Colorimetric Gene assay Using DNA-Modified Nanoparticles

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1. Preparation of the ODN (oligonudeotide) -modified nanoparticles and preliminary study of their aggregationAmmo-terminated ODNs were modified onto carboxylates-modified nanoparticles using water soluble carbodiimide (EDAC). Complementary ODN made the particle aggregate under appropriate conditions. The dimension of the aggregates reached tens ofμm and we could easily observe them using ordinary fluorescence microscopy with corresponding optical filters.2. Aggregates in binary system (red-green)Two 45 mer ODNs, one of which is a part of wild type p53 gene and the other is the mutant containing one base substitution, were used as model gene samples. Two 25 mer ODNs, each of which contains the 15 nudeotide sequence complementary to each of the terminus of fhe wild type 45 mer, were immobilized onto red and green nanoparticles, respectively. The wild type gathered the both spheres to produce aggregate that emits yellow light (yellow=red+green) under the appropriate conditions. On the other hand, the mutant scarcely affects the distribution of the both spheres. Interestingly the aggregates with the wild type also showed characteristic emission based on the FRET (fluorescence resonance energy transfer) from green particles to closely contacted red particles (from green to red).3. Aggregates in ternary system (red-green-blue)Aggregation experiments in binary system mentioned above were extended to the ternary system, in which mixed solution of differently colored (R/G/B) three kinds of tbe ODN-modified nanoparticles were adopted. The wild type and the mutant 45 mers gave the aggregates with yellow (Y=R+G) and magenta (M=R+B) on the whole, respectively.Improvement of the method for ODN modification onto the particles should help to obtain more distinct results. Colorimetric SNP analysis proposed in this study would be an alternative for high-throughput gene diagnosis and analysis.