Nanostructured Microelectrodes Research Articles

Ultradense Electrochemical Chips with Arrays of Nanostructured Microelectrodes to Enable Sensitive Diffusion-Limited Bioassays.

Nanostructured microelectrodes (NMEs) are an attractive alternative to yield sensitive bioassays in unprocessed samples. However, although valuable for different applications, nanoporous NMEs usually cannot boost the sensitivity of diffusion-limited analyses because of the enlarged Debye length within the nanopores, which reduces their accessibility. To circumvent this limitation, nanopore-free gold NMEs were electrodeposited from 45 μm SU-8 apertures, featuring nanoridged microspikes on a recessed surface of gold thin film while carrying interconnected crown-like and spiky structures along the edge of a SU-8 passivation layer. These structures were grown onto ultradense, vertical array chips that offer a promising strategy for translating reproducible, high-resolution, and cost-effective sensors into real-world applications. The NMEs yielded reproducible analyses, while machine learning allowed us to predict the analytical responses from NME electrodeposition data. By taking advantage of the high surface area and accessible structure of the NMEs, these structures provided a sensitivity for [Fe(CN)6]3-/4- that was 5.5× higher than that of bare WEs while also delivering a moderate antibiofouling property in undiluted human plasma. As a proof of concept, these electrodes were applied toward the fast (22 min) and simple determination of Staphylococcus aureus by monitoring the oxidation of [Fe(CN)6]4-, which acted as a cellular respiration rate redox reporter. The sensors also showed a wide dynamic range, spanning 5 orders of magnitude, and a calculated limit of detection of 0.2 CFU mL-1.

Implantable nanostructured microelectrode array with biphasic current stimulator for retinal prostheses.

In retinal prosthetic systems on multi-channel microelectrodes to effectively stimulate retinal neurons, the electrode-electrolyte interface impedance of a microelectrode should be minimized to drive sufficiently large current at a given supply voltage. This paper presents the fabrication of the nanostructured microelectrode array with simplified fabrication and its characteristic evaluation using biphasic current stimulator. The nanostructured microelectrodes with the base diameter of 25 μm, 50 μm, 75 μm are fabricated, and the maximum allowable current injection limits are measured to verify the estimated injection limit. Also, a biphasic stimulator has been fabricated using the 2-stage amplifier and 4 switches based on a stimulator cell. The adjustable load resistance is adopted to control between 5 kΩ to 20 kΩ, and the biphasic stimulator can drive the stimulation current between 50 uA and 200 uA. The proposed electrode-electrolyte interface impedance of the fabricated nanostructured microelectrode is 3178 Ω, 1218 Ω and 798.8 Ω for electrodes with diameter of 25 μm, 50 μm, 75 μm, respectively. This paper shows the advantages of the nanostructured microelectrode arrays for high resolution retinal prostheses, which could be a basic experiment for artificial retina research.

Advanced Biomimetic Nanostructured Microelectrode Arrays for Enhanced Extracellular Recordings of Enteric Neurons

AbstractMicroelectrode surfaces covered with nanostructures derived from components of extracellular matrix, such as collagen fibers, have shown immense beneficial effects in promoting neuronal growth and cellular signaling. Synthetic nanostructures mimicking the features of biological nanostructures with durable conductive materials could promote the cell adhesion on microelectrode surfaces by providing topographical cues and simultaneously improve the charge transfer properties by reducing its global impedance. Therefore, an advanced nanostructuring method mimicking the structural and organizational features of natural collagen fibers onto metallic microelectrode surfaces has been presented here, which is adapted from previous technological achievements of the group and is based on nanoimprint lithography and gold electroplating. Surface characterization methods reveal an increase in surface area between 20% and 68% on the microelectrodes fabricated with two different nanostructure heights. Impedance spectroscopy measurements reveal reduction in impedance magnitude (at 1 kHz) between 22% and 41%, depending upon the nanostructure height and density on the microelectrode, which should subsequently modulate its charge transfer properties for biosensing application. Cell adhesion analysis performed with seal impedance measurements reveals a tighter coupling of enteric neurons on the nanostructured microelectrodes. Finally, extracellular recordings from enteric neurons exhibit a significant improvement in spike detection properties of the nanostructured microelectrodes.

