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Abstract: Based on gold-labeled silver stain (GLSS) method, we developed the visual protein microarray for simultaneous, sensitive, and specific detection of Ureaplasma parvum and Chlamydia trachomatis using N-terminus multiple-banded antigen (NMBA) of U. parvum and major outer membrane protein of C. trachomatis. The specific antigens were immobilized on glass surface that was treated with 3-glycidoxypropyltrimethoxysilane, and they were used as the capturing probes to recognize the complementary target antibodies binding to the detecting probes of Nano-gold-Staphylococcal protein A (SPA). In the “sandwich” format, Nano-gold-SPA probe was used as an indicator and GLSS was applied to amplify the detection signals and produce black image on array spots, which were visible with naked eyes. In our model arrays, the detection limit of protein microarray was as low as 2 ng/mL, and the lowest titer of detectable antibody was 1:128; thus, this sensitivity was comparable to the fluorescent detection method. The visual simultaneous protein microarrays were used to detect total 186 clinical samples, which had been determined by enzyme-linked immunosorbent assay (ELISA) and fluorescence quantitative real-time polymerase chain reaction; the results were identical and no distinct difference (P > 0.05) existed between them. Our results demonstrate that we have developed the visual protein microarray technique, which is of high sensitivity and high specificity, and it may have potential in clinical applications. [Copyright &y& Elsevier]