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Updated: Apr 15, 2026

Fluorescence detection methods for microfluidic droplet platforms
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Rationally Designing a Fluorogenic Substrate of HRP To Bring Benefits for Enzyme Cascade-based Fluorescence-Activated

Guilin Li1,2, Xiaotian Hu1, Xiao Li2

  • 1Key Laboratory of Chinese Medicinal Resource from Lingnan, Ministry of Education, School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, 510006, P. R. China.

Analytical Chemistry
|April 14, 2026
PubMed
Summary
This summary is machine-generated.

A new horseradish peroxidase (HRP) fluorescent substrate, AQHR, enhances hydrophilicity and reduces interference, improving applications in fluorescence-activated droplet sorting (FADS) and enzyme engineering.

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Enzyme Assays

Background:

  • Horseradish peroxidase (HRP) and its fluorogenic substrates are vital for high-throughput analysis.
  • Fluorescence-activated droplet sorting (FADS) offers ultrahigh-throughput potential for single-cell analysis and enzyme engineering.
  • Traditional HRP substrates face limitations in FADS due to nonspecific reactivity and poor droplet retention.

Purpose of the Study:

  • To develop a novel HRP fluorescent substrate with improved properties for FADS.
  • To address the limitations of existing HRP substrates in high-throughput analytical techniques.

Main Methods:

  • Construction of a new HRP fluorescent substrate, AQHR, by modifying the resorufin scaffold.
  • Comparative analysis of AQHR and the commercial substrate ADHP, focusing on hydrophilicity, spectral properties, reactivity, and anti-interference capabilities.
  • Validation of AQHR's advantages through multienzyme cascade fluorescence assays and FADS screening of carboxylesterase (CES) mutants.

Main Results:

  • AQHR demonstrates enhanced hydrophilicity and red-shifted absorption/emission spectra compared to ADHP.
  • AQHR exhibits superior reactivity with HRP/H2O2 and significantly improved anti-interference capability.
  • The efficacy of AQHR was successfully demonstrated in multienzyme cascade assays and FADS screening.

Conclusions:

  • AQHR is a novel HRP fluorescent substrate with enhanced properties suitable for advanced analytical applications.
  • AQHR overcomes the limitations of traditional substrates, showing promise for widespread use in FADS and other analytical fields.
  • The development of AQHR is expected to advance single-cell analysis and enzyme engineering through improved HRP-based assays.