Impedance-Based Nanoporous Anodized Alumina/ITO Platforms for Label-Free Biosensors.

We report an experimental and computational approach for the fabrication and characterization of a highly sensitive and responsive label-free biosensor that does not require the presence of redox couples in electrolytes for sensitive electrochemical detection. The sensor is based on an aptamer-functionalized transparent electrode composed of nanoporous anodized alumina (NAA) grown on indium tin oxide (ITO)-covered glass. Electrochemical impedance changes in a thrombin binding aptamer (TBA)-functionalized NAA/ITO/glass electrode due to specific binding of α-thrombin are monitored for protein detection. The aptamer-functionalized electrode enables sensitive and specific thrombin protein detection with a detection limit of ∼10 pM and a high signal-to-noise ratio. The transient impedance of the alumina film-covered surface is computed using a computational electrochemical impedance spectroscopy (EIS) approach and compared to experimental observations to identify the dominant mechanisms underlying the sensor response. The computational and experimental results indicate that the sensing response is due to the modified ionic transport under the combined influence of steric hindrance and surface charge modification due to ligand/receptor binding between α-thrombin and the aptamer-covered alumina film. These results suggest that alumina film-covered electrodes utilize both steric and charge modulation for sensing, leading to tremendous improvement in the sensitivity and signal-to-noise ratio. The film configuration is amenable for miniaturization and can be readily incorporated into existing portable sensing systems.

A novel photoelectrochemical sensor based on tailoring printable mesoscopic chip for fast and real-time phospholipids oxidation detection.

The detection of phospholipids oxidation is important for meat control and disease prevention. In this paper, a photoelectrochemical sensor based on printable mesoscopic chip (PMC) for fast and real-time monitoring phospholipids oxidation was designed and fabricated. TiO2, ZrO2 and carbon films of PMC were screen-printed onto the FTO glass layer by layer. The PMC and the feasibility for determination of phospholipids oxidation were investigated by scanning electron microscope (SEM), UV-vis spectroscopy, cyclic voltammograms (CVs) and electrochemical impedance spectroscopy (EIS), etc. The short circuit current (Jsc) was used as a signal current, which would decrease if phospholipids in PMC were undergoing oxidation for the change of electrical properties. Compared with other methods, phospholipids in PMC did not require pretreatment, and the process was nondestructive and real-time. Meanwhile, this method showed high sensitivity and good selectivity. The fabricating process of PMC is simple, and the costs are low, relatively.

Micro-Raman Spectroscopy for Monitoring of Deposition Quality of High-k Stack Protective Layer onto Nanowire FET Chips for Highly Sensitive miRNA Detection.

Application of micro-Raman spectroscopy for the monitoring of quality of high-k (h-k) dielectric protective layer deposition onto the surface of a nanowire (NW) chip has been demonstrated. A NW chip based on silicon-on-insulator (SOI) structures, protected with a layer of high-k dielectric ((h-k)-SOI-NW chip), has been employed for highly sensitive detection of microRNA (miRNA) associated with oncological diseases. The protective dielectric included a 2-nm-thick Al2O3 surface layer and a 8-nm-thick HfO2 layer, deposited onto a silicon SOI-NW chip. Such a chip had increased time stability upon operation in solution, as compared with an unprotected SOI-NW chip with native oxide. The (h-k)-SOI-NW biosensor has been employed for the detection of DNA oligonucleotide (oDNA), which is a synthetic analogue of miRNA-21 associated with oncological diseases. To provide biospecificity of the detection, the surface of (h-k)-SOI-NW chip was modified with oligonucleotide probe molecules (oDVA probes) complementary to the sequence of the target biomolecule. Concentration sensitivity of the (h-k)-SOI-NW biosensor at the level of DL~10-16 M has been demonstrated.

A novel 384-multiwell microelectrode array for the impedimetric monitoring of Tau protein induced neurodegenerative processes.

