¹⁵N NMR Relaxation Data Reveal Significant Chemical Exchange Broadening in the α-Domain of Human α-Lactalbumin

Human α-lactalbumin (α-LA), a 123-residue calcium-binding protein, has been studied using 15N NMR relaxation methods in order to characterize backbone dynamics of the native state at the level of individual residues. Relaxation data were collected at three magnetic field strengths and analyzed using the model-free formalism of Lipari and Szabo. The order parameters derived from this analysis are generally high, indicating a rigid backbone. A total of 46 residues required an exchange contribution to T2; 43 of these residues are located in the α-domain of the protein. The largest exchange contributions are observed in the A-, B-, D-, and C-terminal 310-helices of the α-domain; these residues have been shown previously to form a highly stable core in the α-LA molten globule. The observed exchange broadening, along with previous hydrogen/deuterium amide exchange data, suggests that this part of the α-domain may undergo a local structural transition between the well-ordered native structure and a less-ordered molten-globule-like structure

Tailored Ultrastable Core–Shell Au@Ag Nanoparticles for Enhanced Colorimetric Detection in Lateral Flow Assays

In the quest for more effective colorimetric reporters compared with traditional gold nanoparticles (AuNPs), a family of Au@Ag core–shell nanoparticles was designed and synthesized using a seed growth-mediated approach starting from commercial 37 nm AuNPs. This method enabled precise control over the thickness of the silver shell by employing hydroquinone for the reduction of silver and citrate for stabilization of the resulting core–shell particles. Core–shell NPs with an Ag shell of 7 nm (Au@Ag5NPs) and 18 nm (Au@Ag10NPs) were synthesized, resulting in orange and milky yellow suspension, respectively. Additionally, the impact of an external gold layer on Au@Ag10NPs (Au@Ag10@AuNPs), which significantly altered their optical properties from milky yellow to gray, was investigated. The core–shell Au@AgNPs exhibited substantially higher molar extinction coefficients than their parent AuNPs: from 3.5-fold for Au@Ag5NPs and 9-fold for Au@Ag10NPs and Au@Ag10@AuNPs. Subsequently, all core–shell NPs were functionalized with a calix[4]arene layer, imparting superior stability against external stresses, such as dispersion in PBS, when compared to NPs functionalized with traditional ligands. This calixarene coating enabled the covalent bioconjugation of antibodies on all NP types without inducing noticeable aggregation. Their performance as colorimetric reporters was evaluated in a lateral flow assay for troponin I detection, demonstrating positive signals down to 1 ng/mL, surpassing the detection limit of the parent gold NPs (2.5 ng/mL). Notably, the gray color of the core–shell Au@Ag10@AuNPs provided strong contrast against the white NC membrane, facilitating T-line visualization even at low signal intensity. Despite the lack of optimization of our LFA, it competes with the limit of quantification of commercial LFAs for troponin I detection, offering the potential for the development of a highly sensitive assay. The diverse core–shell NPs employed in this study form a library of colorimetric reporters with distinct optical properties, paving the way for multiplexed detection systems targeting multiple proteins simultaneously and enhancing diagnostic reliability. Furthermore, the choice of colorimetric reporters allows tailoring the detection range based on the pertinent limit of quantification desired for the analyte, dictated by the reporter’s light extinction properties

Bifunctional Calix[4]arene-Coated Gold Nanoparticles for Orthogonal Conjugation

Gold nanoparticles (AuNPs) are currently intensively exploited in the biomedical field as they possess interesting chemical and optical properties. Although their synthesis is well-known, their controlled surface modification with defined densities of ligands such as peptides, DNA, or antibodies remains challenging and has generally to be optimized case by case. This is particularly true for applications like in vivo drug delivery that require AuNPs with multiple ligands, for example a targeting ligand and a drug in well-defined proportions. In this context, we aimed to develop a calixarene-modification strategy that would allow the controlled orthogonal conjugation of AuNPs, respectively, via amide bond formation and copper­(I)-catalyzed azide–alkyne cycloaddition (CuAAC). To do this, we synthesized a calix[4]­arene-tetradiazonium salt bearing four PEG chains ended by an alkyne group (C1) and, after optimization of its grafting on 20 nm AuNPs, we demonstrated that CuAAC can be used to conjugate an azide containing dye (N3-cya7.5). It was observed that AuNPs coated with C1 (AuNPs-C1) can be conjugated to approximately 600 N3-cya7.5 that is much higher than the value obtained for AuNPs decorated with traditional thiolated PEG ligands terminated by an alkyne group. The control over the number of molecules conjugated via CuAAC was even possible by incorporating a non-functional calixarene (C2) into the coating layer. We then combined C1 with a calix[4]­arene-tetradiazonium salt bearing four carboxyl groups (C3) that allows conjugation of an amine (NH2-cya7.5) containing dye. The conjugation potential of these bifunctional AuNPs-C1/C3 was quantified by UV–vis spectroscopy: AuNPs decorated with equal amount of C1 and C3 could be conjugated to approximately 350 NH2-dyes and 300 N3-dyes using successively amide bond formation and CuAAC, demonstrating the control over the orthogonal conjugation. Such nanoconstructs could benefit to anyone in the need of a controlled modification of AuNPs with two different molecules via two different chemistries

