Information processing through a bio-based redox capacitor: Signatures for redox-cycling

Bioelectrochemistry 98 (2014) 94–102

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Bioelectrochemistry journal homepage: www.elsevier.com/locate/bioelechem

Information processing through a bio-based redox capacitor: Signatures for redox-cycling Yi Liu a,b, Eunkyoung Kim a,b, Ian M. White b,c, William E. Bentley a,b, Gregory F. Payne a,b,⁎ a b c

Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, MD 20742, USA Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, USA Institute for Systems Research, University of Maryland, College Park, MD 20742, USA

a r t i c l e

i n f o

Article history: Received 27 January 2014 Received in revised form 27 March 2014 Accepted 28 March 2014 Available online 5 April 2014 Keywords: Biofabrication Chitosan Electrodeposition Redox-cycling Signal processing

a b s t r a c t Redox-cycling compounds can significantly impact biological systems and can be responsible for activities that range from pathogen virulence and contaminant toxicities, to therapeutic drug mechanisms. Current methods to identify redox-cycling activities rely on the generation of reactive oxygen species (ROS), and employ enzymatic or chemical methods to detect ROS. Here, we couple the speed and sensitivity of electrochemistry with the molecular-electronic properties of a bio-based redox-capacitor to generate signatures of redox-cycling. The redox capacitor film is electrochemically-fabricated at the electrode surface and is composed of a polysaccharide hydrogel with grafted catechol moieties. This capacitor film is redox-active but non-conducting and can engage diffusible compounds in either oxidative or reductive redox-cycling. Using standard electrochemical mediators ferrocene dimethanol (Fc) and Ru(NH3)6Cl3 (Ru3+) as model redox-cyclers, we observed signal amplifications and rectifications that serve as signatures of redox-cycling. Three bio-relevant compounds were then probed for these signatures: (i) ascorbate, a redox-active compound that does not redox-cycle; (ii) pyocyanin, a virulence factor well-known for its reductive redox-cycling; and (iii) acetaminophen, an analgesic that oxidatively redox-cycles but also undergoes conjugation reactions. These studies demonstrate that the redox-capacitor can enlist the capabilities of electrochemistry to generate rapid and sensitive signatures of biologically-relevant chemical activities (i.e., redox-cycling). Published by Elsevier B.V.

1. Introduction Electrodes provide a simple, sensitive, and rapid means to acquire information. One common goal is to acquire information of an individual chemical species. In this case, the challenge is selectivity and several approaches can be used to improve the selectivity of an electrochemical measurement. For instance, selectivity can be improved by tailoring the electrical inputs (e.g., by pulse voltammetry) or by modifying the electrode to “filter out” unwanted information (e.g., by using molecular recognition elements or selectively-permeable membranes). An alternative goal is to characterize states, functions or activities using measurements from multiple electrodes. The challenge in this case is to process the information to recognize characteristic patterns. For instance, electrocardiograms provide a pattern of electrode measurements that characterize the functioning of a complex organ (i.e., the beating heart). Also, the artificial/electronic nose may provide information from a “breath print” that can be correlated with pathologies [1–4]. ⁎ Corresponding author at: Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, MD 20742, USA. Tel.: + 1 301 405 8389; fax: + 1 301 314 9075. E-mail address: [email protected] (G.F. Payne).

http://dx.doi.org/10.1016/j.bioelechem.2014.03.012 1567-5394/Published by Elsevier B.V.

We are investigating a hybrid approach using a hydrogel redoxcapacitor film to manipulate electron exchange and thereby “process” information acquired by the electrode. Specifically, we focus on biologically-relevant redox-cycling activities. Probably the most familiar redox-cycling couples in biology are NAD(P)+/NAD(P)H which are essential for energy-harvesting and biosynthesis. Yet, redox-cycling may be much more prevalent because many biology systems (e.g., the lungs and intestinal tract) [5] are characterized by steep gradients in redox potential that provides the necessary context (i.e., the thermodynamic driving forces) for redox-cycling. For instance, the opportunistic pathogen Pseudomonas aeruginosa produces a redox-active virulence factor pyocyanin that can accept electrons from host cells (thus disrupting their redox homeostasis) and then donate these electrons to O2 to generate reactive oxygen species (ROS) [6–8]. Another example is acetaminophen where its ability to interrupt the redox-cycling of hemeproteins (e.g., cyclooxygenase; COX) is believed to confer therapeutic benefit [9,10] while its oxidation by liver cytochrome enzymes is believed to be responsible for its sometimes lethal side effects [11,12]. Redox-cycling assays have been developed using enzymatic [13] or chemiluminescence [14–16] methodologies. Such redox-cycling assays can be used for detection/diagnosis [14] or employed in drug screening programs either to eliminate false-positives [17–20] or to discover promising leads (e.g., for anticancer drugs) [21]. Electrochemistry

