Specific Magnetic Isolation of E6 HPV16 Modified Magnetizable Particles Coupled with PCR and Electrochemical Detection

Abstract

The majority of carcinomas that were developed due to the infection with human papillomavirus (HPV) are caused by high-risk HPV types, HPV16 and HPV18. These HPV types contain the E6 and E7 oncogenes, so the fast detection of these oncogenes is an important point to avoid the development of cancer. Many different HPV tests are available to detect the presence of HPV in biological samples. The aim of this study was to design a fast and low cost method for HPV identification employing magnetic isolation, polymerase chain reaction (PCR) and electrochemical detection. These assays were developed to detect the interactions between E6-HPV16 oncogene and magnetizable particles (MPs) using commercial Dynabeads M-280 Streptavidin particles and laboratory-synthesized "homemade" particles called MANs (MAN-37, MAN-127 and MAN-164). The yields of PCR amplification of E6-HPV16 oncogene bound on the particles and after the elution from the particles were compared. A highest yield of E6-HPV16 DNA isolation was obtained with both MPs particles commercial M-280 Streptavidin and MAN-37 due to reducing of the interferents compared with the standard PCR method. A biosensor employing the isolation of E6-HPV16 oncogene with MPs particles followed by its electrochemical detection can be a very effective technique for HPV identification, providing simple, sensitive and cost-effective analysis.

Keywords: PCR; electrochemistry; human papillomavirus; magnetic isolation; magnetizable particles; nucleic acid detection.

Figure 1

Main molecular mechanisms of oncogenesis induced for E6 and E7 proteins of human papillomavirus 16 (HPV16).

Figure 2

(A) Scheme of the pUC-57 vector with cloned E6 gene of HPV16. The construction of human E6-HPV16-pUC57 plasmid was purchased from Genewiz Company (GENEWIZ, South Plainfield, NJ, USA); (B) Nucleotide sequence of E6-HPV16 gene.

Figure 3

Optimization of template E6-HPV16-pUC57 plasmid concentration and scheme of standard PCR conditions: (A) finding the optimal concentration of DNA template for polymerase chain reaction (PCR); and (B) PCR protocol.

Figure 4

Scheme of DNA nanoconstruct of biotin-modified oligonucleotides bound to E6-HPV16 oncogene joined to streptavidin modified MPs : (A) 100 µL (10 mg·mL⁻¹) of commercial Dynabeads M-280 Streptavidin MPs; (B) E6-HPV16 complementary oligonucleotides biotinylated (forward and reverse) (20 µL, 100 µM) using Biotin 3′ end DNA Labeling Kit (Thermo Scientific, Waltham, MA, USA) were successfully conjugated with Dynabeads; and (C) E6-HPV16 DNA was amplified from E6-HPV16-pUC57 synthetic plasmid by PCR, which was subsequently purified and conjugated with the nanoconstruct.

Figure 5

Comparison of PCR assay between E6-HPV16 DNA isolated from synthetic plasmid and E6-HPV16 DNA isolated using commercial M-280 MPs, which they were used to make a PCR with set of E6 primers. The DNA nanoconstructs with commercial MPs particles showed 2.6-fold higher fluorescence of Ethidium bromide (EtBr) stained E6-HPV16 bands than the positive control using synthetic plasmid E6-HPV16-pUC57. Conditions for gel electrophoresis were as follows: 1% agarose gel, 1% TAE buffer.

Figure 6

Isolation and detection of E6-HPV16-pUC57 plasmid using different “homemade” magnetiyable particles (MPs): (A–C) SEM microphotographs of MPs used in DNA isolation procedure (MAN-37, MAN-127 and MAN-164 respectively); (D) PCR amplification of E6-HPV16 gene in pUC-57 plasmid after elution from MPs; (E) PCR amplification of E6-HPV16 gene from 5 µL of MPs with bound DNA; (F) PCR amplification of E6-HPV16 gene from 0.5 µL of MPs with bound DNA; (G) the average size of MAN-37, 127 and 164 before and after binding with DNA; (H) the dependence of MAN-37 zeta potential on pH; and (I) the dependence of zeta potential of MAN-37 with bound DNA on pH.

Figure 7

Optimization of electrochemical detection of E6-HPV16 gene and E6 forward primer (E6 Fw) were as follows: (A) frequency; (B) time of accumulation; and (C) calibration curve for E6-HPV16 gene; (D) frequency; (E) time of accumulation; and (F) calibration curve for E6 forward primer. All electrochemical measurements were provided by AdTS SWV in acetate buffer, pH 5.0, with an initial potential 0.0 V, end potential −1.7 V, amplitude 0.025 V, step potential 0.005 V. 100 Hz of frequency and 120 s of time of accumulation were selected as optimal conditions (yellow asterisks).

Figure 8

(A) Gel electrophoresis and (B) electrochemical analysis of PCR product (PCR of E6-HPV16-pUC57 plasmid set of E6 primers) obtained after 10, 20, 30, 35, and 40 PCR cycles. Expected size was 477 bp, corresponded to E6-HPV16 gene. Gel electrophoresis conditions: 1% agarose gel, 1% TAE buffer. Electrochemical conditions: See optimized conditions from Figure 7B; (C) Dependence of the relative CA peak height on the number of PCR cycles.

Figure 9

(A) Typical SWV voltammograms of E6 forward primer construct released from commercial M-280 MPs; (B) compared with E6 reverse primer construct released from commercial M-280 MPs; (C) calibration curve of E6-HPV16 gene in concentration range from 0.15 to 2.5 µM, real CA peak voltammograms and scheme of accumulation of sample on surface of working electrode are inserted; and (D) concentration of E6-HPV16 gene construct isolated by commercial M-280 MPs by two different primers (E6 forward and E6 reverse). The up arrows show the scale for each voltammograms.