Over the last decades, countless bioelectronic monitoring systems were developed for the analysis of cells as well as complex tissues. Most studies addressed the sensitivity and specificity of the bioelectronic detection method in comparison to classical molecular biological assays. In contrast, the up scaling as a prerequisite for the practical application of these novel bioelectronic monitoring systems is mostly only discussed theoretically. In this context, we developed a novel 384-multiwell microelectrode array (MMEA) based measurement system for the sensitive label-free real-time monitoring of neurodegenerative processes by impedance spectroscopy. With respect to the needs of productive screening systems for robust and reproducible measurements on high numbers of plates, we focused on reducing the critical contacting of more than 400 electrodes for a 384-MMEA. Therefore, we introduced an on top array of immersive counter electrodes that are individually addressed by a multiplexer and connected all measurement electrodes on the 384-MMEA to a single contact point. More strikingly, our novel approach provided a comparable signal stability and sensitivity similar to an array with integrated counter electrodes. Next, we optimized a SH-SY5Y cell based tauopathy model by introducing a novel 5-fold Tau mutation eliminating the need of artificial tauopathy induction. In combination with our novel 384-MMEA based measurement system, the concentration and time dependent neuroregenerative effect of the kinase inhibitor SRN-003-556 could be quantitatively monitored. Thus, our novel screening system could be a useful tool to identify and develop potential novel therapeutics in the field of Tau-related neurodegenerative diseases.

Lytic enzymes as selectivity means for label-free, microfluidic and impedimetric detection of whole-cell bacteria using ALD-Al2O3 passivated microelectrodes.

Point-of-care (PoC) diagnostics for bacterial detection offer tremendous prospects for public health care improvement. However, such tools require the complex combination of the following performances: rapidity, selectivity, sensitivity, miniaturization and affordability. To meet these specifications, this paper presents a new selectivity method involving lysostaphin together with a CMOS-compatible impedance sensor for genus-specific bacterial detection. The method enables the sample matrix to be directly flown on the polydopamine-covered sensor surface without any pre-treatment, and considerably reduces the background noise. Experimental proof-of-concept, explored by simulations and confirmed through a setup combining simultaneous optical and electrical real-time monitoring, illustrates the selective and capacitive detection of Staphylococcus epidermidis in synthetic urine also containing Enterococcus faecium. While providing capabilities for miniaturization and system integration thanks to CMOS compatibility, the sensors show a detection limit of ca. 10(8) (CFU/mL).min in a 1.5 µL microfluidic chamber with an additional setup time of 50 min. The potentials, advantages and limitations of the method are also discussed.

The influence of nanopore dimensions on the electrochemical properties of nanopore arrays studied by impedance spectroscopy.

The understanding of the electrochemical properties of nanopores is the key factor for better understanding their performance and applications for nanopore-based sensing devices. In this study, the influence of pore dimensions of nanoporous alumina (NPA) membranes prepared by an anodization process and their electrochemical properties as a sensing platform using impedance spectroscopy was explored. NPA with four different pore diameters (25 nm, 45 nm and 65 nm) and lengths (5 µm to 20 µm) was used and their electrochemical properties were explored using different concentration of electrolyte solution (NaCl) ranging from 1 to 100 µM. Our results show that the impedance and resistance of nanopores are influenced by the concentration and ion species of electrolytes, while the capacitance is independent of them. It was found that nanopore diameters also have a significant influence on impedance due to changes in the thickness of the double layer inside the pores.

Sensor spacing affects the tissue impedance spectra of rabbit ventricular epicardium.

This study was designed to test the hypothesis that a complex composite impedance spectra develops when stimulation and recording of cardiac muscle with sufficiently fine spatial resolution in a four-electrode configuration is used. With traditional (millimeter scale) separations, the ratio between the recorded interstitial central potential difference and total supplied interstitial current is constant at all frequencies. This occurs because the fraction of supplied current that redistributes to the intracellular compartment depends on effective membrane resistance between electrodes, which is low, to a much greater extent than effective membrane capacitance. The spectra should therefore change with finer separations at which effective membrane resistance increases, as supplied current will remain primarily interstitial at lower frequencies and redistribute between compartments at higher frequencies. To test this hypothesis, we built arrays with sensors separated (d) by 804 µm, 452 µm, and 252 µm; positioned those arrays across myocyte axes on rabbit ventricular epicardium; and resolved spectra in terms of resistivity (ρt) and reactivity (χt) over the 10 Hz to 4,000 Hz range. With all separations, we measured comparable spectra with predictions from passive membrane simulations that used a three-dimensional structural framework in which intracellular, interstitial, and membrane properties were prescribed based on the limited data available from the literature. At the finest separation, we found mean ρt at 100 Hz and 4,000 Hz that lowered from 395 Ω-cm to 236 Ω-cm, respectively, with maximal mean χt of 160 Ω-cm. This experimental confirmation of spectra development in whole heart experiments is important because such development is central to achieve measurements of intracellular and interstitial passive electrical properties in cardiac electrophysiological experiments using only interstitial access.