Y. Liu et al. / Bioelectrochemistry 98 (2014) 94–102

should be particularly well-suited for detecting redox-cycling activities and in fact redox-cycling is a common electrochemical amplification method [22,23]. The challenge is to generate and interpret electrochemical signals capable of discerning redox-cycling from other redoxactivities. Here, we probed redox-cycling activities; (i) using an electrode modified with a redox-capacitor film, (ii) coupling film-charging and film-discharging reactions, (iii) imposing varying chemical and electrical inputs, and (iv) analyzing outputs to generate characteristic signature patterns. 2. Redox-capacitor: fabrication and information processing 2.1. Fabrication of bio-based redox-capacitor film We refer to our film as a redox-capacitor because it can accept, store and donate electrons in a controllable fashion [24]. We use the prefix “bio-based” because the film is prepared from organic components common in biology (e.g., a catechol and a polysaccharide) [25,26] and because it can exchange electrons with biologically-relevant oxidants and reductants [27–30]. Two electrochemical steps are used to fabricate the redox-capacitor film as a coating for the gold electrode. First, a hydrogel film of the aminopolysacccharide chitosan is electrodeposited onto the gold electrode (1% chitosan, pH 5.5, 6 A/m 2, 30 s) [31–33]. Chitosan is pH-responsive and film-forming, and electrodeposits at a cathode by a neutralization mechanism. Once deposited the chitosan film is stable under neutral conditions although it will re-dissolve in mild acid (chitosan's pKa ≈ 6.3). Second, the chitosan film is functionalized by grafting catechol moieties. Grafting is achieved by immersing the chitosan-coated electrode in a catechol-containing solution (5.0 mM)

a) Ru

Solution Film

Ru

Ru

+

2.2. Redox-capacitor properties of film Previous studies have shown that the catechol-chitosan films are non-conducting in that they are unable to exchange electrons directly with the underlying electrode [24]. However, the catechol-chitosan films are redox-active and can rapidly and repeatedly exchange electrons with soluble redox-active species (i.e., with mediators). Scheme 1a illustrates reduction (i.e., charging) of the film by a redoxcycling mechanism with the electrochemical mediator Ru(NH3)6Cl3 (Ru3+). In this case oxidized mediators (Ru3 +) diffuse from the bulk solution through the film and are reduced to Ru2 + at the electrode. The reduced Ru2 + diffuse from the electrode surface into the film where they undergo redox-cycling thereby transferring electrons to the film to convert Q to QH2 moieties. Scheme 1b illustrates oxidation (i.e., discharging) of the film through an analogous redox-cycling mechanism in which the reduced 1,1′-ferrocenedimethanol (Fc) mediators diffuse through the film, are oxidized to Fc+ at the electrode and Fc+ then diffuse into and accept electrons from the film to convert QH2 to Q moieties. The plot in Scheme 1c illustrates that electron transfer to/from the film is controlled by thermodynamics. The Ru3 + and Fc mediators provide convenient models of redoxactive species that can engage the catechol-chitosan film in separate reductive and oxidative redox-cycling mechanisms. Importantly, these

Fc

Thermodynamic Scale

−

+

Solution Film

−0.2 V

+

O Q O

+

Fc

Electrode OH QH2 OH

+ 2Ru2+ +

c)

+

e-

Electrode

+

+ 2Ru

OH

QH2

+

+

Ru

e− Q O

+

e− 2+/3+

eO

OH

+0.2 V +0.25 V

+ 2Fc + 2H+

Oxidative redox-cycling (film discharging)

Reductive redox-cycling (film charging)

d)

and applying an anodic potential to the underlying electrode (0.5 V, 5 min). Catechol diffuses through the chitosan film, is oxidized at the underlying electrode and the oxidized species (e.g., o-quinone) grafts to the amine moieties of the chitosan film. After preparation, the catechol-chitosan-coated electrode is sonicated for 3 min, washed extensively with water.

b) Ru3+

95

QH2/Q

Fc/Fc+

+

E0 vs. Ag/AgCl Redox capacitor (Catechol-chitosan film)

1.5

Ru3+/2+

Catechol-Chitosan Chitosan

Charge Transfer

1.0

=

2

Current (A/m )

Red

0.5

Amplification Ratio (AR)

Fc 0.0

Ox

Ox

-0.5

Fc 0.4

Ru

Fc/Fc+ 0.2

= Scan rate)

Ru

Red

0.0

-0.2

Catechol-Chitosan

Catechol-Chitosan

Chitosan

Chitosan

Rectification Ratio (RR)

-0.4

E (V) vs Ag/AgCl Scheme 1. Schematics of redox-cycling mechanisms and information processing. (a) Reductive redox-cycling. (b) Oxidative redox-cycling. (c) Thermodynamics of electron transfer. (d) Illustrative CVs comparing catechol-chitosan redox-capacitor film and an unmodified chitosan control film, and the associated analysis.

