Dielectrophoretic detection of molecular bindings

The specific molecular binding between the `target´ and the `probe´ molecule, for example between antigen and antibody or DNA and oligonucleotide, is the principle of affinity assays. In the assay, the probe is labeled with fluorescence, mixed with the target to form the target-probe complex, and the free probe is separated and removed (Bound/Free separation). The fluorescence from the residue is proportional to the amount of the target-probe complex and thus is the measure of the target concentration in the original sample. Here, the authors propose and experimentally demonstrate the use of dielectrophoresis (DEP) for such B/F separations. Two methodologies are developed. (1) Using DEP chromatography, separation of λ-DNA (48.5 kbp) and oligonucleotide (22-base), and quantitative detection of biotinylated DNA by anti-biotin antibody, are shown. (2) Using the triple complex formation to facilitate the DEP separation, a method is developed to detect B/F binding by a direct observation of the separation pattern on the micro electrode system. The method is applied for quantitative detection of diagnostic marker of the liver cancer alpha-fetoprotein (AFP) through antigen-antibody reaction, and the detection of DNA sequence through hybridization. The methods developed here is compatible with micro fabrication, and suitable for affinity assays in micro Total Analysis Systems