高熵普魯士藍類似物應用於茶鹼電化學感測之研究

Abstract

甲基黃嘌呤類化合物（methylxanthine derivatives）廣泛存在於茶、咖啡、巧克力及能量飲料中，其中咖啡因（caffeine）、茶鹼（theophylline）與可可鹼（theobromine）因具藥理活性，於臨床治療藥物監測與食品安全管控上具有重要的定量分析需求。傳統儀器分析方法（如 HPLC）雖具備高準確度，但儀器昂貴且前處理繁瑣，難以滿足現場快速篩檢之需求。電化學感測技術以其微型化、低成本與快速響應之優勢，提供了替代方案；然而，普魯士藍（Prussian Blue, PB）及其類似物（PBA）在中性至弱鹼性環境下，常因氰基鍵結受 OH⁻ 攻擊而造成結構崩解與訊號快速衰退，限制其於近生理環境之實際應用。本研究引入高熵材料設計策略，以共沉澱法將 Fe、Mn、Co、Ni 與 Cu 五種過渡金屬同步導入 PBA 氰基框架，合成高熵普魯士藍類似物（High-Entropy Prussian Blue Analogue, HEPBA），並以滴塗方式修飾於玻璃碳電極（GCE）表面，探討其材料特性、電化學行為與甲基黃嘌呤類似物感測性能。材料表徵方面，透過EDS、SEM、FTIR與XRD確認所合成的HEPBA具有均勻且單相的結構，並確認不同的製程條件並不會影響其晶格結構。電化學分析方面，中性循環穩定性測試（pH 7.4，0–1.3 V）顯示 HEPBA 循環 30 圈後仍保留約 70% 的電荷量，遠優於傳統 PB 在 20 圈後僅剩 24% 的表現，定量驗證了高熵化對中性環境電化學穩定性之提升效益。感測測試方面，HEPBA/GCE 對茶鹼於約 1.2 V 呈現最清楚的濃度依賴性氧化峰，咖啡因與可可鹼因結構差異而訊號較不明顯。以 pH 3 對電極進行預掃描可活化 HEPBA 表面金屬位點，使靈敏度提升約 75%，R² 由 0.9040 改善至 0.9493；KCl 電解質在靈敏度、線性與背景穩定性之間提供最佳平衡，對應偵測極限（LOD）為 128.9 μM。干擾物測試顯示，抗壞血酸（AA）與尿酸（UA）對茶鹼感測構成嚴重正干擾（分別達 121.4% 與 98.9%）；10% 胎牛血清（FBS）則透過蛋白質非特異性吸附造成 65.1% 之基質效應，此效應在本質上不同於特定分子的電化學氧化干擾，屬於複雜基質整體環境改變所致。本研究結果表明，HEPBA 作為新型多金屬感測材料，在中性環境穩定性上確實優於傳統 PB，並初步驗證了高熵配位聚合物跨域應用於有機小分子電化學感測的可行性。

Methylxanthine derivatives are widely present in tea, coffee, chocolate, and energy drinks. Among them, caffeine, theophylline, and theobromine possess pharmacological activities and therefore require reliable quantitative analysis for therapeutic drug monitoring and food safety control. Conventional instrumental methods, such as high-performance liquid chromatography (HPLC), provide high analytical accuracy; however, their high cost, complicated pretreatment procedures, and limited suitability for on-site rapid screening restrict their practical use. Electrochemical sensing offers an alternative approach because of its potential for miniaturization, low cost, and rapid response. Nevertheless, Prussian Blue (PB) and Prussian Blue analogues (PBA) often suffer from structural collapse and rapid signal decay in neutral-to-weakly alkaline environments due to hydroxide attack on the cyano-bridged framework, limiting their application under near-physiological conditions.In this study, a high-entropy material design strategy was introduced to synthesize high-entropy Prussian Blue analogues (HEPBA). Five transition metals, Fe, Mn, Co, Ni, and Cu, were simultaneously incorporated into a PBA cyano-bridged framework through a co-precipitation method. The obtained HEPBA was then modified onto a glassy carbon electrode (GCE) by drop casting to investigate its material characteristics, electrochemical behavior, and sensing performance toward methylxanthine derivatives. Material characterization by EDS, SEM, FTIR, and XRD confirmed that the synthesized HEPBA exhibited a uniform and single-phase structure, and that different preparation conditions did not significantly change the PBA-type lattice structure.Electrochemical analysis showed that HEPBA exhibited improved cycling stability under neutral conditions. In pH 7.4 electrolyte within a potential window of 0-1.3 V, HEPBA retained approximately 70% of its charge after 30 cycles, which was markedly better than conventional PB, which retained only 24% after 20 cycles. These results quantitatively demonstrate that the high-entropy strategy can enhance the electrochemical stability of PBA-based materials in neutral environments. For sensing tests, HEPBA/GCE showed the clearest concentration-dependent oxidation peak toward theophylline at approximately 1.2 V, whereas the responses of caffeine and theobromine were less distinct due to structural differences and signal overlap. Pre-scanning the electrode at pH 3 activated the surface metal sites of HEPBA, corresponding to an improvement of approximately 75%. Among the tested electrolytes, KCl provided the best balance among sensitivity, linearity, and background stability, with a corresponding limit of detection (LOD) of 128.9 μM.Interference tests indicated that ascorbic acid (AA) and uric acid (UA) caused severe positive interference in theophylline sensing, with interference ratios of 121.4% and 98.9%, respectively. In contrast, 10% fetal bovine serum (FBS) caused a matrix effect of 65.1% through non-specific protein adsorption. This effect is fundamentally different from the electrochemical oxidation interference caused by specific molecules and should be attributed to the overall change in the complex sample matrix. Overall, this study demonstrates that HEPBA, as a novel multi-metal sensing material, provides better neutral-environment stability than conventional PB and verifies the feasibility of applying high-entropy coordination polymers to electrochemical sensing of organic small molecules.