Wearable piezoelectric mass sensor based on pH sensitive hydrogels for sweat pH monitoring

Colorimetric and electrochemical (bio)sensors are commonly employed in wearable platforms for sweat monitoring; nevertheless, they suffer from low stability of the sensitive element. In contrast, mass-(bio)sensors are commonly used for analyte detection at laboratory level only, due to their rigidity. To overcome these limitations, a flexible mass-(bio)sensor for sweat pH sensing is proposed. The device exploits the flexibility of piezoelectric AlN membranes fabricated on a polyimide substrate combined to the sensitive properties of a pH responsive hydrogel based on PEG-DA/CEA molecules. A resonant frequency shift is recorded due to the hydrogel swelling/shrinking at several pH. Our device shows a responsivity of about 12 kHz/pH unit when measured in artificial sweat formulation in the pH range 3-8. To the best of our knowledge, this is the first time that hydrogel mass variations are sensed by a flexible resonator, fostering the development of a new class of compliant and wearable devices

Label-free biomechanical nanosensor based on LSPR for biological applications

A label-free localized surface plasmon resonance (LSPR)-based biosensor exploiting gold nanorods (ONRs) is proposed and demonstrated. For this aim, 35 +/- 5 nm long and 20 +/- 4 thick GNRs spaced by a few nanometers thick polyelectrolytes (PE) from a gold thin film was analyzed and synthesized. The morphology of the GNRs, the plasmon properties of GNRs, swelling of PE layers and the wettability of the surfaces were characterized by transmission and scanning electron microscopy, spectroscopic reflectivity and contact angle measurements, respectively. Indeed, when immersed in a phosphate buffer saline solution, the GNRs-PE-gold system shows an optical shift of the LSPR wavelength. This shift was found to correspond to a vertical swelling of about 2 nm, demonstrating the extreme sensitivity of the biosensor. Finally, we show that LSPR measurements can be used to detect dynamic resonance changes in response to both thickness and buffer solution, while the hydrophobic behavior of the surface can be exploited for reducing the number of liquid analytes in clinical biosensing application. (C) 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreemen

Methyl-to-Ethyl Replacement Makes the Difference: Structure-Guided Design of a New Cancer Vaccine Based on a Tn An-gen Surrogate

Mucins are large extracellular glycoproteins that exhibit different glycosylation patterns and post-translational modifications between healthy and cancer cells[1]. Mucin 1 (MUC1) is a common glycoprotein in cancer cells that plays a multifaceted role in cancer development, cell proliferation, and migration[2,3]. These properties make MUC1 an excellent antigen for cancer vaccine candidates. Several works[3,4] have used MUC1-derived GalNAc glycopeptides, especially the sequence APDT(-O-GalNAc-Thr)RP, for cancer vaccine development but with limited success due to the low immunogenicity and stability of the glycopeptide. We have developed a novel Tn antigen following a structure-guided design in which the threonine of the above sequence has been replaced by the unnatural amino acid L-4-hydroxynorvaline (Hnv) to increase the antigen/antibody affinity. We have confirmed by X-crystallography analysis of the complex that the ethyl group at the C of the unnatural residue favors the CH/ interactions between the Tn antigen and the SM3 antibody, resulting in a slight increase in affinity due to enthalpy-entropy balance. The chemical modification (HnvThr) allows the synthetic glycopeptide to exhibit similar properties to the naturally occurring derivative, similar serum stability, and a similar conformational landscape in solution. A vaccination campaign in mice is currently underway in which the synthetic antigen has been conjugated to evaluate the biological impact of this chemical modification

Radioactive Phosphorylation of Alcohols to Monitor Biocatalytic Diels-Alder Reactions

Nature has efficiently adopted phosphorylation for numerous biological key processes, spanning from cell signaling to energy storage and transmission. For the bioorganic chemist the number of possible ways to attach a single phosphate for radioactive labeling is surprisingly small. Here we describe a very simple and fast one-pot synthesis to phosphorylate an alcohol with phosphoric acid using trichloroacetonitrile as activating agent. Using this procedure, we efficiently attached the radioactive phosphorus isotope 32P to an anthracene diene, which is a substrate for the Diels-Alderase ribozyme—an RNA sequence that catalyzes the eponymous reaction. We used the 32P-substrate for the measurement of RNA-catalyzed reaction kinetics of several dye-labeled ribozyme variants for which precise optical activity determination (UV/vis, fluorescence) failed due to interference of the attached dyes. The reaction kinetics were analyzed by thin-layer chromatographic separation of the 32P-labeled reaction components and densitometric analysis of the substrate and product radioactivities, thereby allowing iterative optimization of the dye positions for future single-molecule studies. The phosphorylation strategy with trichloroacetonitrile may be applicable for labeling numerous other compounds that contain alcoholic hydroxyl groups

Structure-Based Design of Potent Tumor-Associated Antigens: Modulation of Peptide Presentation by Single-Atom O/S or O/Se Substitutions at the Glycosidic Linkage.

