Journal of Electroanalytical Chemistry 662 (2011) 100–104 Contents lists available at ScienceDirect Journal of Electroanalytical Chemistry journal homepage: www.elsevier.com/locate/jelechem Flexible, micron-scaled superoxide sensor for in vivo applications Rebekah C.K Wilson a, Dao Thanh Phuong b, Edward Chainani a, Alexander Scheeline a,⇑ a b Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, United States Faculty of Chemistry, Hanoi University of Science, VNU 334 Nguyen Trai, Thanh Xuan, Ha Noi, Viet Nam a r t i c l e i n f o Article history: Available online 29 March 2011 Keywords: Superoxide sensor Flexible microelectrode Chronoamperometry Reactive oxygen species sensor Cytochrome c immobilized protein electrode Thiol on gold modified electrode a b s t r a c t Superoxide radical plays an important role in cell signaling However, certain events can result in a large increase in superoxide concentration which has been linked to, among other conditions, inflammation, neurodegenerative diseases, and cancer Consequently, in vivo detection of superoxide is of great interest Previously, due to brittleness, instability, or size, superoxide sensors have been limited in their ability for in vivo work We report the development of a flexible, micron-scale, superoxide sensor Thin gold films are patterned on Kapton™ to form multiple electrodes that constitute the sensor Cytochrome c was covalently anchored to the working electrode using a self-assembled monolayer of 3,30 -Dithiodipropionic acid di(N-hydroxysuccinimide ester) Calibration showed a linear response within the constraints imposed by using xanthine/xanthine oxidase as the superoxide source Testing demonstrated that interference from physiological levels of NADH, citric acid, and uric acid to be insignificant However, minor interference was seen in the presence of H2O2 and glucose, and significant interference arose from ascorbic acid, a known radical scavenger Qualitative observations provide insight into the preparation and cleaning of thin layer gold on Kapton™ Ó 2011 Elsevier B.V All rights reserved Introduction Reactive oxygen species (ROS), or pro-oxidants, are molecules or ions formed by the reduction of oxygen and are highly reactive These species include, but are not limited to, singlet oxygen (1O2), superoxide (OÅÀ ), peroxides (R-O-O-R ), the hydroxyl radical (OH), and hypochlorous acid (HOCl) They can be generated via photochemistry and by toxic chemical or drug exposure Biologically, OÅÀ is the result of a one-electron reduction of molecular oxygen during cell metabolism and also plays an important role in cell signaling [1,2] Enzymes, such as superoxide dismutase (SOD), and antioxidants, such as ascorbic acid, are present biologically to keep the concentration of superoxide manageable However, during times of environmental stress, the levels of OÅÀ can exceed those which can be removed by natural defenses and can lead to destruction of vital cell structures, leading ultimately to cancer, ischemia/ reperfusion damage, diabetes, aging-correlated pathology, or cardiovascular disease [3–5] Because of its damaging effects, the detection and quantification of OÅÀ has been the subject of biomedical interest for decades [6–11] A robust and reliable detection scheme for superoxide would lead to better understanding of its effects However, the detection of OÅÀ is complicated by its rapid dismutation At physi- ⇑ Corresponding author Tel.: +1 217 333 2999; fax: +1 217 265 6290 E-mail address: scheelin@illinois.edu (A Scheeline) 1572-6657/$ - see front matter Ó 2011 Elsevier B.V All rights reserved doi:10.1016/j.jelechem.2011.03.024 ological pHs, the half-life ranges from milliseconds to seconds [12] Therefore detection of OÅÀ also requires sensitivity and selectivity along with rapid detection for in vitro and in vivo applications Previous studies have employed numerous analytical techniques to explore OÅÀ reactions Among these, electron spin resonance (ESR) has been a reliable way to monitor ROS, but requires long averaging times, and is restricted in sensitivity [13] High performance liquid chromatography (HPLC) is another popular form of ROS detection, but requires digestion of the biological samples, which is not