Electrochemical Duplex Detection of E2 and E6 Genes of Human Papillomavirus Type 16 and Determination of Physical Status in High-Risk Cervical Carcinoma

Abstract

Human papillomavirus type 16 (HPV-16) is a key driver in the development of cervical carcinoma, with the integration of its genome into the host DNA marking a critical step in disease progression. Monitoring the physical state of HPV-16, particularly the transition from episomal to integrated forms, is essential for evaluating the risk of malignancy development in cervix. This study presents the development of a duplex electrochemical biosensor for the simultaneous detection of the E2 and E6 genes of HPV-16. Using a one-step sandwich hybridization assay, the biosensor was able to detect HPV-16 E2 and E6 genes with a sensitivity of 8 copies/mL and 12 copies/mL respectively and distinguish between the episomal and integrated forms based on the E2/E6 ratio (cut-off 0.77, 100% sensitivity/specificity). The sensor was validated with 30 clinical cervical tissue samples, providing results comparable to qPCR method. This novel biosensor offers a rapid and efficient platform for the detection and monitoring of HPV-16, with potential applications in cervical cancer screening and prognosis.

Keywords: cervical carcinoma; duplex detection; electrochemical sensor; human papillomavirus type 16; physical state.

Figure 1

Schematic illustration of single, monoplex, and duplex detection formats. (A) Single detection format: A single target was hybridized with its corresponding capture probe‐modified magnetic beads (CP‐MB). After 30 min of incubation at 50°C and a washing step, the specific reporter probe (Si‐RP) was added. Following further incubation and washing, the complex was electrochemically detected using differential pulse voltammetry (DPV). (C) Monoplex detection format: The procedure was similar to the single detection format, but the single target was hybridized with a mixture of E2 and E6 CP‐MB and a combination of SiMB‐E2 and SiAO‐E6 reporter probes. (E) Duplex detection format: Both targets (E2 and E6) were mixed and incubated with the E2 and E6 CP‐MB, along with the SiMB‐E2 and SiAO‐E6 reporter probes. The differential pulse voltammograms for (B) single detection, (D) monoplex detection, and (F) duplex detection showed current peaks at –0.3 V and +0.7 V, corresponding to the oxidation of SiMB‐E2 and SiAO‐E6, respectively. (G) Comparison of current responses across the single, monoplex, and duplex assays. All current responses are presented as mean values ± standard deviation (SD), n = 5.

Figure 2

Comparison of Electrochemical Signals Between Step‐by‐Step and One‐Step Assays. (A) Schematic representation of the step‐by‐step and one‐step assay procedures. The step‐by‐step assay involves four stages: hybridization of the targets (E2 and E6) with CP‐MB, washing to remove excess target, sandwich DNA hybridization with Si‐RP, and a second washing step to eliminate excess Si‐RP. In contrast, the one‐step assay simplifies the process by combining target, CP‐MB, and Si‐RP in a single sandwich hybridization step, followed by a single wash to remove unbound components. (B) Comparison of current values from step‐by‐step and one‐step assays, performed in single, monoplex, and duplex formats. The current values for blank, E2‐LT, E6‐LT, E2‐mLT, and E6‐mLT were comparable across all assay types. All current responses are presented as mean values ± standard deviation (SD), n = 5.

Figure 3

Analytical sensitivity of the assay using linear targets and DNA copies. (A) Differential pulse voltammograms for E2‐LT and E6‐LT show a clear increasing trend in current signals with rising concentrations of the linear targets, from the lowest to the highest levels. (B) Calibration curves for E2‐LT (purple) and E6‐LT (red) demonstrate a logarithmic‐linear correlation between the current signal and the concentration of the linear targets. (C) Differential pulse voltammograms for the E2 and E6 genes reveal a progressive increase in current peaks as the copy numbers rise from 10¹ to 10⁷ copies of plasmid DNA of whole HPV‐16 genome. (D) A logarithmic‐linear relationship is observed between the electrochemical signal and the copy number for both E2 (purple) and E6 (red) genes. All current responses are presented as mean values ± standard deviation (SD), n = 5.

Figure 4

Electrochemical detection of HPV‐16 E2 and E6 genes in clinical samples. Current values from electrochemical detection of HPV‐16 E2 gene (purple bar) and E6 gene (red bar) from 30 cervical tissue samples, HeLa, SiHa, and plasmid of whole genome HPV‐16 are shown. Plasmid of whole genome HPV‐16, harboring intact E2 and E6 genes is represented as a positive control for episomal HPV‐16, whereas SiHa is cervical cancer cell line containing an integrated HPV‐16 is represented as positive control for integrated HPV‐16. HeLa, a HPV‐18 infected cervical cancer cell line, is provided as negative control of HPV‐16. All current responses are presented as mean values ± standard deviation (SD), n = 5.