Rapid ultrasensitive bacteria detection using microelectrode sensors

Nanostructured microelectrodes provide extremely high sensitivity for bacteria detection without compromising on speed.

Highly sensitive and selective nanostructured microbiosensors for glucose and lactate simultaneous measurements in blood serum and in vivo in brain tissue

Highly sensitive and selective nanostructured lactate and glucose microbiosensors for their in vivo simultaneous determination in rat brain were developed based on carbon fiber microelectrodes (CFM) modified with nanoporous gold (NPG) using the Dynamic Hydrogen Bubble Template (DHBT) method. Electrodeposition of platinum nanoparticles (PtNP) onto the NPG film enhances the sensitivity and the electrocatalytic properties towards H2O2 detection. The nanostructured microelectrode platform was modified by glucose oxidase (GOx) and lactate oxidase (LOx) enzyme immobilization. High selective measurements were achieved by covering with a perm-selective layer of electropolymerized m-phenylenediamine, deposition of a Nafion® film and by using a null sensor. The morphological characteristics and electroanalytical performance of the microbiosensors were assessed, by scanning electron microscopy and electrochemical techniques, respectively.The PtNP/NPG/CFM shows a high sensitivity to H2O2 (5.96 A M-1 cm-2) at 0.36 V vs. Ag/AgCl, with a linear range from 0.2 to 200 μM, and an LOD of 10 nM. The microbiosensors were applied to the simultaneous determination of lactate and glucose in blood serum samples. Moreover, the basal extracellular concentrations of lactate and glucose were measured in vivo in four different rat brain structures. These results support the potential of the microbiosensor to be used as a valuable tool to investigate brain neurochemicals in vivo.

Electrochemical Immuno-Biosensors on Nanostructured Electrodes for Rapid Sensitive Detection of Disease Biomarkers

Development of rapid tests for quantitative detection of analytes in complex matrices, plays an important role in medical diagnostics, prognostics and therapeutics. Current methods for molecular diagnostics are time-consuming and multiple-step reliant on well-trained technicians and fully-equipped laboratories to provide quantitative detection of biomarkers. In this case, the challenges to overcome the sensitivity and selectivity of working directly in biological fluids in a timely manner have always been the main step for the development of such rapid tests.Electrochemical sensors, analogous to glucometer, are known to be rapid with the ability to provide direct electronic signal without any interference from the biological phenomena. When functionalized with specific biological substances, they intend to provide the required specificity in addition to signal selectivity in biological fluids. Inspired by nature, we design biorecognition probes that are combined with newest advancement in the engineering for the development of electrochemical immune-biosensors. These molecular recognition probes are designed using the ability of antibodies to bind antigens, and DNA construct to hybridize to the complementary construct, to create specificity for direct detection in complex media. On the other hand, we rely on the innovative approaches in nanomaterials, surface sciences, and self-assembly techniques to improve the conductivity and sensitivity of electrode surface. This includes the incorporation of high-curvature nanostructured microelectrodes that helps with the efficiency of molecular interactions at the surface and the transfer of electrons for the ease of signal transduction.In this regard, we fabricate small-scale nanostructured electrodes and engineer the surface for the immobilization of the biomolecular monolayer at the interface to provide the desired specificity for biomarkers in real biological samples, e.g. whole blood. We adapted our nano-bio-sensors for translational applications such as (1) at-line monitoring of signaling proteins in hematopoietic stem cell expansion, (2) rapid diagnosis of pathogenic infections through quantitative detection of antibodies directly in patient samples, and (3) rapid diagnosis of inner ear disorders based on the detection of blood biomarkers. We showed that our electrochemical immune-biosensors are capable of detecting at therapeutic concentrations with tunable ranges while performing in a 10 µL sample within less than 30 minutes.

Proliferation and Cluster Analysis of Neurons and Glial Cell Organization on Nanocolumnar TiN Sub-Strates.

