Image-based detection of oligonucleotides - a low cost alternative to spectrophotometric or fluorometric methodsElectronic supplementary information (ESI) available. See DOI: 10.1039/c3an02402d

• Main Authors: Ahirwar, Rajesh, Tanwar, Swati, Parween, Shahila, Kumar, Ashok, Nahar, Pradip
• Format: Journal Article
• Language: English
• Published: 31.03.2014
• ISSN: 0003-2654; 1364-5528
• DOI: 10.1039/c3an02402d

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 ( R 2 = 0.8081, p -value < 0.0001) and absorbance ( R 2 = 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′-NH 2 terminus was immobilised covalently to the APPμTP and hybridised with different samples of biotinylated single-stranded por A DNA. We report the simple and low cost image-based quantitative detection of oligonucleotides without using a spectrophotometer or fluorometer.