Quantitative Detection of Cathepsin B Activity in Neutral pH Buffers Using Gold Microelectrode Arrays: Toward Direct Multiplex Analyses of Extracellular Proteases in Human Serum.

Proteases are critical signaling molecules and prognostic biomarkers for many diseases including cancer. There is a strong demand for multiplex bioanalytical techniques that can rapidly detect the activity of extracellular proteases with high sensitivity and specificity. This study demonstrates an activity-based electrochemical biosensor of a 3 × 3 gold microelectrode array for the detection of cathepsin B activity in human serum diluted in a neutral buffer. Proteolysis of ferrocene-labeled peptide substrates functionalized on 200 × 200 µm microelectrodes is measured simultaneously over the nine channels by AC voltammetry. The protease activity is represented by the inverse of the exponential decay time constant (1/τ), which equals to (kcat/KM)[CB] based on the Michaelis-Menten model. An enhanced activity of the recombinant human cathepsin B (rhCB) is observed in a low-ionic-strength phosphate buffer at pH = 7.4, giving a very low limit of detection of 8.49 × 10-4 s-1 for activity and 57.1 pM for the active rhCB concentration that is comparable to affinity-based enzyme-linked immunosorbent assay (ELISA). The cathepsin B presented in the human serum sample is validated by ELISA, which mainly detects the inactive proenzyme, while the electrochemical biosensor specifically measures the active cathepsin B and shows significantly higher decay rates when rhCB and human serum are activated. Analyses of the kinetic electrochemical measurements with spiked active cathepsin B in human serum provide further assessment of the protease activity in the complex sample. This study lays the foundation to develop the gold microelectrode array into a multiplex biosensor for rapid detection of the activity of extracellular proteases toward cancer diagnosis and treatment assessment.

Simultaneous, multiplex quantification of protease activities using a gold microelectrode array.

Proteases are a large family of enzymes involved in many important biological processes. Quantitative detection of the activity profile of specific target proteases is in high demand for the diagnosis and monitoring of diseases such as cancers. This study demonstrates the fabrication and characterization of an individually addressable 3 × 3 Au microelectrode array for rapid, multiplex detection of cathepsin B activity based on a simple electrochemical method. The nine individual microelectrodes in the array show highly consistent cyclic voltammetric signals in Au surface cleaning experiments and detecting benchmark redox species in solution. The individual Au microelectrodes are further selectively functionalized with specific ferrocene-labeled peptide molecules which serve as the cognate substrates for the target proteases. Consistent proteolytic kinetics are measured by monitoring the decay of the AC voltammetry signal from the ferrocene label as the peptide molecules are cleaved by cathepsin B. Accurate activity of cathepsin B is derived with an improved fitting algorithm. Simultaneous detection of the proteolysis of cathepsin B on the microelectrode array functionalized with three different hexapeptides is demonstrated, showing the potential of this sensor platform for rapid detection of the activity profiles of multiple proteases in various diseases including many forms of cancer.

Fully inkjet-printed multilayered graphene-based flexible electrodes for repeatable electrochemical response.

Graphene has proven to be useful in biosensing applications. However, one of the main hurdles with printed graphene-based electrodes is achieving repeatable electrochemical performance from one printed electrode to another. We have developed a consistent fabrication process to control the sheet resistance of inkjet-printed graphene electrodes, thereby accomplishing repeatable electrochemical performance. Herein, we investigated the electrochemical properties of multilayered graphene (MLG) electrodes fully inkjet-printed (IJP) on flexible Kapton substrates. The electrodes were fabricated by inkjet printing three materials - (1) a conductive silver ink for electrical contact, (2) an insulating dielectric ink, and (3) MLG ink as the sensing material. The selected materials and fabrication methods provided great control over the ink rheology and material deposition, which enabled stable and repeatable electrochemical response: bending tests revealed the electrochemical behavior of these sensors remained consistent over 1000 bend cycles. Due to the abundance of structural defects (e.g., edge defects) present in the exfoliated graphene platelets, cyclic voltammetry (CV) of the graphene electrodes showed good electron transfer (k = 1.125 × 10-2 cm s-1) with a detection limit (0.01 mM) for the ferric/ferrocyanide redox couple, [Fe(CN)6]-3/-4, which is comparable or superior to modified graphene or graphene oxide-based sensors. Additionally, the potentiometric response of the electrodes displayed good sensitivity over the pH range of 4-10. Moreover, a fully IJP three-electrode device (MLG, platinum, and Ag/AgCl) also showed quasi-reversibility compared to a single IJP MLG electrode device. These findings demonstrate significant promise for scalable fabrication of a flexible, low cost, and fully-IJP wearable sensor system needed for space, military, and commercial biosensing applications.

