From speciation study to removal of Pb2+ from natural waters by a carnosine-based polyacrylamide/azlactone copolymer.

A deep speciation study on L-carnosine (CAR) and Pb2+ system was performed in aqueous solution with the aim to assess its potential use as a sequestering agent of metal cation. To determine the best conditions for Pb2+ complexation, potentiometric measurements were carried out over a wide range of ionic strength (0.15 ≤ I/≤ 1 mol/L) and temperature (15 ≤ T/°C ≤ 37), and thermodynamic interaction parameters (logß, ΔH, ΔG and TΔS) were determined. The speciation studies allowed us to simulate sequestration ability of CAR toward Pb2+ under different conditions of pH, ionic strength and temperature and to establish a priori the conditions for the best removal performance, i.e., pH > 7 and I = 001 mol/L. This preliminary investigation was very useful in optimizing removal procedures and limiting subsequent experimental measurements for adsorption tests. Therefore, to exploit the binding ability of CAR for Pb2+ removal from aqueous solutions, CAR was covalently grafted on an azlactone-activated beaded-polyacrylamide resin (AZ) using an efficient click coupling reaction (78.3% of coupling efficiency). The carnosine-based resin (AZCAR) was analyzed by ThermoGravimetric Analysis (TGA), Differential Scanning Calorimetry (DSC) and Differential Thermal Analysis (DTA). Morphology, surface area and pore size distribution were studied through a combination of Scanning Electron Microscope (SEM) and adsorption/desorption of N2 analyses according to the Brunauer-Emmett-Teller (BET) and Barret-Johner-Halenda (BJH) approaches. The adsorption capacity of AZCAR toward Pb2+ was investigated under conditions simulating the ionic strength and pH of different natural waters. The time needed to reach equilibrium in the adsorption process was 24 h, and the best performance was obtained at pH > 7, typical of most natural waters, with removal efficiency ranging from 90.8% (at I = 0.7 mol/L) to 99.0 (at I = 0.001 mol/L).

State of the Art on Green Route Synthesis of Gold/Silver Bimetallic Nanoparticles.

Recently, bimetallic nanoparticles (BMNPs) blending the properties of two metals in one nanostructured system have generated enormous interest due to their potential applications in various fields including biosensing, imaging, nanomedicine, and catalysis. BMNPs have been developed later with respect to the monometallic nanoparticles (MNPs) and their physicochemical and biological properties have not yet been comprehensively explored. The manuscript aims at collecting the main design criteria used to synthetize BMNPs focusing on green route synthesis. The influence of experimental parameters such as temperature, time, reagent concentrations, capping agents on the particle growth and colloidal stability are examined. Finally, an overview of their nanotechnological applications and biological profile are presented.

Pseudo-Dipeptide Bearing α,α-Difluoromethyl Ketone Moiety as Electrophilic Warhead with Activity against Coronaviruses.

The synthesis of α-fluorinated methyl ketones has always been challenging. New methods based on the homologation chemistry via nucleophilic halocarbenoid transfer, carried out recently in our labs, allowed us to design and synthesize a target-directed dipeptidyl α,α-difluoromethyl ketone (DFMK) 8 as a potential antiviral agent with activity against human coronaviruses. The ability of the newly synthesized compound to inhibit viral replication was evaluated by a viral cytopathic effect (CPE)-based assay performed on MCR5 cells infected with one of the four human coronaviruses associated with respiratory distress, i.e., hCoV-229E, showing antiproliferative activity in the micromolar range (EC50 = 12.9 ± 1.22 µM), with a very low cytotoxicity profile (CC50 = 170 ± 3.79 µM, 307 ± 11.63 µM, and 174 ± 7.6 µM for A549, human embryonic lung fibroblasts (HELFs), and MRC5 cells, respectively). Docking and molecular dynamics simulations studies indicated that 8 efficaciously binds to the intended target hCoV-229E main protease (Mpro). Moreover, due to the high similarity between hCoV-229E Mpro and SARS-CoV-2 Mpro, we also performed the in silico analysis towards the second target, which showed results comparable to those obtained for hCoV-229E Mpro and promising in terms of energy of binding and docking pose.

Oxazolidinone Antibiotics: Chemical, Biological and Analytical Aspects.

