Chemiluminescence "Add-and-Measure" Sensing Paper Based on the Prussian Blue/Metal-Organic Framework MIL-101 Nanozyme for Rapid Hydrogen Peroxide Detection.

In this work, a chemiluminescent sensing paper has been developed using a peroxidase biomimetic metal-organic framework as a versatile host platform. For the first time, we have explored the use of in situ growth of Prussian Blue nanoparticles (PB-NPs) onto the MIL-101(Fe) structure for the assembly of a ready-to-use sensing paper. In situ growth of PB-NPs has been performed on the surface of the MIL-101(n) family. This novel composite, named PB-NPs@MIL-101(Fe), has been successfully used to develop a sensing paper for one-step detection of H2O2 in real samples (commercial disinfectant solutions and tap water samples). The as-prepared material was fully characterized, including X-ray analysis, Fourier transform infrared, scanning and transmission electron microscopies, nitrogen isotherms, and elemental analysis. After the characterization, the analytical performance of the PB-NPs@MIL-101(Fe) sensing paper was evaluated. The low-cost sensor (0.15 euro per unit) was able to detect down to 8.2 µM (corresponding to 8.2 × 10-11 mol) H2O2 using only 10 µL of sample with satisfactory reproducibility (relative standard deviation 17%).

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.

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.

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.

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.

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.

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.

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.

Precision medicine, bioanalytics and nanomaterials: toward a new generation of personalized portable diagnostics.

The customization of disease treatment focused on genetic, environmental and lifestyle factors of individual patients, including tailored medical decisions and treatments, is identified as precision medicine. This approach involves the combination of various aspects such as the collection and processing of a large amount of data, the selection of optimized and personalized drug dosage for each patient and the development of selective and reliable analytical tools for the monitoring of clinical, genetic and environmental parameters. In this context, miniaturized, compact and ultrasensitive bioanalytical devices play a crucial role for achieving the goals of personalized medicine. In this review, the latest analytical technologies suitable for providing portable and easy-to-use diagnostic tools in clinical settings will be discussed, highlighting new opportunities arising from nanotechnologies, offering peculiar perspectives and opportunities for precision medicine.

Prêt-à-porter nanoYESα and nanoYESß bioluminescent cell biosensors for ultrarapid and sensitive screening of endocrine-disrupting chemicals.

Cell-based assays utilizing reporter gene technology have been widely exploited for biosensing, as they provide useful information about the bioavailability and cell toxicity of target analytes. The long assay time due to gene transcription and translation is one of the main drawbacks of cell biosensors. We report the development of two yeast biosensors stably expressing human estrogen receptors α and ß and employing NanoLuc as the reporter protein to upgrade the widely used yeast estrogen screening (YES) assays. A viability control strain was also developed based on a chimeric green-emitting luciferase, PLG2, expressed for the first time in Saccharomycescerevisiae. Thanks to their brightness, NanoLuc and PLG2 provided excellent sensitivity, enabling the implementation of these biosensors into low-cost smartphone-based devices. The developed biosensors had a rapid (1 h) response and reported on (anti)estrogenic activity via human estrogen receptors α and ß as well as general sample toxicity. Under optimized conditions, we obtained LODs of 7.1 ± 0.4 nM and 0.38 ± 0.08 nM for E2 with nanoYESα and nanoYESß, respectively. As a proof of concept, we analyzed real samples from plants showing significant estrogenic activity or known to contain significant amounts of phytoestrogens. Graphical abstract.

A high susceptibility to redox imbalance of the transmissible stages of Plasmodium falciparum revealed with a luciferase-based mature gametocyte assay.

The goal to prevent Plasmodium falciparum transmission from humans to mosquitoes requires the identification of targetable metabolic processes in the mature (stage V) gametocytes, the sexual stages circulating in the bloodstream. This task is complicated by the apparently low metabolism of these cells, which renders them refractory to most antimalarial inhibitors and constrains the development of specific and sensitive cell-based assays. Here, we identify and functionally characterize the regulatory regions of the P. falciparum gene PF3D7_1234700, encoding a CPW-WPC protein and named here Upregulated in Late Gametocytes (ULG8), which we have leveraged to express reporter genes in mature male and female gametocytes. Using transgenic parasites containing a pfULG8-luciferase cassette, we investigated the susceptibility of stage V gametocytes to compounds specifically affecting redox metabolism. Our results reveal a high sensitivity of mature gametocytes to the glutathione reductase inhibitor and redox cycler drug methylene blue (MB). Using isobologram analysis, we find that a concomitant inhibition of the parasite enzyme glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase, a key component of NADPH synthesis, potently synergizes MB activity. These data suggest that redox metabolism and detoxification activity play an unsuspected yet vital role in stage V gametocytes, rendering these cells exquisitely sensitive to decreases in NADPH concentration.