96

Y. Liu et al. / Bioelectrochemistry 98 (2014) 94–102

and whether the currents are attributed to Fc or Ru3+. The currents in each of these quadrants are then integrated to generate the charge transfer for the individual quadrants. We should emphasize that the assignment of each quadrant to activities of a single mediator is useful for processing the information although such an assignment is a simplification of the underlying chemistries. It should be noted that our analyses rely on expectations of how redox-cycling will impact outputs and can be considered “hypothesis-driven” analyses as compared to “discovery-driven” analyses such as principle component analyses. Scheme 1d also indicates that the information is summarized as two sets of ratios. The amplification ratio (AR) compares the charge transfer (Q) in a given quadrant for the catechol-chitosan film to an equivalent Q for the unmodified chitosan film. This AR is expected to contain information of redox-cycling since the major difference between the films is that the catechol-chitosan film can redox-cycle while the unmodified chitosan film cannot redox-cycle. The second ratio, the rectification ratio (RR), is more subtle and can be rationalized by considering two extremes. In one extreme, if a single Ru3 + mediator was used and no redox-cycling occurred, then it is possible to imagine that each electron transferred to Ru3+ during the reductive sweep of the CV could be recovered by transfer from Ru2+ Ox during the oxidative sweep. In this extreme, Q Red Ru ≈ Q Ru and the rectifi3+ Red Ox cation ratio for Ru (RRRu = Q Ru /Q Ru) would approach 1. In the other

mediators can be incubated together and cycling the imposed electrode potential allows these redox-cycling mechanisms to be engaged sequentially. For instance, Scheme 1d compares illustrative cyclic voltammograms (CVs) for a catechol-chitosan film and a control unmodified chitosan film. When the potential is swept in the reducing direction, the electrochemical reduction of Ru3 + initiates reductive redoxcycling of the film yielding a considerable amplification of the reduction current (compared to the chitosan control film). Similarly, when the potential is swept in the oxidizing direction, the electrochemical oxidation of Fc initiates oxidative redox-cycling of the film yielding an amplified oxidation current. It is important to note that the catechol-chitosan film rapidly and reversibly exchanges electrons with these mediators although the film's redox-capacity is finite. 2.3. Signal analyses The underlying hypothesis of this study is that the CVs for the catechol-chitosan film contain information on redox-cycling activities, and our goal is to analyze this information to extract appropriate signatures of redox-cycling. Because Fc and Ru3+ are well-behaved redoxcyclers, we use these electrochemical mediators as models. To analyze the information, Scheme 1d illustrates that the CVs are divided into 4 quadrants based on whether the currents are oxidizing or reducing,

a)

c)

Potential Input (−0.4 to 0.5 V)

[Fc] µM

Fc

50

3+

[Ru ] = 50 µM [Fc] = 0 - 100 µM

25 0

16 8 0 32

Chit

6

24

4

0.4

16

2

0.0

8

Oxidize Fc

-0.4

Reduce Ru 0

500

1000

1500

2500

0

d) 3+

Cat-Chit Chit

4

Reduce Ru

0 -4

20

40

60

80

100

40

60

80

100

80

100

80

100

[Fc] (µM) 8

ARRu ARFc

6

4

Oxidize Fc 0

500

1000

1500

2000

2500

4

[Fc] = 20 µM

Cat-Chit Chit

20

40

60

[Fc] (µM) Cat-Chit

Reduce 3+

Ru

0 Oxidize Fc

-4

0

e)

[Fc] = 100 µM

[Fc] = 50 µM

Rect. Ratio (RR

[Fc] = 0 µM

2

3000

Time (s)

Current (µA)

20

3000

Time (s)

b) Current (µA)

2000

0

0

3+

Ampl. Ratio (AR)

Potential (V)

Average Charge (µC)

100

Ru3+

Cat-Chit

24

Chemical Input (Fc)

75

32

RRRu

12

Chit

RRRu 9 6 3

0.4

0.0

-0.4

0.4

0.0

-0.4

0.4

0.0

Potential (V) vs. Ag/AgCl

-0.4

0.4

0.0

-0.4 0

20

40

60

Chemical Input, [Fc] (µM)

Fig. 1. Response to chemical inputs of Fc (constant Ru3 + = 50 μM). (a) Chemical input (Fc) and cyclic potential input. (b) Output currents for catechol-chitosan (red) and chitosan (blue) coated electrodes and representative CVs for various Fc concentrations. (c) Calculated charges for the quadrants of Scheme 1d (averaged from at least 4 cycles). (d) Calculated amplification ratios. (e) Calculated rectification ratio for Ru3+. Note: standard deviations in (c)–(e) are typically smaller than the corresponding symbol size. [See Supporting Information for further details.]

