介绍 /Introduction

胰蛋白酶是胰腺功能和病理变化的特异且可靠的诊断生物标志物。因此，检测胰蛋白酶和筛选胰蛋白酶抑制剂对于临床诊断和疾病治疗至关重要。在此，本文提出了一种基于碳量子点（O-CDs）和细胞色素 C（Cyt C）的比色/荧光传感探针，用于灵敏且特异的胰蛋白酶检测和胰蛋白酶抑制剂筛选。所制备的 O-CDs 具有持久荧光和环境耐受性。在胰蛋白酶存在的情况下，Cyt C 被水解成具有氧化酶模拟活性的血红素，可将无色的 TMB 转化为蓝色的 oxTMB，其在 652 纳米处有强吸收，并通过内滤效应淬灭 O-CDs 在 580 纳米处的荧光。该传感探针实现了胰蛋白酶的精确定量，具有宽线性范围（比色和荧光模式下均为 10 - 2000 纳克/毫升）和低检测限（比色模式下为 0.05 纳克/毫升，荧光模式下为 3.44 纳克/毫升）。 所开发的方法随后被用于检测人血清和尿液中的胰蛋白酶，具有良好的准确性和重现性。此外，该传感探针还成功地用于筛选豆类中的胰蛋白酶抑制剂，其依据是这些抑制剂抑制胰蛋白酶活性的能力。因此，这种双模式检测方法在胰蛋白酶相关疾病的临床诊断以及胰蛋白酶抑制剂药物的筛选方面具有巨大的应用潜力。 相关成果以 “Dual-Mode Detection of Trypsin and Screening of Trypsin Inhibitors on the Basis of a Colorimetric/Fluorescence Sensing Probe” 为题发表在 Anal. Chem. 。

研究图文/Introduction

Figure 1. Dual-mode detection of trypsin and screening of trypsin inhibitors on the basis of a colorimetric and fluorescence sensing probe.

在这项研究中，开发了一种基于碳量子点（O-CDs）和Cyt C的新型比色/荧光复合传感探针，用于双模式胰蛋白酶检测（图1）。胰蛋白酶水解Cyt C产生具有氧化酶（OXD）样活性的血红素肽段，由于TMB氧化为oxTMB，在652nm的吸收光谱上呈现蓝色。所得的oxTMB通过内部滤光效应（IFE）在580nm处淬灭O-CDs的荧光。因此，在抑制剂存在或不存在的情况下，胰蛋白酶活性的双模式检测可以通过监测比色/荧光信号的变化来实现。这种双重检测方式可以防止假阴性或假阳性结果，满足各种分析情况的不同需求。此外，该方法还可用于筛选豆科植物中胰蛋白酶抑制剂。

Figure 2. (A) HRTEM image (bar: 5 nm), (B) particle size distribution, and (C) EDS spectra of the O-CDs. (D) Absorption and emission spectra of the O-CDs; the inset is a photo of the O-CDs under daylight and 365 nm light.

Figure 3. (A) Oxidase mimetic activity produced upon tryptic hydrolysis of substrate Cyt C. (B) Absorbance of oxTMB in the TrpCyt C system under O2 and N2. (C) The absorbance of oxTMB gradually decreased with increasing SOD concentration. (D) ESR spectrum of O2•- in the Trp-Cyt C system.

Figure 4. (A) Fluorescence intensities of Trp-Cyt C, Trp, Cyt C, and TMB in the presence of trypsin (1 μg/mL). (B) Fluorescence spectrum of the O-CDs and absorbance spectrum of oxTMB. (C) Fluorescence decay curves of the O-CDs with and without oxTMB. (D) Histograms of the absorbance values of the O-CDs, Trp-Cyt C + TMB, and O-CDs + Trp-Cyt C + TMB at 652 nm.

Figure 5. (A) Absorbance spectra of trypsin at different concentrations (10-2000 ng/mL) at 652 nm. (B) Fluorescence spectra of trypsin at different concentrations (10-2000 ng/mL) at 580 nm. (C) Histogram of the absorbance values of ions and small interfering molecules at 652 nm. (D) Histogram of the F0/F ratios of ions and small interfering molecules at 580 nm. (E) Histogram of the absorbance values of ions and common protein interfering substances at 652 nm. (F) Histogram of the F0/F ratios of common protein interfering substances at 580 nm. The concentration of trypsin was 10 μg/mL, and the concentration of each interfering substance was 100 μg/mL. F0 and F represent the fluorescence intensities at 580 nm in the absence and presence of trypsin, respectively, upon excitation at 350 nm. All of the above experiments were repeated at least three times (n = 3).

Figure 6. (A) Absorption spectra of different concentrations of trypsin (10-6000 ng/mL) in human serum. (B) Fluorescence spectra of different concentrations of trypsin (10-8000 ng/mL) in human serum. (C) Absorption spectra of different concentrations of trypsin (10-10,000 ng/mL) in human urine. (D) Fluorescence spectra of different concentrations of trypsin (10-2000 ng/mL) in human urine. (E) Histogram of the decrease in Trp-Cyt C complex absorption upon treatment with heat-inactivated trypsin. (F) Histogram of the decrease in Trp-Cyt C complex fluorescence upon treatment with heat-inactivated trypsin.

Figure 7. Curves of trypsin inhibition in (A) colorimetric and (B) fluorescence modes. The established (C) colorimetric and (D) fluorescence methods were verified by the application of positive drugs (TIS, GM, and UTI) and negative drugs (D-GLU, ENAL, and NEO). Screening trypsin inhibitors in legume extracts (500 μg/mL) was performed in (E) colorimetric and (F) fluorescence modes.

结论 /Conclusion

在本研究中，成功开发了一种基于细胞色素 C（Cyt C）、3,3',5,5'-四甲基联苯胺（TMB）和 O-碳点（O-CDs）的比色/荧光双模式方法，用于检测胰蛋白酶及其抑制剂。该方法具有超灵敏、选择性好、操作简便且成本低廉的特点，无需任何标记物或复杂操作。此外，所开发的方法成功应用于血清和尿液样本中胰蛋白酶的检测以及胰蛋白酶抑制剂的筛选。因此，该策略不仅可用于生物分析研究和医学诊断中的胰蛋白酶检测，还可用于发现新型胰蛋白酶抑制剂。

文献信息 /Literature information

Dual-Mode Detection of Trypsin and Screening of Trypsin Inhibitors on the Basis of a Colorimetric/Fluorescence Sensing Probe
Jiangyue Ning, Xingyan Bao, Haotian Chen, Zelong Yan, Li Ding,* and Chang Shu*

https://doi.org/10.1021/acs.analchem.5c02187