A Magnetoelectrochemical Bioassay for Highly Sensitive Sensing of Point Mutations in Interleukin-6 Gene Using TMB as a Hybridization Intercalation Indicator

Abstract

Point mutations are common in the human DNA genome and are closely related to higher susceptibility to cancer diseases. Therefore, suitable methods for their sensing are of general interest. In this work, we report on a magnetic electrochemical bioassay using DNA probes tethered to streptavidin magnetic beads (strep-MBs) to detect T > G single nucleotide polymorphism (SNP) within the inteleukin-6 (IL6) gene in human genomic DNA. In the presence of the target DNA fragment and tetramethylbenzidine (TMB), the electrochemical signal related to the oxidation of TMB is observed, which is much higher than the one obtained in the absence of the target. The key parameters affecting the analytical signal, such as the concentration of the biotinylated probe, its incubation time with strep-MBs, DNA hybridization time, and TMB loading, were optimized using the electrochemical signal intensity and signal-to-blank (S/B) ratio as selection criteria. Using spiked buffer solutions, the bioassay can detect the mutated allele in a wide range of concentrations (over six decades) with a low detection limit (7.3 fM). Furthermore, the bioassay displays a high specificity with high concentrations of the major allele (one mismatched), and two mismatched and non-complementary DNA. More importantly, the bioassay can detect the variation in scarcely diluted human DNA, collected from 23 donors, and can reliably distinguish between heterozygous (TG genotype) and homozygous (GG genotype) in respect to the control subjects (TT genotype), where the differences are statistically highly significant (p-value < 0.001). Thus, the bioassay is useful for cohort studies targeting one or more mutations in human DNA.

Keywords: cancer; electrochemistry; human DNA; interleukin-6; ovarian cancer; point mutation; sensing.

Scheme 1

Schematic illustration of the magnetic electrochemical bioassay for the sensing of the single point mutation in genomic IL6 DNA. (a) The probe fully recognizes the mutated target and after TMB intercalation, a strong oxidation signal is obtained and (b) the probe partially recognizes the wild target and a weak redox signal is observed.

Figure 1

(A) SWV responses obtained in presence of the synthetic tIL6 sequence using MB (a and a’) and TMB (b and b’). Current and S/B ratio variation as function of the (B) pIL6 concentration, (C) incubation time of pIL6, (D) hybridization time of tIL6 sequence with pIL6/MBs, (E) dilution ratios of commercial TMB, and (F) incubation time into TMB solution. The symbol represents the signal-to-blank ratio values for each measurement.

Figure 2

(A) SWV curves of pIL6/MBs in presence of increasing concentrations of the tIL6 sequence. (B) Calibration plot for the tIL6 sequence showing the dynamic range equation, (C) currents and percentages obtained with 500 pM of tIL6, or 5.0 nM of tIL6-1m, tIL6-2m, or the noncomplementary sequence compared with the blank response, and (D) stability response of the bioconjugate assayed over a period of three month.

Figure 3

Amperometric response from the analysis (A) and (B) box plot showing the currents and their mean values recorded for real samples of control subjects and patients (diamonds, circles, and squares) having heterozygous or homozygous genotypes in IL6. One-sample t-test statistical analysis p-value is <0.001.