Y. Liu et al. / Bioelectrochemistry 98 (2014) 94–102

a)

97

c)

Potential input (step toward positive)

Cat-Chit

Oxidize Fc

Fc + Ru3+

0.4 0.0 -0.4

3+

Reduce Ru 0

200

400

800

1000

Cat-Chit Chit

10

Reduce Ru

3+

5 0 -5

Oxidize Fc 0

200

400

600

800

1000

1200

Oxidize Fc

0.4

0.0 -0.4 0.4

0.0 -0.4 0.4

0.0 -0.4 0.4

0.0 -0.4 0.4

0.0 -0.4 0.4

Potential (V) vs. Ag/AgCl

0.0 -0.4

0.1

0.2

0.3

0.4

0.5

Potential (V) vs. Ag/AgCl

ARRu

12 9

0

EFc = 0.25 V

6 0.0

0 -4

20

0.1

0.2

0.3

0.4

0.5

Potential (V) vs. Ag/AgCl

e)

Reduce 3+ Ru

4

Chit 40

d)

Rect. Ratio (RR)

Current (µA)

Cat-Chit Chit

0

0 0.0

Time (s) 8

20

1200

Time (s)

b) Current (µA)

600

Ampl. Ratio (AR)

Potential (V)

[Fc] = 50 µM; [Ru3+] = 50 µM

Average Charge (µC)

40

12

Cat-Chit

RRRu

Chit RRRu

8 4 0 0.0

0.1

0.2

0.3

0.4

0.5

Potential (V) vs. Ag/AgCl

Fig. 2. Response to changes in the potential input range in the presence of both Fc and Ru3+ (50 μM each). (a) Imposed potential input cycles from the negative region and steps toward the positive region (scan rate of 0.05 V/s). (b) Output currents for catechol-chitosan (red) and chitosan (blue) coated electrodes and representative CVs. (c) Calculated charges for Ru3+ reduction and Ru2+ oxidation. (d) Calculated amplification ratio for Ru3+. (e) Calculated rectification ratio for Ru3+.

extreme, if every Ru3 + reduced at the electrode during the reductive sweep is re-oxidized by redox-cycling in the film, then there would be no Ru2+ for electrochemical oxidation during the oxidative sweep. In this extreme, Q Ox Ru → 0 and RRRu would approach infinity. This illustrative example indicates that the rectification ratio also provides information of redox-cycling. 3. Materials and methods The following materials were purchased from Sigma-Aldrich: chitosan from crab shells (85% deacetylation, and 200 kDa as reported by the supplier), catechol, Ru(NH3)6Cl3, 1,1′-ferrocenedimethanol, pyocyanin, and acetaminophen. Water was de-ionized with Millipore SUPER-Q to a resistivity N18 MΩ cm. Chitosan was dissolved in dilute HCl solution (pH = 5.5) as previously described [34]. Other chemicals were prepared in phosphate buffer (0.1 M; pH 7.0). The gold working electrode (2 mm diameter) was first cleaned with piranha solution (H2SO4: H2O2 = 7: 3 v/v) for 15 min and washed thoroughly with DI water, followed by drying under N2 stream. Electrodeposition of chitosan was conducted on a 2400 Sourcemeter (Keithley). Electrochemical measurements were carried out with a CHI6273C Electrochemical Analyzer (CH Instruments, Inc., Austin, TX). Measurements were performed using three-electrode configurations with Ag/AgCl (3 M NaCl) as a reference electrode and Pt wire as an auxiliary electrode [24], as illustrated in Scheme S1 in the Supporting Information. Solution was degassed with nitrogen for about 40 min before electrochemical measurement, and during the measurement a stream of nitrogen was gently blown over the surface of the solution. Specific experimental conditions such as the sweeping potential range and scan rate are provided in the text and figure captions.

