Chemistry
Hsiao-Tieh Hsu

Trinuclear Ruthenium Clusters for Electrochemical Biosensor Development

  • Faculty Advisor

    Thomas J. Meade

Published On

May 2014

Originally Published

NURJ 2013-14
Honors Thesis

PHOTO | macaroni 1945

Introduction

1. Biosensors

A biosensor is a non-invasive device that can detect biomolecular analytes associated with diseases. Electrochemical biosensors are useful due to their low cost, ease of use, and remarkable reproducibility. Previous studies show that electron transfer between electrodes and surface-confined redox molecules obeys the Marcus Theory of Electron Transfer. Therefore, changes in the system caused by interactions between redox molecules and the analytes they are sensing could translate into quantifiable changes of electrochemical signals such as electron transfer rate and overpotential.

2. Theoretical Background

To aid the development of electrochemical biosensors, the work presented here studies the weak interactions that govern ligand-receptor binding using trinuclear ruthenium clusters. Weak interactions between proteins and small molecules are important to biological processes such as photosynthesis and DNA damage and repair, but they are difficult to measure directly. Weak interactions affect the static dielectric constant (εs) of a system, which is a part of reorganization energy (λ). The Marcus Equation of Electron Transfer relates λ to electron transfer rate (kET). Therefore, the changes in weak interactions can be inferred by the changes in λ by measuring kET using electrochemistry.

Reorganization energy (λ) is the energy required to force the reactants from their equilibrium configuration into the equilibrium configuration of the products before electron transfer actually occurs. According to The Marcus Theory of Electron Transfer, reorganization energy is the sum of outer sphere (λo) and inner sphere reorganization energy (λi) (Equation 1). λo depends on several variables: 1) the radii of the electron donor and acceptor (rA and rD), 2) the distance between the donor and acceptor (dAD), 3) the charge transferred (e), 4) the optical dielectric constant (εop) and 5) the static dielectric constant (εs) (Equation 2). εop is approximately constant between different systems while εs differs and gives information on weak interactions. According to the Marcus Equation, kET depends on λ (Equation 3).

If the protein binding ligand is modified with a redox center, the potential, current and total amount of charge transferred in a redox event can be measured with cyclic voltammetry (CV). By fitting the CV data, λ and kET can be derived. Upon protein binding, overpotential (E1/2, the average of anodic and cathodic peak potential), λ, and kET are expected to shift.

To investigate the change in electrochemical parameters upon protein binding, the biotin/avidin system was used in the work presented here. This system was chosen because biotin and avidin form specific, strong non-covalent interactions, with a dissociation constant of 10-15 M.18 Avidin is a stable tetrameric glycoprotein that is widely used in biotechnological and medical research. It can bind to multiple ligands, which allows systematic studies for ligand-receptor binding.

3. Experimental Design

In previous work, biotinylated iron- and ruthenium-modified complexes, such as [Fe(BMB)(CN)4]2- and [(4-BMP)NRu(NH3)5]2+,10,15 were used for electrochemical detection of avidin binding. However, due to the small size of the metal complex, the protein decreased the coupling between the metal center and the electrode, subsequently interfering with the electron transfer process and leading to the loss of current signal in the CV. Additionally, these CV experiments were done in solution, so the slow diffusion of the protein to the electrode contributed to the signal loss. To solve these problems, trinuclear ruthenium clusters were used to modify the avidin-binding ligand biotin. Since these clusters are larger, protein binding is less likely to interfere with the coupling between the metal centers and the electrode. The target molecules were incorporated into self-assembled monolayers to overcome the poor diffusion of avidin in solution.

CV was performed to analyze the species on the monolayer. CV is an electrochemical technique that scans the potential and measures the current in a redox event. λ and kET can be derived from plotting the CV data into a Tafel plot. The curvature is indicative of λ, and the y-intercept of the plot is kET. A modified Marcus Equation, which applies to species on monolayers, can be used to simulate the CV data to give electronic coupling (HAB), λ and kET. (Equation 4).