A novel bioluminescent NanoLuc yeast-estrogen screen biosensor (nanoYES) with a compact wireless camera for effect-based detection of endocrine-disrupting chemicals.

The presence of chemicals with estrogenic activity in surface, groundwater, and drinking water poses serious concerns for potential threats to human health and aquatic life. At present, no sensitive portable devices are available for the rapid monitoring of such contamination. Here, we propose a cell-based mobile platform that exploits a newly developed bioluminescent yeast-estrogen screen (nanoYES) and a low-cost compact camera as light detector. Saccharomyces cerevisiae cells were genetically engineered with a yeast codon-optimized variant of NanoLuc luciferase (yNLucP) under the regulation of human estrogen receptor α activation. Ready-to-use 3D-printed cartridges with immobilized cells were prepared by optimizing a new procedure that enables to produce alginate slices with good reproducibility. A portable device was obtained exploiting a compact camera and wireless connectivity enabling a rapid and quantitative evaluation (1-h incubation at room temperature) of total estrogenic activity in small sample volumes (50 µL) with a LOD of 0.08 nM for 17ß-estradiol. The developed portable analytical platform was applied for the evaluation of water samples spiked with different chemicals known to have estrogen-like activity. Thanks to the high sensitivity of the newly developed yeast biosensor and the possibility to wireless connect the camera with any smartphone model, the developed configuration is more versatile than previously reported smartphone-based devices, and could find application for on-site analysis of endocrine disruptors. Graphical abstract Wireless effect-based detection of endocrine-disrupting chemicals with nanoYES platform.

Proteomic analysis and bioluminescent reporter gene assays to investigate effects of simulated microgravity on Caco-2 cells.

Microgravity is one of the most important features in spaceflight. Previous evidence from in-vitro studies has shown that significant changes occur under simulated microgravity. For this reason, human colon adenocarcinoma Caco-2 cells were selected as cell model of intestinal epithelial barrier and their response to altered gravity conditions was investigated, especially on the protein level. In this study, we combined label-free shotgun proteomics and bioluminescent reporter gene assays to identify key proteins and pathways involved in the response of Caco-2 cells under reference and microgravity conditions. A two-dimensional clinostat was modified with 3D-printed adaptors to hold conventional T25 culture flasks. The comparative proteome analysis led to identify 38 and 26 proteins differently regulated by simulated microgravity after 48 and 72 h, respectively. Substantial fractions of these proteins are involved in regulation, cellular and metabolic processes and localization. Bioluminescent reporter gene assays were carried out to investigate microgavity-induced alterations on the transcriptional regulation of key targets, such as NF-kB pathway and CYP27A1. While no significant difference was found in the basal transcription, a lower NF-kB basal activation in simulated microgravity conditions was reported, corroborating the hypothesis of reduced immunity in microgravity conditions.

Exploiting NanoLuc luciferase for smartphone-based bioluminescence cell biosensor for (anti)-inflammatory activity and toxicity.

The availability of smartphones with high-performance digital image sensors and processing power has completely reshaped the landscape of point-of-need analysis. Thanks to the high maturity level of reporter gene technology and the availability of several bioluminescent proteins with improved features, we were able to develop a bioluminescence smartphone-based biosensing platform exploiting the highly sensitive NanoLuc luciferase as reporter. A 3D-printed smartphone-integrated cell biosensor based on genetically engineered Hek293T cells was developed. Quantitative assessment of (anti)-inflammatory activity and toxicity of liquid samples was performed with a simple and rapid add-and-measure procedure. White grape pomace extracts, known to contain several bioactive compounds, were analyzed, confirming the suitability of the smartphone biosensing platform for analysis of untreated complex biological matrices. Such approach could meet the needs of small medium enterprises lacking fully equipped laboratories for first-level safety tests and rapid screening of new bioactive products. Graphical abstract Smartphone-based bioluminescence cell biosensor.

Integrating biochemiluminescence detection on smartphones: mobile chemistry platform for point-of-need analysis.