Biomaterials employed for neural stimulation, as well as brain/machine interfaces, offer great perspectives to combat neurodegenerative diseases, while application of lab-on-a-chip devices such as multielectrode arrays is a promising alternative to assess neural function in vitro. For bioelectronic monitoring, nanostructured microelectrodes are required, which exhibit an increased surface area where the detection sensitivity is not reduced by the self-impedance of the electrode. In our study, we investigated the interaction of neurons (SH-SY5Y) and glial cells (U-87 MG) with nanocolumnar titanium nitride (TiN) electrode materials in comparison to TiN with larger surface grains, gold, and indium tin oxide (ITO) substrates. Glial cells showed an enhanced proliferation on TiN materials; however, these cells spread evenly distributed over all the substrate surfaces. By contrast, neurons proliferated fastest on nanocolumnar TiN and formed large cell agglomerations. We implemented a radial autocorrelation function of cellular positions combined with various clustering algorithms. These combined analyses allowed us to quantify the largest cluster on nanocolumnar TiN; however, on ITO and gold, neurons spread more homogeneously across the substrates. As SH-SY5Y cells tend to grow in clusters under physiologic conditions, our study proves nanocolumnar TiN as a potential bioactive material candidate for the application of microelectrodes in contact with neurons. To this end, the employed K-means clustering algorithm together with radial autocorrelation analysis is a valuable tool to quantify cell-surface interaction and cell organization to evaluate biomaterials’ performance in vitro.

Electrochemical PEDOT Sensor for Antioxidantsin Beverages and Milks

Introduction The conducting polymer PEDOT (poly(3,4-ethylenedioxythiophene)) as an electrochemical sensor element possesses redox-mediating properties that allow excellent separation of signals due to antioxidants present in beverages such as teas and coffee [1]. This includes the phenolic acids present in white wines, with specific features noted for nanostructured microelectrodes [2]. Excellent separation of signals due to ascorbic acid and sulfites, alongside the response due to the oxidation of polyphenols in beverages, has been achieved using the redox-mediating properties of PEDOT [3]. Method PEDOT films were grown by cycling to +1.2 V (Ag/AgCl) on 3 mm dia. glassy carbon electrodes and 10 µm dia. gold microelectrodes in 0.1 M EDOT/ 0.1 M LiClO4 in propylene carbonate at 100 mV s-1. The electrodes were then transferred to undiluted milks (c. pH 6.5), or to an aqueous 0.1 M sodium phosphate buffer (PBS) at pH 6.6 containing uric acid and ascorbic acid standards, prior to testing by cyclic voltammetry (CV). A background PEDOT curve was taken in the PBS solution and subtracted from the voltammograms obtained for the milk samples. Results and Conclusions The PEDOT sensor showed an excellent response for the oxidation of uric acid, with an anodic peak seen at 350 mV (Ag/AgCl), taken at pH 6.6, with both uric acid standards and undiluted milks [4]. A small peak due to ascorbic acid was at times located close to 0 mV, enabling the simultaneous detection of the two antioxidants. Very good linear relationships were obtained between peak current intensities and concentrations in the range of 6-100 μM for uric acid and 30-500 μM for ascorbic acid. The newly developed sensor was then successfully applied to 36 commercial milk samples for a fast voltammetric determination of uric acid and ascorbic acid concentration. An independent HPLC analysis was also performed immediately after running the same milk samples, with a pre-treatment step to remove fats and proteins from the milk. The average difference between the results for uric acid from the two methods was 7%, demonstrating an acceptable degree of agreement between the two measurement methods. At the same time the results were obtained much more rapidly with the electrochemical method than in the lengthy HPLC procedure.The polyphenols present in teas, chocolate and flavoured milks present additional analytical challenges, due to strong adsorption effects. PEDOT-modified electrodes have been successfully developed to quantify flavonoids present in chocolate-flavoured milks, and hydroxycinnamic acids present in coffee-flavoured milks. Strong adsorption effects were seen with regular and flavoured milks at PEDOT microelectrodes. Rather than a diffusion-controlled plateau current, peaks with current densities up to 1000 times greater than observed at the corresponding macroelectrodes were observed, consistent with an extremely high surface area and fractal-type growth of PEDOT at microelectrodes [2].

A multiplexed, electrochemical interface for gene-circuit-based sensors.

