Donato Calabria

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.

Fructose Intake, Serum Uric Acid, and Cardiometabolic Disorders: A Critical Review

There is a direct relationship between fructose intake and serum levels of uric acid (UA), which is the final product of purine metabolism. Recent preclinical and clinical evidence suggests that chronic hyperuricemia is an independent risk factor for hypertension, metabolic syndrome, and cardiovascular disease. It is probably also an independent risk factor for chronic kidney disease, Type 2 diabetes, and cognitive decline. These relationships have been observed for high serum UA levels (>5.5 mg/dL in women and >6 mg/dL in men), but also for normal to high serum UA levels (5–6 mg/dL). In this regard, blood UA levels are much higher in industrialized countries than in the rest of the world. Xanthine-oxidase inhibitors can reduce UA and seem to minimize its negative effects on vascular health. Other dietary and pathophysiological factors are also related to UA production. However, the role of fructose-derived UA in the pathogenesis of cardiometabolic disorders has not yet been fully clarified. Here, we critically review recent research on the biochemistry of UA production, the relationship between fructose intake and UA production, and how this relationship is linked to cardiometabolic disorders.

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.

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.

Single-cell chemiluminescence imaging of parvovirus B19 life cycle.

Human parvovirus B19 (B19V) is a single-stranded DNA virus. The genome encodes a multifunctional non-structural protein (NS), two capsid proteins (VP1, VP2) and other small non-structural proteins (7.5 kDa, 9 kDa, 11 kDa). Within sensitive cells, B19V can achieve a productive replicative cycle or, on the contrary, establish persistence; differences in its expression profile have been yet investigated following in vitro infections by methodologies enabling information on the entire infected cell population. Conversely, the present study reports quantitative data on the production of B19V analytes (DNA, RNAs, and proteins) at single cell-level, underlining cell-to-cell differences through the viral specific macromolecular synthesis process. Microscope imaging assays (in situ hybridization and immunocytochemical assays), exploiting chemiluminescence as principle detection and targeting viral nucleic acids and antigens, have been performed on a permissive cell line following in vitro infection. Chemiluminescence, involving the emission of photons deriving from a chemical reaction, provided the localization and quantitative detection of analytes down to a few molecules within infected cells. In our experimental conditions, B19V transcriptional activity, leading to the production of NS and VP RNAs, has been detected early in the viral cycle (from 12h post-infection, hpi) and before genome replication, starting at 24 hpi. The analysis of VP RNAs and related proteins suggested an inhibitory effect on capsid protein translation, as a post-transcriptional regulation events. Indeed, high levels of VP transcripts have been detected at early stages of infection while the proteins accumulated within cells only at 48-72 hpi.