Comparison of the Thermodynamics and Base-Pair Dynamics of a Full LNA:DNA Duplex and of the Isosequential DNA:DNA Duplex

Locked nucleic acids (LNA), conformationally restricted nucleotide analogues, are known to enhance pairing stability and selectivity toward complementary strands. With the aim to contribute to a better understanding of the origin of these effects, the structure, thermal stability, hybridization thermodynamics, and base-pair dynamics of a full-LNA:DNA heteroduplex and of its isosequential DNA:DNA homoduplex were monitored and compared. CD measurements highlight differences in the duplex structures: the homoduplex and heteroduplex present B-type and A-type helical conformations, respectively. The pairing of the hybrid duplex is characterized, at all temperatures monitored (between 15 and 37 °C), by a larger stability constant but a less favorable enthalpic term. A major contribution to this thermodynamic profile emanates from the presence of a hairpin structure in the LNA single strand which contributes favorably to the entropy of interaction but leads to an enthalpy penalty upon duplex formation. The base-pair opening dynamics of both systems was monitored by NMR spectroscopy via imino protons exchange measurements. The measurements highlight that hybrid G-C base-pairs present a longer base-pair lifetime and higher stability than natural G-C base-pairs, but that an LNA substitution in an A-T base-pair does not have a favorable effect on the stability. The thermodynamic and dynamic data confirm a more favorable stacking of the bases in the hybrid duplex. This study emphasizes the complementarities between dynamic and thermodynamical studies for the elucidation of the relevant factors in binding events

Rapid and Selective Detection of Proteins by Dual Trapping Using Gold Nanoparticles Functionalized with Peptide Aptamers

A colorimetric platform for the fast, simple, and selective detection of proteins of medical interest is presented. Detection is based on the aggregation of two batches of peptide functionalized gold nanoparticles via the dual-trapping of the protein of interest. As proof of concept, we applied our platform to the detection of the oncoprotein Mdm2. The peptide aptamers used for the functionalization are based on the reported binding sequences of proteins p53 and p14 for the oncoprotein. Rapid aggregation, and a color change from red to purple, was observed upon addition of Mdm2 with concentrations as low as 20 nM. The selectivity of the system was demonstrated by the lack of response upon addition of bovine serum albumin (in large excess) or of a truncated version of Mdm2, which lacks one of the peptide binding sites. A linear response was observed between 30 and 50 nM of Mdm2. The platform reported here is flexible and can be adapted for the detection of other proteins when two binding peptide aptamers can be identified. Unlike current immunoassay methods, it is a one-step and rapid method with an easy readout signal and low production costs

Fluorescent Chemosensors for Anions and Contact Ion Pairs with a Cavity-Based Selectivity

The association of a concave macrocyclic compound to one or multiple fluorophores is an appealing strategy for the design of chemosensors. Indeed, as with biological systems, a cavity-based selectivity can be expected with such fluorescent receptors. Examples of calix[6]­arene-based systems using this strategy are rare in the literature, and to our knowledge, no examples of fluorescent receptors that can bind organic contact ion pairs have been reported. This report describes the straightforward synthesis of fluorescent calix[6]­arene-based receptors 4a and 4b bearing three pyrenyl subunits and the study of their binding properties toward anions and ammonium salts using different spectroscopies. It was found that receptor 4a exhibits a remarkable selectivity for the sulfate anion in DMSO, enabling its selective sensing by fluorescence spectroscopy. In CDCl3, the receptor is able to bind ammonium ions efficiently only in association with the sulfate anion. Interestingly, this cooperative binding of ammonium sulfate salts was also evidenced in a protic environment. Finally, a cavity-based selectivity in terms of size and shape of the guest was observed with both receptors 4a and 4b, opening interesting perspectives on the elaboration of fluorescent cavity-based systems for the selective sensing of biologically relevant ammonium salts such as neurotransmitters

Atomic Force Manipulation of Single Magnetic Nanoparticles for Spin-Based Electronics

Magnetic nanoparticles (MNPs) are instrumental for fabrication of tailored nanomagnetic structures, especially where top-down lithographic patterning is not feasible. Here, we demonstrate precise and controllable manipulation of individual magnetite MNPs using the tip of an atomic force microscope. We verify our approach by placing a single MNP with a diameter of 50 nm on top of a 100 nm Hall bar fabricated in a quasi-two-dimensional electron gas (q2DEG) at the oxide interface between LaAlO3 and SrTiO3 (LAO/STO). A hysteresis loop due to the magnetic hysteresis properties of the magnetite MNPs was observed in the Hall resistance. Further, the effective coercivity of the Hall resistance hysteresis loop could be changed upon field cooling at different angles of the cooling field with respect to the measuring field. The effect is associated with the alignment of the MNP magnetic moment along the easy axis closest to the external field direction across the Verwey transition in magnetite. Our results can facilitate experimental realization of magnetic proximity devices using single MNPs and two-dimensional materials for spin-based nanoelectronics