The field of synthetic biology has used the engineered assembly of synthetic gene networks to create a wide range of function in biological systems. As part of this work, gene circuit-based sensors have primarily used optical proteins (e.g. fluorescent, colorimetric) as reporter outputs, which has limited the potential to measure multiple distinct signals. Here we present an electrochemical interface that permits expanded multiplexed reporting for cell-free gene circuit-based sensors. We have engineered a scalable system of reporter enzymes that cleave specific DNA sequences in solution, which results in an electrochemical signal when these newly liberated strands are captured at the surface of a nanostructured microelectrode. We describe the development of this interface and show its utility using a ligand-inducible gene circuit and toehold switch-based sensors, including the detection of multiple antibiotic resistance genes in parallel. This technology has the potential to expand the field of synthetic biology by providing an interface with materials, hardware and software.

Fabrication and evaluation of nanostructured microelectrodes for high-spatial resolution in retinal prostheses

To provide visual information to patients with retinal degenerative disease, retinal prosthetic devices based on multi-channel microelectrode arrays (MEAs) have shown some promising results. For high-quality information, the number of microelectrodes should be increased in the limited area of MEAs, which results in decreased dimensions of the single microelectrode and a limit of dynamic range of injection current. Previously, our research group has presented 3D microelectrodes to overcome the trade-off between high-spatial resolution and injection current range. However, the 3D microelectrode requires a complex fabrication process, including multiple steps in front-side and back-side microfabrication. In this paper, the nanostructured microelectrode with platinum-black is fabricated with a simple process, and its electrical characteristics are evaluated. The nanostructured microelectrode parameters are analyzed by a conventional three-element circuit model. The comprehensive electrical characteristics between planar 2D, 3D, and nanostructured microelectrodes with various base areas are compared by measuring electrode–electrolyte interface impedance and maximum allowable injection current limit. The experimental results show improved interface impedance in nanostructured microelectrodes compared to planar 2D and 3D microelectrodes with same base area. This implies that nanostructured microelectrodes have a large dynamic range for current stimulation, which can be more sufficient in retinal neuron stimulation. This research can be used to estimate the theoretical limit of injection current for high-resolution MEAs.

Metal-Organic Framework Thin Films on High-Curvature Nanostructures Toward Tandem Electrocatalysis.

In tandem catalysis, two distinct catalytic materials are interfaced to feed the product of one reaction into the next one. This approach, analogous to enzyme cascades, can potentially be used to upgrade small molecules such as CO2 to more valuable hydrocarbons. Here, we investigate the materials chemistry of metal-organic framework (MOF) thin films grown on gold nanostructured microelectrodes (AuNMEs), focusing on the key materials chemistry challenges necessary to enable the applications of these MOF/AuNME composites in tandem catalysis. We applied two growth methods-layer-by-layer and solvothermal-to grow a variety of MOF thin films on AuNMEs and then characterized them using scanning electron microscopy, X-ray diffraction, and X-ray photoelectron spectroscopy. The MOF@AuNME materials were then evaluated for electrocatalytic CO2 reduction. The morphology and crystallinity of the MOF thin films were examined, and it was found that MOF thin films were capable of dramatically suppressing CO production on AuNMEs and producing further-reduced carbon products such as CH4 and C2H4. This work illustrates the use of MOF thin films to tune the activity of an underlying CO2RR catalyst to produce further-reduced products.

Biomolecular Steric Hindrance Effects Are Enhanced on Nanostructured Microelectrodes.

The availability of rapid approaches for quantitative detection of biomarkers would drastically impact global health by enabling decentralized disease diagnosis anywhere that patient care is administered. A promising new approach, the electrochemical steric hindrance hybridization assay (eSHHA) has been introduced for quantitative detection of large proteins (e.g., antibodies) with a low nanomolar detection limit within 10 min. Here, we report the use of a nanostructured microelectrode (NME) platform for eSHHA that improves the performance of this approach by increasing the efficiency and kinetics of DNA hybridization. We demonstrated that eSHHA on nanostructured microelectrodes leverages three effects: (1) steric hindrance effects at the nanoscale, (2) a size-dependent hybridization rate of DNA complexes, and (3) electrode morphology-dependent blocking effects. As a proof of concept, we showed that the sensitivity of eSHHA toward a model antibody is enhanced using NMEs as scaffolds for this reaction. We improved the detection limit of eSHHA, taking advantage of nanostructured surfaces to allow the use of longer capture strands for detection of proteins. Finally, we concluded that using the eSHHA approach in conjunction with nanostructured microelectrodes is an advantageous alternative to conventional macroelectrodes as the sensitivity and detection limits are enhanced.

