Image-based detection of oligonucleotides – a low cost alternative to spectrophotometric or fluorometric methods

Abstract

Herein, we report a sensitive and low cost image-based (photocolorimetric) method for the detection of oligonucleotides on an activated polypropylene microtest plate (APPμTP). The assay was developed on the APPμTP by covalently immobilising 20-mer amino-modified oligonucleotides. Biotin-tagged complementary target sequences were then hybridised with the immobilised oligonucleotides. Colour was developed by streptavidin–HRP conjugate and the image of the coloured assay solution was taken by a desktop scanner and analysed using colour saturation. The developed method was analysed for its detection limit, accuracy, sensitivity and interference. The linearity range was found to be 1.7–170 ng mL⁻¹ while the lower limit of detection and limit of quantification were 1.7 and 5.6 ng mL⁻¹ respectively. The method showed comparable sensitivity to fluorometric methods, and was found to be correlated to fluorescence (R² = 0.8081, p-value < 0.0001) and absorbance (R² = 0.9394, p-value < 0.0001)-based quantification. It discriminates mismatched base sequences from perfectly matched sequences efficiently. Validation of the method was carried out by detecting por A DNA of Neisseria meningitidis in bacterial meningitis samples. The por A-specific probe having a 6-carbon spacer at its 5′-NH₂ terminus was immobilised covalently to the APPμTP and hybridised with different samples of biotinylated single-stranded por A DNA.