Colloids and Surfaces B: Biointerfaces 123 (2014) 866–869

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb

Short Communication

Electrochemical hydrogen peroxide sensors fabricated using cytochrome c immobilized on macroelectrodes and ultramicroelectrodes S. Ehsan Salamifar, Stephen Lee, Rebecca Y. Lai ∗ Department of Chemistry University of Nebraska-Lincoln, 651 Hamilton Hall, Lincoln, NE 68588-0304, USA

a r t i c l e

i n f o

Article history: Received 2 August 2014 Received in revised form 4 October 2014 Accepted 15 October 2014 Available online 28 October 2014 Keywords: Cytochrome c Hydrogen peroxide Ultramicroelectrodes Rotating disk electrodes Michaelis–Menten kinetics Electron transfer rate

a b s t r a c t We report the design and fabrication of hydrogen peroxide (H2 O2 ) sensors using heme proteins immobilized on macroelectrodes and ultramicroelectrodes (UMEs). In this sensor design, the heme centers are directly “wired” to the electrode via the use of an imidazole-terminated self-assembled monolayer. We have systematically evaluated the effect of electrode type and size on sensor performance. The limit of detection for H2 O2 determined using a 10-␮m gold UME is significantly lower than that obtained using a stationary macroelectrode. Our results also highlight the advantages of using UMEs for enzyme kinetics analysis; the Km determined using a 10-␮m UME is similar to that obtained from a rotating disk electrode.

Reactive oxygen species (ROS) are continuously produced in aerobic cells as by-products of metabolism. Some of them are highly toxic and thus rapidly scavenged by both enzymatic and nonenzymatic pathways [1,2]. Owing to these detoxifying mechanisms, intracellular ROS level is maintained at a constant level. Faulty control of these chemical processes could result in ROS accumulation inside cells, creating a condition called “oxidative stress”, which has been known to cause cell genome injury [3]. The correlation between “oxidative stress” and pathological conditions such as different types of cancers is well established [4,5]. There is a need for developing rapid and sensitive method for in vivo measurement of ROS in biomedical research. Recently, ultramicroelectrodes (UMEs) and nanoelectrodes, due to their unique properties such as higher rates of mass transport, reduced capacitance and reduced ohmic drop, have found widespread applications in real time analysis of ROS at single cell level [6–8]. While ROS can be detected directly using Pt UMEs, there are merits in developing biosensors capable of measuring ROS. Heme proteins such as hemoglobin, myoglobin (Mb) and cytochrome c (Cyt c) are known to be capable of catalyzing reduction of ROS such as hydrogen peroxide (H2 O2 ) [9]. Biosensors have since been fabricated using these proteins; however, few have been fabricated directly on an imidazole

∗ Corresponding author. Tel.: +1 402 472 5340; fax: +1 402 472-9402. E-mail address: [email protected] (R.Y. Lai). http://dx.doi.org/10.1016/j.colsurfb.2014.10.033 0927-7765/© 2014 Elsevier B.V. All rights reserved.

© 2014 Elsevier B.V. All rights reserved.

(Im)-terminated self-assembled monolayer (SAM) [10–12]. Here we report the design and fabrication of H2 O2 sensors using heme proteins that are directly “wired” to the electrode. This study also focuses on the comparison of heme protein-based H2 O2 sensors fabricated on UMEs and macroelectrodes. Prior to using a gold UME as the sensor electrode, we evaluated the effect of different protein immobilization procedures on the electrochemical signal of the sensor on gold macroelectrodes. We first fabricated sensors using two conventional methods, direct adsorption and covalent attachment via n-hydroxysuccinimide/1ethyl-3-(3-dimethylaminopropyl)-carbodiimide (NHS-EDC) coupling [13,14]. For both systems, the effects of ionic strength, incubation time and concentration of reagents were also investigated. Cyclic voltammograms (CVs) of the sensors fabricated using the optimized protocols are shown in Fig. S1. A set of redox peaks with a half-wave potential (E1/2 ) of ∼−0.05 V was observed in a degassed phosphate buffer solution (PBS), indicating successful immobilization of Cyt c on the 11-mercaptoundecanoic acid (C11 COOH) terminated SAM (Fig. S1A). However, the electrochemical signal was unstable, presumably because of the gradual desorption of proteins from the electrode surface. In contrast, while the use of NHS-EDC coupling resulted in a sensor with a stable electrochemical signal (Fig. S1B), the multi-step process renders this approach less attractive. This protein immobilization method is also less reproducible (i.e., larger sensor-to-sensor variation in probe coverage), thus it is not ideal for sensor fabrication. Because