This review covers the main aspects concerning the chemistry, the biological activity and the analytical determination of oxazolidinones, the only new class of synthetic antibiotics advanced in clinical use over the past 50 years. They are characterized by a chemical structure including the oxazolidone ring with the S configuration of substituent at C5, the acylaminomethyl group linked to C5 and the N-aryl substituent. The synthesis of oxazolidinones has gained increasing interest due to their unique mechanism of action that assures high antibiotic efficiency and low susceptibility to resistance mechanisms. Here, the main features of oxazolidinone antibiotics licensed or under development, such as Linezolid, Sutezolid, Eperezolid, Radezolid, Contezolid, Posizolid, Tedizolid, Delpazolid and TBI-223, are discussed. As they are protein synthesis inhibitors active against a wide spectrum of multidrug-resistant Gram-positive bacteria, their biological activity is carefully analyzed, together with the drug delivery systems recently developed to overcome the poor oxazolidinone water solubility. Finally, the most employed analytical techniques for oxazolidinone determination in different matrices, such as biological fluids, tissues, drugs and natural waters, are reviewed. Most are based on HPLC (High Performance Liquid Chromatography) coupled with UV-Vis or mass spectrometer detectors, but, to a lesser extent are also based on spectrofluorimetry or voltammetry.

Salinomycin-loaded PLA nanoparticles: drug quantification by GPC and wave voltammetry and biological studies on osteosarcoma cancer stem cells.

A new straightforward gel permeation chromatography (GPC) method was developed to calculate the drug encapsulation efficiency and loading content of Poly(lactic acid) nanoparticles (PLA NPs) loaded with Salinomycin (Sal), exploiting the capability of this technique to separate a macromolecular/molecular mixture on the basis of the molecular weight of each component. The proposed GPC method allowed Sal detection until 1% of Sal content in PLA NPs, avoiding sample pre-treatments. The method was validated by wave voltammetry (SW) technique, using a slightly modified literature procedure, useful to detect Sal in the concentration range 0.4 ≤ C/µmol/L ≤ 12 (linear concentration range). PLA-based NPs were prepared by nanoprecipitation with either native and functionalized PLA. Specifically, folate-decorated PLA NPs (PLA-FA NPs) were obtained by CuAAC click functionalization of alkyne-grafted PLA with azide-folate. Sal-loaded NPs were characterized physicochemically and morphologically. They exhibited adequate physicochemical properties, good drug encapsulation efficiency (98 ± 0.5% and 99 ± 0.5%), and loading content (8.8 ± 0.1% and 8.9 ± 0.1% for PLA/Sal and PLA-FA/Sal NPs, respectively). The size of empty PLA NPs resulted smaller (90 ± 3.2 nm and 680 ± 15.3 nm, for PLA NPs and PLA-FA NPs respectively) than the correspondent drug-loaded NPs (110 ± 3.8 nm and 875 ± 20.5 nm, respectively). Their biological activity was assessed on osteosarcoma bulk cells MG63, healthy osteoblast cell line (hFOB1.19), and enriched osteosarcoma cancer stem cells (CSCs), showing cell-depending effect. Entrapped Sal maintained its cytotoxic effect on CSCs and MG63 cells, with a potency comparable to the free drug and no evident benefit was detected for folate-decorated PLA NPs respect to native PLA NPs. Graphical abstract.

Putative Inhibitors of SARS-CoV-2 Main Protease from A Library of Marine Natural Products: A Virtual Screening and Molecular Modeling Study.

The current emergency due to the worldwide spread of the COVID-19 caused by the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a great concern for global public health. Already in the past, the outbreak of severe acute respiratory syndrome (SARS) in 2003 and Middle Eastern respiratory syndrome (MERS) in 2012 demonstrates the potential of coronaviruses to cross-species borders and further underlines the importance of identifying new-targeted drugs. An ideal antiviral agent should target essential proteins involved in the lifecycle of SARS-CoV. Currently, some HIV protease inhibitors (i.e., Lopinavir) are proposed for the treatment of COVID-19, although their effectiveness has not yet been assessed. The main protease (Mpr) provides a highly validated pharmacological target for the discovery and design of inhibitors. We identified potent Mpr inhibitors employing computational techniques that entail the screening of a Marine Natural Product (MNP) library. MNP library was screened by a hyphenated pharmacophore model, and molecular docking approaches. Molecular dynamics and re-docking further confirmed the results obtained by structure-based techniques and allowed this study to highlight some crucial aspects. Seventeen potential SARS-CoV-2 Mpr inhibitors have been identified among the natural substances of marine origin. As these compounds were extensively validated by a consensus approach and by molecular dynamics, the likelihood that at least one of these compounds could be bioactive is excellent.

