Ultrasensitive cDNA detection of dengue virus RNA using electrochemical nanoporous membrane-based biosensor

Abstract

A nanoporous alumina membrane-based ultrasensitive DNA biosensor is constructed using 5'-aminated DNA probes immobilized onto the alumina channel walls. Alumina nanoporous membrane-like structure is carved over platinum wire electrode of 76 µm diameter dimension by electrochemical anodization. The hybridization of complementary target DNA with probe DNA molecules attached inside the pores influences the pore size and ionic conductivity. The biosensor demonstrates linear range over 6 order of magnitude with ultrasensitive detection limit of 9.55×10(-12) M for the quantification of ss-31 mer DNA sequence. Its applicability is challenged against real time cDNA PCR sample of dengue virus serotype1 derived from asymmetric PCR. Excellent specificity down to one nucleotide mismatch in target DNA sample of DENV3 is also demonstrated.

Figure 1. Schematic.

Scheme of construction and operation for nanoporous alumina membrane based DNA biosensor.

Figure 2. Complementary target detection and reproducibility.

(A) Differential pulse voltammetry current signal response of (a) biosensor, (b) preconditioning of biosensor and towards increasing concentration of complementary target: (c) 10⁻¹²,(d) 10⁻¹⁰,(e) 10⁻⁰⁸ and (f)10⁻⁰⁶ M. DPV currents were offset to 0 µA to allow comparison of results and all measuring solutions contain 1×, pH 7.2 PBS electrolyte solution. (B) Averaged normalized current signal response best fitted linearly with log C of complementary target. Error bars and points represent average standard deviations derived from single biosensor with three consecutive measurements (C) Normalized DPV current signal response of different biosensors 1, 2 and 3 towards identical complementary analyte at 10⁻⁶ M concentration. Error bars correspond to standard deviations obtained from 3 consecutive DPV measurements.

Figure 3. Specificity and one base-pair mismatch DENV3 detection.

Changes in normalized differential current signal of the biosensor probe towards 10⁻⁸ M 31-mer complementary target sequence (DENV 1) and single-base mismatch target sequence (DENV3) respectively. Error bars correspond to standard deviations obtained from 3 consecutive DPV measurements.

Figure 4. Regeneration of biosensors.

(A) Differential pulse voltammetry current signal response of (a) BS, biosensor (b) Comp-BS, biosensor towards complementary analyte 10⁻⁰⁶ M (c) R1, Regenerated biosensor after first heating cycle (d) Comp-R1,first regenerated biosensor towards complementary analyte 10⁻⁰⁶ M (e) R2, Regenerated biosensor after second heating cycle. (f) Comp-R2, second regenerated biosensor towards complementary analyte 10⁻⁰⁶ M. DPV currents were offset to 0 µA to allow comparison of results and all measuring solutions contain 1×, pH 7.2 PBS electrolyte solution. (B) Normalized DPV current signal response of biosensor, first regenerated and second regenerated biosensor towards identical complementary analyte 10⁻⁰⁶ M. Error bars correspond to standard deviations obtained from 3 consecutive DPV measurements.

Figure 5. Detection of real time PCR DNA sample derived from DENV1 genomic RNA.

(A) Electrophoresis analysis of the 183 bp region of DENVI amplified using asymmetric PCR. (B) Normalized differential current signal response of biosensor towards this real time cDNA PCR sample of 10⁻¹¹, 10⁻¹⁰, 10⁻⁹ and 10⁻⁸ M, derived from DENV1 genomic sequence using asymmetric PCR. Error bars correspond to standard deviations obtained from 3 consecutive DPV measurements.