Microfluidic biosensor for the detection of DNA by fluorescence enhancement and the following streptavidin detection by fluorescence quenching

• Detection of DNA or streptavidin based on fluorescence switching technique.
• ssDNA-Cy3 optical molecular beacon probes without terminally attached quencher molecule and complicated hairpin-like stem-loop structure are used in this biosensor.
• Specific detection is based on DNA hybridization or biotin–streptavidin reaction.
• Label-free detection DNA and streptavidin in the microfluidic channel.
• Microfluidic channel size and the flow rate are proved to influence the detection efficiency.

We reported an optical DNA/protein microfluidic sensor which consists of single stranded (ss) DNA-Cy3 probes on gold surface and simple line-shape microfluidic channel. These ssDNA-Cy3 probes with random sequence in bulk solution or on gold surface exhibits fluorescence enhancement after binding with complementary ssDNA (cssDNA) targets. Particularly it did not require complicated design or hairpin-like stem-loop conformation, which made it easier to be made and applied in analytes detection by fluorescence switching techniques. Using ssDNA-cy3 probes attached on gold surface in a microfluidic channel, strong fluorescence enhancement was measured by ssDNA with cssDNA binding or ssDNA with cssDNA–biotin binding. The following introduction of streptavidin resulted in fluorescence quenching (fluorescence decrease) because of the binding of hybridized DNA–biotin with streptavidin. This sensor showed strong affinity and high sensitivity toward the streptavidin, the minimum detectable concentration for streptavidin was 1 pM, equating to an absolute detection limit of 60 amol in this microfluidic channel. Microfluidic channel height and flow rate is optimized to increase surface reaction efficiency and fluorescence switching efficiency. In contrast to previously reported optical molecular beacon approach, this sensor can be used not only for the detection of cssDNA target, but also for the detection of streptavidin. This microfluidic sensor offers the promise of analyzing kinds of molecular targets or immunoreactions.