Cellulose Based Genoassays for the Detection of Pathogen DNA

Abstract

Simple, reliable and cost-effective methods for detecting pathogens are a vital part of diagnostics inside and outside the clinic, in particular in the developing world. Paper based colorimetric techniques are a promising approach for biosensors and bioassays as they can be used at the point of sampling and require little equipment. This study reports on the development of a colorimetric cellulose bioassay that can detect pathogen DNA with covalently attached single-stranded DNA probes. Chemical activation of cellulose via tosylation and oxidation was investigated. The successful activation of cellulose was characterised by Fourier transform infrared spectroscopy, scanning electron microscopy and elemental analysis. Sulfhydryl and amine functionalised oligonucleotide probes complementary to a segment of IS6110 element in Mycobacterium tuberculosis genome were covalently immobilised on the cellulose strips for recognition of target nucleic acid. The detection of biotinylated target oligonucleotides was achieved with horseradish peroxidase (HRP) linked to streptavidin that binds biotin with high affinity. HRP catalysed the oxdidation of tetramethylbenzidine by hydrogen peroxide. The successful assay was confirmed by the appearance of blue coloured spots on cellulose strips incubated with biotinylated target oligonucleotides complementary to the surface attached probe. The study also showed that tosylated cellulose is more reliable for the detection of targets. Initial experiments have shown sensitivity upto 0.1 µM and considerable specificity. High probe immobilization efficiencies (>90%) have been observed. The assay was also effectively demonstrated with mycobacterial DNA. Additionally, the development of a label free assay based on a dual-probe approach was investigated, but did not yield conclusive results. The developed assay has the potential for use as a simple test for the detection of pathogen DNA in clinical samples since it requires minimal equipment and is cost effective. In addition, it also shows the potential use of tosylated cellulose as a prospective surface for attaching other types of biomolecules in an active conformation.