CV experiments were performed on trinuclear ruthenium clusters (Table 1) before and after avidin addition to measure the changes in electrochemical properties of the systems upon protein binding. Ru(py)2(C16SH) was used as a control system because it does not have an avidin binding ligand. Bovine Serum Albumin (BSA), a protein that does not bind to biotin, was also added to the trinuclear ruthenium cluster systems as a negative control to verify that the changes in electrochemical properties were caused by biotin-avidin interactions. Finally, the solvent polarity of CV experiments was altered by titrating DMF in the electrochemical cell to simulate the environment after protein binding.

Methodology

A. Modification of Ligands

1. Synthesis of 4-BMP

684.1 mg (2.800 mmol) of biotin and 1019.5 mg (3.386 mmol) of TSTU were dissolved in 10 mL of DMF while stirring. 1 mL of Et3N was added to the stirring solution, and the resulting solution was stirred for 1 hour, during which time the color of the solution turned from orange to dark brown. 288 μL (2.798 mmole) of distilled 4-(aminomethyl)pyridine (4-AMP) was added and the solution was stirred overnight at room temperature under argon. When the reaction was completed, DMF was removed using rotary evaporation. The product was purified with column chromatography on silica in 10% MeOH/CHCl3 and modified by TLC and Pt stain. The first band that stained dark brown was collected. The final product appeared to be a light yellow, oily solid. (Scheme 1).10 Yield: 61 %. 1H NMR (500 MHz, CDCl3) δ 8.44 (t, 2H), 7.28 (d, 1H), 4.48-4.42 (m, 1H), 4.38 (s, 2H), 4.26-4.22 (m, 1H), 3.31-3.23 (m, 2H), 2.88 (dt, 1H), 2.28 (dq, 2H), 1.79-1.51 (m, 4H), 1.38-1.48 (m, 2H). MS (ESI+) m/z calculated for 4-BMP + H+ is 335.15, found 335.1. 4-BMP + Na+ is 357.14, found 357.0.

2. Synthesis of (4-AMP)CO(CH2)15SH

98.2 mg (0.340 mmol) of HS(CH2)15COOH (16-mercaptohexadecanoic acid), 75.8 mg (0.367 mmole) of dicyclohexylcarbodiimide (DCC), and 38 μL (0.369 mmole) of distilled 4-AMP were dissolved in 10 mL 4:1 anhydrous, degassed CH2Cl2/acetone. The solution was stirred under a blanket of argon for 72 hours, during which time a white dicyclohexylurea precipitate and the product, (4-AMP)CO(CH2)15SH, were formed. The product was purified by filtration, followed by column chromatography on silica in 5% MeOH/CHCl3 and monitored by TLC and Pt stain. The first band that stained dark brown was collect. The product appeared to be a white solid (Scheme 2). Yield: 61%. 1H NMR (500 MHz, CDCl3) δ 8.52 (d, 2H), 7.17 (d, 2H), 5.91 (s, 1H), 4.45 (d, 2H), 2.57-2.51 (m, 2H), 2.29-2.25 (m, 2H), 1.85-1.58 (m, 8H), 1.50-1.42 (m, 8H), 1.40-1.32 (m, 22H). MS (ESI+) m/z calculated for (4-AMP)CO(CH2)15SH + H+ is 379.62, found 379.2, (4-AMP)CO(CH2)15SH + Na+ is 401.60, found 401.2.

B. Trinuclear Ruthenium Clusters

The syntheses of trinuclear ruthenium clusters are summarized in the following scheme:

1. Synthesis of Ru(H2O)3

991.0 mg (4.78 mmol) of RuCl3•xH2O, 2.206 g (24.7 mmol) of sodium acetate and 25 mL of glacial acetic acid were dissolved in 25 mL of absolute ethanol and refluxed overnight in the dark, during which time the color of the solution turned from dark brown to dark green. The mixture was cooled to room temperature and centrifuged at 2500 rpm for 20 minutes to remove the excess salt. The supernatant was filtered and EtOH was removed using rotary evaporation. The obtained solid was dissolved in 50 mL MeOH and filtered. MeOH was then removed using rotary evaporation and Ru(H2O)3, a dark green solid, was obtained.21,22 Yield: 67 %. UV-visible (MeOH) λmax = 699 nm.