GalNAc-glycopeptides derived from mucin MUC1 are an important class of tumor-associated antigens. α- O-glycosylation forces the peptide to adopt an extended conformation in solution, which is far from the structure observed in complexes with a model anti-MUC1 antibody. Herein, we propose a new strategy for designing potent antigen mimics based on modulating peptide/carbohydrate interactions by means of O → S/Se replacement at the glycosidic linkage. These minimal chemical modifications bring about two key structural changes to the glycopeptide. They increase the carbohydrate-peptide distance and change the orientation and dynamics of the glycosidic linkage. As a result, the peptide acquires a preorganized and optimal structure suited for antibody binding. Accordingly, these new glycopeptides display improved binding toward a representative anti-MUC1 antibody relative to the native antigens. To prove the potential of these glycopeptides as tumor-associated MUC1 antigen mimics, the derivative bearing the S-glycosidic linkage was conjugated to gold nanoparticles and tested as an immunogenic formulation in mice without any adjuvant, which resulted in a significant humoral immune response. Importantly, the mice antisera recognize cancer cells in biopsies of breast cancer patients with high selectivity. This finding demonstrates that the antibodies elicited against the mimetic antigen indeed recognize the naturally occurring antigen in its physiological context. Clinically, the exploitation of tumor-associated antigen mimics may contribute to the development of cancer vaccines and to the improvement of cancer diagnosis based on anti-MUC1 antibodies. The methodology presented here is of general interest for applications because it may be extended to modulate the affinity of biologically relevant glycopeptides toward their receptors

Discovery of Inhibitors of Leishmania β-1,2-Mannosyltransferases Using a Click-Chemistry-Derived Guanosine Monophosphate Library

Leishmania spp. are a medically important group of protozoan parasites that synthesize a novel intracellular carbohydrate reserve polymer termed mannogen. Mannogen is a soluble homopolymer of β-1,2-linked mannose residues that accumulates in the major pathogenic stages in the sandfly vector and mammalian host. While several steps in mannogen biosynthesis have been defined, none of the enzymes have been isolated or characterized. We report the development of a simple assay for the GDP-mannose–dependent β-1,2-mannosyltransferases involved in mannogen synthesis. This assay utilizes octyl α-d-mannopyranoside to prime the formation of short mannogen oligomers up to 5 mannose residues. This assay was used to screen a focussed library of 44 GMP-triazole adducts for inhibitors. Several compounds provided effective inhibition of mannogen β-1,2-mannosyltransferases in a cell-free membrane preparation. This assay and inhibitor compounds will be useful for dissecting the role of different mannosyltransferases in regulating de novo biosynthesis and elongation reactions in mannogen metabolism

Absolute and Direct MicroRNA Quantification Using DNA-Gold Nanoparticle Probes

DNA-gold nanoparticle probes are implemented in a simple strategy for direct microRNA (miRNA) quantification. Fluorescently labeled DNA-probe strands are immobilized on PEGylated gold nanoparticles (AuNPs). In the presence of target miRNA, DNA-RNA heteroduplexes are formed and become substrate for the endonuclease DSN (duplex-specific nuclease). Enzymatic hydrolysis of the DNA strands yields a fluorescence signal due to diffusion of the fluorophores away from the gold surface. We show that the molecular design of our DNA-AuNP probes, with the DNA strands immobilized on top of the PEG-based passivation layer, results in nearly unaltered enzymatic activity toward immobilized heteroduplexes compared to substrates free in solution. The assay, developed in a real-time format, allows absolute quantification of as little as 0.2 fmol of miR-203. We also show the application of the assay for direct quantification of cancer-related miR-203 and miR-21 in samples of extracted total RNA from cell cultures. The possibility of direct and absolute quantification may significantly advance the use of microRNAs as biomarkers in the clinical praxis

Highly stable cage-like complexes by self-assembly of tetracationic Zn(II) porphyrinates and tetrasulfonatocalix 4 arenes in polar solvents

Tetracationic Zn(II) porphyrinates and tetraanionic calix[4]arenes can be assembled in polar solvents to obtain cage-like complexes in an entropy driven process. These structures are remarkably stable even in the presence of water or competing salts

Self-assembly via ionic interactions in polar solvents. Non-covalent synthesis of calix 4 arene capped porphyrins

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