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Posted: Apr 18, 2011
Sensitive label-free DNA sensing based on metal/fluorophore interactions
(Nanowerk News) With two recent studies, imec scientists contribute to the field of label-free DNA sensing. The measurement technique they have refined is based on the fact that metallic films and nanoparticles absorb the light of nearby light-emitting fluorophores. In one study, the quenching and enhancement of the emitted light was studied and quantified with a wide range of gold nanoparticles and DNA hairpin probes. In a second study and using similar probes, the technique was used to demonstrate a functional label-free genosensor.
Metals are ultra-efficient quenchers of light-emitting fluorophores. When fluorophore molecules are located within a few nanometers from a metal surface, their fluorescence signal is suppressed. When they are moved further away from the metal (e.g. by some spacer molecule), an enhanced fluorescence signal is measured. This phenomenon of clearly distinct quenched and enhanced signals can be used for biosensing applications such as DNA detection.
DNA hairpin probes are single-stranded DNA sequences that are folded through their outer ends that bind to each other. The middle part of the strands is complementary to the DNA sequence that we want to detect. Here we used hairpin probes that have at one end a fluorophore molecule attached while at the other end they are bound to the metal surface (gold). In the absence of the target DNA sequence, the hairpin remains closed and the fluorophore is close to the metal. But when the DNA sequence of the hairpin meets its complimentary strand, the strands hybridize and the hairpin opens. At that moment, the fluorophore molecule is further away from the metal, resulting in an enhanced fluorescence signal.
In one study, we examined fluorescence quenching and enhancement near gold nanoparticles of various sizes, and with DNA hairpin probes of different lengths. As a result, we report a 96.8% quenching efficiency for all particles sizes tested, and a more than complete signal recovery after DNA hybridization. Initial sensing experiments indicate a detection sensitivity of 100pM and below. Previous studies had not been able to relate light quenching and enhancement to the number of fluorescence molecules involved. We were able to quantify the number of fluorophores attached to DNA hairpin probes using a novel thiol exchange method. This allowed us to examine and quantify both the quenching efficiency and enhancement factor.
In a second development, we made a label-free genosensor based on DNA hairpins immobilized on a gold surface under microfluidic conditions. We used a microfluidic integrated instrument based on surface plasmon resonance combined with a gold chip. This instrument was first used to immobilize the DNA-fluorophore hairpins and then to promote the hybridization of the target molecules. The fluorescent signal was visualized with fluorescence microscopy. We experimented with the various design options of this sensor, investigating the detection sensitivity and specificity. With our resulting sensor, we reached a detection sensitivity of 300pM.