2. Synthesis of Ru(CO)(MeOH)2

1.0 g (1.32 mmole) of Ru(H2O)3 was dissolved in 50 mL MeOH and the solution was degassed for 30 minutes. A few pieces of Zn/Hg (~8 g) were added to the solution with stirring. The solution was stirred for 2.5 hours under Ar, during which time the color of the solution turned more yellow. The air-sensitive solution was then cannulated to an argon-purged Schlenk flask to remove the Zn/Hg amalgam. CO was bubbled into the cannulated solution for 45 minutes. The solution was then stirred under a CO atmosphere at room temperature in the dark overnight, during which time [Ru(CO)(MeOH)2]0 was formed and the color of the solution turned from dark green to dark purple. The product was purified with column chromatography on silica in 10% MeOH/CHCl3 and appeared to be a purple solid upon rotary evaporation. 21,22 Yield: 57%. UV-visible (MeOH) λmax = ~ 560 nm.

3. Synthesis of Ru(CO)(L)(MeOH), L = BMP (3a) or pyridine (3b)

0.8 equivalents of L (154.7 mg of 4-BMP, or 12.5 μL of pyridine) was dissolved in 100 mL 1:1 MeOH/CH2Cl2. Once dissolved, 440.9 mg (0.580 mmole) of Ru(CO)(MeOH)2 was added to the stirring solution. The reaction was stirred at room temperature in the dark for 3 days to form Ru(CO)(L)(MeOH). The product was purified with column chromatography on silica in 10% MeOH/CHCl3 and appeared to be a purplish blue solid. L = BMP: Yield: 47%. 1H NMR (500 MHz, MeOD) δ 8.95 (d, 2H), 7.98 (d, 2H), 4.82 (s, 2H), 4.38 (m, 1H), 4.20 (m, 1H), 3.13 (q, 2H), 2.82 (dt, 1H), 2.55 (dq, 1H) 2.35 (t, 4H), 1.85 (s, 6H), 1.80 (s, 6H), 1.75-1.50 (m, 10H), 1.50-1.35 (m, 2H). UV-visible (MeOH) λmax = 568 nm. L = pyridine: Yield: 52.3 %. 1H NMR (500 MHz, MeOD) δ 9.05 (d, 2H), 8.28 (t, 2H), 8.08 (dd, 2H), 1.98 (m, 6H), 1.92 (m, 6H), 1.64 (m, 6H), 1.51 (m, 6H).

4. Synthesis of Ru(CO)(L)(C16SH), L = BMP (4a) or pyridine (4b)

Ru(CO)(L)(MeOH) and 1.2 equivalents of (4-AMP)CO(CH2)15SH were dissolved in 100 mL of degassed 1:1 MeOH/CHCl3 solution. The resulting solution was stirred under a blanket of Ar at room temperature in the dark overnight, during which time the color turned more blue. The final product, Ru(CO)(L)(C16SH), was purified by column chromatography on silica in 5% MeOH/CHCl3 and appeared to be a blue solid. L = BMP: Yield: 79%. 1H NMR (500 MHz, CDCl3) δ 8.93 (d, 2H), 7.98 (s, 2H), 4.85 (s, 1H), 4.38 (m, 1H), 4.22 (m, 1H), 3.13 (q, 1H), 2.82 (dd, 1H), 2.55-2.50 (m, 3H), 2.38-2.23 (m, 4H), 1.86 (s, 12H), 1.75-1.48 (m, 16H), 1.48-1.38 (m, 4H), 1.36-1.10 (m, 22H). UV-visible (MeOH) λmax = 585 nm. L = pyridine: Yield: 45 %. 1H NMR (500 MHz, CDCl3) δ 9.10 (d, 2H), 9.00 (d, 2H), 8.19 (t, 1H), 8.04 (t, 2H), 7.92 (d, 2H), 4.91 (s, 2H), 2.49 (t, 2H), 2.38 (t, 2H), 2.09 (s, 12H), 1.81 (s, 6H), 1.75 (m, 2H), 1.63 (m, br, 4H), 1.4-1.1 (m 20H). UV-visible (MeOH) λmax = 582 nm.

