Issue 22, 2023

New synthetic red- and orange-emitting luciferases to upgrade in vitro and 3D cell biosensing

Abstract

Bioluminescence (BL), i.e., the emission of light in living organisms, has become an indispensable tool for a plethora of applications including bioassays, biosensors, and in vivo imaging. Current efforts are focused on the obtainment of new luciferases having optimized properties, such as improved thermostability at 37 °C, pH-insensitive emission, high quantum yield, extended kinetics and red-shifted emission. To address these issues we have obtained two new synthetic luciferases, an orange and a red-emitting luciferase, which were designed to achieve high sensitivity (BoLuc) and multiplexing capability (BrLuc) for in vitro and in vivo biosensing using as a starting template a recently developed thermostable synthetic luciferase (BgLuc). Both luciferases were characterized in terms of emission behaviour and thermal and pH stability showing promising features as reporter proteins and BL probes. As proof-of-principle application, an inflammation assay based on Human Embryonic Kidney (HEK293T) 3D cell cultures was developed using either the orange or the red-emitting mutant. The assay provided good analytical performance, with limits of detection for Tumor Necrosis Factor (TNFα) of 0.06 and 0.12 ng mL−1 for BoLuc and BrLuc, respectively. Moreover, since these luciferases require the same substrate, D-luciferin, they can be easily implemented in dual-color assays with a significant reduction of total cost per assay.

Graphical abstract: New synthetic red- and orange-emitting luciferases to upgrade in vitro and 3D cell biosensing

Supplementary files

Article information

Article type
Paper
Submitted
21 Jul 2023
Accepted
27 Sep 2023
First published
28 Sep 2023
This article is Open Access
Creative Commons BY-NC license

Analyst, 2023,148, 5642-5649

New synthetic red- and orange-emitting luciferases to upgrade in vitro and 3D cell biosensing

M. M. Calabretta, D. Gregucci and E. Michelini, Analyst, 2023, 148, 5642 DOI: 10.1039/D3AN01251D

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