Steric Hindrance Assay for Secreted Factors in Stem Cell Culture.

The ex vivo expansion of hematopoietic stem cells is significantly inhibited by secreted proteins that induce negative feedback loops. The ability to effectively monitor these factors is critical for their real-time regulation and control and, by extension, enhancing stem cell expansion. Here, we describe a novel monitoring strategy for the detection of soluble signaling factors in stem cell cultures using a DNA-based sensing mechanism on a chip-based nanostructured microelectrode platform. We combine DNA hybridization engineering with antibody-capturing chemistry in an amplified steric hindrance hybridization assay. This method enables the quantification of important secreted proteins, showcased by the detection of 10 pg·mL-1 level concentrations of three proteins in stem cell culture samples. This approach is the first universal nonsandwich technique that permits pg·mL-1 level quantification of small proteins in stem cell culture media without signal loss.

Construction of DNA-based logic gates on nanostructured microelectrodes

Electrochemical logical operations utilizing biological molecules (protein or DNA), which can be used in disease diagnostics and bio-computing, have attracted great research interest. However, the existing logic operations, being realized on macroscopic electrode, are not suitable for implantable logic devices. Here, we demonstrate DNA-based logic gates with electrochemical signal as output combined with gold flower microelectrodes. The designed logic gates are of fast response, enzyme-free, and micrometer scale. They perform well in either pure solution or complex matrices, such as fetal bovine serum, suggesting great potential for in vivo applications.

Mechanistic Control of the Growth of Three-Dimensional Gold Sensors

Three-dimensional (3D) electrodes with large surface areas are highly effective biomolecular sensors. These structures can be generated via the electrodeposition of gold inside microscale apertures patterned on the surface of a microelectronic chip. Such electrodes enable the ultrasensitive analysis of nucleic acids, proteins, and small molecules. Since the performance of these electrodes is directly related to their surface area, the ability to control their microscale morphology is critical. Here, we explore an electrochemical model based on the theory of nucleation and growth to better understand how to control the morphology of these electrodes. The insights gained from this model enabled us to create preferential conditions for the formation of different morphological features. We demonstrate for the first time that electrodeposition of 3D nanostructured microelectrodes inside a microscale aperture is governed by two stages of nucleation and growth. The first stage involves the creation of primary nu...

Nanostructured gold microelectrodes for SERS and EIS measurements by incorporating ZnO nanorod growth with electroplating.

In this paper, a fine gold nanostructure synthesized on selective planar microelectrodes in micro-chip is realized by using an advanced hybrid fabrication approach incorporating growth of nanorods (NRs) with gold electroplating. By this developed nanostructure, integration of in-situ surface-enhanced Raman spectroscopy (SERS) detection with electrochemical impedance spectroscopy (EIS) measurement for label-free, nondestructive, real-time and rapid monitoring on a single cell has been achieved. Moreover, parameters of Au nanostructures such as size of nanoholes/nanogaps can be controllably adjusted in the fabrication. We have demonstrated a SERS enhancement factor of up to ~2.24 × 106 and double-layer impedance decrease ratio of 90% ~ 95% at low frequency range below 200 kHz by using nanostructured microelectrodes. SERS detection and in-situ EIS measurement of a trapped single cell by using planar microelectrodes are realized to demonstrate the compatibility, multi-functions, high-sensitivity and simplicity of the micro-chip system. This dual function platform integrating SERS and EIS is of great significance in biological, biochemical and biomedical applications.

Sample-to-Answer Isolation and mRNA Profiling of Circulating Tumor Cells.

