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Abstract
This study investigates the impact of different incubation times and concentrations of a self-assembled monolayer (SAMs) of 3-mercaptopropionic acid (MPA) on the rate of electron transfer in redox processes. The aim is to understand how these parameters can affect the sensitivity and efficiency of biosensors based on direct electron transfer in redox proteins. Through a series of experiments, different incubation times and concentrations of MPA were examined to determine their impact on the electron-transfer rate. Using methylene blue MB molecules as a model system and employing the EC-SPR technique, the reflectance differences (ΔR) between the reduced and oxidized states of MB were analyzed, serving as an indicator of the electron transfer rate. The results revealed significant variations in the rate depending on the incubation times and concentrations of the MPA. It was determined that a combination of 1 mM MPA concentration and 6-hour incubation time provided optimal conditions for maintaining a significant (ΔR). These findings have important implications for optimizing sensor surfaces in biosensors based on direct electron transfer in redox proteins.
© 2023 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
1. Introduction
The research on modified molecular assemblies is a highly dynamic and active field worldwide. This is mainly driven by the enormous potential of biomolecular films in device technologies, particularly in biosensor applications [1,2]. Alkanethiol self-assembled monolayers (SAMs) are commonly used to modify metal surfaces, such as gold, platinum, and silver, and are a fundamental component in the assembly of many systems and devices in nanotechnology such as surface plasmon resonance (SPR) [3,4]. SAMs are effective in protecting proteins from direct interaction with solid surfaces, preventing protein denaturation, and enhancing electron-transfer between redox proteins and the electrode [5–7]. In biosensing applications, the achievement of direct and rapid electron transfer between the electrode surface and the biological system is crucial for enhancing the activity of immobilized proteins. The chain length of the thiol used in monolayer formation, along with the concentration and preparation time of the monolayer, are critical factors that can significantly impact the stability, selectivity, and sensitivity of sensing films. Over the years, extensive research has been conducted on the electron-transfer phenomenon within molecular assemblies on electrode surfaces. Both theoretical and experimental investigations have been dedicated to understanding the electron-transfer mechanism, exploring the rate constants’ dependence on distance, and examining the impact of orientation on these rate constants [5,6,8–13]. The use of SAMs on modified electrode surfaces has shown great promise in controlling the electron-transfer mechanism, which is crucial for biosensors based on direct electron transfer in redox proteins [13–16]. This mechanism has an extensive impact on both the sensitivity and efficiency of the detection process of these biosensors [17–23]. Therefore, understanding the impact of different parameters, such as incubation times, concentrations of SAMs, and their stability, on the electron-transfer rate is essential for optimizing the performance of such biosensor systems.
In this study, we aim to investigate the influence of different incubation times and concentrations of MPA (3-mercaptopropionic acid from Sigma-Aldrich) on the rate of electron transfer in redox processes. The focus is on biosensors based on direct electron transfer in redox proteins, where the efficiency and sensitivity of the detection heavily rely on the electron-transfer kinetics. By exploring various incubation times and concentrations of MPA, we seek to explain their impact on the electron-transfer rate. Furthermore, we introduce a novel procedure for preparing the MPA monolayer inside the flow cell, specifically designed to prevent its exposure to air. This is crucial because air exposure can lead to the instability of the monolayer [24]. By implementing this new method, we ensure a controlled environment for the MPA monolayer, enhancing its stability throughout the experimental process.
To evaluate the electron-transfer rate, we employed the electrochemical surface plasmon resonance (EC-SPR) technique, which allows real-time monitoring of the ΔR between the reduced and oxidized states of a model redox molecule (MB) [25]. These ΔR serve as an indicator of the electron-transfer rate [26]. By analyzing the variations in reflectance under different experimental conditions, we can gain insights into the influence of incubation times and concentrations of MPA on the electron-transfer kinetics.
2. Experimental methods
2.1 Experimental set-up
Figure 1 illustrates the EC-SPR device.
Fig. 1. Schematic representation of the EC-SPR device. P-polarized light is generated from a (CW) laser with two quarter-wave retarders and linear polarizer to excite plasmons. The reflectance is measured using a photo-detector, while an electrical potential through a potentiostat with three electrodes is applied onto the metallic SPR interface.
