A novel optical nanoprobe for trypsin detection and inhibitor screening based on Mn-doped ZnSe quantum dots
Graphical abstract
Highlights
► A new fluorescence turn-on nanoprobe for detection of trypsin was reported. ► The method was based on the fluorescence quenching and recovery of Mn:ZnSe d-dots. ► Good sensitivity and selectivity were obtained for the determination of trypsin. ► Trypsin in human biological fluids was detected with satisfactory results.
Introduction
As one class of protease, trypsin is the most important digestive enzyme produced in the pancreas as the inactive proenzyme trypsinogen [1]. This self-regulating process can be adversely affected by pathologies, such as pancreatitis, which result in organ damage and release of enzyme into the blood. An immunoassay-based, quantitative study found healthy individuals to have a mean serum trypsin concentration of 0.25 ± 0.1 μg mL−1, whereas acute pancreatitis patients exhibited a higher concentration of 1.4 ± 0.6 μg mL−1 [2]. Although there is no trypsin in healthy person's urine, the level of trypsin in patient's urine was very high (average trypsin concentration was 84.4 μg mL−1 in pancreas transplant patients) [3]. The activity of trypsin is properly suppressed by pancreatic secretory trypsin inhibitor (PSTI). However, if trypsin activation exceeds the capacity of PSTI, it may activate various other proteases to start autodigestion which will damage cells, causing pancreatitis and cystic fibrosis. Trypsin plays a key role in controlling the pancreatic exocrine function. It is known that the trypsin level is increased with some types of pancreatic diseases [4]. Therefore, new convenient assays for trypsin and its inhibitor screening are highly desired for the development of efficient diagnostic and therapeutic methods toward these pancreatic diseases and application in the proteomics area. Traditional methods for trypsin detection involve multiple clinical tests including radioimmunoassay [5], gelatin-based film techniques [6], enzyme-linked immunosorbent assay (ELISA) [7] and colorimetric assay [8]. However, these methods are either time-consuming or require specific instruments. Recently, sensitive amperometric [9] and fluorescent probes [10] have been described for trypsin activity assay and inhibitor screening. Zhang et al. [11] described a new, label-free, continuous assay for trypsin and inhibitor screening by taking advantage of the AIE (aggregation-induced emission) behavior of tetraphenylethene compounds. However, this method did not achieve quantitative analysis for trypsin.
Semiconductor quantum dots (QDs) have been used as biological fluorescent probes due to their long-term photostability, allowing real-time and continuous monitoring. Recently, the use of QDs for simultaneous imaging and therapeutic application has also been reported. However, cadmium-based QDs are toxic to the biological systems and sensitive to heat, chemical, and photochemical disturbances, which limits their use as biomedical probes [12], [13], [14]. Recently, transition metal ion-doped quantum dots (d-dots) without heavy metal ions have attracted a lot of attentions. Manganese-doped QDs especially using ZnSe or ZnS as the hosts are a novel class of luminescent materials, which retain some advantages of the conventional QDs and offer significant solution to the above problems [15]. Recently, the Mn-doped ZnS QDs (Mn:ZnSe d-dots) have been explored to detect DNA [16], pentachlorophenol [17], glucose [18], ascorbic acid [19], 2,4,6-trinitrotoluene (TNT) [20], and protein [21]. As excellent complements to traditional QDs, non-heavy metal-containing doped QDs have been used in a diverse range of biological applications such as cell labeling, genomic and proteomic detection, optical sensors, and so on.
In this paper we report a novel optical nanoprobe (Mn:ZnSe d-dots-Arg6) for trypsin detection and its inhibitor screening based on the fluorescence quenching and recovery of Mn:ZnSe d-dots, as illustrated in Scheme 1. Arg6, which is positively charged at appropriate pH, is selected as the substrate for trypsin. Mn:ZnSe d-dots modified by mercaptopropionic acid (MPA) possess negative charges, and can interact with positively charged Arg6 via electrostatic interaction to form Mn:ZnSe d-dots/Arg6 complex, which will lead to the aggregation of Mn:ZnSe d-dots and quench their fluorescence. Trypsin is a pancreatic serine protease that specifically cleaves the C-terminus of lysine and arginine by hydrolysis [22]. Arg6 can be hydrolyzed into small fragments in the presence of trypsin. The Mn:ZnSe d-dots/Arg6 complex will be dissociated in the present of trypsin, and the fluorescence of Mn:ZnSe d-dots will be restored. Thus, a new optical nanoprobe for trypsin detection can be established. The hydrolysis of Arg6 by trypsin will be retarded in the presence of inhibitors. Thus, aggregation of Mn:ZnSe d-dots would occur and the fluorescence of Mn:ZnSe d-dots would be quenched accordingly. Therefore, the new optical nanoprobe can also be employed for screening the inhibitors of trypsin.
Section snippets
Materials and apparatus
All chemicals were of analytical reagent grade and were used without further purification. Mercaptopropionic acid (MPA) (99%), selenium powder (∼200 mesh, 99.9%), Zn(NO3)2·6H2O (99.9%), MnCl2·4H2O (99.9%), and NaBH4 (99%) were purchased from Aldrich Chemical Co. The Arg6 was purchased from Invitrogen Trading Co. Ltd. (Shanghai) and purified with high-performance liquid chromatography. The trypsin (1:250) and soybean trypsin inhibitor (SBTI) were purchased from Beijing Dingguo Changsheng
Quenching effect of Arg6 on the fluorescence of Mn:ZnSe d-dots
There were some reports on the fluorescence quenching of QDs by aggregation in water [25], [26], so we discussed the influence of aggregation on the fluorescence of Mn:ZnSe d-dots in this study. As shown in Fig. 1, the initial experiments demonstrated that the quenching effect of Arg6 on the fluorescence of Mn:ZnSe d-dots was finished within 30 min. So we recorded the fluorescence intensity of the Mn:ZnSe d-dots/Arg6 system after reaction time of 30 min. As shown in Fig. 2, intensive quenching
Conclusion
In summary, we have developed a novel optical nanoprobe (Mn:ZnSe d-dots-Arg6) for trypsin detection and its inhibitor screening based on the fluorescence quenching and recovery of Mn:ZnSe d-dots. The established method is designed by taking advantage of the aggregation of Mn:ZnSe d-dots induced by Arg6 and hydrolysis of Arg6 in the presence of trypsin. The nanoprobe can be used to detect trypsin of a concentration as low as 40 ng mL−1. Moreover, the nanoprobe can be employed for screening the
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China (No. 21075050) and the science and technology development project of Jilin province, China (No. 20110334).
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