4. Results 4.1. Identifying signatures of redox-cycling: model system 4.1.1. Response to imposed chemical inputs of Fc In our initial study, we examined redox-cycling induced by chemical input [35]. In these experiments, a film-coated gold electrode was immersed in a buffer solution containing Ru3+ (50 μM), a cyclic potential input was imposed (− 0.4 to 0.5 V vs Ag/AgCl; scan rate of 0.05 V/s), and aliquots of the Fc mediator were added as illustrated in Fig. 1a. The output currents were continuously recorded for both a catecholchitosan film and a chitosan control, and the results are shown in Fig. 1b. Initially, when no Fc is present, the current outputs and CVs ([Fc] = 0 μM) show only redox peaks for Ru3+. When Fc is added, the output current for the catechol-chitosan film shows a dramatic increase for both Ru3+ reduction and Fc oxidation, and the CVs show amplification of the output currents. Results for the chitosan control in Fig. 1b show that the addition of Fc results in increases in the Fc peaks but insignificant changes in the Ru3+ region of the CVs. These observations are consistent with the explanation that the catechol-chitosan film engages the mediators in the redox-cycling reactions of Scheme 1. The calculated charge transfer for the four quadrants of Scheme 1d is shown in Fig. 1c. The addition of Fc for the case of the catechol-chitosan film resulted in increases in both the Fc oxidative charge (Q Ox Fc ) and the Ru3 + reductive charge (Q Red Ru ). In contrast, the addition of Fc for the control chitosan film resulted in a comparatively small increase in Fc 3+ oxidation (Q Ox reduction. Fc ) and no change in Ru As illustrated in Scheme 1d, we calculated the amplification ratios (AR) for Fc oxidation and Ru3 + reduction from the output CVs. As shown in Fig. 1d, the addition of Fc resulted in amplified outputs for

Y. Liu et al. / Bioelectrochemistry 98 (2014) 94–102

b) Potential input (−0.4 to 0.5 V)

Fc

Chemical input (Ru3+)

[Fc]=50 µM [Ru3+]=50 µM Fc +

Average Charge (µC)

0

Chit 5

20

0 5

8

12

ARFc

4

20 Cat-Chit

RRFc

10

Chit RRFc

0 0

20

40

60

80

100

Chemical Input, [Ru3+] (µ M)

Ampl. Ratio (AR)

0

6

30

Chemical Input (Ascorbate)

Cat-Chit

b) 8

ARRu

6 4 2

20 10 0 10

Chit

Chit

4 3 2

Cat-Chit

RRRu

Chit

RRRu

1

0 0

0

Rect. Ratio (RR)

Average Charge (µC) Ampl. Ratio (AR) Rect. Ratio (RR)

20

40

Asc

Ru3+

Ru3+

Cat-Chit

Cat-Chit 40

Potential Input (–0.4 to 0.5 V)

Rect. Ratio (RR)

Ru3+

a) Potential input (step)

Average Charge (µC)

a)

Ampl. Ratio (AR)

98

50

100 150 200 250

[Ascorbate] (µM) ARFc

0

50

100 150 200 250

[Ascorbate] (µM)

Fig. 4. Ascorbate does not display the characteristic signatures of redox-cycling. Summary of (a) experiment and calculated charge transfer; and (b) calculated amplification and rectification ratios. Ascorbate was added to a solution containing constant Ru3+ (50 μM). Cyclic potential inputs (−0.4 to 0.5 V) were applied with a scan rate of 0.05 V/s. [See Fig. S4 in Supporting Information for further details.]

9

6 0

ERu = -0.2 V Cat-Chit

20

RRFc

Chit

RRFc 10

0 0.0

-0.1

-0.2

-0.3

-0.4

Potential (V) vs. Ag/AgCl

Fig. 3. Summary of responses to: (a) Ru3+ chemical input (constant Fc); and (b) potential input steps from positive toward negative potential ranges. [See Supporting Information for further details.]

both Fc oxidation and Ru3+ reduction. The simultaneous amplification of both outputs upon Fc inputs indicates that Fc oxidation and Ru3 + reduction are linked. Presumably, Fc's oxidative redox-cycling partially-discharges the film (i.e., by converting some QH2 to Q moieties) providing the oxidized Q sites necessary for Ru 3 + to undergo reductive redox-cycling. Also illustrated in Scheme 1d is the calculation for the Ru 3 + rectification ratio (RR). Inspection of the CVs (Fig. 1b) or charge transfer (Fig. 1c) shows that the addition of Fc resulted in both an 2+ increase in Ru3 + reduction (Q Red oxidation Ru ) and a decrease in Ru 3+ Ox (Q Ru). This rectification of Ru outputs is shown in Fig. 1e. In summary, Fig. 1 shows that the redox-capacitor “processes” information of redox-cycling by generating output signatures (AR and RR) in the regions of the CVs that would otherwise be unaffected by the chemical input. Specifically, Fc additions resulted in dramatic changes in the amplification and rectification of Ru3+ output currents. 4.1.2. Response to imposed changes in the potential range for oxidation Next, we examined the response to changes in the imposed potential range. In the experiment, the film-coated electrode was immersed in a solution containing both Fc and Ru3 + mediators (50 μM each). As shown in Fig. 2a, the potential was initially cycled at negative potentials (−0.4 to 0 V), and then sequentially stepped toward positive potentials (− 0.4 to 0.5 V). Fig. 2b shows that initially when the potential was swept in the negative region, only Ru3 + peaks were observed (the potential did not become sufficiently positive to oxidize Fc and initiate oxidative redox-cycling in the film). When the potential was swept toward more positive potentials (E N 0.25 V), Fc oxidation