Frequency dependent impedimetric cytotoxic evaluation of anticancer drug on breast cancer cell.

The present work reports the impedance characteristics of MCF-7 cell lines treated with anticancer drug ZD6474 to evaluate the cytotoxic effect on cellular electrical behaviour using miniature impedance sensors. Four types of impedance sensing devices with different electrode geometries are fabricated by microfabrication technology. The frequency response characteristics of drug treated cells are studied to evaluate cytotoxic effect of ZD6474 and also to assess the frequency dependent sensitivity variation with electrode area. A significant variation in magnitude of measured impedance data is obtained for drug treated samples above 10 µM dose indicating prominent effect of ZD6474 which results in suppression of cell proliferation and induction of apoptosis process. The results obtained by impedimetric method are correlated well with conventional in vitro assays such as flow cytometry, cell viability assays and microscopic imaging. Finally an empirical relation between cell impedance, electrode area and drug dose is established from impedance data which exhibits a negative correlation between drug doses and impedance of cancer cells.

Dielectric spectroscopy as a viable biosensing tool for cell and tissue characterization and analysis.

The use of dielectric spectroscopy to carry out real time observations of cells and to extract a wealth of information about their physiological properties has expanded in recent years. This popularity is due to the simple, easy to use, non-invasive and real time nature of dielectric spectroscopy. The ease of integrating dielectric spectroscopy with microfluidic devices has allowed the technology to further expand into biomedical research. Dielectric spectra are obtained by applying an electrical signal to cells, which is swept over a frequency range. This review covers the different methods of interpreting dielectric spectra and progress made in applications of impedance spectroscopy for cell observations. First, methods of obtaining specific electrical properties of cells (cell membrane capacitance and cytoplasm conductivity) are discussed. These electrical properties are obtained by fitting the dielectric spectra to different models and equations. Integrating models to reduce the effects of the electrical double layer are subsequently covered. Impedance platforms are then discussed including electrical cell substrate impedance sensing (ECIS). Categories of ECIS systems are divided into microelectrode arrays, interdigitated electrodes and those that allow differential ECIS measurements. Platforms that allow single cell and sub-single cell measurements are then discussed. Finally, applications of impedance spectroscopy in a range of cell observations are elaborated. These applications include observing cell differentiation, mitosis and the cell cycle and cytotoxicity/cell death. Future applications such as drug screening and in point of care applications are then covered.

Nanosensor electrical immunoassay for quantitative detection of NT-pro brain natriuretic peptide.

AIM: To demonstrate a label-free electrical immunoassay for profiling vascular biomarker N-terminal pro-brain natriuretic peptide (NT-proBNP) associated with improved cardiac risk prediction. MATERIALS & METHODS: A high-density nanowell-based electrical immunoassay has been designed by integrating nanoporous aluminum oxide onto printed circuit board chips for the detection of NT-proBNP. The concentration of the biomarker is quantitatively determined by measuring impedance changes to the electrical double layer within the nanowells using electrochemical impedance spectroscopy. Detection sensitivity in the fg/ml range was obtained due to spatial confinement of the target biomarkers in size-matched nanowells. RESULTS & DISCUSSION: Electrical immunoassay performance was determined for the detection of NT-proBNP in phosphate-buffered saline (PBS) and human serum (HS). The lower limit of detection for the sensor was observed to be 10 fg/ml in PBS and 500 fg/ml in HS. The upper limit of detection was observed to be 500 fg/ml in PBS and 500 ng/ml in HS. CONCLUSION: A label-free technique for detection of NT-proBNP at clinically relevant concentrations for evaluating cardiac risk is demonstrated. High sensitivity and specificity, robust detection and low volume (100 µl) per assay project the technology to be a successful competitor to traditional ELISA-based techniques.

Impedance spectroscopy with field-effect transistor arrays for the analysis of anti-cancer drug action on individual cells.

