Maria Maddalena Calabretta

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.

Field-deployable whole-cell bioluminescent biosensors: so near and yet so far

AbstractThe use of smart supports and bioinspired materials to confine living cells and use them for field-deployable biosensors has recently attracted much attention. In particular, bioluminescent whole-cell biosensors designed to respond to different analytes or classes of analyte have been successfully implemented in portable and cost-effective analytical devices. Significant advances in detection technology, biomaterial science, and genetic engineering of cells have recently been reported. Now the challenge is to move from benchtop traditional cell-based assays to portable biosensing devices. Improvement of the analytical performance of these biosensors depends on the availability of optimized bioluminescent reporters, and promising approaches that go beyond reporter gene technology are emerging. To enable handling of cells as ready-to-use reagents, nature-inspired strategies have been used, with the objective of keeping cells in a dormant state until use. Several issues must still be investigated, for example long-term viability of cells, the possibility of performing real-time analysis, and multiplexing capability. FigureConcept of whole-cell bioluminescent biosensor

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.

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.

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

AbstractThe 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 abstractSmartphone-based bioluminescence cell biosensor

Luciferase Genes as Reporter Reactions: How to Use Them in Molecular Biology?

: The latest advances in molecular biology have made available several biotechnological tools that take advantage of the high detectability and quantum efficiency of bioluminescence (BL), with an ever-increasing number of novel applications in environmental, pharmaceutical, food, and forensic fields. Indeed, BL proteins are being used to develop ultrasensitive binding assays and cell-based assays, thanks to their high detectability and to the availability of highly sensitive BL instruments. The appealing aspect of molecular biology tools relying on BL reactions is their general applicability in both in vitro assays, such as cell cultures or purified proteins, and in vivo settings, such as in whole-animal BL imaging. The aim of this chapter is to provide the reader with an overview of state-of-the-art bioluminescent tools based on luciferase genes, highlighting molecular biology strategies that have been applied so far, together with some selected examples.

A high susceptibility to redox imbalance of the transmissible stages ofPlasmodium falciparumrevealed with a luciferase-based mature gametocyte assay: Targeting redox inP. falciparummature gametocytes

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.

Bioluminescence Imaging of Spheroids for High-throughput Longitudinal Studies on 3D Cell Culture Models

Bioluminescent (BL) cell‐based assays based on two‐dimensional (2D) monolayer cell cultures represent well‐established bioanalytical tools for preclinical screening of drugs. However, cells in 2D cultures do not often reflect the morphology and functionality of living organisms, thus limiting the predictive value of 2D cell‐based assays. Conversely, 3D cell models have the capability to generate the extracellular matrix and restore cell‐to‐cell communications; thus, they are the most suitable model to mimic in vivo physiology. In this work, we developed a nondestructive real‐time BL imaging assay of spheroids for longitudinal studies on 3D cell models. A high‐throughput BL 3D cell‐based assay in micropatterned 96‐well plate format is reported. The assay performance was assessed using the transcriptional regulation of nuclear factor K beta response element in human embryonic kidney (HEK293) cells. We compared concentration–response curves for tumor necrosis factor‐α with those obtained using conventional 2D cell cultures. One of the main advantages of this approach is the nonlysing nature of the assay, which allows for repetitive measurements on the same sample. The assay can be implemented in any laboratory equipped with basic cell culture facilities and paves the way to the development of new 3D bioluminescent cell‐based assays.

White grape pomace extracts, obtained by a sequential enzymatic plus ethanol-based extraction, exert antioxidant, anti-tyrosinase and anti-inflammatory activities

The present work aimed at optimizing a two-step enzymatic plus solvent-based process for the recovery of bioactive compounds from white grape (Vitis vinifera L., mix of Trebbiano and Verdicchio cultivars) pomace, the winemaking primary by-product. Phenolic compounds solubilised by water enzyme-assisted and ethanol-based extractions of wet (WP) and dried (DP) pomace were characterised for composition and tested for antioxidant, anti-tyrosinase and anti-inflammatory bioactivities. Ethanol treatment led to higher phenol yields than water extraction, while DP samples showed the highest capacity of releasing polyphenols, most probably as a positive consequence of the pomace drying process. Different compositions and bioactivities were observed between water and ethanol extracts and among different treatments and for the first time the anti-tyrosinase activity of V. vinifera pomace extracts, was here reported. Enzymatic treatments did not significantly improve the total amount of solubilised compounds; Celluclast in DP led to the recovery of extracts enriched in specific compounds, when compared to control. The best extracts (enzymatic plus ethanol treatment total levels) were obtained from DP showing significantly higher amounts of polyphenols, flavonoids, flavanols and tannins and exerted higher antioxidant and anti-tyrosinase activities than WP total extracts. Conversely, anti-inflammatory capacity was only detected in water (with and without enzyme) extracts, with WP samples showing on average a higher activity than DP. The present findings demonstrate that white grape pomace constitute a sustainable source for the extraction of phytochemicals that might be exploited as functional ingredients in the food, nutraceutical, pharmaceutical or cosmetic industries.

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.