Ellipsometric-based novel DNA biosensor for label-free, real-time detection of Bordetella parapertussis

Pertussis (or whooping cough) is a contagious disease mainly affecting infants and children and predominantly caused by Bordetella pertussis followed by Bordetella parapertussis. B. parapertussis causes a milder cough but usually symptomatically appears like B. pertussis infection. Thus the epidemiology of illness caused by B. parapertussis is not well understood. In this study, a sensitive and specific method for the rapid diagnosis of B. parapertussis is presented. The covalent immobilization of thiol-terminated DNA oligonucleotides (ss DNA SAM) on a silicon surface by disulfide bond formation is investigated with atomic force microscopy (AFM) and ellipsometry. The measurements indicated an average layer thickness of 5 ± 0.84 nm for 2 μg/μl concentration and 24 h incubation time. This thickness changed to 8.4 ± 0.92 nm for the same concentration (2 μg/μl) by altering the incubation time to 48 h. Ellipsometric data recorded before and after hybridization of B. parapertussis revealed an increase in mean grain area from 91 nm(2) to 227 nm(2) and a change in the refractive index from 1.489 to 1.648 for 2 μg/μl B. parapertussis, respectively. This change in the refractive index was used to evaluate the amount of adsorbed molecules and their density. The results showed that the density of adsorbed molecules increased from 0.2 to 0.97 g/cm(3) after B. parapertussis attachment, respectively. To confirm the hybridization of B. parapertussis to ss DNA SAM, the ds DNA SAM was denatured and the ss DNA SAM surface was reproduced with an average height variation of 6.42 ± 0.75 nm. This showed the stability of the DNA film that can be tuned by varying the concentration and incubation time, thus providing a robust method for the label-free detection of B. parapertussis other than routinely used PCR detection.