In this study, an electrochemical flowcell was developed for surface plasmon resonance (SPR) analysis, employing three electrodes. The working electrode consisted of an SPR surface, while two gold-plated pins were employed as the reference and counter electrodes, respectively. To ensure optical connectivity, the flowcell incorporated a 60-degree equilateral prism, which was optically coupled to the SPR surface using index matching gel possessing a refractive index of 1.52. To enable adjustment of the angle of incidence, the setup was mounted on a rotational stage. For excitation of the plasmon, a CW laser beam (Obis from Coherent) with a wavelength of 660 nm was used. This laser beam, in combination with two quarter-wave retarders and a linear polarizing element, enabled the generation of linearly polarized light with transverse magnetic (TM) polarization. The polarized light was subsequently directed towards the prism to excite the surface plasmon. A CHI 660D potentiostat from CH Instruments, Inc. was operated to connect to the three electrodes, enabling the control of potential at the working electrode. A power meter was employed to monitor the electrically modulated reflectance at a fixed angle.
2.2 Preparation of the MPA monolayer
To establish a SAMs on the the EC-SPR platform, we used MPA. In order to ensure optimal environmental conditions and enhance the stability of the MPA monolayer, we carried out the incubation of MPA solutions inside the flow cell. This approach ensured a stable environment for the formation of the MPA monolayer on the SPR surface and enhancing its stability during the experimental process. To do so, a solution of MPA with three concentrations of 0.1 mM, 0.5 mM, and 1 mM were prepared on deionized water (DI water). After cleaning process, the EC-SPR platform was dried using N2 gas and then was set up by attaching it to an electrochemical flow cell. Then a solution of 5.0 M phosphate-buffered saline (PBS) with a pH of 7 was introduced into the flow cell. Next, and in order to stabilize the platform, a cyclic voltammetry (CV) potential modulation ranging from -0.1 V to 0.4 V, with a scan rate of 0.02 V/s, was applied. Once the EC-SPR platform was stabilized, a solution of MPA with a concentration of 0.1 mM was incubated in the flow cell for 2 hours. Next, the flow cell was rinsed with a PBS buffer solution to remove any unbound MPA molecules from the surface. Finally, a second CV potential modulation, ranging from -0.1 V to 0.4 V, at a scan rate of 0.02 V/s was applied to stabilize the MPA-(EC-SPR) platform. The same procedure was repeated for the other two MPA concentrations and for 6, 12, and 24 hours incubation times. This allowed for the characterization of the EC-SPR platform with different MPA concentrations and incubation times to observe any variations in the reflectance measurements.
2.3 Measurements
In this study, two experimental procedures were carried out. The initial set of measurements involved using an electrochemical flow cell mounted on a rotation stage to verify the presence of immobilized MPA molecules at the EC-SPR interface. This allowed for precise control of the incident angle, ensuring accurate characterization of the immobilized MPA molecules. The reflectance at various angles of incidence was then monitored using a power meter. Four different incubation times (2, 6, 12, and 24 hours) were examined using 1 mM of MPA.
In the second set of experiments, a CV technique was applied to examine the effect of the MPA concentration at the four incubation times on the electron transfer between MB molecules and the EC-SPR surface. The reflectance differences between the reduced and oxidized states of the MB under potential modulations were used as an indicator of the electron transfer rate.
Initially, MB was introduced onto the MPA- (EC-SPR) platform. A series of potential scans from -0.3 V to 0.3 V were applied at a scan rate of 0.02 V/s. Afterwards, the reflectance was measured at a fixed angle using the power meter. Finally, for each incubation time, the differences in reflectance between the reduced and oxidized states of MB were measured (ΔR). These measurements were performed for three different concentrations of MPA.
3. Results and discussion
3.1 Confirming the MPA monolayer
The immobilization of MPA molecules at the EC-SPR interface was confirmed by detrmining the SPR reflectance curves. For each incubation time, the SPR reflectance curves were determined. Figure 2 shows the SPR curves for 1 mM of MPA after incubation times of 2, 6, 12, and 24 hours.
In Fig. 2, the blue curve represents the SPR reflectance for the PBS buffer solution, while the orange, black, red, and green curves show the SPR reflectance obtained after MPA incubation times of 2, 6, 12, and 24 hours. The results demonstrate a noticeable shift in the position of the minimum reflectance as the monolayer preparation time progresses. This shift indicates not only the successful attachment of MPA molecules to the surface but also an increase in the molecular density within the MPA monolayer over time.
3.2 Cyclic voltammetry (CV) technique
The platform's stability in the electrochemical measurements is crucial in ensuring the reliability and consistency of results for following investigations or analyses. To reach this, the MPA-(EC-SPR) platform was subjected to a stabilization process using a PBS buffer solution. This was achieved by applying CV potential modulation ranging from -0.1 V to 0.4 V at a scan rate of 0.02 V/s. Figure 3 illustrates the reflectance at four different stages of the stabilization process.