The isolation and rapid molecular characterization of circulating tumor cells (CTCs) from a liquid biopsy could enable the convenient and effective characterization of the state and aggressiveness of cancerous tumors. Existing technologies enumerate CTCs using immunostaining; however, these approaches are slow, labor-intensive, and often fail to enable further genetic characterization of CTCs. Here, we report on an integrated circuit that combines the capture of CTCs with the profiling of their gene expression signatures. Specifically, we use a velocity valley chip to efficiently capture magnetic nanoparticle-bound CTCs, which are then directly analyzed for their gene expression profiles using nanostructured microelectrode biosensors. CTCs are captured with 97% efficiency from 2 mL of whole blood, yielding a 500-fold concentration within 1 h. We show efficient capture of as few as 2 cancer cells/(mL of blood) and demonstrate that the gene expression module accurately profiles the expression of prostate-specific genes in CTCs captured from whole blood. This advance provides the first sample-to-answer solution for gene-based testing of CTCs. The approach was successfully validated using samples collected from prostate cancer patients: both CTCs and prostate-specific antigen (PSA) mRNA sequences were detected in all cancer patient samples and not in the healthy controls.

Ultrasensitive visual read-out of nucleic acids using electrocatalytic fluid displacement

Diagnosis of disease outside of sophisticated laboratories urgently requires low-cost, user-friendly devices. Disposable, instrument-free testing devices are used for home and physician office testing, but are limited in applicability to a small class of highly abundant analytes. Direct, unambiguous visual read-out is an ideal way to deliver a result on a disposable device; however, existing strategies that deliver appropriate sensitivity produce only subtle colour changes. Here we report a new approach, which we term electrocatalytic fluid displacement, where a molecular binding event is transduced into an electrochemical current, which drives the electrodeposition of a metal catalyst. The catalyst promotes bubble formation that displaces a fluid to reveal a high contrast change. We couple the read-out system to a nanostructured microelectrode and demonstrate direct visual detection of 100 fM DNA in 10 min. This represents the lowest limit of detection of nucleic acids reported using high contrast visual read-out.

A digital microfluidic device with integrated nanostructured microelectrodes for electrochemical immunoassays.

Nanostructured microelectrodes (NMEs) are three-dimensional electrodes that have superb sensitivity for electroanalysis. Here we report the integration of NMEs with the versatile fluid-handling system digital microfluidics (DMF), for eventual application to distributed diagnostics outside of the laboratory. In the new methods reported here, indium tin oxide DMF top plates were modified to include Au NMEs as well as counter and pseudoreference electrodes. The new system was observed to outperform planar sensing electrodes of the type that are typically integrated with DMF. A rubella virus (RV) IgG immunoassay was developed to evaluate the diagnostic potential for the new system, relying on magnetic microparticles coated with RV particles and analysis by differential pulse voltammetry. The limit of detection of the assay (0.07 IU mL(-1)) was >100× below the World Health Organization defined cut-off for rubella immunity. The sensitivity of the integrated device and its small size suggest future utility for distributed diagnostics.

Simulation and Measurement of Neuroelectrodes Characteristics with Integrated High Aspect Ratio Nano Structures

Improving the interface between electrodes and neurons has been the focus of research for the last decade. Neuroelectrodes should show small geometrical surface area and low impedance for measuring and high charge injection capacities for stimulation. Increasing the electrochemically active surface area by using nanoporous electrode material or by integrating nanostructures onto planar electrodes is a common approach to improve this interface. In this paper a simulation approach for neuro electrodes' characteristics with integrated high aspect ratio nano structures based on a point-contact-model is presented. The results are compared with experimental findings conducted with real nanostructured microelectrodes. In particular, effects of carbon nanotubes and gold nanowires integrated onto microelectrodes are described. Simulated and measured impedance properties are presented and its effects onto the transfer function between the neural membrane potential and the amplifier output signal are studied based on the point-contact-model. Simulations show, in good agreement with experimental results, that electrode impedances can be dramatically reduced by the integration of high aspect ratio nanostructures such as gold nanowires and carbon nanotubes. This lowers thermal noise and improves the signal-to-noise ratio for measuring electrodes. It also may increase the adhesion of cells to the substrate and thus increase measurable signal amplitudes.