compatible with in vivo work [14,15] Chemiluminescence also offers specific targeting of certain ROS [16–18] However, the chemicals involved may easily disrupt the biological system one is attempting to monitor, or may interfere with the reaction to oxidative stress Electrochemical detection of OÅÀ is promising not only for its biocompatibility, fast response times, and selectivity, but can also be easily integrated into portable devices Exploiting the biological function of proteins by integrating them onto, e.g., a gold surface allows for selectivity Cytochrome c (cyt c) is a heme protein that can be found within the inner membrane of mitochondria It can function as a catalyst for hydroxylation and aromatic oxidation, and is an initiator in apoptosis Cytochrome c also can be reduced by OÅÀ and can be anchored to the surface of an electrode using a promoter species Until now, OÅÀ electrodes have been engineered in such a way that the working area is much too large or the support too brittle for some in vivo work, limiting its capabilities Further, calibration of such a sensor has not been found in literature R.C.K Wilson et al / Journal of Electroanalytical Chemistry 662 (2011) 100–104 [19–24] Presented here is the first micron-scale flexible OÅÀ sensor compatible for in vivo detection Here, patterned gold electrodes on a Kapton™ surface were modified using cyt c anchored by a selfassembled monolayer of dithiobis-(succinimidyl) propionate (DTSP) Using the xanthine/xanthine oxidase system to produce OÅÀ , the signal response of this sensor is presented along with calibration and exposure to biological interferents Electrodes were fabricated such that working, counter, and reference electrodes would all fit on a 200 lm wide projection a few millimeters long, as initial applications envisioned included study of noise-induced hearing loss [25–27] in small mammals and monitoring of reactions in ultrasonically-levitated drops [28–30] Materials and methods 2.1 Materials Unless noted, all chemicals are of reagent grade, used as received Mono- and dibasic potassium phosphate, oxalic acid, hydrogen peroxide, potassium carbonate, 3,30 -Dithiodipropionic acid di(N-hydroxysuccinimide ester) (DTSP), xanthine, xanthine oxidase, superoxide dismutase (SOD), b-nicotinamide adenine dinucleotide, reduced disodium salt (NADH), citric acid, ascorbic acid, glucose, cyt c and organic solvents were obtained from Sigma Aldrich (St Louis, Mo) Kapton™ 500 FCP and 500 VN, both 127 micrometers thick (DuPont) were used for the sensor substrate Lithography was achieved using S 1805 positive photoresist (Microchem), MF 319 developer (Shipley), and GE 8111 gold etch (Transene) A 50 mM stock, pH 7.4 phosphate solution was created by dissolving 0.1558 g of monosodium phosphate, monohydrate and 1.037 g disodium phosphate, heptahydrate into 100 mL of deionized (DI) water The stock solution was further diluted to 10:1 to obtain a 25 mM solution, which was used for experimentation A mM xanthine solution was made by dissolving 0.006 g of xanthine in 50 mL of 25 mM phosphate buffer (pH 7.4) and was mechanically stirred on low at low heat to fully dissolve the xanthine The solution is allowed to cool to room temperature before being used Over time, the xanthine precipitates and can be redissolved by heat and stirring A 30 mM solution of SOD was made by dissolving 0.2 mg of SOD in 200 mL of 25 mM phosphate buffer and kept on ice A 0.8 mg/mL solution of xanthine oxidase was made by diluting mL of a 16 mg/mL suspension of xanthine oxidase to 105 lL using 25 mM phosphate buffer (pH 7.4) and kept on ice Artificial perilymph (a mixture chosen for its relevance to future studies of noise-induced hearing loss) contained 112 mM NaCl, 4.2 mM KCl, mM MgCl2, 3.4 mM NaH2PO4, 5.3 mM Na2HPO4, 21 mM NaHCO3 M and 3.6 mM glucose in DI water Specifically, 0.659 g NaCl, 0.0313 g KCl, 0.0203 g MgCl2 Á 6H2O, 0.0475 g NaH2PO4 Á H2O, 0.0764 g Na2HPO4, 0.1764 g NaHCO3, and 0.0649 g glucose for every 100 mL of DI water Unless otherwise noted, a CH660 (CH Instruments, Austin, TX) was the potentiostat used When indicated, a Keithley 6485 Picoammeter (Solon, OH) was used All chronoamperometry data were collected at Hz using the digitizer