Papers by Elisa Michelini
... Nanoparticles 30 Nano and Microsensors for Chemical and Biological Terrorism Surveillance Edi... more ... Nanoparticles 30 Nano and Microsensors for Chemical and Biological Terrorism Surveillance Edited by Jeffrey B.-H. Tok r Royal ... Agents 173 Acronyms 174 References 174 Chapter 8 Conducting Polymer Transistors for Sensor Applications Fabio Cicoira, Daniel A. Bernards ...
Microorganisms
The rice-starch processing industry produces large amounts of a protein-rich byproducts during th... more 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...
Biosensors and Bioelectronics
Analytical and Bioanalytical Chemistry
Analytical and Bioanalytical Chemistry
Proceedings
This work presents a miniaturized lab-on-chip system suitable for monitoring the activity of livi... more This work presents a miniaturized lab-on-chip system suitable for monitoring the activity of living cells through the on-chip detection of their bioluminescence emission. The system integrates amorphous silicon diodes, acting as temperature and light sensors, and indium tin oxide film, acting as heater, on a single glass substrate. During its operation, the glass is thermally and optically coupled to the investigated cells and electrically connected to an electronic board, which controls the lab-on-chip temperature and monitors the sensor photocurrents. The proposed lab-onchip is particularly attractive for the development of portable cell-based biosensors useful for biological activity monitoring as well as for cell cytotoxicity evaluation.
Analytical biochemistry, Jan 4, 2017
Beetle luciferases have been adapted for live cell imaging where bioluminescence is dependent on ... more Beetle luciferases have been adapted for live cell imaging where bioluminescence is dependent on the cellular availability of ATP, O2, and added luciferin. Previous Photinus pyralis red-emitting variants with high Km values for ATP have performed disappointingly in live cells despite having much higher relative specific activities than enzymes like Click Beetle Red (CBR). We engineered a luciferase variant PLR3 having a Km value for ATP similar to CBR and ∼2.6-fold higher specific activity. The red-emitting PLR3 was ∼2.5-fold brighter than CBR in living HEK293T and HeLa cells, an improvement consistent with the importance of the Km value in low ATP environments.
PloS one, 2017
Recently, the isolation of new health-related bioactive molecules derived from agro-food industri... more Recently, the isolation of new health-related bioactive molecules derived from agro-food industrial by-products by means of environment-friendly extraction processes has become of particular interest. In the present study, a protein by-product from the rice starch industry was hydrolysed with five commercial proteolytic enzymes, avoiding the use of solvents or chemicals. The digestion processes were optimised, and the digestates were separated in fractions with four different molecular weight ranges by using a cross-flow membrane filtration technique. Total hydrolysates and fractions were tested in vitro for a wide range of biological activities. For the first time rice-derived peptides were assayed for anti-tyrosinase, anti-inflammatory, cytotoxicity and irritation capacities. Antioxidant and anti-hypertensive activities were also evaluated. Protamex, Alcalase and Neutrase treatments produced peptide fractions with valuable bioactivities without resulting cytotoxic or irritant. Hig...
Analytical and Bioanalytical Chemistry, 2016
The availability of smartphones with high-performance digital image sensors and processing power ... more 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.
Molecular Imaging and Biology, 2010
Chemosphere, Oct 31, 2005
In this paper we describe the construction and use of a set of bioluminescent yeast strains for t... more In this paper we describe the construction and use of a set of bioluminescent yeast strains for the detection of compounds that can affect androgen or estrogen receptor mediated hormonal signalling. The set includes Saccharomyces cerevisiae strains expressing human androgen receptor (AR), estrogen receptor alpha (ERalpha) or estrogen receptor beta (ERbeta), along with firefly luciferase controlled by a respective hormone responsive promoter. A constitutively luminescent strain was included in the set for determining the cytotoxicity of the sample. Yeast cells were incubated with pure chemicals or complex samples for 2.5 h, after which the signal could be detected from the cell-sample mixture after simply adding the D-luciferin substrate. The assays could be completed in one day and they required no cell lysis or centrifugation steps, which makes them suitable for high-throughput analysis of samples. Due to a short incubation time the assays are directly applicable to different sample matrices, requiring no pretreatment of the samples. The assays were used to assess the hormonal activity in moisturizing lotions as an example of a complex sample matrix known to contain endocrine disrupting chemicals. Six out of eight tested moisturisers showed high estrogenic activity, whereas no androgenic activity was observed in the samples.