5. Electrochemical formation of Ru(L1)(L2)(C16SH), L1, L2 = BMP (5a), pyridine (5b)

Gold ball electrodes were made from 7 cm pieces of 0.127 mm diameter, 99% gold wire (Alfa Aesar). The first 2 cm of each 7 cm piece was melted in a Bunsen burner flame to form a gold ball with radius of 0.04 cm. The gold ball electrodes were cleaned in 1 M H2SO4(aq) with CV using a CH Instruments 660A electrochemical workstation to remove contamination on the surface of the gold ball. The potential was cycled between 0V and 1.6V until a constant current was observed. After being cleaned, electrodes were rinsed with H2O and ethanol. The electrodes were soaked in a 20:1 HO(CH2)11SH: Ru(CO)(L1)(C16SH) solution (2 mM total thiol in EtOH, L1 = BMP or pyridine) overnight so that monolayers could be formed. CV was performed using the CH Instruments 660A electrochemical workstation in pH 7 PBS buffer with 100 mM NaCl under room temperature to oxidize the cluster electrochemically. The buffer was degassed with N2 before all experiments. The reference electrode was Ag/AgCl and the counter electrode was a platinum wire. The potential was scanned between -0.6 V and 0.8 V for 20 minutes until the current of newly formed species no longer increased. The CO ligand became and was replaced by a H2O molecule. The gold ball electrodes were then soaked in either 1 mM 4-BMP or pyridine/ H2O solution to form Ru(BMP)2(C16SH), Ru(BMP)(py)(C16SH) (5a), Ru(py)2(C16SH) (5b).

C. CV Experiments

CV experiments were performed on the trinuclear ruthenium clusters in pH 7 PBS buffer with 100 mM NaCl at room temperature on the CH Instruments 660A workstation. The working electrode was the gold ball electrode with the monolayer, the reference electrode was Ag/AgCl, and the counter electrode was a platinum wire. In CV experiments, scan rates ranging from 0.01 V/s to 2500 V/s were applied, and the anodic and cathodic peak potential, peak current, and total amount of charge transferred were measured.

After the initial electrochemical measurements, all the gold electrodes were soaked in 30 μM avidin solution at 37°C overnight. For BSA studies, the Ru(BMP)2(C16SH) system was soaked in 30 μM BSA solution at 37°C overnight. Afterward, the same set of electrochemical experiments was performed on the gold electrodes to see if the electrochemical parameters changed after avidin binding.

D. DMF Study

CV experiments of Ru(BMP)2(C16SH) were performed in 10% ,20%, 30%, 40%, and 50% DMF in pH 7 PBS buffer solution with 100 mM NaCl at room temperature using the CH Instruments 660A electrochemical workstation. Different scan rates, ranging from 0.01 V/s to 2500 V/s were applied, and the anodic and cathodic peak potential, peak current, and total amount of charge transferred were measured.

Results and Discussion

1. CV: peak current

In the CV traces of Ru(BMP)2(C16SH) and Ru(BMP)(py)(C16SH), the peak currents were still observable after avidin addition. This result suggests that the current loss observed in the single-centered biotinylated iron and ruthenium complexes was resolved by using trinuclear ruthenium clusters incorporated into monolayers. Since the clusters are significantly larger than the mononuclear iron and ruthenium complexes, avidin binding is likely to have less effect on coupling between the cluster metal centers and the electrode. In addition, it is known that, in these clusters, the electron density is delocalized over all three metal centers.23 Therefore, electronic coupling and electron transfer were maintained, and current signals could still be observed after avidin addition. Furthermore, since the clusters were incorporated into monolayers on the electrode, the avidin-cluster complexes would not have to diffuse to the electrode for electron transfer. Hence, the slow diffusion problem observed in the previous study was solved. Additionally, attaching the ruthenium clusters to a monolayer facilitates analysis of the electrochemical properties of the system. By using this new system, current signals in CV were maintained after avidin bound to the binding ligand, which allowed further experiments and analyses that were not possible with the previous, mononuclear systems using the various trinuclear ruthenium clusters.

