Electrochemiluminescence-Repurposed Abiological Catalysts in Full Protein Tag for Ultrasensitive Immunoassay

Being announced as one of the "2019 Top Ten Emerging Technologies in Chemistry" by IUPAC, the directed evolution of artificial metalloenzymes has led to a broad scope of abiotic processes. Here, inspired by those key proteins in bioluminescence, a rudimentary expression of bio-electrochemiluminescent (ECL) macromolecules was achieved via the complexation of zinc proto-porphyrin IX (ZnPPIX) within apo-hemoglobin (apo-Hb). A high-yield monochromic irradiation at 644 nm could be provoked potentiostatically from the reconstituted holo-Hb^ZnPPIX in solutions. Its secondary structure integrity was elucidated by UV and circular dichroism spectrometry, while voltammetry-hyphenated surface plasmon resonance authenticated its ligation conservativeness in electrical fields. Further conjugation with streptavidin rendered a homogeneous Janus fusion of both receptor and reporter domains, enabling a new abiological catalyst-linked ECL bioassay. On the other hand, singular ZnPPIX inside each tetrameric subunit of Hb accomplished an overall signal amplification without the bother of luminogenic heterojunctions. This pH-tolerant and non-photobleaching optics was essentialized to be the unique configuration interaction between Zn and O₂, by which the direct electrochemistry of proteins catalyzed the transient progression of O₂ → O₂ → O₂* + hυ selectively. Such principle was implemented as a signal-on strategy for the determination of a characteristic cancer biomarker, the vascular endothelial growth factor, resulting in competent performance at a low detection limit of 0.6 pg·mL^-1 and a wide calibration range along with good stability and reliability in real practices. This simple mutation repurposed the O₂-transport Hb in the erythrocytes of almost all vertebrates into a cluster of oxidoreductases with intrinsic ECL activity, which would enrich the chromophore library. More importantly, its genetically engineered variants may come in handy in biomedical diagnosis and visual electrophysiology.