表面增強拉曼基板之發展與應用

Abstract

利用陽極氧化鋁孔洞來成長奈米銀粒子陣列基板，藉由精確的調整其銀粒子間距（5〜25奈米）使其用於表面增強拉曼光譜，其拉曼增強效果在粒子間距小於10奈米以下開始明顯改變，並在5奈米時達到最大，這樣的結果與表面耦合電漿子理論相符。為了將此增強式拉曼基板應用於細菌檢測上，這種不透明的拉曼增強基板更進一步的借由離子漂移的處理過程中，使其氧化變成透明，這種透明基板擁有拉曼增強能力的同時並有良好的光透射率、表面增強拉曼散射特性和高對比的微生物光學成像，如此的特性可用於檢測水污染物。另一方面，我們也利用萬古黴素塗布於銀粒子基板上使其成為一種特殊類型的基板，它可以用於檢測無標記的單隻細菌的表面增強拉曼光譜，這樣的特殊型基板具有約千倍的捕捉細菌能力，以及約4〜5倍的細菌表面增強拉曼信號增強，且其細菌光譜不會受到萬古黴素的干擾。我們還應用奈米銀粒子拉曼增強基板於辨別細菌細胞壁的精細結構上，以此基板建構的表面增強拉曼光譜平台，可以敏銳和穩定的反映出不同細胞壁結構差異的細菌，如革蘭氏陽性菌、革蘭氏陰性菌或分枝桿菌。此外細菌對抗生素的藥物敏感性，利用細菌的拉曼光譜上在投藥後的變化，可在1個小時左右的時間就可得知，這使得我們可以快速的來區分細菌的耐藥性，這種表面增強拉曼光譜來診斷的方式，也適用於單隻的細菌，使得利用增強拉曼光譜來快速微生物檢測的方式，不再需要長時間靠培養細菌就可以直接對臨床樣品做檢測。

Arrays of silver nanoparticles grown on anodic alumina nanochannels (Ag-NPs/AAO) with precisely tunable gaps (5~25 nm) are fabricated as substrate for using on surface-enhanced Raman spectroscopy. The Raman enhancement becomes significant for gaps below 10 nm and turns dramatically large when gaps reach an unprecedented value of 5 nm. The results are quantitatively consistent with theories based on collectively coupled surface plasmon. These opaque Raman enhancing substrates (Ag-NPs/AAO) have been further rendered transparent by an ion-drift process to complete the oxidation. The transparent substrates exhibit Raman enhancing capability and good optical transmissivity, allowing for concurrent surface-enhanced Raman scattering (SERS) characterization and high contrast transmission-mode optical imaging of microorganisms as well as in-situ detection of dilute water pollutants. On the other hand, we show that vancomycin coating of a special type of substrate covered by Ag-NPs/AAO, which can provide label-free detection of single bacteria via surface enhanced Raman spectroscopy, leads to ~1000 folds increase in its capability to capture bacteria; and 4~5 folds increase in the SERS signal of captured bacteria without introducing significant spectral interference. We also apply the Ag-NPs/AAO SERS substrate to assess the fine structures of the bacterial cell wall. The SERS profiles recorded by such a platform are sensitive and stable, that could readily reflect different bacterial cell walls found in Gram-positive, Gram-negative, or mycobacteria groups. Moreover, characteristic changes in SERS profile were noticed in the drug-sensitive bacteria at the early period (i.e., ~1 hr) of antibiotic exposure, which could be used to differentiate them from the drug-resistant ones. The SERS-based diagnosis could be applied to a single bacterium. The high-speed SERS detection represents a novel approach for microbial diagnostics. The single bacterium detection capability of SERS makes possible analyses directly on clinical specimen instead of pure cultured bacteria.