built into the picoammeter Unless stated, all electrochemical measurements were made using a Ag/AgCl reference (CH Instruments, Austin, TX, [ClÀ] = M) electrode and all potentials are reported as such A Pt wire was used as a counter electrode All ebeam work is done on a Temescal six pocket E-Beam Evaporation System (Livermore, CA) All UV exposures were completed using a Karl Suss MJB3 Mask aligner (SUSS MicroTec Inc., Waterbury, VT) A HI3222 pH/ORP/ISE meter (Hanna Instruments, Woonsocket, RI) was used for all pH measurements Oxygen plasma cleaning is done with a MARCH CS 1701 RIE 101 and RFX 600 generator (Nordson MARCH, Carlsbad,CA) All nitrogen was from tank sources, not house supply The platform of this sensor is inexpensive and disposable with the intention to calibrate, use, validate and dispose without reuse Therefore, all reported experiments use each electrode for only one experiment 2.2 Fabrication and modification of sensor Kapton™ surfaces were cleaned prior to metal deposition by successive washing in acetone, ethanol and isopropyl alcohol, followed by drying with N2 The substrates were then exposed to oxygen plasma (80 sccm, 500 mTorr, 200 W) for 300 s and placed immediately into the ebeam deposition chamber A 60–80 Å adhesion layer of Ti was coated (1 Å sÀ1) followed by 2000 Å of Au Upon removal from the deposition chamber, the newly coated surfaces were flushed with N2 and placed in a dessicator Typical lithography procedures were used to pattern gold electrodes [31] Specifically, S1805 positive photoresist was spun onto the surface of the gold (300 rpm, 30 s) and baked at 110 °C for Once removed, it was allowed to cool A positive mask was placed onto the surface and held in place with a quartz slide The surface was exposed to UV light (12 s, 300 W), placed in MF 319 developer (5–10 s) and quickly rinsed with DI water Ti and Au were etched in GE 8111 (2–4 min) and rinsed generously with DI water Photoresist was removed using acetone Due to the precise dimensions required, laser cutting was used to excise the sensor from the Kapton™ sheet Sensors were kept in a desiccator until modification to reduce delamination of Au Areas where isolation from the environment is necessary, the gold surfaces between sensing and contact pad areas, were coated by hand-painting polyimide onto the surface and cured at 100 °C for h The working area was mm by 50 lm but could have been made smaller by isolating more of the gold lead The electrode surface is then cleaned by placing it in oxygen plasma for 2–4 before being chemically modified Typical electrochemical cleaning in acidic solutions causes the gold to delaminate from the Ti or Cr adhesion layer Covalent binding of cyt c was largely based on a procedure from Chen et al [32] Briefly, a clean gold electrode was placed in a 10 mM (dried over sieves DMSO) solution of DTSP for h at room temperature Once removed, the electrode was lightly rinsed with dry DMSO, then DI water and dried with nitrogen Gold surfaces where DMSO is not desired can be placed in 0.5 M KOH (methanol) and exposed to À1.5 V for 120 s and immediately rinsed with DI water The electrode was then placed in a mg/mL solution of cyt c (25 mM phosphate buffer, pH 7.4) at °C for 24 h DTSP contains the NHS ester which provides a good leaving group allowing amines on the cyt c to covalently bind to the DTSP-modified surface Once removed from the cyt c solution, it was lightly rinsed with cold phosphate buffer and stored in buffer at °C when not in use 2.3 Calibration of superoxide sensor The DTSP/cyt c modified working electrode was removed from 25 mM phosphate buffer (pH 7.4), rinsed with cold 25 mM phosphate buffer (pH 7.4), and placed in a mL solution of xanthine (2 mM) A stir bar was added to the reaction cell along with a Ag/AgCl reference electrode and a Pt wire as the counter electrode A potential was applied to the counter electrode using a potentiostat so that the working electrode was at 200 mV (to ensure proper oxidation of cyt c) with respect to the reference electrode The working electrode was connected to the picoammeter to measure current Background current was recorded for a period of time to establish a stable background level Serial injections of the