2. CV: Overpotential (E1/2)

In the Ru(BMP)2(C16SH) system, there was an observable change in E1/2 upon avidin addition (-43 mV). The negative shift in E1/2 indicated that the oxidized form of the cluster is more stable in the environment of avidin. In the other system, the change in E1/2 was negligible compared to the experimental error range of approximately 10 mV.

In the CV traces, a decrease in peak current was observed upon avidin addition. In order to investigate the cause of this current decrease and to verify that the changes in E1/2 observed in the Ru(BMP)2(C16SH) system was caused by biotin-avidin interactions, the same set of CV experiments were conducted on Ru(py)2(C16SH), a trinuclear ruthenium cluster with no ligand that could bind to avidin. After the first set of CV measurements, electrodes with Ru(py)2(C16SH) monolayer were soaked in avidin solution and water separately overnight, and the CV experiments were repeated. In the CV data of Ru(py)2(C16SH) soaked in avidin solution, the peak potential did not shift. This was expected, since there was no binding between avidin and Ru(py)2(C16SH). This result confirmed that the shift of -43 mV in E1/2 observed in the Ru(BMP)2(C16SH) system was caused by specific binding between avidin and biotin rather than non-specific binding or other changes in the monolayer. Similar to what was observed in the BMP system, the peak current decreased after avidin addition. In the CV data for Ru(py)2(C16SH) soaked in water, the peak potential did not shift, and the peak current remained the same (Figure 2). Since Ru(py)2(C16SH) cannot bind to avidin and the current remained the same at the absence of avidin, this observation suggested that the current decrease in all trinuclear ruthenium cluster systems was caused by nonspecific binding between avidin and the monolayer, rather than interactions between biotin and avidin.

3. CV: Tafel Plots

The Tafel plots of Ru(BMP)2(C16SH) and Ru(BMP)(py)(C16SH) before and after protein binding were overlaid on each other (Figure 3). The overlapping plots showed unobservable changes in curvature and y-intercept, which suggested that there was little change in λ and kET for both systems after avidin binding. These results were different from the expectation that λ and kET would change upon avidin binding. A possible explanation is the metal-centered nature of the +1/0 redox pair of the tri-nuclear ruthenium cluster. It is known that in these clusters, electrons are delocalized over all three metal centers but not to the binding ligands. Since there is little electron density on the binding ligands, protein binding is less likely to affect electron transfer, λ and kET, compared to a system where electron delocalization is extended to the binding ligands. Another possible reason for this observation is that, since the charge is delocalized over three Ru centers, the formal charge difference of the reduced and oxidized forms is 1/3 for each Ru center. This change could be too small for the electrochemical techniques to measure.

4. BSA control

The CV trace for Ru(BMP)2(C16SH) remained almost identical before and after the addition of BSA (Figure 4). Since BSA does not bind to biotin, this experiment verified that the change in E1/2 observed in the previous experiment was caused by the interactions between biotin and avidin. No current decrease was observed after BSA addition, which indicated that there was no nonspecific binding between BSA and the monolayer.

5. DMF Study

In the DMF study, E1/2 decreased as DMF concentration increased (Figure 5). A shift of -43 mV in E1/2 was observed from 0% DMF to 50% DMF at scan rate 250 mV/s, and the shift was consistent between scan rate 50 mV/s and 1000 mV/s (Figure 6). As DMF concentration increases, E1/2 was expected to shift positively. As the polarity of the environment decreases, the neutral, reduced form of this cluster will become more favored than the charged, oxidized form of the cluster. As a result, the redox potential was expected to increase. However, the experimental results showed the opposite. A possible explanation for this observation is that, when DMF concentration is low, solubility of the neutral cluster is not favored in the polar solvent, water, and, hence, it interacts more with the hydrophobic monolayer. As the DMF concentration increases, the polarity of the solvent decreases and the neutral cluster no longer interacts with the monolayer as strongly. Overall, the solvent is expected to be more polar than the monolayer even after DMF addition. As a result, as DMF concentration increases, the polarity of the environment around the cluster actually increases due to decreasing interactions between the cluster and the hydrophobic monolayer, which makes E1/2 shift negatively. Further, this experiment showed that it is possible to simulate protein binding by changing the polarity of the solvent. The results showed that avidin binding is similar to 50% DMF because under both conditions, E1/2 decreased by 43 mV.

