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This research explores the use of the pepN activity fluorescent sensor DCM-Ala in bacterial biofilms, emphasizing its significance due to the critical role of biofilms in various biological processes. Advanced imaging techniques were employed to visualize pepN activity, introducing a novel approach to examining biofilm maturity. We found that the overexpression of pepN increases the ability of E. coli to form biofilm. The findings demonstrate varying levels of pepN activity throughout biofilm development, suggesting potential applications in biofilm research and management. The results indicate that the fluorescent emission from this sensor could serve as a reliable indicator of biofilm maturity, and the imaging techniques developed could enhance our understanding and control of biofilm-related processes. This work highlights the importance of innovative methods in biofilm study and opens new avenues for utilizing chemical emissions in biofilm management.

Reactions in water between the Cu2(µ-EGTA) chelate (EGTA = ethylene-bis(oxyethyleneimino)tetraacetate(4-) ion) and Hdap in molar ratios 1:1 and 1:2 yield only blue crystals of the ternary compound [Cu4(μ-EGTA)2(μ-H(N3)dap)2(H2O)2]·7H2O (1), which has been studied via single-crystal X-ray diffraction and various physical methods (thermal stability, spectral and magnetic properties), as well as DFT theoretical calculations. In the crystal, uncoordinated water is disordered. The tetranuclear complex molecule also has some irrelevant disorder in an EGTA-ethylene moiety. In the complex molecule, both bridging organic molecules act as binucleating ligands. There are two distorted five- and two six-coordinated Cu(II) centers. Each half of EGTA acts as a tripodal tetradentate Cu(II) chelator, with a mer-NO2 + O(ether, distal) conformation. Hdap exhibits the tautomer H(N3)dap, with the dissociable H-atom on its less basic N-heterocyclic atom. These features favor the efficient cooperation between Cu-N7 or Cu-N9 bonds with appropriate O-EGTA atoms, as N6-H···O or N3-H···O interligand interactions, respectively. The bridging role of both organics determines the tetranuclear dimensionality of the complex. In this crystal, such molecules associate in zig-zag chains built by alternating π–π interactions between the five- or six-atom rings of Hdap ligands of adjacent molecules. DFT theoretical calculations (using two different theoretical models and characterized by the quantum theory of “atoms in molecules”) reveal the importance of these π–π interactions between Hdap ligands, as well as those corresponding to the referred hydrogen bonds in the contributed tetranuclear molecule.

Enzyme-linked immunosorbent assay (ELISA) is routinely used to detect biomolecules related to several diseases facilitating diagnosis and monitoring of these, as well as the possibility of decreasing their mortality rate. Several methods have been carried out to improve the ELISA sensitivity through antibodies immobilization on the microtiter plates. Here, we have developed a strategy of antibodies immobilization to improve the ELISA sensitivity increasing the antibody density surface through the tetrazine (Tz)-trans-cyclooctene (TCO) reaction. For this, we prepared surfaces with tetrazine groups while the captured antibody was conjugated with TCO. The tetrazine surfaces were prepared in two different ways: (1) from aminated plates and (2) from Tz-BSA-coated plates. The surfaces were evaluated using two sandwich ELISA models, one of them using the low-affinity antibody anti-c-myc as a capture antibody to detect the c-myc-GST-IL8h recombinant protein, and the other one to detect the carcinoembryonic human protein (CEA). The sensitivity increased in both surfaces treated with tetrazine in comparison with the standard unmodified surface. The c-myc-GST-IL8h detection was around 10-fold more sensible on both tetrazine surfaces, while CEA ELISA detection increased 12-fold on surfaces coated with Tz-BSA. In conclusion, we show that it is possible to improve the ELISA sensitivity using this immobilization system, where capture antibodies bond covalently to surfaces.

In the extensive field of metal ions, their interactions with nucleic acids, and their constituents, the main aim of this work is to develop a metal chelate suitable to recognize two molecules of an adenine nucleoside. For this purpose, the dinuclear chelate Cu2 (µ-EDTA) (ethylenediaminetetraacetate(4-) ion (EDTA)) is chosen as a bicephalic receptor model for N9-(2-hydroxyethyl)adenine (9heade). A one-pot synthesis is reported to obtain the compound [Cu2(µ2-EDTA)(9heade)2(H2O)4]·3H2O, which has been characterized by single-crystal X-ray diffraction and various spectral, thermal, and magnetic methods. The complex unit is a centro-symmetric molecule, where each Cu (II) center is chelated by a half-EDTA, and is further surrounded by an N7-dentate 9heade nucleoside and two non-equivalent trans-O-aqua molecules. The metal chelate-nucleoside molecular recognition is referred to as an efficient cooperation between the Cu-N7(9heade) coordination bond and a (9heade)N6-H···O(carboxyl, EDTA) interligand interaction. Theoretical calculations are also made to account for the relevance of this interaction. The extreme weakness with which each water molecule binds to the metal center disturbs the thermal stability and the infrared (FT-IR) and electron spin resonance (ESR) spectra of the compound.

