Department of Nano-Physics, Gachon University, Seongnam, 13120, Republic of Korea.
Gachon Bionano Research Institute, Gachon University, Seongnam, 13120, Republic of Korea.
Small. 2018 Aug;14(32):e1801385. doi: 10.1002/smll.201801385. Epub 2018 Jul 12.
Plasmonic enhancement of fluorescence from SYBR Green I conjugated with a double-stranded DNA (dsDNA) amplicon is demonstrated on polymerase chain reaction (PCR) products. Theoretical computation leads to use of the bimetallic (Au 2 nm-Ag 50 nm) surface plasmons due to larger local fields (higher quality factors) than monometallic (Ag or Au) ones at both dye excitation and emission wavelengths simultaneously, optimizing fluorescence enhancement with surface plasmon coupled emission (SPCE). Two kinds of reverse Kretschmann configurations are used, which favor, in signal-to-noise ratio, a fluorescence assay that uses optically dense buffer such as blood plasma. The fluorescence enhancement (12.9 fold at maximum) with remarkably high reproducibility (coefficient of variation (CV) < 1%) is experimentally demonstrated. This facilitates credible quantitation of enhanced fluorescence, however unlikely to obtain by localized surface plasmons. The plasmon-induced optical gain of 46 dB due to SPCE-active dye molecules is also estimated. The fluorescence enhancement technologies with PCR enables LOD of the dsDNA template concentration of ≈400 fg µL (CV < 1%), the lowest ever reported in DNA fluorescence assay to date. SPCE also reduces photobleaching significantly. These technologies can be extended for a highly reproducible and sufficiently sensitive fluorescence assay with small volumes of analytes in multiplexed diagnostics.
双金属（Au 2 nm-Ag 50 nm）表面等离激元由于在染料激发和发射波长同时具有更大的局域场（更高的品质因数），因此优于单金属（Ag 或 Au）表面等离激元，从而实现了与表面等离激元耦合发射（SPCE）的荧光增强。使用了两种反向克来希曼配置，这有利于使用光学密度缓冲液（如血浆）进行荧光分析，从而提高信噪比。实验证明了具有高重复性（变异系数（CV）<1%）的荧光增强（最大时为 12.9 倍）。这有助于对增强的荧光进行可信的定量，而通过局域表面等离激元不太可能获得。由于 SPCE 活性染料分子，等离子体诱导的光增益为 46 dB。PCR 结合的荧光增强技术可将 dsDNA 模板浓度的检测限（LOD）降低到 ≈400 fg µL（CV<1%），这是迄今为止 DNA 荧光分析中报告的最低检测限。SPCE 还显著减少了光漂白。这些技术可以扩展到具有高重复性和足够灵敏度的荧光分析，用于在多重诊断中分析小体积的分析物。