Application-Specific Catalyst Layers: Pt-Containing Carbon Nanofibers for Hydrogen Peroxide Detection.

Complete removal of metal catalyst particles from carbon nanofibers (CNFs) and other carbon nanostructures is extremely difficult, and the envisioned applications may be compromised by the left-over impurities. To circumvent these problems, one should use, wherever possible, such catalyst materials that are meant to remain in the structure and have some application-specific role, making any removal steps unnecessary. Thus, as a proof-of-concept, we present here a nanocarbon-based material platform for electrochemical hydrogen peroxide measurement utilizing a Pt catalyst layer to grow CNFs with intact Pt particles at the tips of the CNFs. Backed by careful scanning transmission electron microscopy analysis, we show that this material can be readily realized with the Pt catalyst layer thickness impacting the resulting structure and also present a growth model to explain the evolution of the different types of structures. In addition, we show by electrochemical analysis that the material exhibits characteristic features of Pt in cyclic voltammetry and it can detect very small amounts of hydrogen peroxide with very fast response times. Thus, the present sensor platform provides an interesting electrode material with potential for biomolecule detection and in fuel cells and batteries. In the wider range, we propose a new approach where the selection of catalytic particles used for carbon nanostructure growth is made so that (i) they do not need to be removed and (ii) they will have essential role in the final application.

Plasma jet printing of electronic materials on flexible and nonconformal objects.

We present a novel approach for the room-temperature fabrication of conductive traces and their subsequent site-selective dielectric encapsulation for use in flexible electronics. We have developed an aerosol-assisted atmospheric pressure plasma-based deposition process for efficiently depositing materials on flexible substrates. Silver nanowire conductive traces and silicon dioxide dielectric coatings for encapsulation were deposited using this approach as a demonstration. The paper substrate with silver nanowires exhibited a very low change in resistance upon 50 cycles of systematic deformation, exhibiting high mechanical flexibility. The applicability of this process to print conductive traces on nonconformal 3D objects was also demonstrated through deposition on a 3D-printed thermoplastic object, indicating the potential to combine plasma printing with 3D printing technology. The role of plasma here includes activation of the material present in the aerosol for deposition, increasing the deposition rate, and plasma polymerization in the case of inorganic coatings. The demonstration here establishes a low-cost, high-throughput, and facile process for printing electronic components on nonconventional platforms.

Scalable low-cost fabrication of disposable paper sensors for DNA detection.

Controlled integration of features that enhance the analytical performance of a sensor chip is a challenging task in the development of paper sensors. A critical issue in the fabrication of low-cost biosensor chips is the activation of the device surface in a reliable and controllable manner compatible with large-scale production. Here, we report stable, well-adherent, and repeatable site-selective deposition of bioreactive amine functionalities and biorepellant polyethylene glycol-like (PEG) functionalities on paper sensors by aerosol-assisted, atmospheric-pressure, plasma-enhanced chemical vapor deposition. This approach requires only 20 s of deposition time, compared to previous reports on cellulose functionalization, which takes hours. A detailed analysis of the near-edge X-ray absorption fine structure (NEXAFS) and its sensitivity to the local electronic structure of the carbon and nitrogen functionalities. σ*, π*, and Rydberg transitions in C and N K-edges are presented. Application of the plasma-processed paper sensors in DNA detection is also demonstrated.

Vertically aligned carbon nanofiber nanoelectrode arrays: electrochemical etching and electrode reusability.

Vertically aligned carbon nanofibers in the form of nanoelectrode arrays were grown on nine individual electrodes, arranged in a 3 × 3 array geometry, in a 2.5 cm2 chip. Electrochemical etching of the carbon nanofibers was employed for electrode activation and enhancing the electrode kinetics. Here, we report the effects of electrochemical etching on the fiber height and electrochemical properties. Electrode regeneration by amide hydrolysis and electrochemical etching is also investigated for electrode reusability.

X-ray Absorption Study of Graphene Oxide and Transition Metal Oxide Nanocomposites.