xanthine oxidase solution (20 lL) were added to the reaction cell 102 R.C.K Wilson et al / Journal of Electroanalytical Chemistry 662 (2011) 100–104 Results and discussion followed by sonication in H2SO4 Alumina powder is too abrasive for thin films Piranha (1:3, H2O2:H2SO4) creates too harsh of an environment, causing Kapton™ to warp and gold to delaminate A survey of literature using gold patterned electrodes on Kapton™ found either vague or no instructions for cleaning the gold surfaces prior to being modified [33–35] Gold/Kapton™ electrodes appear to have always been used in an unmodified fashion Therefore, the quality of the gold surface after fabrication was not as critical as when the surface is to be modified As yet, we find that the highest quality modified electrode has been created by cleaning the gold surface using oxygen plasma Cyt c was chosen for the immobilized protein after numerous attempts to immobilize and retain the working function of SOD Using cited procedures [36–38], SOD can be immobilized onto a gold surface electrostatically (3-mercaptopropionic acid, MPA) or via DTSP However, we found that the metal ions in active sites, which are complexed by nearby amino acid ligands to maintain protein shape, easily dissociated from the protein during amperometry, rendering the resultant apoenzyme useless Since certain thiols can yield signal in the presence of OÅÀ without protein present, inactivation is typically not easily detected unless cyclic voltammetry is performed to characterize the electrodes [32] Therefore, cyt c, while not the most selective protein for interacting with OÅÀ , is more stable than SOD when immobilized Cyclic voltammograms of the DTSP/cyt c surface at varying scan rates can be seen in Fig The peak potentials of the sensor near 0.05 V and +0.1 V vs Ag/AgCl remain stable with peak ratio of indicating a quasi reversible reaction The xanthine/xanthine oxidase system for producing OÅÀ was used to calibrate the sensor The response of the sensor (3 mm in length) to multiple additions of xanthine oxidase can be seen in Fig with results summarized in Table 1, with standard deviation being that within the single run Each addition of enzyme generates sufficient OÅÀ that, at steady state, [OÅÀ ] increases by 4.5 lM Data was processed by boxcar averaging the signal using data points (IgorPro, Wavemetrics) to reduce noise Current was then averaged when a stable baseline was reached and again after each addition of xanthine oxidase Shifts in current were calculated by difference using the background current as reference To ensure that the signal was indeed from OÅÀ , SOD was added after the four aliquots of xanthine oxidase, dropping the current 67% from maximum towards baseline Though, these sensors are not intended for repeated use, SOD proved not to alter the sensing surface Large noise fluctuations at 100, 200, 300, 400, and 500 s are due to induced current from magnet mechanical stirring, indicating why mechanical The overall design of the sensor kept in mind the incorporation of a reference electrode and multiple working electrodes on the same substrate with minimal dimensions The overall length of the electrode is 2.5 cm, with a tip width of 200 lm and working length of up to mm The dimensions were governed by the opening of the round window in a Mongolian gerbil, the initial in vivo target of this sensor, to monitor OÅÀ generation in the cochlea Using Kapton™, the substrate is robust and the gold surface malleable enough to produce a flexible sensor However, there are concerns with delamination of gold from the adhesion layer Moisture seems to be the main cause of delamination and can be minimized by placing unmodified electrodes in a desiccator until ready for use After modification, gold surfaces were stable for up to 10 days before delamination rendered the electrodes useless Reliable resolution of the delamination issue is critical if Kapton™ is to be routinely used as a substrate The flexing of the electrode did not appear to cause of any visual delamination The heart of a perfectly modified electrode is a perfectly clean surface Bulk gold electrodes are typically cleaned using a variety of processes such as mechanical polishing