Intracellular Fate and Impact on Gene Expression of Doxorubicin/Cyclodextrin-Graphene Nanomaterials at Sub-Toxic Concentration.

The graphene road in nanomedicine still seems very long and winding because the current knowledge about graphene/cell interactions and the safety issues are not yet sufficiently clarified. Specifically, the impact of graphene exposure on gene expression is a largely unexplored concern. Herein, we investigated the intracellular fate of graphene (G) decorated with cyclodextrins (CD) and loaded with doxorubicin (DOX) and the modulation of genes involved in cancer-associated canonical pathways. Intracellular fate of GCD@DOX, tracked by FLIM, Raman mapping and fluorescence microscopy, evidenced the efficient cellular uptake of GCD@DOX and the presence of DOX in the nucleus, without graphene carrier. The NanoString nCounter™ platform provided evidence for 34 (out of 700) differentially expressed cancer-related genes in HEp-2 cells treated with GCD@DOX (25 µg/mL) compared with untreated cells. Cells treated with GCD alone (25 µg/mL) showed modification for 16 genes. Overall, 14 common genes were differentially expressed in both GCD and GCD@DOX treated cells and 4 of these genes with an opposite trend. The modification of cancer related genes also at sub-cytotoxic G concentration should be taken in consideration for the rational design of safe and effective G-based drug/gene delivery systems. The reliable advantages provided by NanoString® technology, such as sensibility and the direct RNA measurements, could be the cornerstone in this field.

Folate-Decorated Amphiphilic Cyclodextrins as Cell-Targeted Nanophototherapeutics.

Nowadays, active targeting of nanotherapeutics is a challenging issue. Here, we propose a rational design of a ternary nanoassembly (SAP) composed of nonionic amphiphilic ß-cyclodextrins (amphiphilic CD) incorporating pheophorbide (Pheo) as a phototherapeutic and an adamantanyl-folic acid conjugate (Ada-FA) to target tumor cells overexpressing α-folate receptor (FR-α(+)). Dynamic light scattering and ζ-potential pointed out the presence of nanoassemblies bearing a negative surface charge (ζ = -51 mV). Morphology of SAP was investigated by atomic force microscopy and microphotoluminescence, indicating the presence of highly emissive near-spherical assemblies of about 280 nm in size. Complementary spectroscopic techniques such as ROESY-NMR, UV/vis and steady-state fluorescence revealed that the folic acid protrudes out of amphiphilic CD rims, prone for recognition with FR-α. Pheo was strongly loaded in the nanoassembly mostly in monomeric form, thus generating singlet oxygen (1O2) and consequentely showing phototherapeutic action. SAP remained stable until 2 weeks in aqueous solutions. Stability studies in biologically relevant media pointed out the ability of SAP to interact with serum proteins by means of the oligoethylenglycole fringe, without destabilization. Release experiments demonstrated the sustained release of Pheo from SAP in environments mimiking physiological conditions (∼20% within 1 week), plausibly suggesting low Pheo leaking and high integrity of the assembly within 24 h, time spent on average to reach the target sites. Cellular uptake of SAP was confirmed by confocal microscopy, pointing out that SAP was internalized into the tumoral cells expressing FR-α more efficiently than SP. SAP showed improved phototoxicity in human breast MCF-7 cancer cells FR-α(+) (IC50 = 270 nM) with respect to human prostate carcinoma PC3 cells (IC50 = 700 nM) that express a low level of that receptor (FR-α(-)). Finally, an improved phototoxicity in FR-α(+) MCF-7 cells (IC50 = 270 nM) was assessed after treatment with SAP vs SP (IC50 = 600 nM) which was designed without Ada-FA as a targeting unit.

Hydroxamic Acid-Based Histone Deacetylase (HDAC) Inhibitors Bearing a Pyrazole Scaffold and a Cinnamoyl Linker.

