Electrochemical Biosensor

Electrochemical biosensors are a type of biosensor that converts biological or chemical changes into detectable signals for analysis. They are used to measure reference levels of numerous substances in the body, including glucose—the primary analyte in devices used by diabetic patients—as well as lactate, dopamine, CRP, cortisol, cholesterol, and toxins, thereby facilitating diagnosis. In clinical studies, they enable measurement of DNA and RNA sequences, viruses, bacteria, pathogens, and drug concentrations. Additionally, electrochemical biosensors are employed in food safety to detect highly harmful substances such as the mycotoxin aflatoxin B1, heavy metals Pb²⁺ (lead) and Hg²⁺ (mercury), and harmful agricultural pesticides.

Glucose Measurement Sensor (Pixabay)

In this type of biosensor, electrodes serve as transducers, converting biological or chemical events into measurable electrical signals such as current, voltage, or impedance. Electrochemical sensors based on redox reactions—chemical reactions involving electron transfer—generate a detectable signal through the electrodes. In redox terminology, a molecule that gains electrons is reduced, while one that loses electrons is oxidized. These electron transfer processes are known as redox couples. The oxidized and reduced species are described using the Nernst Equation, which forms the fundamental principle of operation for electrochemical biosensors.

Nernst Equation (Generated by Artificial Intelligence)

The symbols in the equation are defined as follows:

E: Actual electrode potential

E∘: Standard electrode potential (1 M, 1 atm, 25°C)

R: Universal gas constant (8.314 J/mol·K)

T: Temperature (in Kelvin)

n: Number of electrons transferred in the reaction

F: Faraday constant (96485 C/mol e⁻)

Q: Ratio of reactant to product concentrations

As in other biosensors, the target substance being measured is called the analyte, and the molecule used to detect it is termed the biorecognition element. The analyte may consist of DNA or RNA sequences, proteins, antibodies, cells, or microorganisms.

Electrode Structure

Metal and carbon-based electrodes are commonly used. Carbon-based materials such as graphite and carbon fiber offer advantages due to their high chemical inertness and low electrical resistance. Other materials used in electrode fabrication include gold, silver, and stainless steel.

Electrode Structure (Generated by Artificial Intelligence)

• Reference electrode provides a stable and known potential for measurements. No current flows through this electrode; it serves as a reference point for potential and is typically made of materials such as silver (Ag) or silver chloride (AgCl).

• Working electrode is the site where the analyte is detected. Reduction and oxidation chemical reactions occur here. Gold, platinum, and carbon are commonly preferred materials.

• Counter electrode completes the current circuit from the working electrode and ensures continuous electron flow in the system. More inert materials such as platinum and carbon are typically chosen for this role.

In this setup, an insulating material is also present to prevent electrical short circuits between the electrodes. This insulator, made of materials such as PVC, PDMS, glass, or ceramic, enhances measurement control by facilitating the immobilization of biomolecules.

Electrochemical System Composed of Electrodes and Solutions (Pixabay)

Electrodes are immersed in an electrolyte solution that contains ions and enables electrical conduction. These solutions are typically composed of potassium ferrocyanide, phosphate-buffered saline (PBS), KCl, Na₂SO₄, HCl, NaOH, or saline water.

Operating Principle

After redox reactions occur, changes in current and potential at the electrode surfaces are measured. The resulting signal, often extremely small at the micro- or nanoscale, is amplified. This analog signal is then converted into a digital format using an analog-to-digital converter for processing by computers and other digital systems. The processed signals are displayed as data on monitors or mobile screens for user interpretation.

Electrochemical biosensors used in point-of-care testing provide not only quantitative analysis but also high-sensitivity qualitative analysis. These features offer advantages over lateral flow assay (LFA) systems.

While certain drawbacks such as electrode fouling, the need for expert interpretation due to complex analyses, high calibration demands, and unwanted parasitic binding to electrodes are considered disadvantages compared to other point-of-care tests, electrochemical biosensors are valued for their ease of use, portability, minimal sample volume requirements, rapid results, and low cost.

Signal Transduction Pathways

The signal generated after a redox reaction is transmitted to the electrode through three main pathways:

1. Direct oxidation-reduction: The redox reaction occurs directly between the analyte and the biorecognition element, with electron transfer occurring directly to the electrode. This mechanism involves simple molecular structures and does not require a mediator.
2. Indirect oxidation-reduction: Electron transfer occurs via a mediator molecule. This approach provides better signal control and greater compatibility with diverse biomolecules.
3. Amplification reactions: When biological signals are weak or present at low concentrations, chemical or biological amplification techniques are employed to enhance detectability. This method achieves high sensitivity.

Voltammetric Analysis Methods

These are methods used in electrochemical biosensors to identify analytes and determine their concentrations.

• Cyclic Voltammetry (CV): The voltage applied to the electrode is varied, and the resulting current is measured. In the resulting redox graph, the upper peak represents oxidation (electron loss), while the lower peak represents reduction (electron gain). Cyclic voltammetry is typically the first test performed when developing an electrochemical biosensor and serves to characterize the electrode and confirm system functionality.

Cyclic Voltammetry Graph (Generated by Artificial Intelligence)

• Differential Pulse Voltammetry (DPV): In this method, a series of voltage pulses are applied at regular intervals, and the current response to each pulse is measured. DPV enables detection of analytes at very low concentrations and is widely used for high-sensitivity calibration and quantitative analysis. In the resulting graph, higher analyte concentrations produce higher peaks, while lower concentrations yield smaller peaks.

Differential Pulse Voltammetry Graph (Generated by Artificial Intelligence)

• Electrochemical Impedance Spectroscopy (EIS): Impedance is the opposition a system presents to alternating current (AC). This method measures resistance changes. Low analyte concentration implies reduced electron transfer efficiency, which increases resistance. Thus, resistance is inversely proportional to analyte concentration. EIS is used to detect biomolecules on the electrode surface and to measure impedance changes.

Electrochemical Impedance Spectroscopy Graph (Generated by Artificial Intelligence)