拉曼光譜於色胺酸代謝物檢測之優化比較與中孔洞氧化石墨烯奈米粒子於細胞探針應用之整合研究

Abstract

本研究旨在建立一套具有高度再現性且具生物應用潛力之拉曼光譜檢測平台，透過多面向的實驗設計優化光譜訊號品質與分析條件。針對常見之拉曼量測不穩定問題，首先導入表面工程技術，採用三甲基矽氯烷 (Trimethylsilane Chloride, TMSCl) 修飾矽晶片之表面親疏水性，藉此提升水相樣品於乾燥後之形貌穩定性與訊號一致性，並成功建立具可預測性之點樣圖樣與重複性條件。更進一步地針對色胺酸 (Tryptophan) 及其代謝產物 (包括：5-Hydroxytryptophan, 5-HTP 與血清素, Serotonin) 進行拉曼光譜訊號的系統性優化，藉由比較多種激發波長 (457、532、785與1064 nm) 對光譜解析度與螢光干擾之影響，發現 785 nm 波長具最佳光譜分辨效果，且 1064 nm 激發條件下的訊雜比可達 250。為進一步強化實驗結果與理論模型間的對應關係，輔以密度泛函理論 (Density Functional Theory, DFT) 進行光譜特徵峰位之理論模擬與比對。此外，本研究亦探討中孔洞氧化石墨烯奈米粒子 (Mesoporous Graphene Oxide Nanoparticles, MGNs) 於不同溶液中之分散性表現，藉由動態光散射 (Dynamic Light Scattering, DLS) 與紫外–可見光吸收光譜 (UV-Vis) 進行分析，證實 MGNs 可於 6 小時內均勻分散於水相環境中。隨後將染劑吸附於分散穩定之 MGNs 表面，進行細胞共培養實驗，並藉由螢光顯微成像與螢光流式細胞儀分析其細胞內輸送效率與螢光表現。初步結果顯示 MGNs 具備進入細胞並攜帶小分子之能力，證實其作為生物探針在細胞層級應用上之可行性。本研究整合表面工程、光譜分析與奈米材料分散性之研究成果，建構一可應用於小分子檢測與細胞追蹤的拉曼光譜平台，為未來生醫檢測與奈米探針開發奠定基礎。This study aims to establish a Raman spectroscopy detection platform with high reproducibility and potential for biological applications. Through multifaceted experimental design, the spectral signal quality and analytical conditions were optimized. To address the common issue of instability in Raman measurements, surface engineering techniques were introduced by using trimethylsilane chloride (TMSCl) to modify the hydrophilic/hydrophobic properties of silicon wafer surfaces, thereby improving the morphological stability and signal consistency of aqueous samples after drying. A predictable spotting pattern and reproducible conditions were successfully established. Furthermore, the Raman spectral signals of tryptophan and its metabolic products (including 5-hydroxytryptophan and serotonin) were systematically optimized. By comparing multiple excitation wavelengths (457, 532, 785, and 1064 nm) on spectral resolution and fluorescence interference, it was found that 785 nm provided the best spectral resolution, while the signal-to-noise ratio under 1064 nm excitation could reach 250. To further strengthen the correlation between experimental results and theoretical models, Density Functional Theory (DFT) was used to simulate and compare the theoretical spectral feature peaks.In addition, this study explored the dispersion performance of mesoporous graphene oxide nanoparticles (MGNs) in different solutions. Dynamic Light Scattering (DLS) and Ultraviolet–Visible (UV-Vis) spectroscopy were used to analyze their stability, confirming that MGNs can be uniformly dispersed in aqueous environments within 6 hours. Subsequently, dyes were adsorbed onto the stably dispersed MGNs and co-cultured with cells. The intracellular delivery efficiency and fluorescence performance were analyzed through fluorescence microscopy and flow cytometry. Preliminary results showed that MGNs have the ability to enter cells and carry small molecules, confirming their feasibility as biological probes in cellular applications.This study integrates the research results of surface engineering, spectral analysis, and nanomaterial dispersion to construct a Raman spectroscopy platform applicable to small molecule detection and cellular tracking, laying the foundation for future biomedical diagnostics and nano-probe development.