Graduate School of Life Science and System Engineering, Kyushu Institute of Technology, 2-4 Hibikino, Wakamatsu-Ku, Kitakyushu-Shi, Fukuoka 808-0196, Japan.
Biosensors (Basel). 2024 Sep 25;14(10):458. doi: 10.3390/bios14100458.
Trypsin enzyme has gained recognition as a potential biomarker in several tumors, such as colorectal, gastric, and pancreatic cancer, highlighting its importance in disease diagnosis. In response to the demand for rapid, cost-effective, and real-time detection methods, we present an innovative strategy utilizing the design and synthesis of NIR-sensitive dye-peptide conjugate () for the sensitive and selective monitoring of trypsin activity by fluorescence ON/OFF sensing. The current research deals with the design and synthesis of three unsymmetrical squaraine dyes , , and along with a dye-peptide conjugate as a trypsin-specific probe followed by their photophysical characterizations. The absorption spectral investigation conducted on both the dye alone and its corresponding dye-peptide conjugates in water, utilizing and respectively, reveals enhanced dye aggregation and pronounced fluorescence quenching compared to observations in DMSO solution. The absorption spectral investigation conducted on dye only and corresponding dye-peptide conjugates in water utilizing and , respectively, reveals not only the enhanced dye aggregation but also pronounced fluorescence quenching compared to that observed in the DMSO solution. The trypsin-specific probe demonstrated a fluorescence quenching efficiency of 61.8% in water attributed to the combined effect of aggregation-induced quenching (AIQ) and fluorescence resonance energy transfer (FRET). FRET was found to be dominant over AIQ. The trypsin-mediated hydrolysis of led to a rapid and efficient recovery of quenched fluorescence (5-fold increase in 30 min). Concentration-dependent changes in the fluorescence at the emission maximum of the dyes reveal that works as a trypsin enzyme-specific fluorescence biosensor with linearity up to 30 nM along with the limit of detection and limit of quantification of 1.07 nM and 3.25 nM, respectively.
胰蛋白酶酶已被确认为几种肿瘤（如结直肠癌、胃癌和胰腺癌）的潜在生物标志物，这突出了其在疾病诊断中的重要性。为了满足对快速、经济高效和实时检测方法的需求，我们提出了一种利用近红外敏感染料-肽偶联物（）设计和合成的创新策略，通过荧光 ON/OFF 传感实现对胰蛋白酶活性的灵敏和选择性监测。本研究涉及三种非对称方酸染料 、 和 以及作为胰蛋白酶特异性探针的染料-肽偶联物 的设计和合成，随后对它们的光物理特性进行了研究。在水中，利用 和 分别对染料单独及其相应的染料-肽偶联物进行吸收光谱研究，与在 DMSO 溶液中的观察结果相比，发现染料聚集增强且荧光猝灭明显。在水中，利用 和 分别对染料单独及其相应的染料-肽偶联物进行吸收光谱研究，发现不仅染料聚集增强，而且与在 DMSO 溶液中的观察结果相比，荧光猝灭明显。胰蛋白酶特异性探针 在水中的荧光猝灭效率为 61.8%，这归因于聚集诱导猝灭（AIQ）和荧光共振能量转移（FRET）的综合效应。发现 FRET 占主导地位。 的胰蛋白酶介导水解导致猝灭荧光迅速而有效地恢复（30 分钟内增加 5 倍）。染料发射峰处的荧光浓度依赖性变化表明， 作为一种具有线性度可达 30 nM 的胰蛋白酶酶特异性荧光生物传感器，其检测限和定量限分别为 1.07 nM 和 3.25 nM。
