, to find the point at which there is high capacitance. During sensor operation, a potential is applied between the two electrodes and the electrical double-layer formed at each electrode results in a localized capacitance. These capacitors may be represented as working and counter electrodes. The surface area of counter electrode is typically many times the area of the working electrode, to maximize the protein binding signal on the working electrode.
The smaller the distance of separation between electrodes, the greater the effect of the capacitive measurements. In many circumstances, a distance of less than 2 um is desired. In one aspect, 10 sets of complimentary electrodes with IrOx nanowires, with sizes in the range of 1-50 microns for each electrode, provide enough surface area to detect proteins down to the picogram per milliliter (pg/ml) range.
In another aspect, the working electrode and counter electrode can be arranged in a multi-fingered interdigital pattern. The sizes of the working and counter electrodes can be the same, or different. The electrodes act as the plates of a capacitor, and the change in the charges from the proteins changes the capacitance measurement in a manner that is unique for each protein.
In
Baseline Measurements: To find and offset the background capacitance, an impedance analyzer may be used. An impedance analyzer measures impedance as a function of frequency. Measurement probes are placed on the working electrode and the counter electrode, and connected to the impedance analyzer after basic calibration. The impedance measurements, cross-referenced to frequency, can be substituted in an equivalent circuit (mathematically) to find the capacitance. AC impedance and, hence, capacitive reactance is observed from the analyzer by varying the frequency sweep and bias voltages, which is then substituted in an equivalent circuit to obtain the effective capacitance.
At a particular frequency the capacitance reaches a local maximum due to the reduction of the double layer thickness. That capacitance value and its corresponding frequency are noted.
PBS is a buffer solution containing sodium chloride, sodium phosphate, and potassium phosphate. It is filled with ions, and the ionic strength of PBS increases with its concentration. To obtain the best capacitance, different concentrations of PBS (1X and 0.1X) are dropped onto the samples and the capacitive measurements are made at prescribed time intervals. De-ionized (DI) water, as the name signifies, is devoid of ions. So, the capacitance can also be measured when DI water acts as the medium to provide another reference marker.
Antibodies are immobilized on to the iridium oxide nanowires by placing them in an incubation chamber for about 30 minutes, at about 60° C. Once they are attached to the iridium oxide nanowires, the nanowires tend to change the equilibrium state of charge by using up some ions for binding. These changes in the surface charge on the nanowires change the overall capacitance measured between the electrodes.
Antibodies are added until the capacitance saturates and stabilizes. That capacitance measured provides a capacitance reference for the characterization for a particular protein.
Using baseline measurements, the substrate capacitance and CIr-subs are known. After adding linkers and adhering the antibodies on to the nanowires, all the capacitances, except the antigen-induced capacitance, can be found. Hence, the overall capacitance before binding is:
1/Cini=1/Csubs+1/CIr-subs+1/Cl-Ir+1/CAb-l
After exposing the nanowires to an environment that contains antigens, Ab—Ag binding takes place to induce the CAb-Ag capacitance to give:
1/Cfin=1/Cini+1/CAb-Ag
Hence, the difference between Cini and Cfin can be used to characterize protein detection. Known concentrations of proteins can be dropped onto the samples, and capacitive measurements taken for different concentrations, for the purpose of calibration.
A blocker or a blocking agent is one which blocks all the unused sites on the nanowires to reduce the amount of nonspecific binding of proteins. When the working electrode is saturated with antibodies, most of the sites are used for binding. However, there may still be certain unoccupied sites that act as active binding sites for proteins when dropped on the electrodes. This condition brings about non-specific binding and increases background interference. Nonspecific binding is relevant due to the presence of other proteins in clinical samples (e.g., blood samples).
In one aspect, a blocking buffer, bovine serum albumin (BSA) is used to bind with these unreacted sites and improve the sensitivity of detection by reducing the background interference. BSA binds well with all potential sites of nonspecific interaction, without altering or obscuring the epitope for antibody binding.
For example, some conventional limits of detection are ˜0.5 nanograms per ml (ng/ml), with a dynamic range of detection in the range from the lower ng/ml to higher micrograms per ml (μg/ml). Conventional frequency sweeps are in the range of ˜10 kHz to ˜3 MHz. The use of blockers as mentioned above, improves the lower limits of detection, which increases the dynamic range, as the blockers reduces background interference and non-specificity. Further, the sweep of frequency is extended to lower frequencies, as the double layer capacitance dominates in the lower frequency range. Again, this result yields higher values and higher sensitivity. As mentioned above, covalent linkers help in covalently binding the antibodies to the nanowires, hence, increasing stability and the dynamic range of detection.
