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.

Current advances in the use of bioluminescence assays for drug discovery: an update of the last ten years.

INTRODUCTION: Bioluminescence is a well-established optical detection technique widely used in several bioanalytical applications, including high-throughput and high-content screenings. Thanks to advances in synthetic biology techniques and deep learning, a wide portfolio of luciferases is now available with tuned emission wavelengths, kinetics, and high stability. These luciferases can be implemented in the drug discovery and development pipeline, allowing high sensitivity and multiplexing capability. AREAS COVERED: This review summarizes the latest advancements of bioluminescent systems as toolsets in drug discovery programs for in vitro applications. Particular attention is paid to the most advanced bioluminescence-based technologies for drug screening over the past 10 years (from 2013 to 2023) such as cell-free assays, cell-based assays based on genetically modified cells, bioluminescence resonance energy transfer, and protein complementation assays in 2D and 3D cell models. EXPERT OPINION: The availability of tuned bioluminescent proteins with improved emission and stability properties is vital for the development of bioluminescence assays for drug discovery, spanning from reporter gene technology to protein-protein techniques. Further studies, combining machine learning with synthetic biology, will be necessary to obtain new tools for sustainable and highly predictive bioluminescent drug discovery platforms.

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.

Super-Stable Metal-Organic Framework (MOF)/Luciferase Paper-Sensing Platform for Rapid ATP Detection.

Adenosine triphosphate (ATP) determination has been used for many decades to assess microbial contamination for hygiene monitoring in different locations and workplace environments. Highly sophisticated methods have been reported, yet commercially available kits rely on a luciferase-luciferin system and require storage and shipping at controlled temperatures (+4 or -20 °C). The applicability of these systems is limited by the need for a secure cold chain, which is not always applicable, especially in remote areas or low-resource settings. In this scenario, easy-to-handle and portable sensors would be highly valuable. Prompted by this need, we developed a bioluminescence paper biosensor for ATP monitoring in which a new luciferase mutant was combined with a metal-organic framework (MOF); i.e., zeolitic imidazolate framework-8 (ZIF-8). A paper biosensor was developed, ZIF-8@Luc paper sensor, and interfaced with different portable light detectors, including a silicon photomultiplier (SiPM) and smartphones. The use of ZIF-8 not only provided a five-fold increase in the bioluminescence signal, but also significantly improved the stability of the sensor, both at +4 and +28 °C. The ATP content in complex biological matrices was analyzed with the ZIF-8@Luc paper sensor, enabling detection down to 7 × 10-12 moles of ATP and 8 × 10-13 moles in bacterial lysates and urine samples, respectively. The ZIF-8@Luc sensor could, therefore, be applied in many fields in which ATP monitoring is required such as the control of microbial contamination.

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.

Looking for peptides from rice starch processing by-product: Bioreactor production, anti-tyrosinase and anti-inflammatory activity, and in silico putative taste assessment.

One of the major challenges for the modern society, is the development of a sustainable economy also aiming at the valorization of agro-industrial by-products in conjunction with at a significant reduction of generated residues from farm to retail. In this context, the present study demonstrates a biotechnological approach to yield bioactive peptides from a protein fraction obtained as a by-product of the rice starch production. Enzymatic hydrolysis, with the commercial proteases Alcalase and Protamex, were optimized in bioreactor up to 2 L of volume. The two best digestates, selected with respect to peptide release and extract antioxidant capacity, were further fractionated (cut-offs of 10, 5, and 1 kDa) via cross-flow filtration. Amino acid composition indicated that most of the fractions showed positive nutritional characteristics, but a putative bitter taste. A fraction obtained with Alcalase enzyme (retentate 8 kDa) exerted anti-inflammatory potential, while the smaller molecular weight fractions (retentate 1-5 kDa and permeate < 1 kDa) were more active in tyrosinase inhibition. The latter were further sub-fractionated by size-exclusion chromatography. From the 15 most anti-tyrosinase sub-fractions, 365 peptide sequences were identified via liquid chromatography coupled with high resolution mass spectrometry. The present data support the possible exploitation of bioactive peptide from rice starch by-product as ingredients into food, nutraceutical, pharmaceutical, and cosmetic formulations.

ATP Sensing Paper with Smartphone Bioluminescence-Based Detection.

Adenosine-5'-triphosphate (ATP) is the primary energy carrier in all living organisms, and its detection in living cells represents a well-established approach. ATP-driven bioluminescence (BL) relying on the D-luciferin-luciferase reaction is a bioanalytical tool widely employed for monitoring hygiene and microbial contamination of foods.Here, we report a straightforward method for ATP BL detection using an ATP sensing paper fabricated with an alternative freeze-dry procedure. The assay can be easily implemented in laboratories equipped with (i) freeze-drying, wax printing, and 3D printing technologies and (ii) instrumentation for BL detection such as benchtop luminometers and portable light detectors including a smartphone camera without the need for additional equipment.

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.

Portable light detectors for bioluminescence biosensing applications: A comprehensive review from the analytical chemist's perspective.