occurred and the redox-cycling in the film was initiated to discharge the film. As a result, the output current and CVs of catechol-chitosan film show dramatic increases in both Fc oxidation and Ru3+ reduction, and also small decreases in Ru3 + oxidation. In contrast, the output current for the chitosan control shows minimal changes in the Ru3 + regions. Fig. 2c shows that as the potential was increased into the region that Fc could be oxidized (E N 0.25 V), the catechol-chitosan film displayed Red Ox increases in Q Ru and decreases in Q Ru , while a minimal change was observed for chitosan control. Fig. 2d summarizes the amplification of Ru3+ reduction and Fig. 2e shows the rectification in the Ru3+ region. These calculations illustrate that imposed changes in the positive potential region yield substantial responses in the negative potential region. Again these responses are consistent with redox-cycling: once Fc is oxidized, its redox-cycling partially-discharges the film providing the opportunity for Ru3 + to undergo reductive redox-cycling. These results also illustrate that the redox-potential (E0) of Fc serves as a voltage “gate” for redox-cycling [28].

4.1.3. Response to imposed chemical inputs of Ru3+ and imposed changes in the potential range for reduction We performed complementary studies to those in Figs. 1 and 2 by imposing chemical inputs of Ru3+ (constant Fc = 50 μM) and potential inputs that cycle into the negative potential range (complete data sets for these experiments are provided in Supporting Information). Results from the Ru 3 + chemical-input study are summarized in Fig. 3a. The addition of Ru3 + resulted in: (i) significant increases in 3+ both Fc-oxidation (Q Ox reduction (Q Red Fc ) and Ru Ru ); (ii) amplification of Fc-oxidation; and (iii) rectification of Fc outputs. These results again illustrate that redox-cycling information appears as output signatures (AR and RR) in regions of the CVs that would otherwise be unaffected by the Ru3+ chemical input. Fig. 3b shows the results obtained when the potential input was cycled from positive (0 to 0.5 V) toward negative potential ranges (− 0.4 to 0.5 V) with constant chemical input (Fc and Ru3 +, 50 μM each). Sweeping into the negative potential range resulted in substantial changes in the positive potential region: (i) increases in Red both Fc-oxidation (Q Ox Fc ) and decreases in Fc-reduction (Q Fc ); (ii) amplification of Fc-oxidation; and (iii) rectification of Fc outputs. Analogous to results in Fig. 2, the oxidative redox-cycling of Fc is linked to the reductive redox-cycling of Ru3+ and the redox-potential (E0) of Ru3+ serves as a voltage gate for redox-cycling.

Y. Liu et al. / Bioelectrochemistry 98 (2014) 94–102

a)

b)

Potential Input

99

Potential Input (Step)

Chemical Input [Fc]=0–50 µM

24

[PYO]=25 µM [Fc]=25 µM

PYO + Fc

Fc

Cat-Chit

24

Average Charge (µC)

Average Charge (µC)

PYO (25 µM)

16 8 0 8

Chit

Ampl.Ratio (AR)

ARPYO

6

4

2 5 Cat-Chit RRPYO Chit RRPYO

4 3 2 0

10

20 30 [Fc] (µM)

40

50

Rect. Ratio (RRFc)

Ampl.Ratio (AR)

16 8 0 8

Chit

0

0

Rect. Ratio (RR)

Cat-Chit

6

ARFc

4 0

EPYO= -0.2 V

2

20

Cat-Chit Chit

10

0 0.0

-0.1 -0.2 -0.3 -0.4 Potential (V) vs. Ag/AgCl

Fig. 5. Pyocyanin (PYO) displays the characteristic signatures of redox-cycling. (a) Redox-cycling of PYO is induced by the addition of Fc inputs. (b) Redox-cycling of PYO induces Fc redox-cycling when the potential input range is increased to include negative potentials. [See Figs. S6 & S7 in Supporting Information for further details.]

4.2. Probing for redox-cycling signatures with bio-relevant molecules 4.2.1. Redox signature with bio-relevant reducing agent—ascorbate Ascorbate is a common reducing agent in biology that can be oxidized but not readily reduced (i.e., it does not readily undergo redox-cycling). We next tested ascorbate to determine whether it displays the characteristic signatures of redox-cycling. In the first experiment, we added aliquots of ascorbate sequentially to a solution containing 50 μM Ru3 + and applied a cyclic potential input to the electrode coated with the catechol-chitosan film. Fig. 4a shows that 3+ the ascorbate oxidation (QOx reduction Asc) increases slightly while Ru Red (QRu ) remains nearly constant. As a result, Fig. 4b shows no amplification of Ru3+ reduction. In addition, the rectification ratio of Ru3 + for both catechol-chitosan film and chitosan control does not increase upon the addition of ascorbate. These results indicate that signatures of redox-cycling are absent when ascorbate is added to a Ru3+ solution. We performed an analogous experiment where aliquots of ascorbate were sequentially added into a solution containing 50 μM Fc and a cyclic potential input was applied (see Fig. S5 in Supporting Information). The results for the catechol-chitosan film show that no amplification of the anodic signal is observed and the RR of the anodic signal is featureless. These results indicate that signatures of redox-cycling are also absent when ascorbate is added to the Fc solution.

