DNA diagnosis in a microseparator based on particle aggregation

A novel aggregation-based biosensing method to achieve detection of oligonucleotides in a pinched-flow fractionation (PFF) microseparator was developed. Employing functionalized polystyrene microspheres, this method is capable of the direct detection of the concentration of a specific DNA sequence. The label-free target DNA hybridizes with probe DNA of two kinds on the surface of the microspheres and causes the formation of an aggregate, thus increasing the average size of the aggregate particles. On introducing the sample into a PFF microseparator, the aggregate particles locate at a specific position depending on the size of the aggregate. Through a multi-outlet asymmetric PFF microseparator, the aggregate particles become separated according to outlets. Because the size of the aggregate particles is proportional to the concentration of the target DNA, a rapid quantitative analysis is achievable with an optical microscope. A biological dose-response curve with concentration in a dynamic range 0.33-10 nM has been achieved; the limit of detection is between 33 and 330 pM. The specificity of the method and the potential to detect single-nucleotide polymorphism (SNP) of known concentration were examined. The method features simple, direct and cheap detection, with a prospect of detecting other biochemical samples with distinct aggregation behavior, such as heavy-metal ions, bacteria and proteins.