In this paper, we report, for the first time, the use of a smartphone to image and quantify biochemiluminescence coupled biospecific enzymatic reactions to detect analytes in biological fluids. Using low-cost three-dimensional (3D) printing technology, we fabricated a smartphone accessory and a minicartridge for hosting biospecific reactions. As a proof-of-principle, we report two assays: a bioluminescence assay for total bile acids using 3α-hydroxyl steroid dehydrogenase coimmobilized with bacterial luciferase system and a chemiluminescence assay for total cholesterol using cholesterol esterase/cholesterol oxidase coupled with the luminol-H2O2-horseradish peroxidase system. These assays can be performed within 3 min in a very straightforward manner and provided adequate analytical performance for the analysis of total cholesterol in serum (limit of detection (LOD) = 20 mg/dL) and total bile acid in serum and oral fluid (LOD = 0.5 µmol/L) with a reasonable accuracy and precision. Smartphone-based biochemiluminescence detection could be thus applied to a variety of clinical chemistry assays.

Multicolor bioluminescence boosts malaria research: quantitative dual-color assay and single-cell imaging in Plasmodium falciparum parasites.

New reliable and cost-effective antimalarial drug screening assays are urgently needed to identify drugs acting on different stages of the parasite Plasmodium falciparum, and particularly those responsible for human-to-mosquito transmission, that is, the P. falciparum gametocytes. Low Z' factors, narrow dynamic ranges, and/or extended assay times are commonly reported in current gametocyte assays measuring gametocyte-expressed fluorescent or luciferase reporters, endogenous ATP levels, activity of gametocyte enzymes, or redox-dependent dye fluorescence. We hereby report on a dual-luciferase gametocyte assay with immature and mature P. falciparum gametocyte stages expressing red and green-emitting luciferases from Pyrophorus plagiophthalamus under the control of the parasite sexual stage-specific pfs16 gene promoter. The assay was validated with reference antimalarial drugs and allowed to quantitatively and simultaneously measure stage-specific drug effects on parasites at different developmental stages. The optimized assay, requiring only 48 h incubation with drugs and using a cost-effective luminogenic substrate, significantly reduces assay cost and time in comparison to state-of-the-art analogous assays. The assay had a Z' factor of 0.71 ± 0.03, and it is suitable for implementation in 96- and 384-well microplate formats. Moreover, the use of a nonlysing D-luciferin substrate significantly improved the reliability of the assay and allowed one to perform, for the first time, P. falciparum bioluminescence imaging at single-cell level.

A 3D-printed device for a smartphone-based chemiluminescence biosensor for lactate in oral fluid and sweat.

Increasingly, smartphones are used as portable personal computers, revolutionizing communication styles and entire lifestyles. Using 3D-printing technology we have made a disposable minicartridge that can be easily prototyped to turn any kind of smartphone or tablet into a portable luminometer to detect chemiluminescence derived from enzyme-coupled reactions. As proof-of-principle, lactate oxidase was coupled with horseradish peroxidase for lactate determination in oral fluid and sweat. Lactate can be quantified in less than five minutes with detection limits of 0.5 mmol L(-1) (corresponding to 4.5 mg dL(-1)) and 0.1 mmol L(-1) (corresponding to 0.9 mg dL(-1)) in oral fluid and sweat, respectively. A smartphone-based device shows adequate analytical performance to offer a cost-effective alternative for non-invasive lactate measurement. It could be used to evaluate lactate variation in relation to the anaerobic threshold in endurance sport and for monitoring lactic acidosis in critical-care patients.

Exploiting in vitro and in vivo bioluminescence for the implementation of the three Rs principle (replacement, reduction, and refinement) in drug discovery.

Bioluminescence-based analytical tools are suitable for high-throughput and high-content screening assays, finding widespread application in several fields related to the drug discovery process. Cell-based bioluminescence assays, because of their peculiar advantages of predictability, possibility of automation, multiplexing, and miniaturization, seem the most appealing tool for the high demands of the early stages of drug screening. Reporter gene technology and the bioluminescence resonance energy transfer principle are widely used, and receptor binding studies of new agonists/antagonists for a variety of human receptors expressed in different cell lines can be performed. Moreover, bioluminescence can be used for in vitro and in vivo real-time monitoring of pathophysiological processes within living cells and small animals. New luciferases and substrates have recently arrived on the market, further expanding the spectrum of applications. A new generation of probes are also emerging that promise to revolutionize the preclinical imaging market. This formidable toolbox is demonstrated to facilitate the implementation of the three Rs principle in the early drug discovery process, in compliance with ethical and responsible research to reduce cost and improve the reliability and predictability of results.