BACKGROUND

Bioelectronics is a rapidly growing, interdisciplinary field that encompasses the integration of biomaterials, cells and tissue with electronic devices. Of the several devices utilized in this field, two important bioelectronic interfaces that are used extensively with electrogenic cells (neural or cardiac cells) are electrodes for electrical impedance spectroscopy and extracellular electrophysiological measurements. Both techniques are label-free methodologies for real-time monitoring of cellular interactions. Impedance spectroscopy measures the frequency-dependent alterations of passive electrical properties of networks of cells by applying defined AC currents or voltages. Since this is a non-invasive method, long-term effects on cellular behavior from external compounds can be realized without invasively entering the cells. Such devices have applications in several in vitro biosensing assays such as cellular proliferation, quality control of cells, compound mediated cytotoxicity, cellular adhesion, cellular morphology, functional monitoring of receptors and viral quantification. Several of these factors are required to be studied during the process of drug discovery both at the cellular network level and at a single cell level. Due to the extent of characterization and the long lead times with animal and human testing, bringing a drug to market is extremely expensive. In the realm of cardiac drugs, cardiotoxicity has been cited as a major reason for 30 percent of all failed drug compounds during development and is a major cause of compound attrition. Early scale detection of the cardiotoxic side effects of drug compounds prevents the disqualification of these compounds at a later stage and as a result reduces the cost and the time to adopt these new pharmacological compounds.

Traditional analysis of the cardiotoxicity of drug compounds has involved ex vivo or in vivo studies with the isolation of these compounds in animal models such as rats and pigs and the electrophysiological and viability studies on the isolated cardiomyocytes. Alternative and newer methodologies rely on in vitro cell-based assays that express specific ion channels in the cardiomyocytes such as the hERG channel or the voltage gated calcium channel. These ion channels serve as possible molecular targets through which the drug compound could induce cytotoxicity and are typically studied with techniques such as patch clamping that are invasive and may lead to cell death after the implementation of the technique. Microelectrode Arrays (MEAs) and impedance arrays are non-invasive, label-free technologies that may be utilized to track the cardiotoxicity of compounds outside the animal model in vitro and in a dish empowering the researcher with greater experimental control. These devices typically have a grid of thin film metal and insulation typically defined on substrates such as glass and silicon in the case of MEAs and a whole variety of interdigitated electrode designs of different thin films defined on mostly transparent substrates in the case of impedance arrays. The interdigitated electrodes (IDEs) report a change in extracellular electrophysiological signature or a change in impedance as the output signal correlated to a change in behavior of a group of cardiomyocytes cultured on the electrodes. Single cell or single ion channel accuracy evades these sensors without complex technologies such as nanoimprint lithography or e-beam lithography that make these sensors unaffordable to most users.

Furthermore, drug-induced cardiotoxicity accounts for one-third of safety-based withdrawn pharmaceuticals, making it the number one cause of drug withdrawal, limitation, and development termination. As of 2016, the Tufts Center for the Study of Drug Development estimates the cost of developing a new drug is on an average 2.89 billion US dollars. Because of this high cost, improved in vitro systems for predicting drug-induced toxicity are of great demand in the pharmaceutical industry to decrease late-stage drug attrition, advance rapid development, and reduce monetary loss.

Such predictive toxicity assays based on human pluripotent stem cells may aid in predicting potential safety issues of drug candidates early in its development process, provide information about the mechanisms of drug-induced organ toxicity, reduce the reliance on animal testing, and increase the relevance of preclinical safety tests. Human induced pluripotent stem cell (iPSC) differentiated cardiomyocytes are the ideal candidate for cardiotoxicity cell-based studies. They exhibit the molecular and functional properties of an intact human heart, and their electrical signatures can be monitored using non-destructive impedance sampling.

Interdigitated electrodes (IDEs), which are comprised of two individually addressable, interwoven, comb-like electrode structures, are one of the most favorable and widely used transducers as chemical and biological sensors because of their low cost, high sensitivity, and ease of fabrication. By affixing a culture well to the IDE substrate, a biosensor can be easily fabricated. This allows for cells to be cultured onto the surface and assessed with label-free electrical and optical assays. A low-voltage signal induces a current between the IDEs. The cells on the electrodes at the bottom of the culture well impede this current, and a change in impedance results. Measuring this impedance change across these electrodes gives an indirect measure of the number of cells in each culture well, as well as an assessment of the interaction between the cells and electrodes. Cellular impedance measurements are useful for studying cell growth and drug interactions in vitro without the use of destructive labelling procedures with fluorescent, chemiluminescent, or radioactive chemicals. Recently, these efforts are gaining industrial acceptance with efforts of collaboration between various tool vendors to introduce rapid assays with uniform standards for testing cardiotoxicity.

Conventional interdigitated electrodes (IDEs) used for impedance spectroscopy represent a label-free, non-invasive technique with a wide range of applications, including in vitro analysis of cells. Such IDEs have remained remarkably similar over the years with metal patterns defined on a silicon or a glass substrate on to which cells and materials are cultured for impedance analysis. Conventional IDEs suffer from undesired background noise.

Several IDE and impedance-based biosensors exist, but many of them require the use of expensive commercial systems for data analysis and involve cost prohibitive cleanroom-based fabrication approaches for the IDE micro and nanostructuring. These systems use very densely packed electrodes, which cover a majority of the substrate surface and prohibit optical tracking of cells. In addition, they tend to only measure cellular activity for hours, not days or weeks. Other approaches integrate microelectrode arrays with IDEs, which allows for more comprehensive measurement at the cost of more complex fabrication processes.

Typical methods for the fabrication of nanostructures include methods like photolithography, e-beam lithography, and focused ion beam lithography. These methods offer high quality nanostructures, but involve tedious procedures, long processing time, limited scalability, and high cost. To achieve scalability, bottom-up approaches like self-assembly have been used, but they are limited to select materials, and pattern versatility cannot be easily achieved using this approach. Sacrificial anodic aluminum oxide (AAO) templates for developing nanostructures are also widely used for fabricating nanostructures; however, the sacrificial nature of AAO and the required use of strong chemical etchants places a serious limitation on this method. Several unconventional lithographic methods have been developed to circumvent the limitations posed by conventional lithographic techniques. Among these techniques, nanoimprinting lithography (NIL) has attracted considerable attention. In NIL technique, many nanostructures can be replicated using an expensive NIL machine from a master mold. The feature size depends on the mold used to print the nanostructures. These nanostructures can subsequently be used as substrates for various applications, including interdigitated electrodes.

BRIEF SUMMARY

Various embodiments relate to interdigitated electrodes, such as nanostructured interdigitated electrodes (nIDEs) and plasmonic interdigitated electrodes (PIDEs). Plasmonic interdigitated electrodes, in particular, may combine the simplicity of an IDE with the sophistication of plasmonics for in vitro biosensing applications. Such PIDEs may separate the excitation source (light) from the detection signal (current), and thereby significantly reduce the undesired background noise, leading to higher sensitivity for bioanalysis compared with conventional IDEs. Various embodiments relate to methods of making plasmonic interdigitated electrodes, which allow the plasmonic substrate to be tuned not only to maximize the targeted interaction of the cells with the nanoscale geometry, but also for the excitation wavelength to minimize biological sample interference. In addition to being useful as cell-based biosensors, various embodiments may be used in the energy storage and distribution field as well. These and other features, aspects, and advantages of various embodiments will become better understood with reference to the following description, figures, and claims.

