DSpace at VNU: Flexible, micron-scaled superoxide sensor for in vivo applications

Calibration showed a linear response within the constraints imposed by using xanthine/xanthine oxidase as the superoxide source.. Here, patterned gold electrodes on a Kapton™ surface wer

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Flexible, micron-scaled superoxide sensor for in vivo applications

Rebekah C.K Wilsona, Dao Thanh Phuongb, Edward Chainania, Alexander Scheelinea,⇑

a

Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, United States

b

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 cova-lently 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 H2O2and 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™

1 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 (O

2), peroxides (R-O-O-R0), the hydroxyl radical (OH),

and hypochlorous acid (HOCl) They can be generated via

photo-chemistry and by toxic chemical or drug exposure Biologically,

O

2 is the result of a one-electron reduction of molecular oxygen

during cell metabolism and also plays an important role in cell

sig-naling [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 O2 can exceed those

which can be removed by natural defenses and can lead to

destruc-tion of vital cell structures, leading ultimately to cancer, ischemia/

reperfusion damage, diabetes, aging-correlated pathology, or

car-diovascular disease[3–5]

Because of its damaging effects, the detection and

quantiﬁca-tion of O

2 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 O

2 is complicated by its rapid dismutation At

physi-ological pHs, the half-life ranges from milliseconds to seconds[12] Therefore detection of O

2 also requires sensitivity and selectivity along with rapid detection for in vitro and in vivo applications Pre-vious studies have employed numerous analytical techniques to explore O

2 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 perfor-mance 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] Chemilumines-cence 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 O

2 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 O

2 and can be anchored to the surface of an electrode using a promoter species Until now, O

2 electrodes have been engineered

in such a way that the working area is much too large or the sup-port too brittle for some in vivo work, limiting its capabilities Fur-ther, calibration of such a sensor has not been found in literature

⇑Corresponding author Tel.: +1 217 333 2999; fax: +1 217 265 6290.

E-mail address: scheelin@illinois.edu (A Scheeline).

Contents lists available atScienceDirect Journal of Electroanalytical Chemistry

j o u r n a l h o m e p a g e : w w w e l s e v i e r c o m / l o c a t e / j e l e c h e m

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[19–24] Presented here is the ﬁrst micron-scale ﬂexible O

2 sensor compatible for in vivo detection Here, patterned gold electrodes on

a Kapton™ surface were modiﬁed using cyt c anchored by a

self-assembled monolayer of dithiobis-(succinimidyl) propionate

(DTSP) Using the xanthine/xanthine oxidase system to produce

O

2, the signal response of this sensor is presented along with

cal-ibration and exposure to biological interferents

Electrodes were fabricated such that working, counter, and

ref-erence electrodes would all ﬁt on a 200lm wide projection a few

millimeters long, as initial applications envisioned included study

of noise-induced hearing loss[25–27]in small mammals and

mon-itoring of reactions in ultrasonically-levitated drops[28–30]

2 Materials and methods

2.1 Materials

Unless noted, all chemicals are of reagent grade, used as

re-ceived 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

dis-solving 0.1558 g of monosodium phosphate, monohydrate and

1.037 g disodium phosphate, heptahydrate into 100 mL of

deion-ized (DI) water The stock solution was further diluted to 10:1 to

obtain a 25 mM solution, which was used for experimentation

A 2 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

xan-thine The solution is allowed to cool to room temperature before

being used Over time, the xanthine precipitates and can be

redis-solved 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

5 mL of a 16 mg/mL suspension of xanthine oxidase to 105lL

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, 1 mM MgCl2, 3.4 mM NaH2PO4, 5.3 mM Na2HPO4,

21 mM NaHCO3M and 3.6 mM glucose in DI water Speciﬁcally,

0.659 g NaCl, 0.0313 g KCl, 0.0203 g MgCl2 6H2O, 0.0475 g

NaH2PO4 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 3 Hz using the digitizer built into the

picoamme-ter Unless stated, all electrochemical measurements were made

using a Ag/AgCl reference (CH Instruments, Austin, TX, [Cl] = 3 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

