Poster D1 - Cyclic Voltammetry and Differential Pulse Voltammetry for Electrochemical Evaluation of Enzyme and Enzyme-Based Complexes

Thursday, October 24, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Emily Weber (she/her/hers)

Undergaduate research student
West Virginia University
Mays Landing, New Jersey, United States

Co-Author(s)

GP

Gia Huy Pham

Graduate Student
West Virginia University
Bruceton Mills, West Virginia, United States

Primary Investigator(s)

CD

Cerasela Zoica Dinu

Professor
Chemical and Biomedical Engineering Department, West Virginia University, United States

Introduction: Cyclic voltammetry (CV) is a versatile electrochemical method that allows for monitoring electron transfer at interfaces. The method was previously applied to identify biosensorial activity or for characterization of organic and inorganic complexes, just to name a few. In particular, previous research from our lab showed that CV could be used to detect and evaluate complex formation and functionality as imposed by biologically-driven self-assembly and self-recognition events, all under controlled chemical energy of adenosine triphosphate (ATP). Our analysis demonstrated not only that a CV-based biosensor is easy to create, fast, convenient and economic in its design and implementation, but further, is capable to detect single reaction events. Herein we used CV and integrated differential pulse voltammetry (DPV) to detect the attachment and functionality of molecules adsorbed to commercially available electrode interfaces envisioned to be further used for biosensor applications. Specifically, we used a screen printed electrode (SPE), a lost-cost electrochemical interface, and ferrocenecarboxylic acid (FCA) or analytes associated with (i.e., FCA-Rubisco or FCA-Mg-based RuBisCO), to detect reversible oxidation in aqueous solutions and determine molecule kinetics and mechanisms of chemical reactions at its interface. We describe both the efficiency of binding and how sensitivity of the biosensor was evaluated, as well as highlight how the electrochemical reactions at electrode interfaces are controlling overall sensitivity of the formed biosensor. We envision that the strategy presented herein could be extended to understanding signal transduction pathways at interfaces formed from single target molecules for applications ranging from detection to lab-on-chip for biomedical studies.

Materials and

Methods: Ferrocenecarboxylic Acid (FCA, Sigma-Aldrich, USA), 1-(3-dimethylaminopropyl)-3-ethylcarbomiide hydrochloride (EDC, Thermo Fisher, USA), N-Hydroxysulfosuccinimide sodium salt (NHS, Sigma-Aldrich, USA), and D-Ribulose 1,5-Diphosphate Carboxylase from spinach (RuBisCO, Sigma-Aldrich, USA) were used to perform this research. A VeraSTAT 3F, Princeton Applied Research, machine made by Ametek (USA) was used to perform the esperiments and data analysis. A Hyper value 501 carbon sensor (SPE) made by Zimmer and Peacock (ZPS, Switzerland) was used as support for analytes attachment and for data collection. The reference electrode (RE) is made of silver/silver chloride, both the working electrode (WE) and counter electrode (CE) are made of carbon, and the substrate (SUB) is made from PET. The binding of FCA with EDC/NHS-Mg/RuBisCO equilibrating EDC and NHS to room temperature by using a 1 mg/mL solution of FCA in 0.1 M 4-morpholinoethanesulfonic acid at pH 4.5-5. Upon vortexing and sonicatinge the FCA solution to ensure it is completely homogenized, 0.4 mg of EDC followed by 1.1 mg of suffo-NHS was added and allowed to react for 15 minutes at room temperature. Subsequently, 1.2 g/mL of 2-mercaptoethanol and an equimolar amount of Mg/RuBisCO in comparison to FCA was added and allowed to react at room temperature for at least 2 hours. The mixture was then centrifuged at 20,000 rpm for 3 times and supernatants and washes volumes recorded. The synthesis was repeated 5 times for viable statistics. Controls with only Rubisco were performed using the above steps.

Results, Conclusions, and Discussions: Our analysis showed detection ability for FCA as low as 3.9 micro g/mL (Figure 1a); further, our analysis allowed for determination of the electrical impedance of all analytes tested and the characterization of the reversibility of electron transfer and its dependence on the attachment and molecules functionality at the electrode-solution interface, respectively. The average concentration of FCA that was immobilized onto the Mg-RuBisCO was also determined with a concentration as high as 15.6 micro g/mL (Figure 1b). Our analysis also allowed for determining the affinity of binding of FCA to the enzyme. For FCA-EDC/NHS-RuBisCO, a concentration under 3.9 g/mL of FCA was found to bind to the complex with analysis also demonstrating a lower affinity most likely due to the absence of Mg known to improve enzyme functionality at interfaces (Figure 1c). DPV results supported the validity and sensitivity of the created biosensor upon its immobilization onto the commercially
available electrode and how oxidation/reduction peaks changes based on the overall analyte chemical structure. Taken together, the results from this study have demonstrated the robustness and effectiveness of CV for characterization of different analytes, from small molecules (FCA) to large complexes (Mg-based Rubisco); we envision that the method could be extended for to detection of chemicals in food or of cancer biomarkers.

References:
Thomas C. Taylor and Inger Andersson et al., 1997- Structure of a Product Complex of Spinach Ribulose-1,5-bisphosphate Carboxylase/Oxygenase.
Elgrishi, Rountree et al., 2017- A Practical Beginner’s Guide to Cyclic Voltammetry.

Koichi Jeremiah Aoki et al., 2020- Peak potential shift of fast cyclic voltammograms owing to capacitance of redox reactions.

Xiao Hu et al., 2019- Biomolecular interfaces based on self-assembly and self-recognition form biosensors capable of recording molecular binding and release.

Xiao Hu et al., 2019-Nanoporous gold electrode for ultrasensitive detection of neurotoxin fasciculin.

Acknowledgements (Optional):