Hydrogen peroxide sensing with horseradish peroxidase-modified polymer single conical nanochannels

Inspired from the funtioning and responsiveness of biological ion channels, researchers attempt to develop biosensing systems based on polymer and solid-state nanochannels. The applicability of these nanochannels for detection/sensing of any foreign analyte in the surrounding environment depends critically on the surface characteristics of the inner walls. Attaching recognition sites to the channel walls leads to the preparation of sensors targeted at a specific molecule. There are many nanochannel platforms for the detection of DNA and proteins, but only a few are capable of detecting small molecules. Here, we describe a nanochannel platform for the detection of hydrogen peroxide, H(2)O(2), which is not only a toxic waste product in the cellular systems but also a key player in the redox signaling pathways. The sensor is based on single conical nanochannels fabricated in an ion tracked polymer membrane. The inner walls of the channel are decorated with horseradish peroxidase (HRP) enzyme using carbodiimide coupling chemistry. The success of the HRP immobilization on the channel surface is confirmed by measuring the pH-dependent current-voltage (I-V) curves of the system. The reported HRP-nanochannel system detects nanomolar concentrations of H(2)O(2) with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as the substrate. The immobilized HRP enzyme is thus capable of inducing redox reactions in a subfemtoliter volume of single nanochannels. We demonstrate that functioning of the designed biosensor is reversible and can be used multiple times to detect H(2)O(2) at various concentrations.     

H(2)O(2)-generating peroxidase electrodes as reagentless cyanide sensors

Inexpensive, reagentless, and simple (single-electrode) cyanide biosensors are developed using a pyrolytic graphite (PG) electrode on which horseradish peroxidase (HRP) is adsorbed. The electrode is poised at -300 mV vs Ag/AgCl for 40 s to reduce dissolved O(2) to H(2)O(2) at the PG surface. The generated H(2)O(2) accumulates in the diffusion layer. The potential is then stepped to 0 mV, at which the accumulated H(2)O(2) is reduced, though the O(2) reduction does not proceed. Since the H(2)O(2) reduction is catalyzed by HRP, the transient cathodic current is inhibited by cyanide. Therefore, the transient current is a function of the cyanide concentration. A HRP/PG electrode with saturated HRP coverage is reliable, and it can determine 10(-)(5)-10(-)(3) M cyanide. On the other hand, the electrode with lower HRP coverage is less reliable, though it is so sensitive as to detect 2 × 10(-)(7) M cyanide because the system is under kinetic control.     

AlGaN/GaN-based diodes and gateless HEMTs for gas and chemical sensing

The characteristics of Pt/GaN Schottky diodes and Sc/sub 2/O/sub 3//AlGaN/GaN metal-oxide semiconductor (MOS) diodes as hydrogen and ethylene gas sensors and of gateless AlGaN/GaN high-electron mobility transistors (HEMTs) as polar liquid sensors are reported. At 25/spl deg/C, a change in forward current of /spl sim/6 mA at a bias of 2 V was obtained in the MOS diodes in response to a change in ambient from pure N/sub 2/ to 10% H/sub 2// 90% N/sub 2/. This is approximately double the change in forward current obtained in Pt/GaN Schottky diodes measured under the same conditions. The mechanism appears to be formation of a dipole layer at the oxide/GaN interface that screens some of the piezo-induced channel charge. The MOS-diode response time is limited by the mass transport of gas into the test chamber and not by the diffusion of atomic hydrogen through the metal/oxide stack, even at 25/spl deg/C. Gateless AlGaN/GaN HEMT structures exhibit large changes in source-drain current upon exposing the gate region to various polar liquids, including block co-polymer solutions. The polar nature of some of these polymer chains lead to a change of surface charges in gate region on the HEMT, producing a change in surface potential at the semiconductor/liquid interface. The nitride sensors appear to be promising for a wide range of chemicals, combustion gases and liquids.     

