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ALESSANDRO PARADISI

Assegnista di ricerca
Dipartimento di Scienze della Vita sede ex-Chimica
COLLABORATORE COORDINATO CONTINUATIVO
Dipartimento di Scienze della Vita sede ex Chimica V.Campi 103
Docente a contratto
Dipartimento Educazione e Scienze Umane

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Pubblicazioni

2024 - Dynamic studies of antibody-antigen interactions with an electrolyte-gated organic transistor [Articolo su rivista]
Manco Urbina, P. A.; Paradisi, A.; Hasler, R.; Sensi, M.; Berto, M.; Saygin, G. D.; Dostalek, J.; Pinti, M.; Greco, P.; Borsari, M.; Knoll, W.; Bortolotti, C. A.; Biscarini, F.
abstract

Affinity-based biosensors employing surface-bound biomolecules for analyte detection are important tools in clinical diagnostics and drug development. In this context, electrolyte-gated organic transistors (EGOTs) are emerging as ultrasensitive label-free biosensors. In this study, we present an EGOT sensor integrated within a microfluidic system. The sensor utilizes the cytomegalovirus (CMV) phosphoprotein 65 as a biorecognition element to detect the pathological biomarker human anti-cytomegalovirus antibody in solution. The biorecognition element is grafted onto the gate electrode by exploiting the polyhistidine-tag technology. Real-time monitoring of the EGOT response, coupled with a twocompartment kinetic model analysis, enables the determination of analyte concentration, binding kinetics, and thermodynamics of the interaction. The analysis of the relevant kinetic parameters of the binding process yields a reliable value for the thermodynamic equilibrium constant and suggests that the measured deviations from the Langmuir binding model arise from the co-existence of binding sites with different affinities toward the antibodies.

2024 - How Biorecognition Affects the Electronic Properties of Reduced Graphene Oxide in Electrolyte‐Gated Transistor Immunosensors [Articolo su rivista]
Sensi, Matteo; de Oliveira, Rafael Furlan; Berto, Marcello; Paradisi, Alessandro; Greco, Pierpaolo; Bortolotti, Carlo Augusto; Samorì, Paolo; Biscarini, Fabio
abstract

Ambipolar electrolyte-gated transistors (EGTs) based on reduced graphene oxide (rGO) have been demonstrated as ultra-sensitive and highly specific immunosensors. However, the physics and chemistry ruling the device operation are still not fully unraveled. In this work, the aim is to elucidate the nature of the observed sensitivity of the device. Toward this aim, a physical–chemical model that, coupled with the experimental characterization of the rGO-EGT, allows one to quantitatively correlate the biorecognition events at the gate electrode and the electronic properties of rGO-EGT is proposed. The equilibrium of biorecognition occurring at the gate electrode is shown to determine the apparent charge neutrality point (CNP) of the rGO channel. The multiparametric analysis of the experimental transfer characteristics of rGO-EGT reveals that the recognition events modulate the CNP voltage, the excess carrier density Δn, and the quantum capacitance of rGO. This analysis also explains why hole and electron carrier mobilities, interfacial capacitance, the curvature of the transfer curve, and the transconductances are insensitive to the target concentration. The understanding of the mechanisms underlying the transistor transduction of the biorecognition events is key for the interpretation of the response of the rGO-EGT immunosensors and to guide the design of novel and more sensitive devices.

2024 - Investigation of transcription factor–DNA binding with electrolyte-gated organic transistors [Articolo su rivista]
Sensi, Matteo; Ricci, Andrea; Rigillo, Giovanna; Paradisi, Alessandro; Berto, Marcello; Gnesutta, Nerina; Imbriano, Carol; Biscarini, Fabio; Bortolotti, Carlo Augusto
abstract

Nuclear transcription factor Y (NF-Y) is a CCAAT-binding trimeric protein. The overexpression of the DNA-binding subunit A (NF-YA) results in deregulation of many CCAAT-dependent pro-growth genes in multiple tumor types. Exon 3 alternative splicing of NF-YA results in two different isoforms, NF-YAs (short) and NF-YAl (long), which can promote tumor proliferation or metastasis, respectively. In this work, we developed an electrolyte-gated organic transistor (EGOT) biosensor to study the binding of a NF-YAl-composed NF-Y complex to its consensus sequence. We show that by using the target DNA sequence as a probe, the device detects NF-Y in the range of 1 pM to 10 nM. Control experiments performed with oligonucleotide probes mutated in the consensus sequence exhibit weaker, though not fully hindered, binding to NF-Y compared to the response to unmutated DNA. This behavior confirms that the base pairs near the CCAAT-box also have a role in the transcription factor recognition. Furthermore, we contributed to the advancement of the present state of the art by demonstrating the ability of the EGOT biosensor to detect NF-Y in cell lysate, a fundamental step towards the development of point-of-care (POC) devices for the analysis of biopsies.The first electrolyte-gated organic transistor biosensor for the detection of a transcription factor (NF-Y) in buffer and cell lysate.

