Microchimica Acta

, 186:272 | Cite as

A fluorometric and colorimetric method for determination of trypsin by exploiting the gold nanocluster-induced aggregation of hemoglobin-coated gold nanoparticles

  • Zhengming Zhou
  • Wei Liu
  • Yanying Wang
  • Fang Ding
  • Xiaopeng Liu
  • Qingbiao ZhaoEmail author
  • Ping Zou
  • Xianxiang Wang
  • Hanbing RaoEmail author
Original Paper


A dual-signal assay is described for the determination of trypsin based on the use of gold nanoparticles (AuNPs) that aggregate in the presence of gold nanoclusters (AuNCs) due to electrostatic interaction. This is accompanied by a color change from red to blue. However, if hemoglobin (Hb) is present in the solution, it will attach to the surface of AuNPs, thus preventing aggregation. The Hb-coated AuNPs quench the fluorescence of AuNCs. Trypsin can hydrolyze Hb and destroy the protective coating of Hb on the AuNPs. As a result, AuNP aggregation will occur after the addition of AuNCs, and the blue fluorescence of the AuNCs with 365 nm excitation and 455 nm maximum emission peak is recovered. Thus, trypsin can be determined by measurement of fluorescence emission intensity. Additionally, trypsin can be determined by the maximum absorption peak wavelength between 530 nm and 610 nm. Fluorescence increases linearly in the 10–2500 ng⋅mL−1 concentration range, and absorbance in the 20–2000 ng·mL−1 concentration range. The limits of detection are 4.6 ng·mL−1 (fluorometry) and 8.4 ng·mL−1 (colorimetry), respectively. The assay is sensitive and selective, and can be applied to the determination of trypsin in serum.

Graphical abstract

Schematic presentation of a fluorometric and colorimetric method for determination of trypsin. The presence of hemoglobin (Hb) protects AuNPs from agglomeration after adding AuNCs and the fluorescence of AuNCs is quenched. With trypsin present, trypsin destroys the coating of AuNPs by Hb. AuNPs aggregate again and the fluorescence recovers after the addition of AuNCs.


Dual-signal assay Fluorescence resonance energy transfer Hemoglobin coated gold nanoparticles Fluorescence Colorimetry Electrostatic interaction Serum analysis 



This work was supported by a grant from the Two-Way Support Programs of Sichuan Agricultural University (Project No.03570113), the Education Department of Sichuan Provincial, P. R. China (Grant No. 16ZA0039), National Natural Science Foundation of China (Grant No. 11404358).

Compliance with ethical standards

The author(s) declare that they have no competing interests.

Supplementary material

604_2019_3380_MOESM1_ESM.doc (2.5 mb)
ESM 1 (DOC 2547 kb)


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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  • Zhengming Zhou
    • 1
  • Wei Liu
    • 1
  • Yanying Wang
    • 1
  • Fang Ding
    • 2
  • Xiaopeng Liu
    • 3
  • Qingbiao Zhao
    • 4
    Email author
  • Ping Zou
    • 1
  • Xianxiang Wang
    • 1
  • Hanbing Rao
    • 1
    Email author
  1. 1.College of ScienceSichuan Agricultural UniversityYa’anPeople’s Republic of China
  2. 2.Nanshan District Key Lab for Biopolymers and Safety Evaluation, Shenzhen Key Laboratory of Polymer Science and Technology, Guangdong Research Center for Interfacial Engineering of Functional Materials, College of Materials Science and EngineeringShenzhen UniversityShenzhenPeople’s Republic of China
  3. 3.The Affiliated High School of Shanxi UniversityTaiyuanPeople’s Republic of China
  4. 4.Key Laboratory of Polar Materials and Devices, Ministry of Education, Department of OptoelectronicsEast China Normal UniversityShanghaiPeople’s Republic of China

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