A Phototransistor-Based High-Sensitivity Biosensing System Using 650-nm Light

A miniature optical biosensing system based on a PMOS phototransistor and absorption photometry is proposed. The phototransistor was manufactured in a standard 0.35-$mu{rm m}$ CMOS process, and it exhibited a responsivity higher than 1000 A/W for 650-nm light. For biochemical applications, the ${rm TMB/H}_{2}{rm O}_{2}/{rm HRP}$ method was adopted as a useful basis in our system. A sample volume of only 10 $mu{rm l}$ was required to be dropped on the slide above the phototransistor. Experimental results demonstrated that a high sensitivity of 2.5 $mu{rm A/pM}$ was achieved, and the minimum HRP concentration successfully detected was 2.7 pM. This detection limit is three orders of magnitude better than that of a lately reported silicon biosensor, and is even comparable to that of a commercial spectrophotometer.      

Biodegradation of single-walled carbon nanotubes through enzymatic catalysis

We show here the biodegradation of single-walled carbon nanotubes through natural, enzymatic catalysis. By incubating nanotubes with a natural horseradish peroxidase (HRP) and low concentrations of H2O2 (approximately 40 microM) at 4 degrees C over 12 weeks under static conditions, we show the increased degradation of nanotube structure. This reaction was monitored via multiple characterization methods, including transmission electron microscopy (TEM), dynamic light scattering (DLS), gel electrophoresis, mass spectrometry, and ultraviolet-visible-near-infrared (UV-vis-NIR) spectroscopy. These results mark a promising possibility for carbon nanotubes to be degraded by HRP in environmentally relevant settings. This is also tempting for future studies involving biotechnological and natural (plant peroxidases) ways for degradation of carbon nanotubes in the environment.     

Preparation of an enzyme/inorganic nanosheet/magnetic bead complex and its enzymatic activity

A complex composed of enzyme (horseradish peroxidase, HRP), layered titanate (TiO_x), and magnetic beads (MB) was prepared, and the enzymatic activity of the complex and the repeatable use for the enzyme in the complex were investigated. The HRP/TiO_x/MB complex was formed in acetate buffer (pH 4) by electrostatic interaction among the components where HRP and MB have positive charge, whereas TiO_x is negatively charged. The resultant complex was sufficiently stable in an aqueous solution, and HRP immobilized on the layered titanate in the complex kept a certain enzymatic activity in which typical enzymatic relationship was observed between substrate concentration and initial reaction rate. By giving a magnetic field using commercial magnets, the HRP/TiO_x/MB complex was easily and rapidly recovered, and the recovered complex was able to be used for the next enzymatic reactions. The enzyme/inorganic nanosheet/MB complex proposed in this study is expected to be applied as a new tool in the bioengineering and nanobiotechnological fields.     

A CMOS rotary encoder using magnetic sensor arrays

A new type of small magnetic rotary encoder is presented. The device detects the magnetic field of a permanent magnet attached to the end of the rotating shaft using complementary metal-oxide semiconductor (CMOS) magnetic sensors [magnetic field effect transistor (MAGFET) arrays] set in a square arrangement. The sensor array is integrated onto a CMOS chip along with angle-detection circuits, leading to the realization of a compact, cost-effective rotary encoder. A prototype sensor chip with dimensions of 4.3/spl times/4.3 mm/sup 2/ is shown to provide error as low as 3.5/spl deg/ without offset calibration and 0.36/spl deg/ with offset calibration, based on an angle calculation method with mean square estimation. This result shows that the CMOS rotary encoder can achieve resolution of 10 bits/rotation at the cost of calibration.     

