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Bright Single-Cell Fluorescent Reporter Enables Ultrasensitive Target Detection for Microbial Cell-Based Biosensors
Faying Zhang1,2, Xuting Sun1, Hui Zheng3
1State Key Laboratory of Green Biomanufacturing, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China.
ACS Sensors
|March 10, 2026
Summary
Researchers developed an ultrasensitive fluorescent protein (FP) reporter, NGFP4, using N-terminal peptide fusion. This enhances whole-cell biosensor (WCB) sensitivity and speed for detecting pollutants and toxins.
Area of Science:
- Synthetic Biology
- Biotechnology
- Analytical Chemistry
Background:
- Whole-cell biosensors (WCBs) are crucial for detecting trace analytes but are often limited by reporter gene sensitivity.
- Fluorescent proteins (FPs) are common reporters but generally show lower sensitivity and slower response than enzymatic reporters.
- Existing FP reporters can limit the practical application and field deployment of WCBs due to sensitivity and speed constraints.
Purpose of the Study:
- To develop an ultrasensitive fluorescent protein reporter for enhanced whole-cell biosensor performance.
- To improve reporter gene characteristics including expression speed and intracellular stability.
- To enable faster and more sensitive detection of analytes using microbial cell-based biosensors.
Main Methods:
- Engineered an NGFP4 variant by fusing an N-terminal decapeptide to a superfolder green fluorescent protein (sfGFP).
- Validated NGFP4's reporter characteristics in four microbial hosts: E. coli, Bacillus subtilis, Pichia pastoris, and Saccharomyces cerevisiae.
- Integrated NGFP4 into whole-cell biosensors for detecting salicylic acid and 2-chlorobiphenyl.
Main Results:
- NGFP4 demonstrated significantly enhanced single-cell fluorescence intensity, ranging from 6.4- to 28-fold across different microbial hosts.
- The reporter exhibited rapid expression and robust intracellular stability, improving upon standard sfGFP.
- WCBs utilizing NGFP4 achieved rapid detection times of 1 hour for salicylic acid (LOD 0.36 μM) and 2-chlorobiphenyl (LOD 18.2 μM).
Conclusions:
- The NGFP4 reporter provides a cross-species compatible solution for ultrasensitive detection in microbial biosensors.
- This engineered FP reporter overcomes limitations of traditional reporters, enabling faster and more sensitive analyte detection.
- The developed reporter facilitates field deployment of WCBs with minimal genetic modification and reduced detection times.
