Electrochemical protection of thin film electrodes in solid state nanopores.

Solid state nanopores are a core element of next-generation single molecule tools in the field of nano-biotechnology. Thin film electrodes integrated into a pore can interact with charges and fields within the pore. In order to keep the nanopore open and thus functional electrochemically induced surface alteration of electrode surfaces and bubble formation inside the pore have to be eliminated. This paper provides electrochemical analyses of nanopores drilled into TiN membranes which in turn were employed as thin film electrodes. We studied physical pore integrity and the occurrence of water decomposition yielding bubble formation inside pores by applying voltages between -4.5 and +4.5 V to membranes in various protection stages continuously for up to 24 h. During potential application pores were exposed to selected electrolyte-solvent systems. We have investigated and successfully eliminated electrochemical pore oxidation and reduction as well as water decomposition inside nanopores of various diameters ranging from 3.5 to 25 nm in 50 nm thick TiN membranes by passivating the nanopores with a plasma-oxidized layer and using a 90% solution of glycerol in water as KCl solvent. Nanopore ionic conductances were measured before and after voltage application in order to test for changes in pore diameter due to electrochemical oxidation or reduction. TEM imaging was used to confirm these observations. While non-passivated pores were electrochemically oxidized, neither electrochemical oxidation nor reduction was observed for passivated pores. Bubble formation through water decomposition could be detected in non-passivated pores in KCl/water solutions but was not observed in 90% glycerol solutions. The use of a protective self-assembled monolayer of hexadecylphosphonic acid (HDPA) was also investigated.

Electrochemical characterization of thin film electrodes toward developing a DNA transistor.

The DNA-Transistor is a device designed to control the translocation of single-stranded DNA through a solid-state nanopore. Functionality of the device is enabled by three electrodes exposed to the DNA-containing electrolyte solution within the pore and the application of a dynamic electrostatic potential well between the electrodes to temporarily trap a DNA molecule. Optimizing the surface chemistry and electrochemical behavior of the device is a necessary (but by no means sufficient) step toward the development of a functional device. In particular, effects to be eliminated are (i) electrochemically induced surface alteration through corrosion or reduction of the electrode surface and (ii) formation of hydrogen or oxygen bubbles inside the pore through water decomposition. Even though our motivation is to solve problems encountered in DNA transistor technology, in this paper we report on generic surface chemistry results. We investigated a variety of electrode-electrolyte-solvent systems with respect to their capability of suppressing water decomposition and maintaining surface integrity. We employed cyclic voltammetry and long-term amperometry as electrochemical test schemes, X-ray photoelectron spectroscopy, atomic force microscopy, and scanning, as well as transmission electron microscopy as analytical tools. Characterized electrode materials include thin films of Ru, Pt, nonstoichiometric TiN, and nonstoichiometric TiN carrying a custom-developed titanium oxide layer, as well as custom-oxidized nonstoichiometric TiN coated with a monolayer of hexadecylphosphonic acid (HDPA). We used distilled water as well as aqueous solutions of poly(ethylene glycol) (PEG-300) and glycerol as solvents. One millimolar KCl was employed as electrolyte in all solutions. Our results show that the HDPA-coated custom-developed titanium oxide layer effectively passivates the underlying TiN layer, eliminating any surface alterations through corrosion or reduction within a voltage window from -2 V to +2 V. Furthermore, we demonstrated that, by coating the custom-oxidized TiN samples with HDPA and increasing the concentration of PEG-300 or glycerol in aqueous 1 mM KCl solutions, water decomposition was suppressed within the same voltage window. Water dissociation was not detected when combining custom-oxidized HDPA-coated TiN electrodes with an aqueous 1 mM KCl-glycerol solution at a glycerol concentration of at least 90%. These results are applicable to any system that requires nanoelectrodes placed in aqueous solution at voltages that can activate electrochemical processes.

Prion protein detection in serum using micromechanical resonator arrays.

Prion proteins that have transformed from their normal cellular counterparts (PrP(c)) into infectious form (PrP(res)) are responsible for causing progressive neurodegenerative diseases in numerous species, such as bovine spongiform encephalopathy (BSE) in cattle (also known as mad cow disease), scrapie in sheep, and Creutzfeldt-Jakob disease (CJD) in humans. Due to a possible link between BSE and CJD it is highly desirable to develop non-invasive and ante mortem tests for the detection of prion proteins in bovine samples. Such ante mortem tests of all cows prior to slaughter will help to prevent the introduction of PrP(res) into the human food supply. Furthermore, detection of PrP(res) in donated blood will also help to prevent the transmission of CJD among humans through blood transfusion. In this study, we have continued development of a micromechanical resonator array that is capable of detecting PrP(c) in bovine blood serum. The sensitivity of the resonators for the detection of PrP(c) is further enhanced by the use of secondary mass labels. A pair of antibodies is used in a sandwich immunoassay format to immobilize PrP(c) on the surface of resonators and attach nanoparticles as secondary mass labels to PrP(c). Secondary mass labeling is optimized in terms of incubation time to maximize the frequency shifts that correspond to the presence of PrP(c) on the surface of resonators. Our results show that a minimum of 200 pg mL(-1) of PrP(c) in blood serum can be detected using micromechanical resonator arrays.

Detection of prostate specific antigen with nanomechanical resonators.

In this work, we use arrays of nanomechanical resonators to detect prostate specific antigen (PSA), a protein biomarker associated with prostate cancer. The surfaces of our very thin, trampoline-like devices are functionalized for immunospecific capture of PSA molecules, and the mass of bound material can be detected as a reduction in the resonant frequency. Fetal bovine serum was spiked with known concentrations of PSA, and in conjunction with a nanoparticle-based sandwich assay, concentrations as low as 50 fg mL(-1), or 1.5 fM, could be detected from the realistic samples. The presence of non-specific proteins in the serum did not significantly affect the sensitivity of our assay.

Prion protein detection using nanomechanical resonator arrays and secondary mass labeling.

Nanomechanical resonators have shown potential application for mass sensing and have been used to detect a variety of biomolecules. In this study, a dynamic resonance-based technique was used to detect prion proteins (PrP), which in conformationally altered forms are known to cause neurodegenerative diseases in animals as well as humans. Antibodies and nanoparticles were used as mass labels to increase the mass shift and thus amplify the frequency shift signal used in PrP detection. A sandwich assay was used to immobilize PrP between two monoclonal antibodies, one of which was conjugated to the resonator's surface while the other was either used alone or linked to the nanoparticles as a mass label. Without additional mass labeling, PrP was not detected at concentrations below 20 microg/mL. In the presence of secondary antibodies the analytical sensitivity was improved to 2 microg/mL. With the use of functionalized nanoparticles, the sensitivity improved an additional 3 orders of magnitude to 2 ng/mL.

Micro- and nanomechanical sensors for environmental, chemical, and biological detection.

Micro- and nanoelectromechanical systems, including cantilevers and other small scale structures, have been studied for sensor applications. Accurate sensing of gaseous or aqueous environments, chemical vapors, and biomolecules have been demonstrated using a variety of these devices that undergo static deflections or shifts in resonant frequency upon analyte binding. In particular, biological detection of viruses, antigens, DNA, and other proteins is of great interest. While the majority of currently used detection schemes are reliant on biomarkers, such as fluorescent labels, time, effort, and chemical activity could be saved by developing an ultrasensitive method of label-free mass detection. Micro- and nanoscale sensors have been effectively applied as label-free detectors. In the following, we review the technologies and recent developments in the field of micro- and nanoelectromechanical sensors with particular emphasis on their application as biological sensors and recent work towards integrating these sensors in microfluidic systems.