The surface properties of the electrode materials play a crucial role in determining the performance and efficiency of energy storage devices. Graphene oxide and nanostructures of 3d transition metal oxides were synthesized for construction of electrodes in supercapacitors, and the electronic structure and oxidation states were probed using near-edge X-ray absorption fine structure. Understanding the chemistry of graphene oxide would provide valuable insight into its reactivity and properties as the graphene oxide transformation to reduced-graphene oxide is a key step in the synthesis of the electrode materials. Polarized behavior of the synchrotron X-rays and the angular dependency of the near-edge X-ray absorption fine structures (NEXAFS) have been utilized to study the orientation of the σ and π bonds of the graphene oxide and graphene oxide-metal oxide nanocomposites. The core-level transitions of individual metal oxides and that of the graphene oxide nanocomposite showed that the interaction of graphene oxide with the metal oxide nanostructures has not altered the electronic structure of either of them. As the restoration of the π network is important for good electrical conductivity, the C K edge NEXAFS spectra of reduced graphene oxide nanocomposites confirms the same through increased intensity of the sp2-derived unoccupied states π* band. A pronounced angular dependency of the reduced sample and the formation of excitonic peaks confirmed the formation of extended conjugated network.

Label-free detection of C-reactive protein using a carbon nanofiber based biosensor.

We report the sensitive detection of C-reactive protein (CRP), a biomarker for cardiac disease, using a carbon nanofiber based biosensor platform. Vertically aligned carbon nanofibers were grown using plasma enhanced chemical vapor deposition to fabricate nanoelectrode arrays in a 3×3 configuration. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used for the CRP detection. The CV responses show a 25% reduction in redox current upon the immobilization of anti-CRP on the electrode where as a 30% increase in charge transfer resistance is seen from EIS. Further reduction in redox current and increase in charge transfer resistance result from binding of CRP on anti-CRP immobilized surface, proportional to the concentration of the CRP target. The detection limit of the sensor is found to be ~90 pM or ~11 ng/ml, which is in the clinically relevant range. Control tests using non-specific myoglobin antigen confirmed the specificity of the present approach.

Carbon nanofiber electrode for neurochemical monitoring.

The ability to rapidly detect neurotransmitter release has broad implications in the study of a variety of neurodegenerative diseases. Electrochemical detection methods using carbon nanofiber nanoelectrodes integrated into the Wireless Instantaneous Neurotransmitter Concentration Sensing System (WINCS) offer many important advantages including biocompatibility, selectivity, sensitivity, and rapid adsorption kinetics. Carbon nanofiber nanoelectrodes exhibit greater selectivity and sensitivity in the electrochemical detection of neurotransmitters compared to macroelectrodes and are able to resolve a ternary mixture of dopamine (DA), serotonin (5-HT), and ascorbic acid as well as to detect individual neurotransmitters in concentrations as low as 50 nM for DA and 100 nM for 5-HT using differential pulse voltammetry. Adsorption kinetics studies and isopropyl alcohol treatments modeled on previous studies on carbon fiber microelectrodes were conducted to investigate the analogous properties on carbon nanofiber electrodes using fast-scan cyclic voltammetry with WINCS and showed analogous results in carbon nanofiber electrodes compared with carbon fiber microelectrodes.

Label-free detection of cardiac troponin-I using carbon nanofiber based nanoelectrode arrays.

A label-free biosensor is presented using carbon nanofiber (CNF) nanoelectrode arrays for the detection of cardiac troponin-I in the early diagnosis of myocardial infarction. Immobilization of anti-cTnI Ab on CNFs and the detection of human-cTnI were examined using electrochemical impedance spectroscopy and cyclic voltammetry techniques. Each step of the modification process was monitored, and the results show changes in electrical capacitance or resistance to charge transfer due to the specificity of corresponding adsorption of Ab-Ag interaction. The immunosensor demonstrates a good selectivity and high sensitivity against human-cTnI analytes and is capable of detecting cTnI at concentrations as low as ∼0.2 ng/mL, which is 25 times lower than that possible by conventional methods. Analysis of the electrode at various stages using atomic force microscopy and X-ray reflectivity provides information on the surface roughness and orientation of the antibody.

A carbon nanofiber based biosensor for simultaneous detection of dopamine and serotonin in the presence of ascorbic acid.

