A fluorometric assay of thrombin using magnetic nanoparticles and enzyme-free hybridization chain reaction

Zhongming Huang, Dinggeng He, Hung Wing Li*

*Corresponding author for this work

Research output: Contribution to journalJournal articlepeer-review

14 Citations (Scopus)

Abstract

A fluorescence method based on functionalized magnetic nanoparticles (FMNPs) and hybridization chain reaction (HCR) is developed for the enzyme-free amplified determination of thrombin. In the proposed design, aptamer against thrombin was hybridized with the capture DNA-modified magnetic nanoparticles to yield the FMNPs. In the presence of thrombin, aptamers are released due to the specific and high-affinity binding between thrombin and its aptamer. The exposed capture DNA subsequently hybridized with the partial sequence of helper DNA, and the vacant sequence of helper DNA further hybridized with HCR products which is pre-formed by the alternate hybridization of single-stranded DNAs (H1 and H2). The immobilized HCR products were then labeled with YOYO-1 for fluorescence measurement. Fluorescence signal intensity of labeled YOYO-1 was measured at an emission wavelength of 519 nm (excitation under 488 nm) and used for calibration. By taking advantage of HCR amplification, this direct assay strategy showed a linear response in the 20- to 200-pM concentration range, and the limit of detection is 9.2 pM which is about 3-orders of magnitude lower than the serum thrombin concentration (10 nM) that triggers blood clotting. This developed method can efficiently differentiate the target protein from a protein matrix, and it is verified by determination of thrombin in spiked serum samples with recoveries in the range of 94.5–103.3%.

Original languageEnglish
Article number295
Number of pages8
JournalMicrochimica Acta
Volume187
Issue number5
DOIs
Publication statusPublished - 1 May 2020

Scopus Subject Areas

  • Analytical Chemistry

User-Defined Keywords

  • Aptamer
  • Fluorometric assay
  • Magnetic separation
  • Protein determination
  • Signal amplification

Fingerprint

Dive into the research topics of 'A fluorometric assay of thrombin using magnetic nanoparticles and enzyme-free hybridization chain reaction'. Together they form a unique fingerprint.

Cite this