Genetic abnormalities have been conventionally considered as hallmarks of cancer. However, recent studies have demonstrated that epigenetic mechanisms are also implicated in the insurgence and development of cancer. Patterns of the epigenetic component include DNA methylation and histone modifications. Acetylation of histones is controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Imbalance of these two enzymatic systems is known to be a key factor in tumor progression. Because HDACs have been found to function incorrectly in cancer, various HDAC inhibitors (HDACIs) are being investigated to act as cancer chemotherapeutics. Herein, we report the synthesis, docking studies and biological activity of a series of hydroxamic acid-based HDACIs bearing an N¹-aryl or N¹-H pyrazole nucleus as surface recognition motif and a cinnamoyl group as a linker to the hydroxamic acid zinc-binding group (ZBG). Some of the tested compounds exhibited inhibitory properties towards HDACs and antiproliferative activity against neuroblastoma SH-SY5Y tumor cell line both at micromolar concentrations.

Cellular Signaling Pathways Activated by Functional Graphene Nanomaterials.

The paper reviews the network of cellular signaling pathways activated by Functional Graphene Nanomaterials (FGN) designed as a platform for multi-targeted therapy or scaffold in tissue engineering. Cells communicate with each other through a molecular device called signalosome. It is a transient co-cluster of signal transducers and transmembrane receptors activated following the binding of transmembrane receptors to extracellular signals. Signalosomes are thus efficient and sensitive signal-responding devices that amplify incoming signals and convert them into robust responses that can be relayed from the plasma membrane to the nucleus or other target sites within the cell. The review describes the state-of-the-art biomedical applications of FGN focusing the attention on the cell/FGN interactions and signalosome activation.

Efficient synthesis of highly substituted tetrahydroindazolone derivatives.

A straightforward and efficient method for the synthesis of novel highly substituted and diversely functionalized indazolone derivatives has been developed. The transformation consists of a cyclocondensation of selected 1,3,3'-tricarbonyls with monosubstituted hydrazines. The starting ß-triketones were prepared by an efficient chemo- and regioselective method under MW irradiation, exploiting the oxazolone chemistry. The reaction is easily accomplished under mild conditions and appears versatile, providing a synthetic diversification method with potential for drug-like compounds preparation.

Synthesis of C3/C1-Substituted Tetrahydroisoquinolines.

A broad biological screening of the natural alkaloid N-methylisosalsoline (2) extracted from Hammada scoparia leaves against a panel of human and parasitic proteases revealed an interesting activity profile of 2 towards human 20S proteasome. This outcome suggests that the 1,2,3,4-tetrahydroisoquinoline skeleton may be exploited as a template for the development of novel anticancer agents. In this article, we report the synthesis and chemical characterization of a new series of isosalsoline-type alkaloids (10-11) with variations at N2 and C3 positions with respect to the natural Compound 2, obtained by a synthetic strategy that involves the Bischler-Napieralski cyclization. The substrate for the condensation to the tetrahydroisoquinoline system, i.e., a functionalized ß-arylethyl amine, was obtained through an original double reduction of nitroalkene. The synthetic strategy can be directed to the construction of highly substituted and functionalized 1,2,3,4-tetrahydroisoquinolines.

Direct synthesis of C3-mono-functionalized oxindoles from N-unprotected 2-oxindole and their antileishmanial activity.

A novel approach for the synthesis of unprecedented C3-mono-functionalized indolin-2-ones is reported, starting from 2-oxindole and chalcones. The reactions proceed regioselectively under mild conditions, without di- and tri-alkylated side products. The new compounds have been evaluated in vitro for their antiproliferative effects against the protozoan Leishmania infantum. Interestingly, they appear able to kill L. infantum promastigotes and amastigotes, without significant cytotoxic effects.

Targeting Viral and Cellular Cysteine Proteases for Treatment of New Variants of SARS-CoV-2.

The continuous emergence of SARS-CoV-2 variants caused the persistence of the COVID-19 epidemic and challenged the effectiveness of the existing vaccines. The viral proteases are the most attractive targets for developing antiviral drugs. In this scenario, our study explores the use of HIV-1 protease inhibitors against SARS-CoV-2. An in silico screening of a library of HIV-1 proteases identified four anti-HIV compounds able to interact with the 3CLpro of SARS-CoV-2. Thus, in vitro studies were designed to evaluate their potential antiviral effectiveness against SARS-CoV-2. We employed pseudovirus technology to simulate, in a highly safe manner, the adsorption of the alpha (α-SARS-CoV-2) and omicron (ο-SARS-CoV-2) variants of SARS-CoV-2 and study the inhibitory mechanism of the selected compounds for cell-virus interaction. The results reported a mild activity against the viral proteases 3CLpro and PLpro, but efficient inhibitory effects on the internalization of both variants mediated by cathepsin B/L. Our findings provide insights into the feasibility of using drugs exhibiting antiviral effects for other viruses against the viral and host SARS-CoV-2 proteases required for entry.