Versatile Self-Adapting Boronic Acids for H‑Bond Recognition: From Discrete to Polymeric Supramolecules

Because of the peculiar dynamic covalent reactivity of boronic acids to form tetraboronate derivatives, interest in using their aryl derivatives in materials science and supramolecular chemistry has risen. Nevertheless, their ability to form H-bonded complexes has been only marginally touched. Herein we report the first solution and solid-state binding studies of the first double-H-bonded DD·AA-type complexes of a series of aromatic boronic acids that adopt a <i>syn</i>–<i>syn</i> conformation with suitable complementary H-bonding acceptor partners. The first determination of the association constant (<i>K</i>_a) of <i>ortho</i>-substituted boronic acids in solution showed that <i>K</i>_a for 1:1 association is in the range between 300 and 6900 M^–1. Crystallization of dimeric 1:1 and trimeric 1:2 and 2:1 complexes enabled an in-depth examination of these complexes in the solid state, proving the selection of the −B­(OH)₂ <i>syn</i>–<i>syn</i> conformer through a pair of frontal H-bonds with the relevant AA partner. Non-<i>ortho</i>-substituted boronic acids result in “flat” complexes. On the other hand, sterically demanding analogues bearing <i>ortho</i> substituents strive to retain their recognition properties by rotation of the ArB­(OH)₂ moiety, forming “T-shaped” complexes. Solid-state studies of a diboronic acid and a tetraazanaphthacene provided for the first time the formation of a supramolecular H-bonded polymeric ribbon. On the basis of the conformational dynamicity of the −B­(OH)₂ functional group, it is expected that these findings will also open new possibilities in metal-free catalysis or organic crystal engineering, where double-H-bonding donor boronic acids could act as suitable organocatalysts or templates for the development of functional materials with tailored organizational properties

Ultrastable Silver Nanoparticles for Rapid Serology Detection of Anti-SARS-CoV‑2 Immunoglobulins G

Dipstick assays using silver nanoparticles (AgNPs) stabilized by a thin calix[4]­arene-based coating were developed and used for the detection of Anti-SARS-CoV-2 IgG in clinical samples. The calixarene-based coating enabled the covalent bioconjugation of the SARS-CoV-2 Spike Protein via the classical EDC/sulfo-NHS procedure. It further conferred remarkable stability to the resulting bioconjugated AgNPs, as no degradation was observed over several months. In comparison with lateral-flow immunoassays (LFIAs) based on classical gold nanoparticles, our AgNP-based system constitutes a clear step forward, as the limit of detection for Anti-SARS-CoV-2 IgG was reduced by 1 order of magnitude and similar signals were observed with 10 times fewer particles. In real clinical samples, the AgNP-based dipstick assays showed impressive results: 100% specificity was observed for negative samples, while a sensitivity of 73% was determined for positive samples. These values match the typical sensitivities obtained for reported LFIAs based on gold nanoparticles. These results (i) represent one of the first examples of the use of AgNP-based dipstick assays in the case of real clinical samples, (ii) demonstrate that ultrastable calixarene-coated AgNPs could advantageously replace AuNPs in LFIAs, and thus (iii) open new perspectives in the field of rapid diagnostic tests

Empirical Optimization of Peptide Sequence and Nanoparticle Colloidal Stability: The Impact of Surface Ligands and Implications for Colorimetric Sensing

Surface ligands play a critical role in controlling and defining the properties of colloidal nanocrystals. These aspects have been exploited to design nanoparticle aggregation-based colorimetric sensors. Here, we coated 13-nm gold nanoparticles (AuNPs) with a large library of ligands (e.g., from labile monodentate monomers to multicoordinating macromolecules) and evaluated their aggregation propensity in the presence of three peptides containing charged, thiolate, or aromatic amino acids. Our results show that AuNPs coated with the polyphenols and sulfonated phosphine ligands were good choices for electrostatic-based aggregation. AuNPs capped with citrate and labile-binding polymers worked well for dithiol-bridging and π–π stacking-induced aggregation. In the example of electrostatic-based assays, we stress that good sensing performance requires aggregating peptides of low charge valence paired with charged NPs with weak stability and vice versa. We then present a modular peptide containing versatile aggregating residues to agglomerate a variety of ligated AuNPs for colorimetric detection of the coronavirus main protease. Enzymatic cleavage liberates the peptide segment, which in turn triggers NP agglomeration and thus rapid color changes in <10 min. The protease detection limit is 2.5 nM