Fabrication of an electrochemical platform based on the self-assembly of graphene oxide-multiwall carbon nanotube nanocomposite and horseradish peroxidase: direct electrochemistry and electrocatalysis

A novel hybrid nanomaterial (GO-MWNTs) was explored based on the self-assembly of multiwall carbon nanotubes (MWNTs) and graphene oxide (GO). Compared with pristine MWNTs, such a nanocomposite could be well dispersed in aqueous solution and exhibit a negative charge. Driven by the electrostatic interaction, positively charged horseradish peroxidase (HRP) could then be immobilized onto GO-MWNTs at the surface of a glassy carbon (GC) electrode to form a HRP/GO-MWNT/GC electrode under mild conditions. TEM was used to characterize the morphology of the GO-MWNT nanocomposite. UV-vis and FTIR spectra suggested that HRP was immobilized onto the hybrid matrix without denaturation. Furthermore, the immobilized HRP showed enhanced direct electron transfer for the HRP-Fe(III)/Fe(II) redox center. Based on the direct electron transfer of the immobilized HRP, the HRP/GO-MWNT/GC electrode exhibited excellent electrocatalytic behavior to the reduction of H(2)O(2) and NaNO(2), respectively. Therefore, GO-MWNTs could provide a novel and efficient platform for the immobilization and biosensing of redox enzymes, and thus may find wide potential applications in the fabrication of biosensors, biomedical devices, and bioelectronics.     

Hematin as a peroxidase substitute in hydrogen peroxide determinations.

Hematin can substitute for horseradish peroxidase (HRP) as the catalyst in the determination of hydrogen peroxide using phenolic substrates such as p-hydroxyphenylacetate or p-cresol. Although the peroxidatic activity of hematin from bovine blood is not as great as HRP in terms of unit iron content, the activity per unit weight is substantially greater. Hematin is 500 times less expensive than HRP per unit peroxidatic activity. In hematin-catalyzed systems, reaction development and fluorescence measurement can both be conducted optimally in the same ammoniacal buffer. Hydroxyalkyl hydroperoxides are rapidly hydrolyzed to H2O2 at this pH and are also determined. On the other hand, for methyl hydroperoxide, hematin exhibits only approximately 10% of the sensitivity exhibited by HRP. Hematin is significantly more stable in solution than HRP. The use of hematin as catalyst and p-cresol as the substrate leads to a particularly inexpensive and sensitive system, permitting a limit of detection (LOD) of 7 nM H2O2 in a flow-injection configuration.     

Enzyme-based colorimetric detection of nucleic acids using peptide nucleic acid-immobilized microwell plates

The development of label-free or nonlabeling assays for nucleic acids is important in basic biological research and biomedical diagnosis. In this study, we have developed an enzyme-based colorimetric assay for nucleic acids, which combines the robustness of nonlabeling of DNA and RNA samples and the adequate sensitivity of enzymatic reactions. The core of this assay is the use of neutral peptide nucleic acid (PNA) as capture probe and the electrostatic adsorption of horseradish peroxidase (HRP) on hybridized, negatively charged nucleic acids to report the hybridization events, through HRP-catalyzed color reactions of 3,3',5,5'-tetramethylbenzidine and H(2)O(2). The proposed assay has been validated with fully complementary and single base-mismatched DNAs of different chain lengths. The proposed assay has also been validated with total RNA samples extracted from two human cancer cell lines (A 549 lung cancer cell and HeLa cell) for microRNA detection in real samples. Through extensive optimizations of HRP adsorption and nucleic acid hybridization conditions, detection limits of 0.1-0.2 nM for DNA (depending on chain length) and approximately 2 microg of total RNA have been achieved. Surface plasmon resonance spectroscopy has been used to elucidate the HRP adsorption and PNA-nucleic acid hybridizations through real-time measurements and to provide guidance for the development of the colorimetric assay.     

Chemical sensing systems using xerogel-based sensor elements and CMOS photodetectors

We present the first example of an integrated complementary metal-oxide-semiconductor (CMOS) photodetector coupled with a solid-state xerogel-based thin-film sensor to produce a compact chemical sensor system. We compare results using two different CMOS-based detector systems to results obtained by using a standard photomultiplier tube (PMT) or charge-coupled device (CCD) detector. Because the chemical sensor elements are governed by a Stern-Volmer relationship, the Stern-Volmer quenching constant is used as the primary comparator between the different detectors. All of the systems yielded Stern-Volmer constants from 0.042 to 0.049 O/sub 2/%/sup -1/. The results show that the CMOS detector system yields analytical data that are comparable to the CCD- and PMT-based systems. The disparity between the data obtained from each detector is primarily associated with the difference in how the signals are obtained by each detector as they presently exist. We have also observed satisfactory reversibility in the operation of the sensor system. The CMOS-based system exhibits a response time that is faster than the chemical sensor element's intrinsic response time, making the CMOS suitable for time-dependent measurements. The CMOS array detector also uses less than 0.1% the power in comparison to a standard PMT or CCD. The combined xerogel/CMOS system represents an important step toward the development of a portable, efficient sensor system.     