Analytical Chemistry, 2004
A new bioluminescence resonance energy transfer (BRET) homogeneous assay to evaluate the presence... more A new bioluminescence resonance energy transfer (BRET) homogeneous assay to evaluate the presence of estrogenlike compounds has been developed and optimized. The assay is based on the direct evaluation of estrogen r receptor (ERr) homodimerization as a result of estrogenlike compound binding. ERr monomer was genetically fused either to Renilla luciferase (Rluc) or to enhanced yellow fluorescent protein (EYFP). In the presence of estrogens, ERr dimerization brings Rluc and EYFP molecules close enough for an energy transfer. An in vitro BRET assay was first developed using purified fusion proteins (ERr-Rluc and ERr-EYFP) expressed in Escherichia coli to evaluate and optimize the analytical performances of the assay in the presence of 17-estradiol. The "in vivo" BRET quantitative assay was then developed by coexpressing the two fusion proteins in live HepG2 cells. The assay can be performed in 96-well microplate format with a 30-min incubation and allows detection with adequate accuracy and precision of as low as 1 nM of 17-estradiol. This new "in vivo" BRET assay allows evaluating the estrogen-like activity and synthetic xenoestrogens from biological and environmental samples.
Analytical Chemistry, 2002
Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology, Oct 31, 2006
The cDNA encoding the luciferase from the Italian firefly Luciola italica was cloned using revers... more The cDNA encoding the luciferase from the Italian firefly Luciola italica was cloned using reverse transcriptase-PCR and a gene-specific primer set based on the DNA sequence of Luciola mingrelica. The cDNA sequence of L. italica luciferase was determined to be 1647 base pairs in length with an open reading frame of 548 amino acids. Phylogenetic analysis of the protein sequence demonstrated that this luciferase is closely related to that of other fireflies of the Lampyridae family, particularly within the Luciolinae subfamily, showing 96% homology to luciferases from the fireflies Hotaria unmunsana and Hotaria parvula. The specific activity of the L. italica luciferase was 78% of the North American enzyme, after correction for emission color differences. The bioluminescence emission of the Italian firefly is pH sensitive with maxima at 566 nm and 614 nm at pH 7.8 and 6.0, respectively. Interestingly, the total bioluminescence output was approximately 2-fold greater than that of P. pyralis luciferase due to differences in turnover characteristics evidenced by extended light emission decay kinetics. We expect that this newly discovered luciferase will be suitable for a wide range of bioluminescence applications including in vivo imaging and multiplex assays.
New Biotechnology, 2016
The present work aimed at the recovery and characterization of polyphenolic compounds extracted f... more The present work aimed at the recovery and characterization of polyphenolic compounds extracted from red grape pomace (Vitis vinifera L.), a winemaking by-product. Polyphenolic compounds of wet (WP) and dried (DP) red pomace were recovered by enzymatic digestions and ethanol-based extractions. Fungamyl and Celluclast enzymes were found to be the most effective in enhancing polyphenol release from WP. WP samples showed the highest capacity of releasing polyphenols with 2h control 24°C and 2h 1% Celluclast resulting as the best treatments. A significantly lower amount of polyphenols was recovered from DP most probably as a consequence of the pomace drying. The best extracts contained high amounts of total polyphenols, flavonoids, tannins and anthocyanins and exerted antioxidant and cholesterol-lowering activities. The results support the possibility of exploiting the extracts coming from grape processing by-products as ingredients for functional and innovative products in the nutraceutical, pharmaceutical or cosmetic fields.
Analytical Chemistry, Feb 1, 2008
The availability of new bioluminescent proteins, obtained by cDNA cloning and mutagenesis of wild... more The availability of new bioluminescent proteins, obtained by cDNA cloning and mutagenesis of wild-type genes, expanded the applicability of these reporters from the perspective of using more proteins emitting at different wavelengths in the same cell-based assay. By spectrally resolving the light emitted by different reporter proteins, it is in fact possible to simultaneously monitor multiple targets. A new luciferase isolated from Luciola italica has been recently cloned, and thermostable red- and green-emitting mutants were obtained by random and site-directed mutagenesis. Different combinations of luciferases were used in vitro as purified proteins and expressed in bacterial and mammalian cells to test their suitability for multicolor assays. A mammalian triple-color reporter model system was then developed using a green-emitting wild-type Photinus pyralis luciferase, a red thermostable mutant of L. italica luciferase, and a secreted Gaussia princeps luciferase (GLuc) to monitor the two main pathways of bile acid biosynthesis. The two firefly luciferases were used to monitor cholesterol 7-alpha hydroxylase and sterol 27-hydroxylase, while secreted constitutively expressed GLuc was used as an internal vitality control. By treating the cells with chenodeoxycholic acid, it was possible to obtain dose-dependent inhibitions of the two specific signals together with a constant production of GLuc, which allowed for a dynamic evaluation of the metabolic activity of the cells. This is the first triple-color mammalian reporter assay that combines secreted and nonsecreted luciferases requiring different substrates, thus avoiding reciprocal interference between different BL signals. This approach is suitable for high content analysis of gene transcription in living cells to shorten the time for screening assays, increasing throughput and cost-effectiveness.
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Papers by Elisa Michelini