建構一以奈米金球為基礎的光學生物感測平台用於蛋白質酶 活性之檢測

Abstract

奈米金球（gold nanoparticles, AuNPs）具有獨特的物理特性，並且在光學上具有表面電漿共振吸收（surface plasmon resonance, SPR）的現象而備受注目。由於奈米金球的吸收光譜會因大小、形狀，或表面修飾上有機分子而有所差異，因此本研究利用AuNPs之SPR特性，建構出一光學式生物感測平台用於蛋白質酶（proteinase）活性之檢測。 本研究首先將明膠（gelatin）修飾於13 nm的AuNPs上作為proteinase之受質，同時修飾上6-醯基己-1-醇（6-mercapto-1-hexanol, MCH）當作誘導子。當AuNPs修飾上gelatin與MCH時，gelatin所造成的空間障礙可防止金球彼此間的距離拉近，避免AuNPs產生聚集。因此AuNPs/MCH-gelatin可穩定存在於極端的檢測環境中。當proteinase中的胰蛋白酶（trypsin）或基質金屬蛋白質酶-2（matrix metalloproteinase-2, MMP-2）降解AuNPs/MCH-gelatin表面上的受質後，AuNPs失去保護，同時MCH增強AuNPs彼此間的吸引力，AuNPs彼此間距離逐漸靠近，因而產生聚集。AuNPs聚集時，其SPR會有紅位移（red-shift）現象，並且AuNPs的呈色會由原先的酒紅色轉變為紫色。此外，AuNPs之顏色變化可直接以肉眼觀察，且最大吸收波峰值（λmax）可經由UV/Vis spectroscopy量測之。 在此以奈米金球為基礎的光學式生物感測平台中，利用AuNPs的吸光值比值（A625 nm/A525 nm）估計proteinase之活性，針對trypsin之偵測線性範圍為5 × 10-1至5 × 102 U，偵測極限為5 × 10-1 U；而針對MMP-2測得的線性範圍為50 ng/mL到600 ng/mL，偵測極限為50 ng/mL。此外利用兩種MMP-2抑制劑，galardin及ONO-4817進行藥物篩檢方面研究。利用此奈米金球系統針對galardin及ONO-48進行檢測，其IC50值分別為1.87 nM 以及17.76 nM，而以酶譜分析法（zymography）所測得之IC50值分別為3.48與14.33 nM，結果顯示此兩種方式所測得之結果有高度一致性。然而此光學式生物感測系統可將偵測時間縮短到30分鐘內。因此，此快速且敏感性高的奈米金球光學生物感測平台具有相當潛力運用於anti-MMPs藥物篩檢及MMPs相關疾病診斷上。

Gold nanoparticles (AuNPs) have interesting physical and optical property on surface plasmon resonance (SPR). Especially, AuNPs display different absorption spectra when the AuNPs possess different sizes, shapes, or being functionalized with chemical molecules. In this study, an optical biosensing platform was established and was based on the SPR property of AuNPs for the measurement of proteinase activity. First, the 13 nm AuNPs was modified with gelatin as proteinase substrate and then modified with 6-mercapto-1-hexanol (MCH) as an inducer. When AuNPs was modified with gelatin and MCH (AuNPs/MCH-gelatin), the gelatin increased the steric repulsion of AuNPs, and prevented the AuNPs surfaces from coming into close contact. For this reason, the AuNPs/MCH-gelatin could disperse in solution, and exhibited a dramatic stability in strict environment. After the proteinase (trypsin or matrix metalloproteinase-2 [MMP-2]) digested the substrate on AuNPs/MCH-gelatin, the AuNPs lost shelter and MCH increased the attraction force between AuNPs. Therefore, the AuNPs were close to each other and gradually resulted in aggregation. The AuNPs aggregation could be monitored by a red-shift in surface plasmon absorption and color would change from wine-red to violet-purple. Furthermore, the color change can be observed by naked eyes, and the maximum wavelength (λmax) was measured by the UV/Vis spectroscopy. In the AuNPs-based optical biosensing platform, the absorption ratio (A625 nm/A525 nm) of AuNPs was used to quantitively estimate the proteinase activity, and a linear range of trypsin detected by the AuNPs-based optical biosensing platform was from 5 × 10-1 to 5 × 102 U and with a detection limit of 5 × 10-1 U. A linear correlation was established when the MMP-2 activity was from 50 ng/mL to 600 ng/mL, and with a detection limit of 50 ng/mL. Besides, two MMP-2 inhibitors, galardin and ONO-4817, were used in this study for feasibility analysis of drugs screening. In the AuNPs-based method, the MMPs activity for IC50 values of galardin and ONO-4817 were 1.87 nM and 17.76 nM, respectively. In comparision with zymography, MMP-2 activity for IC50 values of galardin and ONO-4817 were 3.48 and 14.33 nM, respectively. The result of the AuNPs-based method was consistent with that of zymography method. Furthermore, the AuNPs-based method can shorten detection time to less than 30 min. Thus, the rapid and sensitive novel AuNPs-based optical biosensing platform had potential for further applications in anti-MMPs drug screening and for MMP-related diseases diagnosis.