measure-ments Oxygen plasma cleaning is done with a MARCH CS 1701 RIE

and RFX 600 generator (Nordson MARCH, Carlsbad,CA) All nitro-gen 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, fol-lowed by drying with N2 The substrates were then exposed to oxy-gen plasma (80 sccm, 500 mTorr, 200 W) for 300 s and placed immediately into the ebeam deposition chamber A 60–80 Å adhe-sion layer of Ti was coated (1 Å s1) followed by 2000 Å of Au Upon removal from the deposition chamber, the newly coated surfaces were flushed with N2and placed in a dessicator Typical lithogra-phy procedures were used to pattern gold electrodes[31] Specifi-cally, S1805 positive photoresist was spun onto the surface of the gold (300 rpm, 30 s) and baked at 110 °C for 3 min 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 re-moved using acetone Due to the precise dimensions required, la-ser cutting was used to excise the sensor from the Kapton™ sheet Sensors were kept in a desiccator until modiﬁcation to re-duce 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 1 h The working area was 3 mm by 50lm but could have been made smaller by isolating more of the gold lead The electrode sur-face is then cleaned by placing it in oxygen plasma for 2–4 min be-fore 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 2 h at room temperature Once removed, the electrode was lightly rinsed with dry DMSO, then DI water and dried with nitro-gen Gold surfaces where DMSO is not desired can be placed in 0.5 M KOH (methanol) and exposed to 1.5 V for 120 s and imme-diately rinsed with DI water The electrode was then placed in a

2 mg/mL solution of cyt c (25 mM phosphate buffer, pH 7.4) at

4 °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 buf-fer at 4 °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 phos-phate buffer (pH 7.4), and placed in a 2 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 potentio-stat 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 xan-thine oxidase solution (20lL) were added to the reaction cell

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and the mechanical stirring was turned on for 20 s and then turned

off A ﬁnal 50lL injection of the SOD solution was then added to

the reaction cell to demonstrate the extent to which removal of

O

2 returns signal to the background level Again, mechanical

stir-ring was employed for a brief period and then stopped while

cur-rent 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 3 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 50lL xanthine oxidase solution was

added to the solution and the solution was mechanically stirred

for a brief time 50lL of interferent (ﬁnal concentration of

3.75 mM) was then pipetted into the solution, mechanically

stir-ring brieﬂy Finally, 50lL SOD solution was added and mixed for

a brief time to void the solution of O

2

3 Theory

The evolution of O

2 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 O

2 found in Eq.(2) Xanthine þ O2þ H2O !½XOUrate þ O2 þ 2Hþ ð1Þ

2O

Assuming no interference, the concentration of O

2 at steady state is proportional to [XO]1/2 This presumes that dismutation,

rather than consumption of O

2 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 O

2 formation, and making the steady-state approximation,

d½O2

dt ¼ keff½XO k2½O

½O

2SS¼ keff½XO

k2

ð4Þ with keffthe effective rate constant for reaction(1)and k2the

pH-dependent rate constant for reaction(2)

4 Results and discussion

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 200lm and working

length of up to 3 mm The dimensions were governed by the

open-ing of the round window in a Mongolian gerbil, the initial in vivo

target of this sensor, to monitor O

2 generation in the cochlea

Using Kapton™, the substrate is robust and the gold surface

mal-leable enough to produce a ﬂexible sensor However, there are

con-cerns 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

res-olution of the delamination issue is critical if Kapton™ is to be

rou-tinely 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,

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 high-est 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 amper-ometry, rendering the resultant apoenzyme useless Since certain thiols can yield signal in the presence of O

2 without protein pres-ent, inactivation is typically not easily detected unless cyclic vol-tammetry is performed to characterize the electrodes [32] Therefore, cyt c, while not the most selective protein for interacting with O

2, is more stable than SOD when immobilized Cyclic vol-tammograms of the DTSP/cyt c surface at varying scan rates can

be seen inFig 1 The peak potentials of the sensor near 0.05 V and +0.1 V vs Ag/AgCl remain stable with peak ratio of 2 indicating

a quasi reversible reaction The xanthine/xanthine oxidase system for producing O2 was used to calibrate the sensor

The response of the sensor (3 mm in length) to multiple addi-tions of xanthine oxidase can be seen inFig 2with results summa-rized in Table 1, with standard deviation being that within the single run Each addition of enzyme generates sufﬁcient O

2 that,

at steady state, [O

2] increases by 4.5lM Data was processed by boxcar averaging the signal using 3 data points (IgorPro, Wavemet-rics) to reduce noise Current was then averaged when a stable baseline was reached and again after each addition of xanthine oxi-dase Shifts in current were calculated by difference using the background current as reference To ensure that the signal was in-deed from O

