Elsevier

Analytica Chimica Acta

Volume 743, 19 September 2012, Pages 131-136
Analytica Chimica Acta

A novel optical nanoprobe for trypsin detection and inhibitor screening based on Mn-doped ZnSe quantum dots

https://doi.org/10.1016/j.aca.2012.07.007Get rights and content

Abstract

In this paper, a novel optical nanoprobe (Mn:ZnSe d-dots-Arg6) for trypsin detection and its inhibitor screening has been constructed successfully based on the fluorescence quenching and recovery of Mn:ZnSe d-dots. Mn:ZnSe d-dots would aggregate in the presence of positively charged Arg6 (six arginine residues) due to electrostatic interactions that result in the fluorescence quenching. Arg6 can be hydrolyzed into small fragments in the presence of trypsin, and accordingly, the aggregation of Mn:ZnSe d-dots can be prohibited, which lead to the fluorescence recovery. Experimental results show that the recovery in fluorescence intensity is linearly proportional to the concentration of trypsin within the range of 0.1–12.0 μg mL−1 with a detection limit of 40 ng mL−1 under the optimized experimental conditions. We also prove the feasibility of fluorescence recovery of Mn:ZnSe d-dots for trypsin detection through the resonance light scattering (RLS) technique. Additionally, the optical nanoprobe can be employed for screening the inhibitors of trypsin. The optical nanoprobe was successfully applied for the determination of trypsin in human serum and urine samples with good accuracy and satisfactory recovery.

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).

References (31)

  • M. Noh et al.

    Colloids Surf. A: Physicochem. Eng. Aspects

    (2010)
  • Y. Zhang et al.

    J. Lumin.

    (2008)
  • H.X. Wang et al.

    Biochem. Biophys. Res. Commun.

    (2006)
  • M. Hirota et al.

    J. Gastroenterol.

    (2006)
  • J.M. Artigas et al.

    Postgrad. Med. J.

    (1981)
  • W.A. See et al.

    Transplantation

    (1991)
  • M.F. Byrne et al.

    Can. J. Gastroenterol.

    (2002)
  • J.M. Argtigas et al.

    Postgrad. Med. J.

    (1981)
  • A.D. Kersey et al.

    Opt. Lett.

    (1993)
  • M.B. Rhodes et al.

    Anal. Chem.

    (1957)
  • G.M. Homer et al.

    Clin. Chem.

    (1963)
  • R.E. Ionescu et al.

    Anal. Chem.

    (2006)
  • J.M. Mu et al.

    ACS Nano

    (2010)
  • W.X. Xue et al.

    Org. Lett.

    (2010)
  • V. Bagalkot et al.

    Nano Lett.

    (2007)
  • Cited by (50)

    • Self-assembled diphenylalanine-zinc oxide hybrid nanostructures as a highly selective luminescent biosensor for trypsin detection

      2021, Applied Surface Science
      Citation Excerpt :

      In order to test the sensing mechanism of the FF-ZnO system, trypsin, a serine protease enzyme, was used as an analyte to examine. Trypsin belongs to serine proteases family of enzymes present in the digestive system where hydrolysis of proteins takes place [50]. It cleaves the peptide bond, particularly at the carboxyl end of amino acids.

    • The recent biological applications of selenium-based nanomaterials

      2021, Nano Today
      Citation Excerpt :

      This label-free detection strategy might pave the way for point-of-care molecular diagnostic technology. Selenium-based nanomaterials can also be applied for the fluorometric determination of target proteins [63–66]. For example, Su’s group designed a Mn:ZnSe d-dots-Arg6 nanoprobe to achieve the fluorescence detection of trypsin [63].

    • Application of high-resolution ultrasonic spectroscopy for real-time monitoring of trypsin activity in β-casein solution

      2021, Food Chemistry
      Citation Excerpt :

      Traditional methods for trypsin detection involve multiple clinical tests including radioimmunoassay, gelatin-based film techniques, enzyme-linked immunosorbent assay (ELISA) and colorimetric assay. However, these methods are time-consuming and costly (Gao, Tang, Li & Su, 2012). To overcome the limitations of traditional discontinuous methods, a variety of novel techniques providing the real-time data of trypsin activity have been reported.

    View all citing articles on Scopus
    View full text