We synthesized and studied the polymeric compound {[Cu2(µ4-EDTA)(Him)2] 2H2O}n (1). The single-crystal structure is reported along with an in depth characterization of its thermal stability (TGA), spectral properties (FT-IR, Vis-UV and RSE), and magnetic behavior. The crystal consists of infinite 2D-networks built by centrosymmetric dinuclear motifs, constructed by means of a bridging anti,syn-carboxylate group from each asymmetric unit. Each layer guides Him ligands toward their external faces. They are connected by intermolecular (Him)N-H···O(carboxylate) bonds and antiparallel π–π stacking between symmetry related pairs of Him ligands, and then pillared in a 3D-network with parallel channels, where disordered water molecules are guested. About half of the labile water is lost from these channels over a wide temperature range (r.t. to 210 °C) before the other one, most strongly retained by the cooperating action of (water)O1-H(1A)···O(carboxylate) and (water) O1-H(1B)···π(Him) interactions. The latter is lost when organic ligands start to burn. ESR spectra and magnetic measurements indicated that symmetry related Cu(II) centers connected by the bridging carboxylate groups behave magnetically not equivalently, enabling an exchange interaction larger than their individual Zeeman energies.

Three mixed-ligands of Cd(II) coordination polymers were unintentionally obtained: {[Cd(µ3-EDTA)(Him)·Cd(Him)(H2O)2]·H2O}n (1), {[Cd(µ4-CDTA)(Hade)·Cd(Hade)2]}n (2), and {[Cd(µ3-EDTA)(H2O)·Cd(H9heade)(H2O)]·2H2O}n (3), having imidazole (Him), adenine (Hade) or 9-(2-hydroxyethyl)adenine (9heade) as the N-heterocyclic coligands. Compounds 2 and 3 were obtained by working with an excess of corresponding N-heterocyclic coligands. The single-crystal X-ray diffraction structures and thermogravimetric analyses are reported. The chelate moieties in all three compounds exhibit hepta-coordinated Cd centers, whereas the non-chelated Cd center is five-coordinated in 1 and six-coordinated in 2 and 3. Him and Hade take part in the seven-coordinated chelate moieties in 1 and 2, respectively. In contrast, 9heade is unable to replace the aqua ligand of the chelate [Cd (EDTA) (H2O)] moiety in 3. The thermogravimetric analysis (TGA) behavior of [Cd (H2EDTA) (H2O)]·2H2O in 1 and 3 leads to a residue of CdO, whereas the N-rich compound 2 yields CdO·Cd(NO3)2 as a residue. Density functional theory (DFT) calculations along with molecular electrostatic potential (MEP) and quantum theory of atoms-in-molecules computations were performed in adenine (compound 2) and (2-hydroxyethyl)adenine (compound 3) to analyze how the strength of the H-bonding and π-stacking interactions, respectively, are affected by their coordination to the Cd-metal center.

In a peer-review of teaching (PRT) program, we aimed to i) use pre- and post-observation questionnaires to enhance self-reflection, and ii) perform a nominal group technique (NGT) to agree on improvements. The questionnaires showed that PRT helped discovering new weaknesses but not new strengths, and that preconceived negative feelings vanished during the teaching observation. The NGT highlighted that ensuring the pairs of teachers belong to different knowledge areas and offering information to future participants may improve the PRT program and increase faculty participation. In conclusion, questionnaires along the PRT program promoted self-reflection and important improvements were made by the NGT.

Melanoma is a type of highly malignant and metastatic skin cancer. In situ molecular imaging of endogenous levels of the melanoma biomarker tyrosinase (TYR) may decrease the likelihood of mortality. In this study, we proposed the weakly fluorescent probe 1-(4-(2-(4-(dicyanomethylene)-4H-chromen-2-yl)vinyl)phenyl)-3-(4-hydroxybenzyl)urea (DCM-HBU), which releases a strong red-shifted fluorescent signal after a TYR-mediated oxidation followed by hydrolysis of the urea linkage. The large Stokes shift of the dye is owed to the recovery of the intramolecular charge transfer (ICT) effect. The resulting probe derivate shows a highly ratiometric fluorescence output. Furthermore, the simultaneous excitation by two near-infrared (NIR) photons of the released derivative of dicyanomethylene-4H-pyran (DCM-NH2) fluorophore could avoid the usual drawbacks, such as cellular absorption, autofluorescence, and light scattering, due to an usually short wavelength of the excitation light on biological systems, resulting in images with deeper tissue penetration. In addition, the probe is useful for the quantitative sensing of TYR activity in vivo, as demonstrated in zebrafish larvae. This new ratiometric two-photon NIR fluorescent probe is expected to be useful for the accurate detection of TYR in complex biosystems at greater depths than other one-photon excited fluorescent probes.