A Novel Saw Device in CMOS: Design, Modeling, and Fabrication

The design, finite element modeling, fabrication, and characterization of a novel surface acoustic wave (SAW) delay line for bio/chemical and telecommunication applications in CMOS technology are introduced. A full modeling was carried out. The devices are designed in a standard semiconductor foundry 1.5-mum two-metal two-poly process. A unique maskless postprocessing sequence is designed and completed. The three postprocessing steps are fully compatible with any standard integrated circuit technology such as CMOS. This allows any signal control/processing circuitry to be easily integrated on the same chip. ZnO is used as the piezoelectric material for SAW generation. A thorough characterization and patterning optimization of the sputtered ZnO was carried out. The major novelties that are introduced in the SAW delay line features are the embedded heater elements for temperature control, compensation, and acoustic absorbers that are designed to eliminate edge reflections and minimize triple transit interference that is amplified by edge reflections. Both of these attributes are designed by using CMOS materials without disturbing SAW performance     

Optical and electrical H/sub 2/- and NO/sub 2/-sensing properties of Au/In/sub x/O/sub y/N/sub z/ films

In this paper, we describe the optical and electrical gas-sensing properties of In/sub x/O/sub y/N/sub z/ films with an ultrathin gold promoter overlayer. We have fabricated In/sub x/O/sub y/N/sub z/ films with a nanocrystalline porous structure by RF-sputtering in Ar/N/sub 2/ followed by an annealing process. Gold particles with 20-30-nm diameter have been formed on top of the In/sub x/O/sub y/N/sub z/ films by dc sputtering and an annealing process. We have investigated the optical H/sub 2/and NO/sub 2/-sensing properties (change of absorbance) and also the electrical sensing effect (change of electrical resistance) for these two gases. A combined optical/electrical sensor for H/sub 2//NO/sub 2/ is proposed.     

Chip-based P450 drug metabolism coupled to electrospray ionization-mass spectrometry detection

A chip-based P450 in vitro metabolism assay coupled with ESI-MS and ESI-MS/MS detection is described in this paper. The chips were made of a cyclic olefin polymer using a hot embossing process. The introduction of reagent solutions into the chip was carried out using fused-silica capillaries coupled to two syringes with the flow rate controlled by a syringe pump. Initial experiments described here employed a small commercial guard column in an off-chip format to desalt and concentrate the products of the enzymatic reaction prior to ESI-MS analysis. The system was used both to yield the Michaelis constant (K(m)) of the P450 biotransformation of imipramine into desipramine and to determine the IC50 value of a chemical inhibitor (tranylcypromine) for this CYP2C19-mediated reaction. The results demonstrated that the kinetics of the reaction inside the 4-microL volume within the channels of the cyclic olefin polymer chip provided results in agreement with those reported in the literature using conventional assays. The above reactions were carried out using human liver microsomes, and the metabolites were detected by ESI-MS showing the potential of the chip-based P450 reaction for metabolite screening studies as well as for P450 inhibition assays. A porous monolithic column was subsequently integrated into the chip to perform the reaction mixture cleanup process in an integrated fashion on the chip that is necessary for ESI-MS detection. The miniature monolithic SPE column was prepared in situ inside the chip via UV-initiated polymerization. The results obtained using the integrated system demonstrated the possibility of performing P450 enzymatic reactions in a microvolume reaction chamber coupled directly to ESI-MS detection and required less than 4 microg of HLM protein.     

