Development of an electrochemical sensing technique for rapid genotyping of hepatitis B virus

Abstract

Objective: To develop a convenient; sensitive; accurate; and economical technique for genotyping of hepatitis B viruses (HBVs).

Methods: The mercapto-modified B1; B2; C1; and C2-specific genotyping probes consisted of two probes for each HBV genotype that served as a double verification system. These probes were fixed on the surface of No. 1; 2; 3; and 4 gold electrodes; respectively; via Au-S bonds. Different charge generated by the binding of RuHex to phosphate groups of the DNA backbone before and after hybridization was used for distinguishing the different genotypes.

Results: During hybridization with genotype B; the charges detected at the No. 1 and 2 electrodes were significantly increased; while the charge at the No. 3 and 4 electrodes did not change significantly. During hybridization with genotype C; the charges detected at No. 3 and 4 electrodes were significantly increased; while the signals remained unchanged at the No. 1 and 2 electrodes. During hybridization with mixed genotypes (B and C); the charges detected at all four electrodes were significantly increased. The linear range of detection was 10(-7) to 10(-10) mol/L and the sensitivity for detecting mixed B (10%) or C (10%).

Conclusions: Rapid genotyping of HBVs based on electrochemical sensing is simple, has good specificity; and can greatly reduce the cost. This method can be used for sensitive detection of mixed B and C HBV genotypes.

Figure 1.

Schematic representation of the chronocoulometric DNA biosensor. B type HBV is determined when the signals of electrodes No. 1 and No. 2 increase simultaneously after hybridization. C type is determined when the signals of electrodes No. 3 and No. 4 increase simultaneously after hybridization. The mixed type is determined when the signals of No. 1, No. 2, No. 3 and No. 4 electrodes increase simultaneously after hybridization. The type cannot be confirmed when the signals of electrodes No. 1 and No. 3 or No. 2 and No. 3 or No. 1 and No. 4 or No. 2 and No. 4 increase simultaneously after hybridization.

Figure 2.

Cyclic voltammograms of electrodes. Bare AuE (A), MCH/AuE (B), DNA/MCH/AuE (C). The electrolyte was 10.0 mM Tris buffer (pH 7.4) containing 50 μM RuHex. Pulse period, 250 ms.

Figure 3.

(A) Cyclic voltammograms and chronocoulometry curves of the concentration of probe is 2.0 μmol/L. Cyclic voltammograms: dsDNA/RuHex/MCH/AuE(a), ssDNA/RuHex/MCH/AuE (b), chronocoulometry curves ssDNA/RuHex/MCH/ AuE (c), ssDNA /RuHex/MCH/ AuE (d). (B) Cyclic voltammograms and chronocoulometry curves of the concentration of probe is 0.5 μmol/L. Cyclic voltammograms: dsDNA/RuHex/MCH/ AuE(a), ssDNA/RuHex/MCH/AuE(b), chronocoulometry curves: ssDNA/RuHex/MCH/ AuE (c), ssDNA /RuHex/MCH / AuE(d).

Figure 4.

(A) Chronocoulometry curves of probe B1 and B2 using 50 μM RuHex as redox indicator. Curves of probe B1 hybridization with noncomplementary sequence (10 nmol/L C1 or C2 target) (a); the blank hybridization (b); curves of probe B2 using 50 μM RuHex as redox indicator: the blank hybridization (c); hybridization with noncomplementary sequence (10 nmol/L C1 or C2 target) (d); complementary target sequence (10 nmol/L B1 target) (e); complementary target sequence (10 nmol/L B1 target) (f). (B) Chronocoulometry curves of probe C1 and C2 using 50 μM RuHex as redox indicator. Curves of probe C1 hybridization: the blank hybridization (a); hybridization with noncomplementary sequence (10 nmol/L B1 or B2 target) (b); hybridization with noncomplementary sequence (10 nmol/L B1 or B2 target) added complementary target sequence (10 nmol/L C2 target) (e); chronocoulometry curves of probe C1 using 50 μM RuHex as redox indicator: the blank hybridization (c); hybridization with noncomplementary sequence (10nmol/L B1 or B2 target) (d); hybridization with noncomplementary sequence (10 nmol/L B1 or B2 target); added complementary target sequence (10 nmol/L C1 target) (f).

Figure 5.

Chronocoulometry curves using 50 μM RuHex as redox indicator for a probe modified electrode after hybridization with different target sequence: 0 nmol/L (a); 0.1 nmol/L (b); 1 nmol/L (c); 10 nmol/L (d); 100 nmol/L (e). The figure (A) is for the B2 probe and the figure (B) for the C2 probe, respectively. Inset shows the plot of the charge increment of RuHex as a function of the target concentration. Signal was defined as the difference in the redox charge of RuHex after and before hybridization (signal = Q_after − Q_before).