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Background: The sensitivity and selectivity of traditional methods limits ultramicro detection of proteins. Bio-barcode amplification detection methods based on nanotechnology enables ultramicro detection of protein. However, bio-barcode amplification detection depends on the oligonucleotides being fixed on a glass chip. It also requires specialized equipment, which limits its application. We introduce a nano-nucleic acid barcode dot detection technology to determine ultramicro concentrations of protein. The method is simple, quick and accurate. Methods: Magnetic probe (IgG-M) and dual-labeled gold nanoparticle bio-probe (IgG-Au-DNA) were prepared. Protein was captured using a sandwich assay technique and magnetic separation was used. The DNA barcode was released with dithiothreitol (DTT) and detected directly without the requirement for polymerase chain reaction (PCR). Serum prostate-specific antigen (PSA) from 135 patients was detected with this method and compared with enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Results: Each IgG-Au-DNA could be covered with 138±47 oligonucleotides and 11±3 antibodies. The IgG-M could bind 118 μg of antibody per mg. The sensitivity of nano-nucleic acid barcode dot detection technology might allow detection of 1 fg/mL. There were no significant differences in serum PSA from 135 patients when comparing the three methods (compared with ELISA, r=0.950; and with RIA, r=0.967). Conclusions: The nucleic acid barcode dot method does not require special equipment or complex procedures, but its detection limit is 2–3 orders of magnitude lower than ELISA. Clin Chem Lab Med 2010;48:279–83. [ABSTRACT FROM AUTHOR]