RGD-tagging of star-shaped PLA-PEG micellar nanoassemblies enhances doxorubicin efficacy against osteosarcoma.

We developed cyclic RGD-tagged polymeric micellar nanoassemblies for sustained delivery of Doxorubicin (Dox) endowed with significant cytotoxic effect against MG63, SAOS-2, and U2-OS osteosarcoma cells without compromising the viability of healthy osteoblasts (hFOBs). Targeted polymeric micellar nanoassemblies (RGD-NanoStar@Dox) enabled Dox to reach the nucleus of MG63, SAOS-2, and U2-OS cells causing the same cytotoxic effect as free Dox, unlike untargeted micellar nanoassemblies (NanoStar@Dox) which failed to reach the nucleus and resulted ineffective, demonstrating the crucial role of cyclic RGD peptide in driving cellular uptake and accumulation mechanisms in osteosarcoma cells. Micellar nanoassemblies were obtained by nanoformulation of three-armed star PLA-PEG copolymers properly synthetized with and without decoration with the cyclic-RGDyK peptide (Arg-Gly-Asp-D-Tyr-Lys). The optimal RGD-NanoStar@Dox nanoformulation obtained by nanoprecipitation method (8 % drug loading; 35 % encapsulation efficiency) provided a prolonged and sustained drug release with a rate significantly lower than the free drug under the same experimental conditions. Moreover, the nanosystem preserved Dox from the natural degradation occurring under physiological conditions (i.e., dimerization and consequent precipitation) serving as a slow-release "drug reservoir" ensuring an extended biological activity over the time.

Harnessing the power of PLA-PEG nanoparticles for Linezolid delivery against methicillin-resistant Staphylococcus aureus.

This study deals with the development of novel poly(lactic acid)-poly(ethylene glycol) nanoparticles (PLA-PEG NPs) for the efficient and prolonged delivery of Linezolid (LNZ), a synthetic antibacterial agent used against methicillin-resistant Staphylococcus aureus (MRSA). A two-step synthetic strategy based on carbodiimide coupling and copper-catalyzed azide-alkyne cycloaddition was first exploited for the conjugation of PLA with PEG. The encapsulation of LNZ into medium-molecular-weight PLA-PEG NPs was carried out by different methods including nanoprecipitation and dialysis. The optimal PLA-PEG@LNZ nanoformulation resulted in 3.5% LNZ payload (15% encapsulation efficiency, with a 10:3 polymer to drug mass ratio) and sustained release kinetics with 65% of entrapped antibiotic released within 80 h. Moreover, the zeta potential values (from -31 to -39 mV) indicated a good stability without agglomeration even after freeze-drying and lyophilization. The PLA-PEG@LNZ NPs exerted antimicrobial activity against a panel of Gram-positive bacteria responsible for human infections, such as Staphylococcus aureus including MRSA, Staphylococcus epidermidis, Staphylococcus lugdunensis and vancomycin-resistant Enterococcus faecium (VREfm). Moreover, PLA-PEG@LNZ NPs showed inhibitory activity on both planktonic growth and preformed biofilm of MRSA. The antibacterial activity of LNZ incorporated in polymeric NPs was well preserved and the nanosystem served as an antibiotic enhancer with a potential role in MRSA-associated infections management.

Recent Advances in SARS-CoV-2 Main Protease Inhibitors: From Nirmatrelvir to Future Perspectives.

The main protease (Mpro) plays a pivotal role in the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is considered a highly conserved viral target. Disruption of the catalytic activity of Mpro produces a detrimental effect on the course of the infection, making this target one of the most attractive for the treatment of COVID-19. The current success of the SARS-CoV-2 Mpro inhibitor Nirmatrelvir, the first oral drug for the treatment of severe forms of COVID-19, has further focused the attention of researchers on this important viral target, making the search for new Mpro inhibitors a thriving and exciting field for the development of antiviral drugs active against SARS-CoV-2 and related coronaviruses.

Thermodynamic and voltammetric study on carnosine and ferrocenyl-carnosine.