2023 - Effects of removal of the axial methionine heme ligand on the binding of S. cerevisiae iso-1 cytochrome c to cardiolipin. [Articolo su rivista]
Paradisi, Alessandro; Bellei, Marzia; Bortolotti, Carlo Augusto; DI ROCCO, Giulia; Ranieri, Antonio; Borsari, Marco; Sola, Marco; Battistuzzi, Gianantonio
abstract

The cleavage of the axial S(Met)-Fe bond in cytochrome c (cytc) upon binding to cardiolipin (CL), a glycerophospholipid of the inner mitochondrial membrane, is one of the key molecular changes that impart cytc with (lipo)peroxidase activity essential to its pro-apoptotic function. In this work, UV-VIS, CD, MCD and fluorescence spectroscopies were used to address the role of the Fe−M80 bond in controlling the cytc-CL interaction, by studying the binding of the Met80Ala (M80A) variant of S. cerevisiae iso-1 cytc (ycc) to CL liposomes in comparison with the wt protein [Paradisi et al. J. Biol. Inorg. Chem. 25 (2020) 467–487]. The results show that the integrity of the six-coordinate heme center along with the distal heme site containing the Met80 ligand is a not requisite for cytc binding to CL. Indeed, deletion of the Fe-S(Met80) bond has a little impact on the mechanism of ycc-CL interaction, although it results in an increased heme accessibility to solvent and a reduced structural stability of the protein. In particular, M80A features a slightly tighter binding to CL at low CL/cytc ratios compared to wt ycc, possibly due to the lift of some constraints to the insertion of the CL acyl chains into the protein hydrophobic core. M80A binding to CL maintains the dependence on the CL-to-cytc mixing scheme displayed by the wt species

2022 - Mixed ionic-electronic conductance across naphthalenediimide-functionalized biopolymers [Articolo su rivista]
Ramanthrikkovil Variyam, A.; Agam, Y.; Paradisi, A.; Bortolotti, C. A.; Amdursky, N.
abstract

Mixed ionic-electronic conductive polymers are gaining high momentum for several electronic and bioelectronic applications. These polymers are composed of a synthetic conjugated polymer for electronic conduction and a synthetic ionomer for the ionic one. To make these materials more environmentally sustainable, much effort is being put in replacing synthetic polymers with biopolymers. However, to date, the only strategy for making mixed conductors with biopolymers is to blend them with synthetic polymers. Here, we show that by targeting certain amino acids of a protein-based biopolymer, we can modify them with naphthalenediimide (NDI) after polymerization, resulting in an improved electronic transport, which is in addition to the native ionic transport of the biopolymer. We further show that by reducing the NDI moieties we can reach conductivity values in the order of 40 mS cm−1, though NDI can re-oxidize depending on the environment of the biopolymer. The abundant nature of the protein building blocks together with the easy post-polymerization functionalization chemistry of NDI, which is very much different than any previous use of NDI in conductive polymers, is making our new strategy for making mixed ionic-electronic conductive biopolymers highly attractive.

2021 - Activity and substrate specificity of lytic polysaccharide monooxygenases: An ATR FTIR-based sensitive assay tested on a novel species from Pseudomonas putida [Articolo su rivista]
Serra, Ilenia; Piccinini, Daniele; Paradisi, Alessandro; Ciano, Luisa; Bellei, Marzia; Bortolotti, Carlo Augusto; Battistuzzi, Gianantonio; Sola, Marco; Walton, Paul H.; DI ROCCO, Giulia
abstract

Pseudomonas putida W619 is a soil Gram-negative bacterium commonly used in environmental studies thanks to its ability in degrading many aromatic compounds. Its genome contains several putative carbohydrate-active enzymes such as glycoside hydrolases and lytic polysaccharide monooxygenases (PMOs). In this study, we have heterologously produced in Escherichia coli and characterized a new enzyme belonging to the AA10 family, named PpAA10 (Uniprot: B1J2U9), which contains a chitin-binding type-4 module and showed activity toward β-chitin. The active form of the enzyme was produced in E. coli exploiting the addition of a cleavable N-terminal His tag which ensured the presence of the copper-coordinating His as the first residue. Electron paramagnetic resonance spectroscopy showed signal signatures similar to those observed for the copper-binding site of chitin-cleaving PMOs. The protein was used to develop a versatile, highly sensitive, cost-effective and easy-to-apply method to detect PMO's activity exploiting attenuated total reflection-Fourier transform infrared spectroscopy and able to easily discriminate between different substrates.