In this study, impedance spectroscopy measurements of silicon-based open-gate field-effect transistor (FET) devices were utilized to study the adhesion status of cancer cells at a single cell level. We developed a trans-impedance amplifier circuit for the FETs with a higher bandwidth compared to a previously described system. The new system was characterized with a fast lock-in amplifier, which enabled measuring of impedance spectra up to 50 MHz. We studied cellular activities, including cell adhesion and anti-cancer drug induced apoptosis of human embryonic kidney (HEK293) and human lung adenocarcinoma epithelial (H441) cells. A well-known chemotherapeutic drug, topotecan hydrochloride, was used to investigate the effect of this drug to tumor cells cultured on the FET devices. The presence of the drug resulted in a 20% change in the amplitude of the impedance spectra at 200 kHz as a result of the induced apoptosis process. Real-time impedance measurements were performed inside an incubator at a constant frequency. The experimental results can be interpreted with an equivalent electronic circuit to resolve the influence of the system parameters. The developed method could be applied for the analysis of the specificity and efficacy of novel anti-cancer drugs in cancer therapy research on a single cell level in parallelized measurements.

Electrochemical impedance spectroscopy characterization of nanoporous alumina dengue virus biosensor.

The Faradaic electrochemical impedance technique is employed to characterize the impedance change of a nanoporous alumina biosensor in response towards the specific binding of dengue serotype 2 (Denv2) viral particles to its serotype 2-specific immunoglobulin G antibody within the thin alumina layer. The optimal equivalent circuit model that matches the impedimetric responses of the sensor describes three distinct regions: the electrolyte solution (R(s)), the porous alumina channels (including biomaterials) (Q(1), R(1)) and the conductive electrode substrate layer (Q(2), R(2)). Both channel resistance R(1) and capacitance Q(1) change in response to the increase of the Denv2 virus concentration. A linear relationship between R(1) and Denv2 concentration from 1 to 900 plaque forming unit per mL (pfu mL(-1)) can be derived using Langmuir-Freundlich isotherm model. At 1pfu mL(-1) Denv2 concentration, R(1) can be distinguished from that of the cell culture control sample. Moreover, Q(1) doubles when Denv2 is added but remains unchanged in the presence of two other non-specific viruses - West Nile virus and Chikungunya virus indicates biosensor specificity can be quantitatively measured using channel capacitance.

Electrochemical impedimetric biosensor based on a nanostructured polycarbonate substrate.

This study integrates the techniques of nanoelectroforming, hot-embossing, and electrochemical deposition to develop a disposable, low-cost, and high sensitivity nanostructure biosensor. A modified anodic aluminum oxide barrier-layer surface was used as the template for thin nickel film deposition. After etching the anodic aluminum oxide template off, a three-dimensional mold of the concave nanostructure array was created. The fabricated three-dimensional nickel mold was further used for replica molding of a nanostructure polycarbonate substrate by hot-embossing. A thin gold film was then sputtered onto the polycarbonate substrate to form the electrode, followed by deposition of an orderly and uniform gold nanoparticle layer on the three-dimensional gold electrode using electrochemical deposition. Finally, silver nanoparticles were deposited on the uniformly deposited gold nanoparticles to enhance the conductivity of the sensor. Electrochemical impedance spectroscopy analysis was then used to detect the concentration of the target element. The sensitivity of the proposed scheme on the detection of the dust mite antigen, Der p2, reached 0.1 pg/mL.

Nanoporous impedemetric biosensor for detection of trace atrazine from water samples.

Trace contamination of ground water sources has been a problem ever since the introduction of high-soil-mobility pesticides, one such example is atrazine. In this paper we present a novel nanoporous portable bio-sensing device that can identify trace contamination of atrazine through a label-free assay. We have designed a pesticide sensor comprising of a nanoporous alumina membrane integrated with printed circuit board platform. Nanoporous alumina in the biosensor device generates a high density array of nanoscale confined spaces. By leveraging the size based immobilization of atrazine small molecules we have designed electrochemical impedance spectroscopy based biosensor to detect trace amounts of atrazine. We have calibrated the sensor using phosphate buffered saline and demonstrated trace detection from river and bottled drinking water samples. The limit of detection in all the three cases was in the femtogram/mL (fg/mL) (parts-per-trillion) regime with a dynamic range of detection spanning from 10 fg/mL to 1 ng/mL (0.01 ppt to 1 ppm). The selectivity of the device was tested using a competing pesticide; malathion and selectivity in detection was observed in the fg/mL regime in all the three cases.