Filter-protected photodiodes for high-throughput enzymatic analysis

This paper relates to the use of a thin film of re-crystallized (polycrystalline) silicon as a low-pass rejection filter in the ultraviolet light range and, more particularly, to the use of this layer as a protective layer for semiconductor diodes. The polycrystalline silicon filters were fabricated by laser annealing a thin film of amorphous silicon deposited by an LPCVD process. A standard component of the polysilicon-gate CMOS process is the boron phosphor silicate glass (BPSG) planarization layer. Since this layer is always applied, the possibility of using it as the isolator between the diode and the filter (and, thereby, omit one SiO/sub 2/ layer) is considered. Using scanning electron microscopy, we compared the crystallization process of the LPCVD silicon film deposited on a glass substrate and on a BPSG layer. The fabrication and the characterization of the filter-protected photodiodes are described in the paper.     

Microfabricated channel array electrophoresis for characterization and screening of enzymes using RGS-G protein interactions as a model system

A microfluidic chip consisting of parallel channels designed for rapid electrophoretic enzyme assays was developed. Radial arrangement of channels and a common waste channel allowed chips with 16 and 36 electrophoresis units to be fabricated on a 7.62 x 7.62 cm(2) glass substrate. Fluorescence detection was achieved using a Xe arc lamp source and commercial charge-coupled device (CCD) camera to image migrating analyte zones in individual channels. Chip performance was evaluated by performing electrophoretic assays for G protein GTPase activity on chip using BODIPY-GTP as enzyme substrate. A 16-channel design proved to be useful in extracting kinetic information by allowing serial electrophoretic assays from 16 different enzyme reaction mixtures at 20 s intervals in parallel. This system was used to rapidly determine enzyme concentrations, optimal enzymatic reaction conditions, and Michaelis-Menten constants. A chip with 36 channels was used for screening for modulators of the G protein-RGS protein interaction by assaying the amount of product formed in enzyme reaction mixtures that contained test compounds. Thirty-six electrophoretic assays were performed in 30 s suggesting the potential throughput up to 4320 assays/h with appropriate sample handling procedures. Both designs showed excellent reproducibility of peak migration time and peak area. Relative standard deviations of normalized peak area of enzymatic product BODIPY-GDP were 5% and 11%, respectively, in the 16- and 36-channel designs.     

Horseradish peroxidase functionalized fluorescent gold nanoclusters for hydrogen peroxide sensing

The fluorescence of metal nanoclusters provides an amusing optic feature to be applied in various fields. However, rational design of dual functional fluorescent metal nanoclusters directed by active enzyme with targeted application remains little explored. In this work, we report a new strategy to construct enzyme functionalized fluorescent gold nanoclusters via a biomineralization process for the detection of hydrogen peroxide. Horseradish peroxidase (HRP) was used as a model functional template to direct the synthesis of fluorescent gold nanoclusters (Au NCs) at physiological conditions to form HRP-Au NCs bioconjugates. We found that the fluorescence of HRP-Au NCs can be quenched quantitatively by adding H(2)O(2), indicating that HRP enzyme remains active and enables catalytic reaction of HRP-Au NCs and H(2)O(2). Upon the addition of H(2)O(2) under optimal conditions, the fluorescence intensity quenched linearly over the range of 100 nM to 100 μM with high sensitivity (LOD = 30 nM, S/N = 3). This study would be potentially extended to other functional proteins to generate dual functional nanoclusters and applied to real time monitoring of biologically important targets in living cells.     