S.E. Salamifar et al. / Colloids and Surfaces B: Biointerfaces 123 (2014) 866–869

867

Scheme 1. Schematic representation of direct “wiring” of heme proteins onto a gold electrode modified with C11-Im and C8-OH.

of these negative results, immobilization of Mb was not attempted using these two approaches. To effectively anchor Cyt c and Mb for our current application, we employed a recently developed immobilization method that relies on “direct-wiring” of the heme centers to the electrode (Scheme 1 and Fig. S2) [10–12]. In brief, a gold electrode was first modified with 1-(11-mercaptoundecyl)imidazole (C11-Im) and 8-mercapto-1-octanol (C8-OH), the SAM-modified electrode was then exposed to a solution containing either Cyt c or Mb. The electrochemical signal of the immobilized proteins was recorded in a protein-free PBS solution. Other passivating diluents, including 6mercapto-1-hexanol (C6-OH) and 4-mercapto-1-butanol (C4-OH), were also used; C8-OH, however, was found to be best suited for protein immobilization. Sensors passivated with C6-OH or C4-OH were noticeably less stable, which is not unexpected based on our previous studies on other SAM-based biosensors [15]. In addition to the choice of passivating diluent, the concentration ratio between the two alkanethiols used in SAM formation was found to have an effect on the resultant protein coverage. Highest electrochemical signal (i.e., highest protein coverage) was achieved using a C11-Im:C8-OH ratio of 1:1 for Cyt c and 7:3 for Mb (Fig. S3). Three hours of exposure time to the protein solution (50 ␮M for Cyt c and 65 ␮M for Mb) resulted in the highest electrochemical signal. The sensors fabricated using the optimal protocol showed a set of redox peaks, verifying successful immobilization of proteins onto the C11-Im/C8-OH SAM. The E1/2 was more negative than that recorded from sensors fabricated via direct adsorption or NHS-EDC coupling; this could be due to the strong interaction between Im and the heme center [10,11,16]. The linear relationship between the peak current (Ip ) and scan rate (), as well as the non-linear correlation between Ip and 1/2 , confirms that the electron transfer process is surface-confined in both cases (Figs. S4 and S5). Furthermore, the peak separation (Ep = Epa − Epc ) for both systems was proportional to log  at scan rates higher than 2 V s−1 (Fig. S6). According to the Laviron theory [17], the electron transfer rate constant (ks ) was 443 ± 36 s−1 and 2136 ± 353 s−1 for Cyt c and Mb, respectively [18,19]. Despite the larger ks , Mb is not ideal for this application because of its sensitivity toward dissolved O2 (data not shown). Even though all the experiments were performed in degassed solutions, there are advantages in using a protein that is less prone to interferences from O2 . Thus, Cyt c, the protein that is less sensitive to O2 , was used for the rest of the study. The electrocatalytic characteristic of heme proteins toward H2 O2 reduction is well established [20]. To verify the activity of the immobilized Cyt c, we added a total of 800 ␮M H2 O2 to the cell. Fig. S7 shows CVs of the Cyt c-modified electrode before and after several additions of H2 O2 . Addition of H2 O2 resulted in a simultaneous increase and decrease in the cathodic peak current (Ipc ) and

anodic peak current (Ipa ), respectively. These results verify that the heme center of the protein, even after being bound to Im, retains its electrocatalytic properties toward reduction of H2 O2 . Addition of H2 O2 to sensors without the heme protein only resulted in a minor change in the background current, proving the role of Cyt c in the electrocatalytic reduction of H2 O2 . The limit of detection (LOD) of this sensor was found to be 200 ␮M. Despite the relatively high LOD, these sensors showed good stability; we observed

Electrochemical hydrogen peroxide sensors fabricated using cytochrome c immobilized on macroelectrodes and ultramicroelectrodes.

We report the design and fabrication of hydrogen peroxide (H2O2) sensors using heme proteins immobilized on macroelectrodes and ultramicroelectrodes (...
695KB Sizes 4 Downloads 6 Views