Nanoimmunosensor based on atomic force spectroscopy to detect anti-myelin basic protein related to early-stage multiple sclerosis.

Multiple Sclerosis (MS) is a chronic inflammatory disorder in the central nervous system for which biomarkers for diagnosis still remain unknown. One potential biomarker is the myelin basic protein. Here, a nanoimmunosensor based on atomic force spectroscopy (AFS) successfully detected autoantibodies against the MBP85-99 peptide from myelin basic protein. The nanoimmunosensor consisted of an atomic force microscope tip functionalization with MBP85-99 peptide, which was made to interact with a mica surface coated either with a layer of anti-MBP85-99 (positive control) or samples of cerebrospinal fluid (CSF) from five multiple sclerosis (MS) patients at different stages of the disease and five non-MS subjects. The adhesion forces obtained from AFS pointed to a high concentration of anti-MBP85-99 for the two patients at early stages of relapsing-remitting multiple sclerosis (RRMS), which were indistinguishable from the positive control. In contrast, considerably lower adhesion forces were measured for all the other eight subjects, including three MS patients with longer history of the disease and under treatment, without episodes of acute MS activity. We have also shown that the average adhesion force between MBP85-99 and anti-MBP85-99 is compatible with the value estimated using steered molecular dynamics. Though further tests will be required with a larger cohort of patients, the present results indicate that the nanoimmunosensor may be a simple tool to detect early-stage MS patients and be useful to understand the molecular mechanisms behind MS.

Peptide-Conjugated Silver Nanoparticle for Autoantibody Recognition.

In this work, we considered the autoantibodies proposed as putative biomarkers of demyelination taking into account their reactivity towards myelin oligodendrocyte glycoprotein (MOG) and myelin basic protein (MBP). These myelin proteins are among the most commonly researched targets in the immunopathology of demyelinating diseases. In this context, the development of assays for autoantibody detection can contribute as a predictive value for the early diagnosis of demyelinating diseases. Hence, we aimed to address the application of silver nanoparticles (AgNPs) as a sensing device of autoantibodies. AgNPs were synthesized via a chemical reduction method and characterized using atomic force microscopy (AFM), X-ray diffractometry, dynamic light scattering, and UV-visible spectrophotometry. The process of peptide conjugation on the nanoparticles was also analyzed. The autoantibody recognition by the peptide-conjugated AgNPs was evaluated with UV-visible spectrophotometry, atomic force spectroscopy (AFS), and color changing. AgNPs exhibited spherical morphology, low polydispersity, face-centered cubic crystal structure, and an average size of 29.3±3.0 nm. The hydrodynamic diameter variation and AFM showed that the MBP peptide induced greater agglomeration, compared to MOG peptide. The AFS measurements indicated the efficient binding of peptides to the AgNPs maintaining their activity, revealed by typical adhesion force and shapes of curves. The absorption spectrum features were more affected by the interaction with the specific autoantibodies, which also caused a visible color change in suspension providing a qualitative response. We described a preliminary study of MOG- and MBP-conjugated AgNPs which showed to be applicable in the autoantibody recognition. These have promising implication in the searching for biological markers for diagnostic purposes in the demyelination context, in which the nanoscale sensing exploitation is recent.

Fluorescent and Photosensitizing Conjugates of Cell-Penetrating Peptide TAT(47-57): Design, Microwave-Assisted Synthesis at 60 °C, and Properties.

Conjugates based on cell-penetrating peptides (CPPs) are scientifically relevant owing to their structural complexity; their ability to enter cells and deliver drugs, labels, antioxidants, bioactive compounds, or DNA fragments; and, consequently, their potential for application in research and biomedicine. In this study, carboxyamidated fluorescently labeled conjugates FAM-GG-TAT(47-57)-NH2 and FAM-PEG6-TAT(47-57)-NH2 and photosensitizer-labeled conjugate Chk-PEG6-TAT(47-57)-NH2 [where TAT(47-57) is the CPP, 5(6)-carboxyfluorescein is the (FAM) fluorophore, chlorin k (Chk) is the photosensitizer, and the dipeptide glycyl-glycine (GG) or hexaethylene glycol (PEG6) is the spacer] were originally designed, prepared, and fully characterized. Practically, all chemical reactions of the synthetic steps (peptide synthesis, spacer incorporation, and conjugation) were microwave-assisted at 60 °C using optimized protocols to give satisfying yields and high-quality products. Detailed analyses of the conjugates using spectrofluorimetry and singlet oxygen detection showed that they display photophysical properties typical of FAM or Chk. Anticandidal activity assays showed that not only this basic property of TAT(47-57) was preserved in the conjugates but also that the minimal inhibitory concentration was slightly reduced for cells incubated with PS-bearing conjugate Chk-PEG6-TAT(47-57)-NH2. Overall, these results indicated that the synthetic approach on-resin assisted by microwaves at 60 °C is simple, straightforward, selective, metal-free, sufficiently fast, cleaner, and more cost-effective than those previously used for preparing this type of macromolecule. Furthermore, such new data show that microwaves at 60 °C and/or conjugation did not harm the integrity of the conjugates' constituents. Therefore, FAM-GG-TAT(47-57)-NH2, FAM-PEG6-TAT(47-57)-NH2, and Chk-PEG6-TAT(47-57)-NH2 have high potential for practical applications in biochemistry, biophysics, and therapeutics.