2020 - Binding of S. cerevisiae iso‑1 cytochrome c and its surface lysine‑to‑alanine variants to cardiolipin: charge effects and the role of the lipid to protein ratio [Articolo su rivista]
Paradisi, Alessandro; Bellei, Marzia; Paltrinieri, Licia; Bortolotti, Carlo Augusto; Di Rocco, Giulia; Ranieri, Antonio; Borsari, Marco; Sola, Marco; Battistuzzi, Gianantonio
abstract

The interaction of cytochrome c with cardiolipin (CL) is a critical step in the initial stages of apoptosis and is mediated by a positively charged region on the protein surface comprising several lysine residues (site A). Here, the interaction of wt S. cerevisiae cytochrome c (ycc) and its K72A/K73A, K72A/K79A, K73A/K79A and K72A/K73A/K79A variants with CL was studied through UV–Vis and MCD spectroscopies at pH 7 and molecular dynamics (MD) simulations, to clarify the role of the mutated lysines. Moreover, the influence of the lipid to protein ratio on the interaction mechanism was investigated using low (0.5–10) and high (5–60) CL/ycc molar ratios, obtained with small and gradual or large and abrupt CL additions, respectively. Although all proteins bind to CL, switching from the native low-spin His/Met-ligated form to a low-spin bis-His conformer and to a high-spin species at larger CL concentrations, the two schemes of CL addition show relevant differences in the CL/ycc molar ratios at which the various conformers appear, due to differences in the interaction mechanism. Extended lipid anchorage and peripheral binding appear to prevail at low and high CL/ycc molar ratios, respectively. Simultaneous deletion of two or three surface positive charges from Site A does not abolish CL binding, but instead increases protein affinity for CL. MD calculations suggest this unexpected behavior results from the mutation-induced severe weakening of the H-bond connecting the Nε of His26 with the backbone oxygen of Glu44, which lowers the conformational stability compared to the wt species, overcoming the decreased surface electrostatic interaction.

2020 - Met80 and Tyr67 affect the chemical unfolding of yeast cytochrome c: comparing solution vs. immobilized state [Articolo su rivista]
Paradisi, Alessandro; Lancellotti, Lidia; Borsari, Marco; Bellei, Marzia; Bortolotti, Carlo Augusto; DI ROCCO, Giulia; Ranieri, Antonio; Sola, Marco; Battistuzzi, Gianantonio
abstract

Urea-induced denaturation of the Met80Ala and Met80Ala/Tyr67Ala variants of S. cerevisiae iso-1 cytochrome c (ycc) was studied through variable temperature diffusive cyclic voltammetry and electronic absorption, CD and MCD spectroscopies. The susceptibility to unfolding of both variants - represented by the free energy of unfolding at denaturant infinite dilution, ∆〖G°〗_u^(H_2 O)is greater compared to the species showing an intact Met/His coordination, as observed previously for the same species immobilized onto a functionalized electrode. This is consistent with the role of the axial Fe-(S)Met bond and the H-bond network involving Tyr67 in stabilizing the polypeptide matrix in the heme crevice. Notably, we find that the unfolding propensity and axial heme iron coordination of the present Fe-(S)Met bond-deprived variants is affected by the motional regime of the protein. In particular, electrostatic adsorption onto a negatively charged SAM surface - that would mimic the phospholipidic inner mitochondrial membrane - facilitates unfolding compared to the solution state, especially at room temperature. This finding has a physiological relevance related to the cytochrome c interaction with cardiolipin at the IMM in the early stages of apoptosis. Moreover, while both immobilized variants maintain the His/OH- axial heme iron coordination up to 7 M urea, the same species in solution are subjected to urea-induced replacement of the axial hydroxide ligand by a His ligand. The contribution of the enthalpic and entropic terms to ∆〖G°〗_u^(H_2 O) were found to be opposite (H-S compensation) indicating that unfolding thermodynamics are strongly affected by changes in the hydrogen bonding network in the hydration sphere of the protein.