A biosensor based on an array of vertically aligned carbon nanofibers (CNFs) grown by plasma enhanced chemical vapor deposition is found to be effective for the simultaneous detection of dopamine (DA) and serotonin (5-HT) in the presence of excess ascorbic acid (AA). The CNF electrode outperforms the conventional glassy carbon electrode (GCE) for both selectivity and sensitivity. Using differential pulse voltammetry (DPV), three distinct peaks are seen for the CNF electrode at 0.13 V, 0.45 V, and 0.70 V for the ternary mixture of AA, DA, and 5-HT. In contrast, the analytes are indistinguishable in a mixture using a GCE. For the CNF electrode, the detection limits are 50 nM for DA and 250 nM for 5-HT.

Carbon nanofiber multiplexed array and Wireless Instantaneous Neurotransmitter Concentration Sensor for simultaneous detection of dissolved oxygen and dopamine.

PURPOSE: While the mechanism of Deep Brain Stimulation (DBS) remains poorly understood, previous studies have shown that it evokes release of neurochemicals and induces activation of functional magnetic resonance imaging (fMRI) blood oxygen level-dependent signal in distinct areas of the brain. Therefore, the main purpose of this paper is to demonstrate the capabilities of the Wireless Instantaneous Neurotransmitter Concentration Sensor system (WINCS) in conjunction with a carbon nanofiber (CNF) multiplexed array electrode as a powerful tool for elucidating the mechanism of DBS through the simultaneous detection of multiple bioactive-molecules. METHODS: Patterned CNF nanoelectrode arrays were prepared on a 4-inch silicon wafer where each device consists of 3 × 3 electrode pads, 200 µm square, that contain CNFs spaced at 1µm intervals. The multiplexed carbon nanofiber CNF electrodes were integrated with WINCS to detect mixtures of dopamine (DA) and oxygen (O2) using fast scan cyclic voltammetry (FSCV) in vitro. RESULTS: First, simultaneous detection of O2 at two spatially different locations, 200 um apart, was demonstrated. Second, simultaneous detection of both O2 and DA at two spatially different locations, using two different decoupled waveforms was demonstrated. Third, controlled studies demonstrated that the waveform must be interleaved to avoid electrode crosstalk artifacts in the acquired data. CONCLUSIONS: Multiplexed CNF nanoelectrode arrays for electrochemical detection of neurotransmitters show promise for the detection of multiple analytes with the application of time independent decoupled waveforms. Electrochemistry on CNF electrodes may be helpful in elucidating the mechanism of DBS, and may also provide the precision and sensitivity required for future applications in feedback modulated DBS neural control systems.

Detection of ricin using a carbon nanofiber based biosensor.

We report ricin detection using antibody and aptamer probes immobilized on a nanoelectrode array (NEA) consisting of vertically aligned carbon nanofibers (VACNFs). These biosensor chips are fabricated on a wafer scale using steps common in integrated circuit manufacturing. Electrochemical impedance spectroscopy is used to characterize the detection event and the results indicate that the electron transfer resistance changes significantly after the ricin protein binds to the probe. Further confirmation is obtained from evaluation of the electrode surface by atomic force microscopy which clearly shows a change in height from the bare electrode to the surface bound by the probe-protein.

Carbon nanofiber electrode array for electrochemical detection of dopamine using fast scan cyclic voltammetry.

A carbon nanofiber (CNF) electrode array was integrated with the Wireless Instantaneous Neurotransmitter Concentration Sensor System (WINCS) for the detection of dopamine using fast scan cyclic voltammetry (FSCV). Dopamine detection performance by CNF arrays was comparable to that of traditional carbon fiber microelectrodes (CFMs), demonstrating that CNF arrays can be utilized as an alternative carbon electrode for neurochemical monitoring.

Vertically aligned carbon nanofibers: interconnecting solid state electronics with biosystems.

Vertically aligned carbon nanofibers (VACNFs) are grown directly on prefabricated electronic circuits with nanoscale precision. Utilizing the free-standing nanofiber array geometry, we have demonstrated the detection of nucleic acids to construct an ultrasensitive electrochemical sensor. Extending this technology towards in vivo applications, we have modified the free-standing VACNF arrays in order to achieve a multifunctional three dimensional (3-D) matrix that interpenetrates the neuronal network of PC12 cells. We found that PC12 cells cultured on the nanofiber arrays can form an extended neural network upon proper chemical and biochemical modification. The soft 3-D nanofiber array architecture provides a novel platform to fine-tune the topographical, mechanical, chemical, and electrical cues at sub-cellular scales. This biomaterial platform can be used for both fundamental studies of nanomaterial-cell interactions and the development of multifunctional, chronically stable implantable devices. The application of these devices and potential utility as a multifunctional platform for neurophysiology and biochemical studies will be discussed.

