Nucleic acid sensor for M. tuberculosis detection based on surface plasmon resonance

Abstract

Cysteine modified NH₂-end peptide nucleic acid (PNA) (24-mer) probe and 5′-thiol end labeled deoxyribonucleic acid (DNA) probes specific to Mycobacterium tuberculosis have been immobilized onto BK-7 gold coated glass plates for the detection of complementary, one-base mismatch, non-complementary targets and complementary target sequence in genomic DNA of Mycobacterium tuberculosis using a surface plasmon resonance (SPR) technique. The DNA/Au and PNA/Au bio-electrodes have been characterized using contact angle, atomic force microscopy (AFM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetric (CV) techniques, respectively. It is revealed that there is a 252 millidegrees SPR angle change in the case of PNA immobilization and 205 millidegrees for DNA immobilization, indicating increased amount of immobilized PNA molecules. Hybridization studies reveal that there is no binding of the non-complementary target to DNA/Au and PNA/Au electrode. Compared to the DNA/Au bioelectrode, PNA/Au electrode has been found to be more efficient for detection of one-base mismatch sequence. The PNA/Au bioelectrode shows better detection limit (1.0 ng ml⁻¹) over the DNA-Au bioelectrode (3.0 ng ml⁻¹). The values of the association (k_a) and dissociation rate constant (k_d) for the complementary sequence in case of the PNA/Au bioelectrode have been estimated as 8.5 × 10⁴ m⁻¹ s⁻¹ and 3.6 × 10⁻³ s⁻¹, respectively.