using alumina powder, Fig Cyclic voltammograms of cyt c/DTSP/Au Kapton™ working electrode varying scan rates from innermost to outermost scan: 0.1, 0.2, 0.3 and 0.4 V sÀ1 in 25 mM phosphate buffer (pH 7.4) and the mechanical stirring was turned on for 20 s and then turned off A final 50 lL injection of the SOD solution was then added to the reaction cell to demonstrate the extent to which removal of OÅÀ returns signal to the background level Again, mechanical stirring was employed for a brief period and then stopped while current data continued to be collected until the end of the analysis 2.4 Interferent studies The DTSP/cyt c electrode was rinsed with phosphate buffer and placed in mL xanthine solution (2 mM in artificial perilymph) The sensor was then held at 200 mV vs the reference until a stable background was obtained 50 lL xanthine oxidase solution was added to the solution and the solution was mechanically stirred for a brief time 50 lL of interferent (final concentration of 3.75 mM) was then pipetted into the solution, mechanically stirring briefly Finally, 50 lL SOD solution was added and mixed for a brief time to void the solution of OÅÀ Theory The evolution of OÅÀ from the reaction between xanthine and xanthine oxidase (XO), in the presence of O2, can be seen in Eq (1) with the dismutation reaction of OÅÀ found in Eq (2) ẵXO ỵ Xanthine ỵ O2 ỵ H2 O ! Urate þ ỔÀ þ 2H ð1Þ þ 2ỔÀ þ 2H ! H2 O ỵ H2 O2 2ị O Assuming no interference, the concentration of at steady state is proportional to [XO]1/2 This presumes that dismutation, rather than consumption of OÅÀ by the electrode, is the main sink for the radical Presuming [O2] and [xanthine] are high enough that xanthine oxidase concentration limits the rate of OÅÀ formation, and making the steady-state approximation, d½ỔÀ 2 ¼ keff ½XO À k2 ½ỔÀ ¼ dt 3ị 1=2 keff ẵXO k2 4ị ẵO SS ¼ with keff the effective rate constant for reaction (1) and k2 the pHdependent rate constant for reaction (2) R.C.K Wilson et al / Journal of Electroanalytical Chemistry 662 (2011) 100–104 Fig Chronoamperometry response (at 200 mV) of cyt c to additions of 25 lL of xanthine oxidase solution in mM xanthine solution, followed by addition of 50 lLSOD (2 mg/mL) to bring signal back to background current Data (obtained at Hz) has been box averaged for every three data points Table Calibrated response of DTSP/cyt c electrode lM OÅÀ Average Di from baseline (pA) Standard deviation (pA) 4.72 6.4 7.8 8.9 14 20 22 24 4 stirring cannot be carried out throughout the analysis A calibration plot for the sensor can be found in Fig The linear regression does not intersect at origin, which is not very surprising OÅÀ is dismutating, the electrode itself is consuming OÅÀ , and interferents are most likely present The inability of the signal to return to baseline upon the addition of SOD also hints at interference from H2O2 Recently, Kelly et al studied the xanthine/xanthine oxidase reaction and found H2O2 to be the major product even at short reaction times, prior to when the long-known suicide reaction occurs [39] H2O2 is a known interferent when working with cyt c and could possibly be the cause for the shift in baseline and is the basis for studying its effects on the sensors ability to work It is important to note that the xanthine/xanthine oxidase system is far from ideal The basis for using this system relies on Fig Calibration of data collected depicted in Fig Change in current (pA) is plotted against OÅÀ concentration (lM) 103 achieving a steady state concentration of OÅÀ The xanthine/xanthine oxidase reaction deteriorates over time, causing the concentration of OÅÀ to gradually drop to zero after h, by which time XO generates only H2O2 [12] Also, xanthine oxidase activity decreases gradually even when stored as a solid suspension Therefore it cannot be assumed that the same supply of xanthine oxidase will create the same concentration of OÅÀ from day to day Because of this, the xanthine oxidase activity must be calibrated spectrophotometrically using cyt c absorbance at 550.5 nm each time it is used [11] It is not meaningful to compute standard deviations for each data point, as it is impossible to precisely replicate concentration increments of OÅÀ Therefore, as