Background: Bioluminescence imaging (BLI) is one of the most widely used optical platforms in molecular imaging, but it suffers from severe tissue attenuation and autoluminescence in vivo.

Enzymatic reactions that involve a luminescent substrate (luciferin) and enzyme (luciferase) from luminous organisms enable a luminescence detection of target proteins and cells with high specificity, albeit that conventional assay design requires a prelabeling of target molecules with luciferase. Here, we report a luciferase-independent luminescence assay in which the target protein directly catalyzes the oxidative luminescence reaction of luciferin. The SARS-CoV-2 antigen (spike) protein catalyzes the light emission of Cypridina luciferin, whereas no such catalytic function was observed for salivary proteins. This selective luminescence reaction is due to the enzymatic recognition of the 3-(1-guanidino)propyl group in luciferin at the interfaces between the units of the spike protein, allowing a specific detection of the spike protein in human saliva without sample pretreatment. This method offers a novel platform to detect virus antigens simply and rapidly without genetic manipulation or antibodies.

Serum copper levels are biomarkers for copper-related diseases. Quantification of levels of free copper (not bound to proteins) in serum is important for diagnosing Wilson's disease, in which the free copper concentration is elevated. Bioluminescence is commonly used in point-of-care diagnostics, but these assays require genetically engineered luciferase. Here, we developed a luciferase-independent copper detection platform. A luminogenic caged coelenterazine analogue (TPA-H1) was designed and synthesized to detect copper ions in human serum. TPA-H1 was developed by introducing a tris[(2-pyridyl)-methyl]amine (TPA) ligand, which is a Cu⁺ cleavable caging group, to the carbonyl group at the C-3 position of the imidazopyrazinone scaffold. The luciferin, named HuLumino1, is the product of the cleavage reaction of TPA-H1 with a copper ion and displays “turn-on” bioluminescence signals specifically with human serum albumin, which can be used to quantitatively analyse copper ions. TPA-H1 exhibited a fast cleavage of the protective group, high specificity, and high sensitivity for copper over other metal ions. This novel caged coelenterazine derivative, TPA-H1, can detect free copper ions in serum in a simple “mix-and-read” manner.

Encapsulation of the firefly bioluminescence system into biomolecular condensates enhanced the bioluminescence quantum yield, an index of brightness.