Designing an Electrochemical Biosensor Based on Voltammetry for Measurement of Human Chorionic Gonadotropin

Abstract

Background: Human chorionic gonadotropin (hCG) is a polypeptide hormone synthesized during pregnancy and is also upregulated in some pathologic conditions such as certain tumors. Its measurement is essential for diagnosing pregnancy and malignancies. Despite numerous attempts to introduce an accurate method capable of detecting hCG levels, several limitations are found in previous techniques. This study aimed to address the limitations of current hCG assay methods by designing an electrochemical biosensor based on voltammetry for the rapid, selective, inexpensive, and sensitive measurement of hCG levels.

Methods: A carbon paste electrode was prepared and functionalized by para-aminobenzoic acid. The primary anti-β-hCG monoclonal antibody was immobilized on the electrode surface by activating the carboxyl groups with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide solutions. The study also involved optimizing parameters such as the time for primary antibody fixation, the time for hCG attachment, and the pH of the hydrogen peroxide solution to maximize the biosensor response. Different concentrations of hCG hormone were prepared and loaded on the electrode surface, the secondary antibody labeled with HRP enzyme was applied, thionine in phosphate-buffered saline solution was placed on the electrode surface, and the differential pulse electrical signal was recorded.

Results: The linear range ranged from 5 to 100 mIU/ml, and the limit of detection was calculated as 0.11 mIU. The relative standard deviation was 3% and 2% for five repeated measurements of commercial standard samples with concentrations of 2 and 20 mIU/mL, respectively. The percent recovery was obtained from 98.3% to 101.5%.

Conclusion: The sensor represents a promising advancement in hCG level measurement, offering a potential solution to overcome the existing limitations in current diagnostic strategies. Simple and inexpensive design, detecting hCG in its important clinical range during early pregnancy, and successful measurement of hCG in real serum samples are the advantages of this sensor.

Keywords: Biosensor; electrochemistry; voltammetry; β-human chorionic gonadotropin.

Scheme 1

Fabrication and sensing approach of the developed electrochemical biosensor

Figure 1

Primary antibody concentration optimization (The experiments were conducted in a 5.0 mM ferro/ferricyanide redox solution in phosphate-buffered solution buffer, with a pH of 7.4.)

Figure 2

Optimization of H₂O₂ pH Value by differential pulse voltammetry technique (at 5.0 μM hydrogen peroxide)

Figure 3

Primary antibody attachment optimization

Figure 4

Antigen attachment time optimization

Figure 5

Differential pulse voltammograms for different concentrations of human chorionic gonadotropin (5.0–100 mIU) in the presence of fabricated electrochemical biosensor (phosphate-buffered solution of 0.10 M, pH 7.4)

Figure 6

Differential pulse voltammetry calibration plot of I_p (μA) versus (human chorionic gonadotropin). The error bars depict the standard deviations of three measurements (n = 3)