Development of a novel fluorescent protein-based probe for efficient detection of Pb2+ in serum inspired by the metalloregulatory protein PbrR691

Abstract

Background: The accurate and rapid detection of blood lead concentration is of paramount importance for assessing human lead exposure levels. Fluorescent protein-based probes, known for their high detection capabilities and low toxicity, are extensively used in analytical sciences. However, there is currently a shortage of such probes designed for ultrasensitive detection of Pb²⁺, and no reported probes exist for the quantitative detection of Pb²⁺ in blood samples. This study aims to fill this critical void by developing and evaluating a novel fluorescent protein-based probe that promises accurate and rapid lead quantification in blood.

Results: A simple and small-molecule fluorescent protein-based probe was successfully constructed herein using a peptide PbrBD designed for Pb²⁺ recognition coupled to a single fluorescent protein, sfGFP. The probe retains a three-coordinate configuration to identify Pb²⁺ and has a high affinity for it with a K_d' of 1.48 ± 0.05 × 10^-17 M. It effectively transfers the conformational changes of the peptide to the chromophore upon Pb²⁺ binding, leading to fast fluorescence quenching and a sensitive response to Pb²⁺. The probe offers a broad dynamic response range of approximately 37-fold and a linear detection range from 0.25 nM to 3500 nM. More importantly, the probe can resist interference of metal ions in living organisms, enabling quantitative analysis of Pb²⁺ in the picomolar to millimolar range in serum samples with a recovery percentage of 96.64%-108.74 %.

Significance: This innovative probe, the first to employ a single fluorescent protein-based probe for ultrasensitive and precise analysis of Pb²⁺ in animal and human serum, heralds a significant advancement in environmental monitoring and public health surveillance. Furthermore, as a genetically encoded fluorescent probe, this probe also holds potential for the in vivo localization and concentration monitoring of Pb²⁺.

Keywords: Fluorescence quenching; Heavy metal pollutants; Low detection limit; Metalloprotein; Quick testing-technology.