Bioluminescence, that is the emission of light in living organisms, has been extensively explored and applied for diverse bioanalytical applications, spanning from molecular imaging to biosensing. The unprecedented technological evolution of portable light detectors opened new possibilities to implement bioluminescence detection into miniaturized devices. We are witnessing a number of applications, including DNA sequencing, reporter gene assays, DNA amplification for point-of care and point-of need analyses relying on BL. Several photon detectors are currently available for measuring low light emission, such as photomultiplier tubes (PMT), charge-coupled devices (CCD), complementary metal oxide semiconductors (CMOS), single photon avalanche diodes (SPADs), silicon photomultipliers (SiPMs) and smartphone-integrated CMOS. Each technology has pros and cons and several issues, such as temperature dependence of the instrumental specific noise, the power supply, imaging capability and ease of integration, should be considered in the selection of the most appropriate detector for the selected BL application. These issues will be critically discussed from the perspective of the analytical chemist together with relevant examples from the literature with the goal of helping the reader in the selection and use of the most suitable detector for the selected application and to introduce non familiar readers into this exciting field.

Orthogonal paper biosensor for mercury(II) combining bioluminescence and colorimetric smartphone detection.

Mercury contamination in the environment has reached alarming levels. Due to its persistence and bioaccumulation, mercury is one of the most widespread toxic heavy metals found in air, water and food. Thus, it is mandatory to monitor mercury and its compounds, and the availability of sensitive and rapid biosensors is highly valuable. We developed a low-cost biosensor for orthogonal detection of mercury(II) integrating three different biorecognition principles on a three-leaf paper: i) a mercury-specific bioluminescent Escherichia coli bioreporter strain expressing NanoLuc luciferase as reporter protein, ii) a purified ß-galactosidase (ß-gal) enzyme which is irreversibly inhibited by mercury and other metal ions, and iii) an Aliivibrio fischeri bioluminescent strain which is used to quantitatively assess sample toxicity and correct the analytical signal accordingly. Both sensory elements and substrates, Furimazine for the bioluminescent reporter strain and chlorophenol red-ß-D-galactopyranoside for colorimetric detection of ß-gal, were integrated in the paper sensor to provide a stable all-in-one disposable cartridge which can be easily snapped into a smartphone with a clover-shaped 3D printed housing. This is the first integration of bioluminescence and colorimetric detection on a smartphone-paper sensor, providing a readout within 15 and 60 min for the colorimetric and bioluminescent detection respectively. The biosensor was applied to water samples spiked with different concentrations of mercury, interferents and toxic chemicals providing a limit of detection for Hg(II) at the ppb levels.

Paper-Based Immunosensors with Bio-Chemiluminescence Detection.

Since the introduction of paper-based analytical devices as potential diagnostic platforms a few decades ago, huge efforts have been made in this field to develop systems suitable for meeting the requirements for the point-of-care (POC) approach. Considerable progress has been achieved in the adaptation of existing analysis methods to a paper-based format, especially considering the chemiluminescent (CL)-immunoassays-based techniques. The implementation of biospecific assays with CL detection and paper-based technology represents an ideal solution for the development of portable analytical devices for on-site applications, since the peculiarities of these features create a unique combination for fitting the POC purposes. Despite this, the scientific production is not paralleled by the diffusion of such devices into everyday life. This review aims to highlight the open issues that are responsible for this discrepancy and to find the aspects that require a focused and targeted research to make these methods really applicable in routine analysis.

Recent Advancements in Enzyme-Based Lateral Flow Immunoassays.

Paper-based lateral-flow immunoassays (LFIAs) have achieved considerable commercial success and their impact in diagnostics is continuously growing. LFIA results are often obtained by visualizing by the naked eye color changes in given areas, providing a qualitative information about the presence/absence of the target analyte in the sample. However, this platform has the potential to provide ultrasensitive quantitative analysis for several applications. Indeed, LFIA is based on well-established immunological techniques, which have known in the last year great advances due to the combination of highly sensitive tracers, innovative signal amplification strategies and last-generation instrumental detectors. All these available progresses can be applied also to the LFIA platform by adapting them to a portable and miniaturized format. This possibility opens countless strategies for definitively turning the LFIA technique into an ultrasensitive quantitative method. Among the different proposals for achieving this goal, the use of enzyme-based immunoassay is very well known and widespread for routine analysis and it can represent a valid approach for improving LFIA performances. Several examples have been recently reported in literature exploiting enzymes properties and features for obtaining significative advances in this field. In this review, we aim to provide a critical overview of the recent progresses in highly sensitive LFIA detection technologies, involving the exploitation of enzyme-based amplification strategies. The features and applications of the technologies, along with future developments and challenges, are also discussed.

Ultrasensitive On-Field Luminescence Detection Using a Low-Cost Silicon Photomultiplier Device.

