Electrochemical impedance spectroscopy (EIS) has emerged as a powerful analytical technique that offers kinetic and mechanistic data of electrochemical systems. It is widely used in several fields, including the life sciences, where it is used to study various biological processes.
Specific processes can influence the properties of an electrochemical system, namely conductance, resistance, and capacitance. Hence, EIS can be used to study semiconductors, corrosion, chemical sensing, and biorecognition events, including substrate-enzyme interactions and antibody-antigen recognition.
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Foundations of Electrochemical Impedance Spectroscopy
EIS measures the impedance properties of an electrochemical system. Measurements take 2-3 min and span over a large range of frequencies (100 kHz-0.1 Hz). The core components of EIS are an electrochemical cell, electrodes, and a frequency response analyzer.
The electrochemical cell typically consists of a working electrode, a reference electrode, and a counter electrode immersed in an electrolyte solution. At the same time, the frequency response analyzer measures the impedance across the system.
Impedance sensors can be classified as either Faradaic or capacitive sensors. The electrodes have conductive surfaces in Faradaic impedance sensors, and the analysis requires redox-active molecules in solution.
The parameter measured is the charge transfer resistance, which decreases when conductive molecules bind to the sensor's surface.
Conversely, capacitive (non-Faradaic) sensors cover the sensing surface with an insulating layer. The main parameter measured is the double-layer capacitance, which decreases when molecules bind to the surface.
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Applications in Life Science Research
There are several ways EIS can be exploited in life science research, ranging from environmental applications to diagnostics.
EIS is a non-invasive technique, and based on the interactions at the electrode, it can be used to characterize antigen-antibody interactions and cell growth.1
It also allows the detection of pathogens or cancer-related biomarkers and probes biological interactions between cells adherent to the electrode in real-time. Electrodes modified with aptamers and gold nanoparticles allowed the detection of prostate-specific antigen (PSA), a biomarker for prostate cancer, achieving a good detection limit (LOD, ten pg/mL).
Similarly, electrodes functionalized with graphene oxide and reduced graphene oxide were used to design impedimetric sensors for the hepatitis E virus (HEV) with high sensitivity and a linearity response in serum samples between 10 fg/mL and 100 pg/mL.[2]
EIS using gold electrodes coated with human laminin has also been applied to monitor cell adhesion and differentiation from midbrain floor plate progenitors into midbrain neurons.[3]
Advantages of EIS in Life Science Research
Its non-destructive nature is one of the key advantages of EIS. This allows for continuous monitoring of biological samples without damaging them.
The technique is also very sensitive and capable of detecting small changes in the electrical properties, allowing the observation of events that might not be observable with other analytical methods.
Another key benefit is the ability to provide real-time monitoring of biological processes. Unlike other methods that may require extensive sample preparation and time-consuming analysis, EIS delivers immediate insights into the state of the sample.
Other analytical techniques commonly used in the life sciences, such as fluorescence microscopy, enzyme-linked immunosorbent assay (ELISA), and chromatography, are very accurate and have well-established protocols.
At the same time, they can also be expensive and time-consuming, and they might require extensive sample preparation and skilled personnel. Conversely, EIS-based sensors are simple, easy to use, portable, and cost-effective.
Case Studies and Success Stories
EIS was reported as a successful method for detecting endocrine disruptors – compounds of natural or synthetic origins that can cause human cancer and infertility.
A sensor for detecting zearalenone was developed based on molecularly imprinted polymers where o-phenylenediamine was polymerized on a gold electrode. The EIS measurement was based on a simple modification protocol and a short incubation time, with the sensor showing high selectivity and a limit of detection of 2.5 ng/mL.[4]
An impedimetric sensor for total calcium detection in saliva was developed using a gold nanoparticle self-assembled monolayer. The sensor was highly sensitive and selective, with a linear range between 10−12 M and 10−6 M and a low detection limit (3.6 × 10−12 M).
The sensor allowed the non-invasive analysis of real samples of human saliva, exhibiting Ca2+ selectivity over a variety of common interfering ions, proving to be a potential tool for point-of-care diagnostics.[5]
Challenges and Limitations
One of the main challenges of EIS is the complexity of data interpretation. In some cases, impedance spectra require advanced modeling and analysis techniques to extract meaningful information accurately.
In addition, it is possible to misinterpret specific interactions and have false positive results, compromising the analysis outcome.
This can be caused by a lack of control of the experimental conditions and variations caused by drift or non-specific binding. Variability in the analysis setup or environmental factors can also lead to inconsistent results, highlighting the need for meticulous experimental design and control.[6]
Ongoing research efforts are focused on addressing these challenges by developing computational algorithms to enhance the accuracy and simplify impedance data analysis.
Moreover, developing more robust and user-friendly EIS equipment can make the technique more accessible to a broader range of users.
Future Directions
EIS is an emerging tool that can significantly improve understanding of biomolecular interactions and other biological processes. Its non-destructive nature, high sensitivity, and real-time monitoring capabilities offer distinct advantages over other analytical techniques.
Despite some limitations and challenges, there are several successful examples of how EIS can drive scientific discoveries and advance life science research.
Ongoing research and technological advancements are contributing to the growth of EIS applications in the life sciences and other research fields.
References
- Magar, H. S., Hassan, R. Y. A. & Mulchandani, A. (2021). Electrochemical Impedance Spectroscopy (EIS): Principles, Construction, and Biosensing Applications. Sensors (Basel), 21.10.3390/s21196578.
- Chowdhury, A. D., Takemura, K., Li, T. C., Suzuki, T. & Park, E. Y. (2019). Electrical pulse-induced electrochemical biosensor for hepatitis E virus detection. Nat Commun, 10, 3737.10.1038/s41467-019-11644-5.
- Elghajiji, A., Wang, X., Weston, S. D., Zeck, G., Hengerer, B., Tosh, D. & Rocha, P. R. F. (2021). Electrochemical Impedance Spectroscopy as a Tool for Monitoring Cell Differentiation from Floor Plate Progenitors to Midbrain Neurons in Real Time. Advanced Biology, 5, 2100330. https://doi.org/10.1002/adbi.202100330. Available: https://onlinelibrary.wiley.com/doi/abs/10.1002/adbi.202100330
- Zamfir, L.-G., Puiu, M. & Bala, C. (2020). Advances in Electrochemical Impedance Spectroscopy Detection of Endocrine Disruptors. Sensors, 20, 6443. Available: https://www.mdpi.com/1424-8220/20/22/6443
- Magar, H. S., Abbas, M. N., Ali, M. B. & Ahmed, M. A. (2020). Picomolar-sensitive impedimetric sensor for salivary calcium analysis at POC based on SAM of Schiff base–modified gold electrode. Journal of Solid State Electrochemistry, 24, 723-737.10.1007/s10008-020-04500-w. Available: https://doi.org/10.1007/s10008-020-04500-w
- Bogomolova, A., Komarova, E., Reber, K., Gerasimov, T., Yavuz, O., Bhatt, S. & Aldissi, M. (2009). Challenges of Electrochemical Impedance Spectroscopy in Protein Biosensing. Analytical Chemistry, 81, 3944-3949.10.1021/ac9002358. Available: https://doi.org/10.1021/ac9002358
Further Reading