Enteropeptidase (ENTK), a serine protease with strict substrate specificity, plays a dual role in organisms. Physiologically, it regulates protein digestion in the digestive tract by activating trypsinogen; pathologically, its specific high expression in the mitochondria of cancer cells such as HeLa provides a potential biomarker for cancer diagnosis. However, the interference of high concentrations of thiol biomolecules (e.g., glutathione) in cells and the technical barriers to mitochondrial targeted delivery have long restricted the accurate detection of enteropeptidase. In recent years, the emergence of a fluorescent sensor based on diselenide-modified gold nanoparticles (AuNP@DSe-PlinkerTMR) has offered an innovative solution to this predicament.
The uniqueness of enteropeptidase lies in its specific recognition ability for the DDDDK polypeptide sequence, a feature that makes it a "molecular switch" in biological cascade reactions. In normal physiological processes, it is a key initiator of digestive enzyme activation; in the tumor microenvironment, the abnormal high expression of mitochondrial enteropeptidase is closely related to cancer cell proliferation and energy metabolism reconstruction, and its expression level can serve as a potential monitoring indicator for cancer progression.
Existing detection methods (such as ELISA and HPLC) have obvious shortcomings: they are unable to achieve mitochondrial in-situ detection and are severely interfered by biological thiols. Most gold nanosensors rely on Au-S bonds to modify functional groups, while biological thiols can competitively bind to the gold surface, leading to sensor structure damage and false positive signals. Meanwhile, conventional methods lack sufficient sensitivity to capture the expression differences of low-abundance enteropeptidase in cancer cells.
The core breakthrough of this sensor is the use of diselenide (-Se-Se-) bonds instead of traditional Au-S bonds. Experimental data show that when incubated in a 10 mM glutathione environment (far exceeding physiological concentration) for 12 hours, the fluorescence signal of the Au-S bond-modified sensor increases by 16.4 times (indicating structural disintegration), while the fluorescence intensity of AuNP@DSe-PlinkerTMR remains almost unchanged. This confirms that diselenide bonds can effectively resist biological thiol attacks, solving the long-standing stability problem.
The DDDDK polypeptide modified on the sensor surface plays a dual role: it is not only a specific substrate for enteropeptidase, but the negative charge generated by its carboxyl group can also form electrostatic attraction with the positive charge (H+ enrichment) in the mitochondrial intermembrane space, achieving active targeting. Confocal imaging shows that the co-localization coefficient between the sensor and mitochondrial dyes reaches 0.91-0.96, much higher than that of conventional carriers, ensuring the spatial specificity of detection.
A "quenching-activation" design is adopted: gold nanoparticles quench the surface fluorescent groups (TMR). When enteropeptidase is present, the specific cleavage of the DDDDK sequence causes the fluorescent groups to detach, the signal is restored, and the intensity is positively correlated with the enteropeptidase concentration, enabling quantitative detection.
Under physiological conditions, the sensor exhibits three major characteristics: first, excellent anti-interference ability, with its response to enteropeptidase unaffected in high concentrations of glutathione or serum, while the signal of traditional Au-S sensors decreases by more than 60%; second, high sensitivity, with a detection limit as low as 0.18 U·mL⁻¹, meeting the needs of low-abundance detection; third, strong specificity, with no cross-reaction to other proteases.
In cell experiments, AuNP@DSe-PlinkerTMR shows strong fluorescence in HeLa cells with high enteropeptidase expression, and weak signal in MCF-7 cells with low expression. Even in the co-culture system of the two cell types, it can still clearly distinguish them, with a fluorescence signal difference of up to 10.95 times; while the Au-S sensor, due to false positive interference, has a difference of only 2.51 times. After treatment with a biological thiol scavenger, the signal of this sensor shows no significant change, further confirming the high fidelity of its detection.
This diselenide-modified nanosensor, through structural innovation and targeted design, successfully solves the stability and specificity problems in enteropeptidase detection, achieving accurate mitochondrial in-situ, highly sensitive, and anti-interference analysis. It not only provides a new tool for enteropeptidase research but also pioneers new design ideas for organelle-targeted detection.
In the future, by optimizing response speed and tissue penetration, it is expected to expand applications in in-vivo tumor imaging, drug screening, and other fields, providing strong support for the early diagnosis and precise treatment of diseases such as cancer, and promoting the development of bioanalytical technology towards a more microscopic and accurate direction.
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