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.

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.

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.

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.

High-Throughput Bioluminescence Imaging and Reporter Gene Assay with 3D Spheroids from Human Cell Lines.

3D cell culture models represent an attractive approach to decode intracellular and intercellular signaling, providing biologically relevant information and predictive data. Bioluminescent reporter gene assays and bioluminescence imaging in 3D cell models are very promising bioanalytical tools for several applications.Here we report a very straightforward method for bioluminescence imaging and bioluminescent reporter gene assays in 3D cell-culture models. Both the assays can be easily implemented in laboratories equipped with basic cell culture facilities and instrumentation for bioluminescence detection, that is, low-light detectors connected to inverted microscopes and luminometers, without the need for additional equipment.

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.