Light scattering optical waveguide method for detecting specific binding events

A method for determining base differences between at least two nucleic acid sequences, the method comprising:

• (a) providing a waveguide device, the waveguide device comprising (i) a transparent element having a refractive index greater than that of the fluid sample;
• (ii) a light receiving edge;
• and (iii) a reactive surface comprising a plurality of sites having oligonucleotide immobilized thereon, said sites defining an array of oligonucleotides, said reactive surface further comprising other non-situs portions having no oligonucleotides immobilized thereon;
• (b) contacting the reactive surface under hybridizing conditions with said at least two nucleic acid sequences wherein said nucleic acid sequences, either directly or through intermediate cognate binding pairs, is labeled with a light scattering label;
• thereby forming light scattering label complexes attached at those sites of the reactive surface having an oligonucleotide complementary to said nucleic acid sequences immobilized thereon;
• (c) illuminating the light receiving edge of the waveguide with light effective to create total internal reflection within the waveguide, thereby simultaneously illuminating the entire reactive surface;
• (d) collecting light scattered across the entire reactive surface by said light scattering label;
• (e) comparing the degree of light scattering at each situs with either (i) the degree of light scattering at a non-situs portion;
• or (ii) the degree of light scattering at another situs;
• (f) incrementally increasing the stringency conditions at the reactive surface of the waveguide device to initiate dissociation of bound nucleic acid from the sites wherein said comparing of step (e) is continued during the incremental stringency increase;
• whereby base differences between the at least two oligonucleotides can be distinguished by differences in dissociation properties.