By applying molecular logic, researchers at Tufts University have demonstrated what could be the foundation for intelligent medical diagnostics. Their fluorescence-based approach could directly screen for medical conditions where multiple abnormalities are present. An example of how such a combination test might work would be the simultaneous detection of the protein coat and the DNA of a virus. Another possibility could be the detection of the DNA and a protein from a cancer cell. The approach would pay dividends because it increases diagnostic certainty, said chemistry professor David R. Walt, lead researcher. “Just detecting genetic material does not necessarily constitute a positive diagnosis. Measuring both DNA and protein significantly improves the confidence of a positive diagnosis.” Key to the technique is the relatively new idea of molecular logic, which is equivalent to the electronic logic that forms the basis for computers. In molecular logic, the inputs are molecules of interest, and there are logic gates that produce an output dependent on the inputs. An example is an AND gate, which outputs a “1” only if both inputs are present. In the method developed by Walt and former postdoctoral researcher Tania Konry, one input is a protein and the other, a target DNA. The output is fluorescence from a dye. Thus, if both the protein and target DNA are present at a high enough concentration, the result is fluorescence above a threshold at given wavelengths. In a proof of the concept, described in the Journal of the American Chemical Society on the Web on Aug. 28, 2009, the researchers polished and etched the ends of bundled optical fibers, creating microwells in the 3.1-µm-diameter fibers. They loaded the wells with monoclonal antibody-functionalized microspheres, which had the same diameter as the wells. Each microsphere had a unique optical bar code – courtesy of europium dye encoding – enabling identification through image processing using a custom-built epifluorescence system. For the detection step, the investigators used two fluorescent probes, one labeled with the fluorophore Cy3 and the other, with Cy5. Because of its construction, the first probe hybridized when both the protein IL-8 and the target DNA were present. The second was constructed so that Cy5 fluorescence occurred only if the protein was present and the target DNA was not. By looking at the fluorescence and assigning a 0 to everything below a threshold and a 1 to everything above, the researchers could determine whether both protein and DNA were present. Achieving the right response required some tweaking of the assay conditions to avoid false positives, while still reacting when appropriate concentrations of DNA or proteins were present. As for the future and applications outside of the lab, the all-or-nothing nature of the technique offers an advantage because the strength of the fluorescence is not critical. As Walt noted, “Our goal was to take things from an intensity-based measurement to an on-off signal. Consequently, the sensitivity and specificity are built into the assay, enabling a simple array readout.”