Step 1602 provides a substrate from a material such as Si, SiO2, quartz, glass, or polyimide. Step 1604 forms a working electrode and a counter electrode overlying the substrate, from a material such as ITO, SnO2, ZnO, TiO2, doped ITO, doped SnO2, doped ZnO, doped TiO2, TiN, TaN, Au, Pt, or Ir. Step 1606 forms a dielectric layer overlying the working and counter electrodes, from a material such as SiO2 or SiN. Step 1608 forms contact holes in the dielectric layer, exposing regions of the working and counter electrodes. Typically, the contact holes have openings about equal to, and aligned with top surfaces of the underlying working and counter electrodes. Step 1610 grows IrOx (0<X≦2) nanowires from exposed regions of the working electrode. In one aspect, Step 1610 also grows IrOx nanowires from exposed regions of the counter electrode. Optionally, Step 1612 coats the IrOx nanowires with antibody linker molecules, antibodies, or protein blocker agents. In some aspects, the nanowires may be covered with combinations of the above-mentioned materials. For example, linkers may be used with blockers to reduce the nonspecific binding of proteins. In some aspects, an antibody coating may act as a binding for a specific protein.
In one aspect, forming contact holes in the dielectric layer includes substeps. Step 1608a selectively etches the dielectric layer, and Step 1608b exposes the working and counter electrodes. Then, growing IrOx nanowires from the exposed regions of the working electrode includes substeps. Step 1610a grows IrOx nanowires from the regions of the working electrode exposed by the contact hole, and also grows IrOx nanowires on the dielectric. Step 1610b etches the IrOx nanowires grown on the dielectric.
In a different aspect, Step 1608c selectively etches the dielectric layer. Step 1608d partially opens contact holes overlying the working electrode. Step 1608e selectively etches the dielectric layer. Step 1608f opens contact holes overlying the working electrode, and partially opens contact holes overlying the counter electrode. Then, Step 1610c grows IrOx nanowires from the exposed regions of the working electrode, and also grows IrOx nanowires on the dielectric. Unlike Step 1610a, dielectric still covers the counter electrode. Step 1610d etches the IrOx nanowires grown on the dielectric, and Step 1610e opens contact holes overlying the counter electrode.
In another aspect, providing the substrate in Step 1602 includes providing a substrate chip with edges. Then, forming the working electrode and counter electrode includes substeps. Step 1604a conformally deposits a conductive layer overlying the substrate. Prior to forming the dielectric layer, Step 1604b selectively etches the conductive layer to form working and counter electrodes, probe pads along the chip edges, and traces connecting the electrodes to the probe pads. The working electrode and the counter electrode made be formed in an orientation such as adjacent, the counter electrode substantially surrounding the working electrode, and in an interdigital separation pattern. Typically, the working electrode and the counter electrodes are separated by a distance in a range between 0.1 and 10 microns.
The counter electrode may have a top surface area in a range of 1 square micron to 1 square millimeter (mm2), with a shape such as a circle, rectangle, hexagon, or oval. The working electrode likewise may be shaped as a circle, rectangle, hexagon, or oval, with a top surface area in the range of about 1 to 1000 times smaller than the counter electrode top surface area.
In one aspect, providing the substrate chip in Step 1602 includes providing a chip having a surface area in the range of 1 mm2 to 1000 mm2. Forming the working and counter electrodes in Step 1604 includes forming an array of working/counter electrode pairs on the substrate, where the array includes between 2 and 128 electrode pairs. Each electrode pair is separated by a (minimum) distance in the range of 1 to 500 microns, and arranged in a pattern such as a circle, concentric rings, or a grid.
Step 1706 exposes the IrOx nanowires (with or without coating) to an ambient environment including antigen molecules. In response to the antigen molecules binding to the nanowires, Step 1708 measures a change in impedance between the working and counter electrodes. As noted in more detail above, Step 1708 measures a change in impedance (e.g., a maximum impedance) at a first frequency. Alternately stated, the method measures local minimum or maximum electrical characteristics at particular frequencies, which are known to be associated with particular proteins.
In one aspect, coating the IrOx nanowires with antibody linker molecules in Step 1704 includes coating with a material such as alkanethiols, carboxylic acids, organosilicon derivatives, or diphosphonates. Alternately, if the coating is a blocker, it may be BSA. After coating, Step 1704 may include heating the substrate to a temperature in the range of 20° to 60° C., for a duration in the range of about 15 to 60 minutes.
Additional details of the IrOx nanowire fabrication process can be found in the following related pending applications:
OPTICAL DEVICE WITH IrOx NANOSTRUCTURE ELECTRODE NEURAL INTERFACE, invented by Zhang et al, Ser. No. 11/496,157, filed Jul. 31, 2006, Attorney Docket No. SLA8084;
Iridium Oxide Nanotubes and Method for FORMING SAME, invented by Zhang et al., Ser. No. 10/971,280, filed Oct. 21, 2004, Attorney Docket No. SLA0901; and,
Iridium Oxide Nanowire and Method for FORMING SAME, invented by Zhang et al., Ser. No. 10/971,330, filed Oct. 21, 2004, Attorney Docket No. SLA0903.
IrOx NANOWIRE NEURAL SENSOR, invented by Zhang et al., Ser. No. 11/809,959, filed Jun. 4, 2007, Attorney Docket No. SLA2145.
The four above-mentioned applications are incorporated herein by reference.
An IrOx nanowire protein sensor array and corresponding fabrication processes have been provided. Examples of specific materials, process steps, and structures have been presented to illustrate the invention. However, the invention is not limited to merely these examples. Other variations and embodiments of the invention will occur to those skilled in the art.
