Low-cost and sustainable smartphone-based tissue-on-chip device for bioluminescence biosensing.

Several organ-on-chip and cell-on-chip devices have been reported, however, their main drawback is that they are not interoperable (i.e., they have been fabricated with customized equipment, thus cannot be applied in other facilities, unless having the same setup), and require cell-culture facilities and benchtop instrumentation. As a consequence, results obtained with such devices do not generally comply with the principles of findability, accessibility, interoperability, and reusability (FAIR). To overcome such limitation, leveraging cost-effective 3D printing we developed a bioluminescent tissue on-a-chip device that can be easily implemented in any laboratory. The device enables continuous monitoring of cell co-cultures expressing different bioluminescent reporter proteins and, thanks to the implementation of new highly bioluminescent luciferases having high pH and thermal stability, can be monitored via smartphone camera. Another relevant feature is the possibility to insert the chip into a commercial 24-well plate for use with standard benchtop instrumentation. The suitability of this device for 3D cell-based biosensing for monitoring activation of target molecular pathways, i.e., the inflammatory pathway via nuclear factor kappa-B (NF-κB) activation, and general cytotoxicity is here reported showing similar analytical performance when compared to conventional 3D cell-based assays performed in 24-well plates.

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

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.

Illuminating Progress: The Contribution of Bioluminescence to Sustainable Development Goal 6-Clean Water and Sanitation-Of the United Nations 2030 Agenda.

The United Nations Agenda 2030 Sustainable Development Goal 6 (SDG 6) aims at ensuring the availability and sustainable management of water and sanitation. The routine monitoring of water contaminants requires accurate and rapid analytical techniques. Laboratory analyses and conventional methods of field sampling still require considerable labor and time with highly trained personnel and transport to a central facility with sophisticated equipment, which renders routine monitoring cumbersome, time-consuming, and costly. Moreover, these methods do not provide information about the actual toxicity of water, which is crucial for characterizing complex samples, such as urban wastewater and stormwater runoff. The unique properties of bioluminescence (BL) offer innovative approaches for developing advanced tools and technologies for holistic water monitoring. BL biosensors offer a promising solution by combining the natural BL phenomenon with cutting-edge technologies. This review provides an overview of the recent advances and significant contributions of BL to SDG 6, focusing attention on the potential use of the BL-based sensing platforms for advancing water management practices, protecting ecosystems, and ensuring the well-being of communities.

Colorimetric Paper Sensor for Food Spoilage Based on Biogenic Amine Monitoring.

Biogenic amines (BAs), nitrogenous molecules usually present in different foods, can be considered an indicator of freshness and food quality since their amount increases during food spoilage. Their detection, possibly in real time via the use of smart packaging, is therefore of crucial importance to ensure food safety and to fulfill consumers' demand. To this end, colorimetric sensors are considered one of the most feasible solutions. Here, we report a user-friendly colorimetric sensing paper able to detect BAs via the naked eye. The sensing molecule is the aglycone genipin, a natural cross-linking agent extracted from gardenia fruit, able to bind BAs producing water-soluble blue pigments. The paper sensor was applied to chicken meat quality monitoring and a quantitative analysis was performed with image acquisition via a smartphone camera, achieving a limit of detection equivalent to 0.1 mM of putrescine. The suitability of the BA sensing paper was assessed by integrating the sensor into smart packaging and analyzing commercial chicken meat samples stored at different temperatures; the results of the sensor paralleled the "best before date" indicated on the label, confirming the potential applicability of the sensor as a smart label.

Novel Nanozeolitic Imidazolate Framework (ZIF-8)-Luciferase Biocomposite for Nanosensing Applications.

The identification of new strategies to improve the stability of proteins is of utmost importance for a number of applications, from biosensing to biocatalysis. Metal-organic frameworks (MOFs) have been shown as a versatile host platform for the immobilization of proteins, with the potential to protect proteins in harsh conditions. In this work, a new thermostable luciferase mutant has been selected as a bioluminescent protein model to investigate the suitability of MOFs to improve its stability and prompt its applications in real-world applications, for example, ATP detection in portable systems. The luciferase has been immobilized onto zeolitic imidazolate framework-8 (ZIF-8) to obtain a bioluminescent biocomposite with enhanced performance. The biocomposite ZIF-8@luc has been characterized in harsh conditions (e.g., high temperature, non-native pH, etc.). Bioluminescence properties confirmed that MOF enhanced the luciferase stability at acidic pH, in the presence of organic solvents, and at -20 °C. To assess the feasibility of this approach, the recyclability, storage stability, precision, and Michaelis-Menten constants (Km) for ATP and d-luciferin have been also evaluated. As a proof of principle, the suitability for ATP detection was investigated and the biocomposite outperformed the free enzyme in the same experimental conditions, achieving a limit of detection for ATP down to 0.2 fmol.

A Luciferase Mutant with Improved Brightness and Stability for Whole-Cell Bioluminescent Biosensors and In Vitro Biosensing.

The availability of new bioluminescent proteins with tuned properties, both in terms of emission wavelength, kinetics and protein stability, is highly valuable in the bioanalytical field, with the potential to improve the sensitivity and analytical performance of the currently used methods for ATP detection, whole-cell biosensors, and viability assays among others. We present a new luciferase mutant, called BgLuc, suitable for developing whole-cell biosensors and in vitro biosensors characterized by a bioluminescence maximum of 548 nm, narrow emission bandwidth, favorable kinetic properties, and excellent pH- and thermo-stabilities at 37 and 45 °C and pH from 5.0 to 8.0. We assessed the suitability of this new luciferase for whole-cell biosensing with a cell-based bioreporter assay for Nuclear Factor-kappa B (NF-kB) signal transduction pathway using 2D and 3D human embryonic kidney (HEK293T) cells, and for ATP detection with the purified enzyme. In both cases the luciferase showed suitable for sensitive detection of the target analytes, with better or similar performance than the commercial counterparts.

Bioluminescence Sensing in 3D Spherical Microtissues for Multiple Bioactivity Analysis of Environmental Samples.

The development of predictive in vitro sensing tools able to provide rapid information on the different bioactivities of a sample is of pivotal importance, not only to monitor environmental toxicants, but also to understand their mechanisms of action on diverse molecular pathways. This mechanistic understanding is highly important for the characterization of toxicological hazards, and for the risk assessment of chemicals and environmental samples such as surface waters and effluents. Prompted by this need, we developed and optimized a straightforward bioluminescent multiplexed assay which enables the measurement of four bioactivities, selected for their relevance from a toxicological perspective, in bioluminescent microtissues. The assay was developed to monitor inflammatory, antioxidant, and toxic activity, and the presence of heavy metals, and was successfully applied to the analysis of river water samples, showing potential applicability for environmental analyses. The assay, which does not require advanced equipment, can be easily implemented in general laboratories equipped with basic cell culture facilities and a luminometer.