Conclusion and Future Work

In the work presented here, trinuclear ruthenium clusters Ru(BMP)2(C16SH), Ru(BMP)(py)(C16SH), and Ru(py)2(C16SH) were successfully synthesized and incorporated into self-assembled monolayers on gold ball electrodes. These large clusters and monolayer systems successfully overcame the current signal loss in CV observed previously in the biotinylated iron and ruthenium complexes.

The CV traces for Ru(BMP)2(C16SH) after avidin binding showed a change of -43 mV in E1/2. However, in the Ru(BMP)(py)(C16SH) system, the CV traces showed no observable change in E1/2 upon avidin addition. In addition to these two clusters, CV experiments were performed on Ru(py)2(C16SH), a cluster that cannot bind to avidin. A decrease in peak current was observed after Ru(py)2(C16SH) was soaked in avidin overnight, but the current remained the same after it was soaked in water. This result showed that the decrease in peak current was caused by the nonspecific binding between avidin and the monolayer rather than the interactions between avidin and biotin. The lack of shift in E1/2 indicated that the shift observed for the Ru(BMP)2(C16SH) system is due to avidin binding.

The CV data were used to generate Tafel plots for Ru(BMP)2(C16SH) and Ru(BMP)(py)(C16SH). No significant change in the curvature or the y-intercept of the plots was observed after avidin addition, which indicated that, upon protein binding, there was no change in λ and kET, respectively.

In order to confirm that the change in E1/2 in the Ru(BMP)2(C16SH) was caused by the avidin/biotin interaction, CV experiments were performed on Ru(BMP)2(C16SH) before and after the clusters were soaked in BSA, a protein that does not bind to biotin. The CV traces remained the same after BSA addition, which verified that the change in E1/2 was caused by binding interaction. Additionally, the peak current did not decrease, which suggested that there is no nonspecific binding between BSA and the monolayer.

To simulate the conditions of protein binding, CV experiments were performed on Ru(BMP)2(C16SH) in various DMF concentrations. The results show that E1/2 decreased linearly as DMF concentration increased between 0% and 50%. In 50% DMF, a shift of -43 mV in E1/2 was observed (as compared to 0% DMF), which was similar to avidin binding. The linear change in E1/2 was consistent between different scan rates. E1/2 was expected to increase with increasing DMF concentration (and decreasing solvent polarity), but the experimental result showed the opposite. This could be caused by decreasing interaction of the cluster with the hydrophobic monolayer as DMF concentration increases.

In the future the sensitivity of the Ru(BMP)2(C16SH) system, will be improved by tuning electrochemical parameters of the system to enhance the changes in electrochemical signals upon avidin binding. The greater the changes are, the more sensitive the system is, and the more likely it can be applied to aid the development of electrochemical biosensors. Ru(NO)(4-BMP)20/-, which is isoeletronic to Ru(BMP)2(C16SH)+/0, can be used as well, because its electrons are known to be delocalized over the entire molecule. Finally, the biotin-avidin model system can be extended to molecules that are more biologically relevant, such as the Crizotinib-anaplastic lymphoma kinase (ALK) system. Anaplastic lymphoma kinase (ALK) is an oncological biomarker because overexpression of ALK can cause lung cancer. Crizotinib is a commercially available ALK inhibiter, and the binding between Crizotinib and ALK is well studied. Therefore, the Crizotinib-ALK ligand-receptor system can be utilized in the trinuclear ruthenium cluster system for biosensor design.

In the Ru(BMP)2(C16SH) system, there was an observable change in E1/2 upon avidin addition (-43 mV). The negative shift in E1/2 indicated that the oxidized form of the cluster is more stable in the environment of avidin. In the other system, the change in E1/2 was negligible compared to the experimental error range of approximately 10 mV.

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Hsiao-Tieh Hsu