4.2.2. Redox-signature with metabolite—pyocyanin The redox-active bacterial metabolite pyocyanin (PYO) is a well-known redox-cycler. As illustrated in Fig. 5a, a film-coated

gold electrode was immersed in a buffer solution containing PYO (25 μM); the potential was swept continuously (− 0.4 to 0.5 V vs Ag/AgCl; scan rate of 0.05 V/s), and aliquots of the Fc mediator were sequentially added to the PYO solution. Fig. 5a shows that Ox the addition of Fc resulted in: (i) increases in both Q Red PYO and Q Fc for catechol-chitosan film; (ii) amplified reduction in the PYO region, and (iii) rectification of the PYO outputs. Thus the redox-cycling signatures are clearly evident with PYO. Fig. 5b shows redox-cycling of PYO induced by potential input steps from the positive toward the negative region. Fig. 5b shows that when the potential became sufficiently negative to reduce PYO (E0 ~ −0.2 V), Red then: (i) Fc oxidation (Q Ox Fc ) increased while Fc reduction (Q Fc ) decreased; (ii) an amplification of Fc oxidation occurred, and (iii) a rectification of Fc outputs was observed. Again, results from this experiment display the characteristic signatures for redox-cycling, while PYO's redox-potential (E0) serves to “gate” the redox-cycling. 4.3. Redox-cycling signature with drug—acetaminophen Finally, we probed the redox-active analgesic acetaminophen. Because of irreversibilities of acetaminophen's oxidation, it is expected to display intermediate signatures of redox-cycling. As shown in Fig. 6a, a film-coated electrode was immersed in a buffer solution containing acetaminophen and Ru3 + (50 μM each), and the potential input was cycled from negative potential range toward a morepositive range. Fig. 6b shows the output currents and representative Ox CVs for the experiment. Fig. 6c shows that changes in Q Red Ru and Q Ru are observed to occur near E = ~ 0.55 V where acetaminophen starts

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a)

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Potential (V) vs. Ag/AgCl Fig. 6. Acetaminophen (Acet) displays intermediate signatures of redox-cycling. (a) The imposed potential input range is increased to include positive potentials (scan rate of 0.05 V/s). (b) Output currents for catechol-chitosan and chitosan coated electrodes and representative CVs. (c) Calculated charge transfer for Ru3+ reduction and Ru2+ oxidation. (d) Calculated amplification ratios for Ru3+ show little evidence for redox-cycling. (e) Calculated rectification ratios for Ru3+show strong evidence for redox-cycling.

to become oxidized and this observation is consistent with acetaminophen serving as a gate for Ru3+outputs. Fig. 6d shows minimal amplification in Ru3 + reduction currents while Fig. 6e shows significant increases in the rectification of Ru3+ outputs. Thus acetaminophen displays output signatures intermediate between the non-redox-cycler ascorbate and the known redox-cycler pyocyanin. 5. Discussion Compounds capable of redox-cycling can have substantial impacts on biology by altering cellular redox-homeostasis, inducing the formation of reactive oxygen species (ROS) and contributing to oxidative stress [36,37]. For instance, redox-cycling has been implicated in the toxicity of environmental pollutants [38] and agricultural chemicals [14], and also suggested as potential modes-of-action for anticancer therapeutics [39]. Common in vitro-assays to detect redox-cycling activities employ a reducing agent (e.g., dithiothreitol; DTT) in the presence of air to detect whether a sample can redox-cycle to “catalyze” the transfer electrons from the reducing agent to O2 to generate reactive oxygen (e.g., H2O2) [13–16,18,20]. Our approach does not require ROS generation (our incubations are performed in the absence of O2) but employs the electrode as the ultimate source and sink of electrons. Thus, we enlist the sensitivity and selectivity of electrochemistry to detect redox-cycling. Our approach for assaying redox-cycling activities integrates two capabilities. First, we use the capabilities of an electrode to impose the appropriate potentials to initiate redox-cycling. Importantly, electrodeimposed potentials can span biologically-relevant ranges and thus electrochemistry has emerged as an important tool for the in vitro investigation of biological redox-reactions. For instance, electrochemical oxidation has been used to model the metabolism of drugs [40–47] and