it stands, each electrode can only be calibrated approximately by running a spectrometric analysis of the xanthine/xanthine oxidase solution and quickly moving onto the calibration of the cyt c electrode Though not the intended goal, these sensors have proven to give signal for up to five days of use Interferent studies focused on species anticipated to be present during upcoming noise-induced hearing loss studies, and on species present during calibration Uric acid and H2O2 are products generated in the xanthine/xanthine oxidase reaction and/or a product of OÅÀ dismutation, Eq (1) While a significant concentration of uric acid is not typical biologically, it will be present during the calibration process until a better method for calibration (E Chainani, A Keith, personal communication) of OÅÀ can be perfected NADH, citric acid and glucose are also biologically important in metabolism and are most certainly present if a cell were to undergo apoptosis Ascorbic acid is a common radical scavenger and is essential for such scavenging in biological settings The particular interferents listed above were analyzed A typical interferent study can be seen in Fig Here, once the background current became stable, 5.2 lM OÅÀ (final concentration) was added to the system Intense noise fluctuations occur during the brief stirring followed by an increase of 100 pA At about 300 s 3.75 mM glucose (final concentration) was added to the system, again with brief noise fluctuations due to mechanical stirring, resulting in a 20% drop in current Addition of SOD allowed signal to drop 77% back to baseline, similar to studies without interferents The same sensor was then used repeatedly with no further complications, proving no biofouling from glucose This procedure was repeated for each interferent and a summary of results can be found in Table NADH, citric acid, and uric acid showed no significant interference Expected ascorbic acid scavenging abilities were demonstrated, Table Upon addition, signal dropped 60% followed by a relatively insignificant change upon addition of SOD While H2O2 is known to reduce cyt c, the effects in the Fig Chronoamperometry (at 200 mV) of DTSP/cyt c electrode (2 mM xanthine solution) in the presence of 50 lL xanthine oxidase solution (5.2 lM OÅÀ ), followed by 50 lL of glucose (3.75 mM), and 50 lL of SOD (2 mg/mL) 104 R.C.K Wilson et al / Journal of Electroanalytical Chemistry 662 (2011) 100–104 Table Interference of common biological molecules with electrode response Interference NADH Citric acid Uric acid Ascorbic acid H2O2 Glucose Change in current from baseline (pA), (% signal drop) XO XO + interferent SOD added 159 90 158 130 162 110 139 (13%) 84 (7%) 157 (1.4%) 52 (60%) 150 (8.2%) 88 (20%) 22 (86%) 36 (60%) 52.2 (67%) 39 (70%) 140 (17%) 25 (77%) presence OÅÀ are nearly immeasurable However, H2O2 hinders the ability of SOD to return signal to baseline This could be due to an exchange of OÅÀ response for a H2O2 response while maintaining the same current Adding catalase might not clarify this issue, as a signal could easily then be generated by OÅÀ oxidation The difference in current response between data shown in Fig versus Fig can be explained in two parts First, the calibration of OÅÀ is time dependent, therefore the concentration of OÅÀ at the time of use always has a margin of error Second, the working area of the electrode varies as the isolated regions are hand painted with polyimide Future efforts are being put forth to create reproducible working area by utilizing lithography of polyimide Conclusion Thin film gold on Kapton™ was used to create the first flexible OÅÀ sensor for in vivo applications The calibration showed a linear response within the constraints established by using xanthine/xanthine oxidase as a OÅÀ source Interferent studies demonstrated that H2O2 might prove to be a concern while other interferents showed expected results Further work involves incorporation a reference electrode onto the sensor along with a sensor specific for H2O2 Acknowledgements This work was supported by The National Organization for Hearing Research Foundation, the 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stable for up to 10 days before delamination