Stable and non-toxic fluorescent markers are gaining attention in molecular diagnostics as powerful tools for enabling long and reliable biological studies. Such markers should not only have a long half-life under several assay conditions showing no photo bleaching or blinking but also, they must allow for their conjugation or functionalization as a crucial step for numerous applications such as cellular tracking, biomarker detection and drug delivery. We report the functionalization of stable fluorescent markers based on nanodiamonds (NDs) with a bifunctional peptide. This peptide is made of a cell penetrating peptide and a six amino acids long β-sheet breaker peptide that is able to recognize amyloid β (Aβ) aggregates, a biomarker for the Alzheimer disease. Our results indicate that functionalized NDs (fNDs) are not cytotoxic and can be internalized by the cells. The fNDs allow ultrasensitive detection (at picomolar concentrations of NDs) of in vitro amyloid fibrils and amyloid aggregates in AD mice brains. The fluorescence of functionalized NDs is more stable than that of fluorescent markers commonly used to stain Aβ aggregates such as Thioflavin T. These results pave the way for performing ultrasensitive and reliable detection of Aβ aggregates involved in the pathogenesis of the Alzheimer disease.

Biomolecular interactions are at the base of all physical processes within living organisms; the study of these interactions has led to the development of a plethora of different methods. Among these, single-molecule (in singulo) experiments have become relevant in recent years because these studies can give insight into mechanisms and interactions that are hidden for ensemble-based (in multiplo) methods. The focus of this review is on optical tweezer (OT) experiments, which can be used to apply and measure mechanical forces in molecular systems. OTs are based on optical trapping, where a laser is used to exert a force on a dielectric bead; and optically trap the bead at a controllable position in all three dimensions. Different experimental approaches have been developed to study protein–protein interactions using OTs, such as: (1) refolding and unfolding in trans interaction where one protein is tethered between the beads and the other protein is in the solution; (2) constant force in cis interaction where each protein is bound to a bead, and the tension is suddenly increased. The interaction may break after some time, giving information about the lifetime of the binding at that tension. And (3) force ramp in cis interaction where each protein is attached to a bead and a ramp force is applied until the interaction breaks. With these experiments, parameters such as kinetic constants (k_off, k_on), affinity values (K_D), energy to the transition state ΔG^≠, distance to the transition state Δx^≠ can be obtained. These parameters characterize the energy landscape of the interaction. Some parameters such as distance to the transition state can only be obtained from force spectroscopy experiments such as those described here.

Immunoglobulin heavy-chain-binding protein (BiP protein) is a 75-kDa Hsp70 monomeric ATPase motor that plays broad and crucial roles maintaining proteostasis inside the cell. Its malfunction has been related with the appearance of many and important health problems such as neurodegenerative diseases, cancer, and heart diseases, among others. In particular, it is involved in many endoplasmic reticulum (ER) processes and functions, such as protein synthesis, folding, and assembly, and also it works in the posttranslational mechanism of protein translocation. However, it is unknown what kind of molecular motor BiP works like, since the mechanochemical mechanism that BiP utilizes to perform its work during posttranslational translocation across the ER is not fully understood. One novel approach to study both structural and catalytic properties of BiP considers that the viscoelastic regime behavior of the enzymes (considering them as a spring) and their mechanical properties are correlated with catalysis and ligand binding. Structurally, BiP is formed by two domains, and to establish a correlation between BiP structure and catalysis and how its conformational and viscoelastic changes are coupled to ligand binding, catalysis, and allosterism (information transmitted between the domains), optical tweezers and nano-rheology techniques have been essential in this regard.

This study presents the design and development of an ultrasonic sensor as a fundamental tool for characterizing the properties of fluids and biofluids. The analysis primarily focuses on measuring the electrical parameters of the system, which correlate with the density and viscosity of the solutions, in sample volumes of microliters and with high temporal resolution (up to 1 data point per second). The use of this sensor allows the fast and non-destructive evaluation of the viscosity and density of fluids deposited on its free surface. The measurements are based on obtaining the impedance versus frequency curve and the phase difference curve (between current and voltage) versus frequency. In this way, characteristic parameters of the transducer, such as the resonance frequency, phase, minimum impedance, and the quality factor of the resonant system, can characterize variations in density and viscosity in the fluid under study. The results obtained revealed the sensor’s ability to identify two parameters sensitive to viscosity and two parameters sensitive to density. As a proof of concept, the unfolding of the bovine albumin protein was studied, resulting in a curve that reflects its unfolding kinetics in the presence of urea.

Optical tweezers have enabled the exploration of picoNewton forces and dynamics in single–molecule systems such as DNA and molecular motors. In this work, we used optical tweezers to study the folding/unfolding dynamics of the APTSTX1–aptamer, a single-stranded DNA molecule with high affinity for saxitoxin (STX), a lethal neurotoxin. By measuring the transition force during (un)folding processes, we were able to characterize and distinguish the conformational changes of this aptamer in the presence of magnesium ions and toxin. This work was supported by molecular dynamics (MD) simulations to propose an unfolding mechanism of the aptamer–Mg⁺² complex. Our results are a step towards the development of new aptamer-based STX sensors that are potentially cheaper and more sensitive than current alternatives.