Jun 3, 2005
Detecting biomolecules with fluorescently-labelled DNA
A variety of applications would benefit from the ability to detect and identify single biological molecules, such as a rare target molecule, in a complex mixture. We are particularly interested in exploring the possibilities for medical diagnosis, such as detecting chemical indicators of disease in body fluids or analyzing the components of small volumes of material such as that from a single cell.
Nucleic acid engineers have developed nanoscale fluorescent labels using DNA as a structural material. These labels contain several fluorescent molecules of two or more colours. They can be used to uniquely identify single biomolecules by selective chemical binding to colour-coded labels. In order for these labels to be applicable for single molecule technology, they must be individually detectable and identifiable by their fluorescence.
In our experiment, we used a submicrometre fluidic channel in glass to guide the labels, one at a time, through two focused laser beams. Here the two colours of fluorescent dye molecules were detected using a confocal microscope and photon counting. The small fluidic channel is a suitable architecture for this type of single molecule study. Because of the submicrometre channel width and depth, labels are confined to move through a small detection volume of almost uniform laser intensity. This means that all labels were uniformly analyzed, which is an important factor to consider when fluorescence emission is being quantified.
Because the fluid flow and particle motion are controllable and can be done in parallel, channels also help to detect a statistically significant number of molecules in a short time. And the small optical excitation volume reduces the amount of extraneous material that may, particularly in a mixture, contribute to noise in the optical measurements.
We tested several labels, including a label with one green and three red dye molecules, 1G3R, and its inverse, 3G1R. We found in these initial results that 1G3R and 3G1R were approximately 80% identifiable based on their fluorescent emission. This suggests that with a large enough difference in the number of dye molecules, single molecule labels can be used to identify biomolecules with a reasonably high degree of accuracy.
Future work includes tagging biomolecules of interest with a variety of labels, and identifying mixtures of such labeled molecules with the help of submicrometre fluidic channels. This could ultimately be incorporated as part of a read-out method for an integrated microfluidic system involving sampling, filtering, concentration and separation of complex samples.
About the author
Harold Craighead is professor of applied and engineering physics at Cornell University, US.