The availability of portable analytical devices for on-site monitoring and rapid detection of analytes of forensic, environmental, and clinical interest is vital. We report the development of a portable device for the detection of biochemiluminescence relying on silicon photomultiplier (SiPM) technology, called LuminoSiPM, which includes a 3D printed sample holder that can be adapted for both liquid samples and paper-based biosensing. We performed a comparison of analytical performance in terms of detectability with a benchtop luminometer, a portable cooled charge-coupled device (CCD sensor), and smartphone-integrated complementary metal oxide semiconductor (CMOS) sensors. As model systems, we used two luciferase/luciferin systems emitting at different wavelengths using purified protein solutions: the green-emitting P. pyralis mutant Ppy-GR-TS (λmax 550 nm) and the blue-emitting NanoLuc (λmax 460 nm). A limit of detection of 9 femtomoles was obtained for NanoLuc luciferase, about 2 and 3 orders of magnitude lower than that obtained with the portable CCD camera and with the smartphone, respectively. A proof-of-principle forensic application of LuminoSiPM is provided, exploiting an origami chemiluminescent paper-based sensor for acetylcholinesterase inhibitors, showing high potential for this portable low-cost device for on-site applications with adequate sensitivity for detecting low light intensities in critical fields.

A Genetically Encoded Bioluminescence Intracellular Nanosensor for Androgen Receptor Activation Monitoring in 3D Cell Models.

In recent years, there has been an increasing demand for predictive and sensitive in vitro tools for drug discovery. Split complementation assays have the potential to enlarge the arsenal of in vitro tools for compound screening, with most of them relying on well-established reporter gene assays. In particular, ligand-induced complementation of split luciferases is emerging as a suitable approach for monitoring protein-protein interactions. We hereby report an intracellular nanosensor for the screening of compounds with androgenic activity based on a split NanoLuc reporter. We also confirm the suitability of using 3D spheroids of Human Embryonic Kidney (HEK-293) cells for upgrading the 2D cell-based assay. A limit of detection of 4 pM and a half maximal effective concentration (EC50) of 1.7 ± 0.3 nM were obtained for testosterone with HEK293 spheroids. This genetically encoded nanosensor also represents a new tool for real time imaging of the activation state of the androgen receptor, thus being suitable for analysing molecules with androgenic activity, including new drugs or endocrine disrupting molecules.

Bioluminescence goes portable: recent advances in whole-cell and cell-free bioluminescence biosensors.

Recent advancements in synthetic biology, organic chemistry, and computational models have allowed the application of bioluminescence in several fields, ranging from well established methods for detecting microbial contamination to in vivo imaging to track cancer and stem cells, from cell-based assays to optogenetics. Moreover, thanks to recent technological progress in miniaturized and sensitive light detectors, such as photodiodes and imaging sensors, it is possible to implement laboratory-based assays, such as cell-based and enzymatic assays, into portable analytical devices for point-of-care and on-site applications. This review highlights some recent advances in the development of whole-cell and cell-free bioluminescence biosensors with a glance on current challenges and different strategies that have been used to turn bioassays into biosensors with the required analytical performance. Critical issues and unsolved technical problems are also highlighted, to give the reader a taste of this fascinating and challenging field.

Microbial Fermentation of Industrial Rice-Starch Byproduct as Valuable Source of Peptide Fractions with Health-Related Activity.

The rice-starch processing industry produces large amounts of a protein-rich byproducts during the conversion of broken rice to powder and crystal starch. Given the poor protein solubility, this material is currently discarded or used as animal feed. To fully exploit rice's nutritional properties and reduce this waste, a biotechnological approach was adopted, inducing fermentation with selected microorganisms capable of converting the substrate into peptide fractions with health-related bioactivity. Lactic acid bacteria were preferred to other microorganisms for their safety, efficient proteolytic system, and adaptability to different environments. Peptide fractions with different molecular weight ranges were recovered from the fermented substrate by means of cross-flow membrane filtration. The fractions displayed in vitro antioxidant, antihypertensive, and anti-tyrosinase activities as well as cell-based anti-inflammatory and anti-aging effects. In the future, the peptide fractions isolated from this rice byproduct could be directly exploited as health-promoting functional foods, dietary supplements, and pharmaceutical preparations. The suggested biotechnological process harnessing microbial bioconversion may represent a potential solution for many different protein-containing substrates currently treated as byproducts (or worse, waste) by the food industry.

Multienzyme chemiluminescent foldable biosensor for on-site detection of acetylcholinesterase inhibitors.

The rapid hydrolysis of acetylcholine (ACh), one of the key neurotransmitters in the human body, by the enzyme acetylcholinesterase (AChE) is fundamental for the termination of ACh impulse transmission. Several chemicals, including organophosphorus (OP) pesticides, warfare agents and drugs, are AChE reversible or irreversible inhibitors, thus their rapid on-site detection is of primary importance. Here we report for the first time a chemiluminescence (CL) foldable paper-based biosensor for detection of AChE inhibitors. The biosensor exploits three coupled enzymatic reactions catalyzed by AChE, choline oxidase and horseradish peroxidase, leading to production of hydrogen peroxide, which is measured with an optimized luminol substrate. The origami approach allows to add reagents and trigger the sequential reactions in separate steps. A compact 3D-printed device including a mini dark box was created to enable smartphone detection. The CL foldable paper-based biosensor showed suitable for the rapid detection of OP pesticides in food matrices with a total assay time of 25 min. It is thus a rapid, low cost portable test suitable for point-of-need detection of chemicals inhibiting AChE.