Arrays of carbon nanofibers as a platform for biosensing at the molecular level and for tissue engineering and implantation.

Arrays of Carbon nanofibers (CNFs) harness the advantages of individual CNF as well the collective property of assemblies, which made them promising materials in biosensing and tissue engineering or implantation. Here, we report two studies to explore the applications of vertically aligned CNFs. First, a nanoelectrode array (NEA) based on vertically aligned CNFs embedded in SiO(2) is used for ultrasensitive DNA detection. Oligonucleotide probes are selectively functionalized at the open ends of the CNFs and specifically hybridized with oligonucleotide targets. The guanine groups are employed as the signal moieties in the electrochemical measurements. Ru(bpy)(3)(2+) mediator is used to further amplify the guanine oxidation signal. The hybridization of less than approximately 1000 molecules of PCR amplified DNA targets are detected electrochemically by combining the CNF nanoelectrode array with the Ru(bpy)(3)(2+) amplification mechanism. Second, the SiO(2) matrix was etched back to produce needle-like protruding nanoelectrode arrays to be used as cell interfacing fibers for investigating gene transfection, electrical stimulation and detection of cellular processes. Our goal is to take advantage of the nanostructure of CNFs for unconventional biomolecular studies requiring ultrahigh sensitivity, high-degree of miniaturization and selective biofunctionalization.

Miniaturized multiplex label-free electronic chip for rapid nucleic acid analysis based on carbon nanotube nanoelectrode arrays.

BACKGROUND: Reducing cost and time is the major concern in clinical diagnostics, particularly in molecular diagnostics. Miniaturization technologies have been recognized as promising solutions to provide low-cost microchips for diagnostics. With the recent advancement in nanotechnologies, it is possible to further improve detection sensitivity and simplify sample preparation by incorporating nanoscale elements in diagnostics devices. A fusion of micro- and nanotechnologies with biology has great potential for the development of low-cost disposable chips for rapid molecular analysis that can be carried out with simple handheld devices. APPROACH: Vertically aligned multiwalled carbon nanotubes (MWNTs) are fabricated on predeposited microelectrode pads and encapsulated in SiO2 dielectrics with only the very end exposed at the surface to form an inlaid nanoelectrode array (NEA). The NEA is used to collect the electrochemical signal associated with the target molecules binding to the probe molecules, which are covalently attached to the end of the MWNTs. CONTENT: A 3 x 3 microelectrode array is presented to demonstrate the miniaturization and multiplexing capability. A randomly distributed MWNT NEA is fabricated on each microelectrode pad. Selective functionalization of the MWNT end with a specific oligonucleotide probe and passivation of the SiO2 surface with ethylene glycol moieties are discussed. Ru(bpy)2+ -mediator-amplified guanine oxidation is used to directly measure the electrochemical signal associated with target molecules. SUMMARY: The discussed MWNT NEAs have ultrahigh sensitivity in direct electrochemical detection of guanine bases in the nucleic acid target. Fewer than approximately 1000 target nucleic acid molecules can be measured with a single microelectrode pad of approximately 20 x 20 microm2, which approaches the detection limit of laser scanners in fluorescence-based DNA microarray techniques. MWNT NEAs can be easily integrated with microelectronic circuitry and microfluidics for development of a fully automated system for rapid molecular analysis with minimum cost.

System optimization for the development of ultrasensitive electronic biosensors using carbon nanotube nanoelectrode arrays.

Vertically aligned multi-walled carbon nanotubes (MWCNTs) have been reported in fabricating nanoelectrode arrays. Further studies on optimizing this system for the development of ultrasensitive DNA sensors are reported here. The mechanical stability of the as-grown MWCNT array can be improved by polymer coating or SiO2 encapsulation. The latter method provides excellent electronic and ionic insulation to the sidewall of MWCNTs and the underlying metal layer, which is investigated with electrochemical impedance spectroscopy. The insulation ensures well-defined nanoelectrode behavior. A method is developed for selectively functionalizing biomolecules at the open end of MWCNTs while keeping the SiO2 surface passivated, using the unique graphitic chemistry. An ultrahigh sensitivity approaching the limit of fluorescence techniques is obtained with this system for DNA detection.