Optical biosensor based on hollow integrated waveguides

The first absorbance biosensor based on pure silicon hollow integrated waveguides is presented in this work. With the use of horseradish peroxidase (HRP) as a model recognition element, an enzymatic sensor for the measurement of hydrogen peroxide was fabricated, numerically simulated, and experimentally characterized. Waveguides with widths ranging from 50 to 80 microm, having a depth of 50 microm and lengths up to 5 mm were easily fabricated by just one photolithographic step. These were further modified by covalent immobilization of HRP using silanization chemistry. Simulation studies of the proposed approach showed a sensor linear behavior up to 300 microM H2O2 and a sensitivity of 2.7 x 10(-3) AU/microM. Experimental results were in good agreement with the simulated ones. A linear behavior between 10 and 300 microM H2O2, a sensitivity of 3 x 10(-3) AU/microM, and a signal-to-noise ratio around 20 dB were attained. Also, kinetic studies of the activity of the immobilized enzyme on the silicon waveguide surface gave an apparent Michaelis-Menten constant of 0.44 mM. The simple technology proposed in this work enables the fabrication of cost-effective, easy-to-use, miniaturized biosensor generic platforms, these being envisioned as excellent candidates for the development of lab-on-a-chip systems.     

Highly Flexible Hybrid CMOS Inverter Based on Si Nanomembrane and Molybdenum Disulfide

2D semiconductor materials are being considered for next generation electronic device application such as thin‐film transistors and complementary metal–oxide–semiconductor (CMOS) circuit due to their unique structural and superior electronics properties. Various approaches have already been taken to fabricate 2D complementary logics circuits. However, those CMOS devices mostly demonstrated based on exfoliated 2D materials show the performance of a single device. In this work, the design and fabrication of a complementary inverter is experimentally reported, based on a chemical vapor deposition MoS₂ n‐type transistor and a Si nanomembrane p‐type transistor on the same substrate. The advantages offered by such CMOS configuration allow to fabricate large area wafer scale integration of high performance Si technology with transition‐metal dichalcogenide materials. The fabricated hetero‐CMOS inverters which are composed of two isolated transistors exhibit a novel high performance air‐stable voltage transfer characteristic with different supply voltages, with a maximum voltage gain of ≈16, and sub‐nano watt power consumption. Moreover, the logic gates have been integrated on a plastic substrate and displayed reliable electrical properties paving a realistic path for the fabrication of flexible/transparent CMOS circuits in 2D electronics.     

Mechanistic and molecular investigations on stabilization of horseradish peroxidase C

The enzyme horseradish peroxidase (HRP) shows a decreasing activity when the enzyme's substrate hydrogen peroxide is present with the degree of inactivation being dependent on the incubation time and the hydrogen peroxide concentration. Incubation times of some minutes do not inactivate the enzyme independent of the H2O2 concentration. After several hours, only 50% of the activity is found for a medium H2O2 excess, and a >100-fold excess of H2O2 completely inactivates the enzyme. Polymeric additives, in particular Gafquat, lead to higher residual activities, whereas stabilizers, such as aminopyrine, preserve the full activity. Circular dichroism (CD) measurements reveal that the enzyme structure remains more or less unchanged when hydrogen peroxide is added. Only when a 1000-fold excess of hydrogen peroxide is present are structural changes observed. UV spectra highlight that the heme group in the enzyme is affected by hydrogen peroxide in a first step. Without any prolonged incubation, a decrease of the Soret band to approximately 50% is found for low hydrogen peroxide concentrations (HRP/H2O2 from 1:1 to 1:100). Higher H2O2 concentrations lead to the formation of catalytically inactive HRP forms. Preincubation of Gafquat, which is a copolymer from vinylpyrrolidone and derivatized methyl methacrylate, with hydrogen peroxide shifts the influence of hydrogen peroxide to higher concentrations, the shift being dependent on the Gafquat concentration. This effect is not observed for other polymers, such as dextrans, but it is also found for the stabilizer aminopyrine. Extended incubation times (24 h) of HRP together with H2O2, however, lead to an at least partial recovery of the Soret band for lower H2O2 concentrations (H2O2/HRP from 1:1 to 1:100). When hydrogen peroxide is used in a >100 fold excess, the heme group is irreversibly destroyed, and even the characteristic band of cpd III is not found. Here, the presence of Gafquat only reduces the degree of destruction. Computer modeling of the interaction between the polymers and the enzyme shows no specific binding sites for the functional groups of the vinylpyrrolidone-methacrylate copolymer Gafquat or of DEAE-dextran on the enzyme, whereas for the only activating polymer, polyethylenimine clustering of binding sites is observed.