2, SOD was added after the four aliquots of xanthine oxidase, dropping the current 67% from maximum towards base-line Though, these sensors are not intended for repeated use, SOD proved not to alter the sensing surface Large noise ﬂuctua-tions at 100, 200, 300, 400, and 500 s are due to induced current from magnet mechanical stirring, indicating why mechanical

Fig 1 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 s 1 in 25 mM

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stirring cannot be carried out throughout the analysis A

calibra-tion plot for the sensor can be found inFig 3 The linear regression

does not intersect at origin, which is not very surprising O

2 is dis-mutating, the electrode itself is consuming O

2, 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

Re-cently, 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

sys-tem is far from ideal The basis for using this syssys-tem relies on

achieving a steady state concentration of O

2 The xanthine/xan-thine oxidase reaction deteriorates over time, causing the concen-tration of O2 to gradually drop to zero after 3 h, by which time XO generates only H2O2[12] Also, xanthine oxidase activity decreases gradually even when stored as a solid suspension Therefore it can-not be assumed that the same supply of xanthine oxidase will cre-ate the same concentration of O

2 from day to day Because of this, the xanthine oxidase activity must be calibrated spectrophotomet-rically 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 incre-ments of O

2 Therefore, as it stands, each electrode can only be cal-ibrated 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 spe-cies present during calibration Uric acid and H2O2 are products generated in the xanthine/xanthine oxidase reaction and/or a product of O

2 dismutation, Eq.(1)While a signiﬁcant concentra-tion 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 O

2 can be per-fected NADH, citric acid and glucose are also biologically impor-tant 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 inFig 4 Here, once the background current became stable, 5.2lM O

2 (ﬁnal concentration) was added

to the system Intense noise fluctuations occur during the brief stir-ring 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 2 NADH, citric acid, and uric acid showed no signiﬁcant interference Expected ascorbic acid scavenging abilities were demonstrated, Table 2 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 2 Chronoamperometry response (at 200 mV) of cyt c to 4 additions of 25lL of

xanthine oxidase solution in 2 mM xanthine solution, followed by addition of

50lLSOD (2 mg/mL) to bring signal back to background current Data (obtained at

3 Hz) has been box averaged for every three data points.

Table 1

Calibrated response of DTSP/cyt c electrode.

lM O

2 Average Di from baseline (pA) Standard deviation (pA)

Fig 3 Calibration of data collected depicted in Fig 2 Change in current (pA) is

plotted against O

concentration (lM).

Fig 4 Chronoamperometry (at 200 mV) of DTSP/cyt c electrode (2 mM xanthine solution) in the presence of 50lL xanthine oxidase solution (5.2lM O

2 ), followed

by 50lL of glucose (3.75 mM), and 50lL of SOD (2 mg/mL).

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presence O

2 are nearly immeasurable However, H2O2hinders the

ability of SOD to return signal to baseline This could be due to an

exchange of O2 response for a H2O2response while maintaining

the same current Adding catalase might not clarify this issue, as

a signal could easily then be generated by O

2 oxidation The differ-ence in current response between data shown inFig 2versusFig 4

can be explained in two parts First, the calibration of O

2 is time dependent, therefore the concentration of O

2 at the time of use al-ways has a margin of error Second, the working area of the

elec-trode 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

5 Conclusion

Thin film gold on Kapton™ was used to create the first flexible

O

2 sensor for in vivo applications The calibration showed a linear

response within the constraints established by using

xanthine/xan-thine oxidase as a O2 source Interferent studies demonstrated

that H2O2might 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 Deafness Research Foundation,

the US Army Corps of Engineers (cooperative agreements

W911NF-07-1-0005 and W9132T-08-2-0009), the US Army

Re-search Ofﬁce (Grant W911NF-07-1-0075), and ReRe-search

Corpora-tion (Grant RA0333) We further thank Donna S Whitlon for

drawing our attention to the correlation between NIHL and ROS

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Table 2

Interference of common biological molecules with electrode response.

Interference Change in current from baseline (pA), (% signal drop)

XO XO + interferent SOD added

NADH 159 139 (13%) 22 (86%)

Citric acid 90 84 (7%) 36 (60%)

Uric acid 158 157 (1.4%) 52.2 (67%)

Ascorbic acid 130 52 (60%) 39 (70%)

H 2 O 2 162 150 (8.2%) 140 (17%)

Glucose 110 88 (20%) 25 (77%)

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