A potentiometric study on the interactions of L-carnosine (CAR) (2-[(3-aminopropanoyl)amino]-3-(1H-imidazol-5-yl)propanoic acid) with two toxic metal cations, Hg2+ and Cd2+, is reported here. The elucidation of the metal (M2+)-CAR interactions in aqueous solution highlighted the speciation model for each system, the dependence of the formation constants of the complex species on ionic strength (0.15 ≤ I/mol L-1 ≤ 1) and temperature (288.15 ≤ T/K ≤ 310.15) and changes in enthalpy and entropy. The sequestering ability of CAR towards the two metal ions was quantified and compared with that with Pb2+, previously determined. Considering the complexing ability of CAR and its unclear electrochemical properties, a more electroactive derivative, the ferrocenyl-carnosine (FcCAR), was synthesized and its complexing ability was evaluated by UV-vis spectroscopy. FcCAR electrochemical properties were investigated by Cyclic Voltammetry (CV) and Differential Pulse Voltammetry (DPV) on Screen-Printed Electrodes (SPEs), to evaluate its sensing properties. Electrochemical responses in the presence of Hg2+ and Pb2+ have been shown to be promising for the electrochemical detection of these metal cations in aqueous environment.

An Integrated In Silico and In Vitro Approach for the Identification of Natural Products Active against SARS-CoV-2.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has provoked a global health crisis due to the absence of a specific therapeutic agent. 3CLpro (also known as the main protease or Mpro) and PLpro are chymotrypsin-like proteases encoded by the SARS-CoV-2 genome, and play essential roles during the virus lifecycle. Therefore, they are recognized as a prospective therapeutic target in drug discovery against SARS-CoV-2 infection. Thus, this work aims to collectively present potential natural 3CLpro and PLpro inhibitors by in silico simulations and in vitro entry pseudotype-entry models. We screened luteolin-7-O-glucuronide (L7OG), cynarin (CY), folic acid (FA), and rosmarinic acid (RA) molecules against PLpro and 3CLpro through a luminogenic substrate assay. We only reported moderate inhibitory activity on the recombinant 3CLpro and PLpro by L7OG and FA. Afterward, the entry inhibitory activity of L7OG and FA was tested in cell lines transduced with the two different SARS-CoV-2 pseudotypes harboring alpha (α) and omicron (o) spike (S) protein. The results showed that both compounds have a consistent inhibitory activity on the entry for both variants. However, L7OG showed a greater degree of entry inhibition against α-SARS-CoV-2. Molecular modeling studies were used to determine the inhibitory mechanism of the candidate molecules by focusing on their interactions with residues recognized by the protease active site and receptor-binding domain (RBD) of spike SARS-CoV-2. This work allowed us to identify the binding sites of FA and L7OG within the RBD domain in the alpha and omicron variants, demonstrating how FA is active in both variants. We have confidence that future in vivo studies testing the safety and effectiveness of these natural compounds are warranted, given that they are effective against a variant of concerns.

Screen-Printed Carbon Electrodes with Cationic Cyclodextrin Carbon Nanotubes and Ferrocenyl-Carnosine for Electrochemical Sensing of Hg(II).

The study reports the use of nanoassembly based on cationic cyclodextrin carbon nanotubes (CNT-CDs) and ferrocenylcarnosine (FcCAR) for electrochemical sensing of Hg(II) in aqueous solution. ß-cyclodextrins (CDs) were grafted onto CNTs by a click chemistry reaction between heptakis-(6-azido-6-deoxy)-ß-cyclodextrin and alkyne-terminated CNTs. The cationic amine groups on the CD units were produced by the subsequent reduction of the residual nitrogen groups. The chemical composition and morphology of CNT-CDs were analyzed by X-ray photoelectron spectroscopy, scanning electron microscopy, and thermogravimetric analysis. A N,N-dimethylformamide dispersion of CNT-CDs was cast on the surface of screen-printed carbon electrodes (SPCEs), and the electrochemical response was evaluated by cyclic voltammetry (CV) using [Fe(CN)6]3- as the redox probe. The ability of SPCE/CNT-CD to significantly enhance the electroactive properties of the redox probe was combined with a suitable recognition element (FcCAR) for Hg(II). The electrochemical response of the CNT-CD/FcCAR nanoassembly was evaluated by CV and electrochemical impedance spectroscopy. The analytical performance of the Hg(II) sensor was evaluated by differential pulsed voltammetry and chronoamperometry. The oxidative peak current showed a linear concentration dependence in the range of 1-100 nM, with a sensitivity of 0.12 µA/nM, a limit of detection of 0.50 nM, and a limit of quantification of 1 nM.