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

Abstract

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-HbZnPPIX 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.

Electrochemiluminescent Ion-Channeling Framework for Membrane Binding and Transmembrane Activity Assays

“…In regard to the PL performances in Figure D, the emissions of both TFPP (curve a ) and PC (curve c ) culminate at a main (λ em ) max,1 = 656 nm and a shoulder (λ em ) max,2 = 718 nm when being excited optimally at λ S = (λ ex ) opt . Simultaneously, (λ em ) max of ZnTFPP (curve b ) and ZnPC (curve d ) blue-shifts to 620/668 nm, all in good accordance with their Q waves that unravel an intersystem crossing-dictated pathway . The smaller Stokes shifts in the latter two implies less nonradiative hoppings.…”

“…Simultaneously, (λ em ) max of ZnTFPP (curve b) and ZnPC (curve d) blue-shifts to 620/668 nm, all in good accordance with their Q waves that unravel an intersystem crossingdictated pathway. 37 The smaller Stokes shifts in the latter two implies less nonradiative hoppings. As a trade-off, the original porphines behave superior in PL efficiency to their metalloderivatives, reflecting splits of excitonic degeneracy by multidentate Zn 2+ .…”

Sorting and Screening of Quaternary Ammonium Lipoids for Membrane-Binding Assays Based on Electrochemiluminescent Cocrystalline Nanosheets

“…Its incorporation with ZnTPP opened a more energy-saving deactivation route rather than traditional back-to-the-ground hoppings via nonradiative or intersystem crossing . Being an electro-stimulated equal to PL, the ECL spectrograms seem analogous to the forms of PL with ∼30 nm blue-shift of double extrema at 640/690 nm (Figure B), that stemmed from the hopping d z 2 → d xz ,d yz of zinc 3d 10 -electrons under electrolytic perturbation and coreactive O 2 mingling . Notably, the area integral of ZnTPP-C 70 (curve b) outnumbers that of ZnTPP (curve a) by roughly 1.5 times.…”

“…That is, the greater is the surge of i P1 , the more boost there is to luminosity. Abnormally, the i P1 of DDDAB and DODAB were found extraordinarily large, for that a considerable portion of O 2 undertook 2/4 – e – reduction into ECL-inactive H 2 O 2 /H 2 O on DO­(D)­DAB-assembled dielectric double layer, , which would rather be employed for the direct electrochemistry of hemoglobin . More-over, E P2 moves away from zero as the carbon chain elongates, i.e., THXAB (−1.18 V) > THPAB (−1.20 V) > TOAB (−1.21 V) > MTOAB (−1.24 V) > DDDAB (−1.27 V) > DODAB (−1.28 V).…”

“…Three derivative moieties, tetrabutyl (TBAB), tetra- n -octyl (TOAB), and didodecyldimethyl (DODAB), had early on demonstrated their prevailing potency in the enhancement of voltammetric peak resolution and excitonic life expectancy of porphyrin assemblies, − which often comes in exceptionally handy in the roles as electrocatalyst and ECL luminophor . Of note, we corroborated rivalrous abiological ECL irradiations from mimicking aldolase, Na + -I – symporter, reconstituted hemoglobin, and native pigment metabolon under the help of the above lipoidal dispersions . Moving forward, their contributions to ECL brightness will hereby turn to be tested on a state-of-the-art organic frameworkthe cocrystalline nanosheet of Buckyball C 70 and zinc meso -tetraphenylporphine (ZnTPP) via simple liquid–liquid interfacial deposition (Scheme ).…”