cosmetics [48,49], while electrochemical reduction has been used to model bioreductions (e.g., of antitumor prodrugs) [50,51]. Second, we use the capabilities of a catechol-chitosan redox-capacitor to engage diffusible species in redox-cycling mechanisms. Importantly, the film's catechol-quinone redox potential is physiologically-appropriate to engage redox-cycling mechanisms that are initiated by either oxidation or reduction reactions [24,29,30]. The redox-cycling between the film and diffusible species perturbs the electrochemical signal by amplifying and partially-rectifying the output currents [28]. We integrate the capabilities of the electrode and redox-capacitor by imposing oscillating electrode potentials in the presence of both oxidative and reductive redox-cyclers to generate near-steady oscillating outputs. Steady, sinusoidally oscillating inputs and outputs are commonly used in signal processing to acquire and transmit information. In contrast to signal processing, we do not use frequency and phase shift as the key information-containing parameters: rather we use amplification and rectification ratios to provide signatures for redox-cycling. In this study we examined three compounds that display characteristic features of redox-cycling. Pyocyanin is a well-known redox-cycling metabolite of the opportunistic pathogen P. aeruginosa [52–54]. Common to such reductive redox-cyclers, the reduced pyocyanin can be re-oxidized by donating its electrons to O2 to generate reactive oxygen species (ROS) [7,8]. Interestingly, pathogenic Pseudomonas are often localized in niches where substantial gradients in O2 are expected (e.g., the lungs [55,56], burn wounds [57,58] and the gastrointestinal tract [59,60]). Thus, only short diffusion paths are required for this redox-active metabolite to transverse redox environments that allow cycling between their reduced and oxidized states. Results with pyocyanin displayed the strongest amplification and rectification signatures for redox-cycling.

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Ascorbate is a biological reductant [61] known to reduce quinones [62], quinone based drugs [63–65], and the quinone moieties of the catechol-chitosan redox-capacitor [29,35]. While quinone redox-cycling can catalyze the transfer of electrons from ascorbate to O2 to generate ROS [62,66], the oxidized ascorbate is not as readily reduced (although mechanisms do exist for ascorbate recycling [67]). Thus, ascorbate typically acts as a reductant and not a redoxcycler. Consistent with expectations, we observed ascorbate's redoxactivity but the amplification and rectification signatures of redoxcycling were absent. Our third example, acetaminophen, is an example of a molecule that can undergo redox-cycling upon oxidation. However, oxidation also yields reactive intermediates (e.g., free radicals or quinones) that can also undergo coupling and conjugation reactions (e.g., with glutathione). Oxidation is important both for acetaminophen's beneficial activities and also for its harmful side effects [9,10,12]. As expected, acetaminophen displayed redox-cycling signatures intermediate between those of pyocyanin and ascorbate. The broad vision of this work is to apply the power of electrochemistry to problems in redox-biology. Sensitive electrochemical measurements allow information to be acquired rapidly and simply, and in a format that is convenient for information processing. Traditionally, electrode measurements are used either to detect a single chemical species or to provide patterns of global function (e.g., electrocardiograms can detect abnormalities in the beating heart). Here, we use a hybrid approach in which a capacitor film “processes” information to reveal signatures that are characteristic of redox-cycling activities. These signatures appear to be generic (and not molecule specific) for an important mechanism in chemical biology. The most immediate application of this work is as a method to detect redox-cycling activities [13–16] in drug screening programs either to eliminate false leads [18–20] or to identify molecules with potential therapeutic benefits (e.g., redox-cycling anticancer activities) [21,68]. Our longer term goal is to extend electrochemical approaches to acquire global system-level information of more complex activities. For instance, oxidative stress is hypothesized to be important for a range of maladies yet there is little agreement of what measurements best characterize oxidative stress (e.g., which ROS or what biomarker) [37,69–71]. Potentially, electrochemistry could provide a simple system-level measure of oxidative stress [28] to complement more detailed measures of individual ROS, host defense mechanisms or biomarkers.

6. Conclusions The catechol-chitosan redox-capacitor is redox-active and nonconducting, and can rapidly and reversibly exchange electrons in either oxidative or reductive redox-cycling reactions. These features result in dramatic signal amplifications and rectifications if: (i) the film is exposed to redox-cyclers with E0's above and below the film's E0 (≈ 0.2 V vs Ag/AgCl); and (ii) the electrode potential is cycled to sequentially engage the oxidative and reductive redoxcycling mechanisms. For the compounds tested, these amplifications and rectifications serve as redox-cycling signatures that appear to be chemically non-specific and thus provide a generic means to detect redox-cycling activities. Potentially, this redox-capacitor provides a unique opportunity to apply electrochemical methods to redox-biology.

Acknowledgments The authors gratefully acknowledge financial support from Robert W. Deutsch Foundation, and the Defense Threat Reduction Agency (BO085PO008).

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Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bioelechem.2014.03.012.

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