Fig. 3. Reflectance under a CV scan of a PBS solution on the MPA-(EC-SPR) platform at a scan rate of 0.02 V/s.
In Fig. 3(a), the measurement was taken after rinsing the flow cell with 1 mL of PBS buffer solution, followed by two-minutes waiting period. This procedure was repeated for Figs. 3(b), 3(c), and 3(d), representing following stages of the stabilization process. Figure 3(d) indicates that the MPA-(EC-SPR) platform achieved a high level of stability, demonstrating its suitability for the next experiments.
In order to characterize and optimize the monolayer assemblies for functionalizing the surface with MPA, different concentrations of MPA (5 mM, 10 mM, and 20 mM) were investigated at four different incubation times (2, 6, 12, and 24 hours). A 2.6 M solution of MB was injected into the flow cell, and CV was employed to study the electron transfer between the MB and the surface. Figure 4 illustrates the reflectance under CV for the MB solution at four different MPA incubation times: (a) 2 hours, (b) 6 hours, (c) 9 hours, and (d) 24 hours.
Fig. 4. Reflectance response during a CV scan of a MB solution on the MPA-(EC-SPR) platform at a scan rate of 0.02 V/s with an incubation time of (a) 2 hours, (b) 6 hours, (c) 12 hours, and (d) 24 hours.
In Fig. 4. (a), (b), (c), and (d), the results obtained with the three concentrations of MPA demonstrate a reversible transition in reflectance. This transition indicates a change in electrons transfer between the MB and the EC-SPR surface as the modulation potential crosses the formal potential of the MB. Next, the ΔR was detemined by subtracting the reflectance of MB in the oxidized state from the reflectance of MB in the reduced state. The results are listed in Table 1.
Table 1. Reflectance difference between the oxidized and reduced states of MB at concentrations of 0.1 mM, 0.5 mM, and 1 mM, for incubation times of 2, 6, 12, and 24 hours.
At an MPA incubation time of 2 hours, the ΔR were determined to be 0.08, 0.11, and 0.16 for concentrations of 0.1 mM, 0.5 mM, and 1.0 mM, respectively. These results indicate that higher concentrations of MPA lead to an increased attachment of MB molecules to the surface. Consequently, the greater presence of MB on the surface enables more efficient electron transfer between MB and the EC-SPR surface. This explains the observed increase in ΔR with increasing MPA concentration. Similar trends were observed at an MPA incubation time of 6 hours, where ΔR were determined to be 0.13, 0.20, and 0.28 for concentrations of 0.1 mM, 0.5 mM, and 1.0 mM, respectively.
However, at an MPA incubation time of 12 hours, the ΔR were found to be 0.25, 0.18, and 0.1 for concentrations of 0.1 mM, 0.5 mM, and 1.0 mM, respectively. Surprisingly, despite the increased number of MB molecules attached to the surface, the electron transfer between MB and the EC-SPR surface decreased. One possible explanation for this observation is the formation of a densely packed MPA monolayer on the surface due to the higher concentration and longer incubation time of MPA. This densely packed monolayer may have block the efficiency of electron transfer between MB and the EC-SPR surface, resulting in reduced ΔR. Similar trends were observed at an MPA incubation time of 24 hours, with lower ΔR determined.
These findings show that the combination of a 1.0 mM MPA concentration and a 6-hour incubation time provides optimal conditions for optimizing the EC-SPR surface. This combination ensures maximum coverage of the active EC-SPR interface and maintains a significant difference ΔR in reflectance.
4. Conclusion
In conclusion, this study examined the influence of different incubation times and concentrations of a self-assembled monolayer (SAM) of MPA on the rate of electron transfer in redox processes. The goal was to evaluate their impact on the sensitivity and efficiency of biosensors relying on direct electron transfer in redox proteins. Through experimental investigations using MB molecules and the EC-SPR technique, the reflectance differences between the reduced and oxidized states of MB were analyzed as a measure of the electron transfer rate.
The results demonstrated significant variations in the electron transfer rate based on the incubation times and concentrations of MPA. Optimal conditions for achieving a substantial reflectance difference were determined to be a 1 mM MPA concentration and a 6-hour incubation time. These findings hold important implications for the optimization of sensor surfaces in biosensors that rely on direct electron transfer in redox proteins.
Overall, this study provides valuable understandings into the relationship between MPA monolayer preparation parameters and electron transfer rate, contributing to the advancement of biosensor technology. By understanding and optimizing these factors, the sensitivity and efficiency of biosensors can be improved, thereby enhancing their performance in various applications.
Disclosures
The authors declare no conflicts of interest.
Data availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
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