Rapid, multistep on-chip DNA hybridisation in continuous flow on magnetic particles.
DNA hybridisation is an important tool for bioanalytical research and clinical diagnostics; conventional methods, however, require long incubation times and numerous washing steps, rendering the procedure time consuming and labour intensive. In this paper, we report on a rapid method for DNA hybridisation and isolation within a microfluidic device, where all reaction and washing steps are performed in continuous flow in an automated fashion within less than two minutes. Magnetic particles were used as a solid support and manipulated through laminar flow streams containing reagents and buffers by means of an external magnet. Thus, hybridisation, washing, intercalation, fluorescence detection and isolation were performed in continuous flow on the surface of the particles. Initially, the sensitivity of the system was investigated for a one-step DNA hybridisation of Alexa Fluor 555 labelled target DNA to a capture probe immobilised on the particle surface. Hybridisation and washing steps were performed in half a minute and target DNA was readily detected down to 20 nmol L(-1). Then a two-step assay, label-free DNA hybridisation followed by intercalation with PicoGreen was performed. All reaction and washing steps were carried out in continuous flow with a total assay time of about 1 min. This is a significant reduction in procedural time compared to conventional methods and opens the door for developing fully automated continuous flow integrated DNA analysis platforms.
Vojtísek
Martin
M
The University of Hull, Department of Chemistry, Cottingham Road, Hull HU67RX, UK.
Iles
Alexander
A
Pamme
Nicole
N
D001483:Base Sequence; D001710:Biotin; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D015342:DNA Probes / Q000235:genetics; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D008280:Magnetics; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D009693:Nucleic Acid Hybridization; D019809:Streptavidin
D015342:DNA Probes; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D001710:Biotin; D004247:DNA; D019809:Streptavidin