Single-molecule immunoassay and DNA diagnosis.
Many assays relevant to disease diagnosis are based on electrophoresis, where the migration velocity is used for distinguishing molecules of different size or charge. However, standard gel electrophoresis is not only slow but also insensitive. We describe a single-molecule imaging procedure to measure the electrophoretic mobilities of up to 100000 distinct molecules every second. The results correlate well with capillary electrophoresis (CE) experiments and afford confident discrimination between normal (16.5 kbp) and abnormal (6.1 kbp) mitochondrial DNA fragments, or beta-phycoerythrin-labeled digoxigenin (BP-D) and its immunocomplex (anti-D-BP-D). This demonstrates that virtually all electrophoresis diagnostic protocols from slab gels to CE should be adaptable to single-molecule detection. This opens up the prossibility of screening single copies of DNA or proteins within single biological cells for disease markers without performing polymerase chain reaction (PCR) or other biological amplification.
Department of Chemistry, Iowa State University, Ames 50011, USA.
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Ma
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Shortreed
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Li
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Huang
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Yeung
D000936:Antigen-Antibody Complex; D001583:Benzoxazoles; D004272:DNA, Mitochondrial / Q000032:analysis*; D004076:Digoxigenin / Q000276:immunology; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D007118:Immunoassay; D010799:Phycoerythrin / Q000276:immunology; D011806:Quinolinium Compounds
D000936:Antigen-Antibody Complex; D001583:Benzoxazoles; D004272:DNA, Mitochondrial; D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; D010799:Phycoerythrin; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; D004076:Digoxigenin