Molten globule-like transition state of protein barnase measured with calorimetric force spectroscopy.

SignificanceUnderstanding the molecular forces driving the unfolded polypeptide chain to self-assemble into a functional native structure remains an open question. However, identifying the states visited during protein folding (e.g., the transition state between the unfolded and native states) is tricky due to their transient nature. Here, we introduce calorimetric force spectroscopy in a temperature jump optical trap to determine the enthalpy, entropy, and heat capacity of the transition state of protein barnase. We find that the transition state has the properties of a dry molten globule, that is, high free energy and low configurational entropy, being structurally similar to the native state. This experimental single-molecule study characterizes the thermodynamic properties of the transition state in funneled energy landscapes.

Explosive electrostatic instability of ferroelectric liquid droplets on ferroelectric solid surfaces.

We investigated the electrostatic behavior of ferroelectric liquid droplets exposed to the pyroelectric field of a lithium niobate ferroelectric crystal substrate. The ferroelectric liquid is a nematic liquid crystal, in which almost complete polar ordering of the molecular dipoles generates an internal macroscopic polarization locally collinear to the mean molecular long axis. Upon entering the ferroelectric phase by reducing the temperature from the nematic phase, the liquid crystal droplets become electromechanically unstable and disintegrate by the explosive emission of fluid jets. These jets are mostly interfacial, spreading out on the substrate surface, and exhibit fractal branching out into smaller streams to eventually disrupt, forming secondary droplets. We understand this behavior as a manifestation of the Rayleigh instability of electrically charged fluid droplets, expected when the electrostatic repulsion exceeds the surface tension of the fluid. In this case, the charges are due to the bulk polarization of the ferroelectric fluid, which couples to the pyroelectric polarization of the underlying lithium niobate substrate through its fringing field and solid-fluid interface coupling. Since the ejection of fluid does not neutralize the droplet surfaces, they can undergo multiple explosive events as the temperature decreases.

Opto-microfluidic coupling between optical waveguides and tilted microchannels in lithium niobate.

This work presents a reconfigurable opto-microfluidic coupling between optical waveguides and tilted microfluidic channels in monolithic lithium niobate crystal. The light path connecting two waveguide arrays located on opposite sides of a microfluidic channel depends on the refractive index between the liquid phase and the hosting crystal. As a result, the optical properties of the flowing fluid, which is pumped into the microfluidic channel on demand, can be exploited to control the light pathways inside the optofluidic device. Proof-of-concept applications are herein presented, including microfluidic optical waveguide switching, optical refractive index sensing, and wavelength demultiplexing.

Opto-Microfluidic Integration of the Bradford Protein Assay in Lithium Niobate Lab-on-a-Chip.

This paper deals with the quantification of proteins by implementing the Bradford protein assay method in a portable opto-microfluidic platform for protein concentrations lower than 1.4 mg/mL. Absorbance is measured by way of optical waveguides integrated to a cross-junction microfluidic circuit on a single lithium niobate substrate. A new protocol is proposed to perform the protein quantification based on the high correlation of the light absorbance at 595 nm, as commonly used in the Bradford method, with the one achieved at 633 nm with a cheap commercially available diode laser. This protocol demonstrates the possibility to quantify proteins by using nL volumes, 1000 times less than the standard technique such as paper-analytical devices. Moreover, it shows a limit of quantification of at least 0.12 mg/mL, which is four times lower than the last literature, as well as a better accuracy (98%). The protein quantification is obtained either by using one single microfluidic droplet as well by performing statistical analysis over ensembles of several thousands of droplets in less than 1 min. The proposed methodology presents the further advantage that the protein solutions can be reused for other investigations and the same pertains to the opto-microfluidic platform.

Opto-Microfluidic System for Absorbance Measurements in Lithium Niobate Device Applied to pH Measurements.

The aim of Lab-on-a-chip systems is the downscaling of analytical protocols into microfluidic devices, including optical measurements. In this context, the growing interest of the scientific community in opto-microfluidic devices has fueled the development of new materials. Recently, lithium niobate has been presented as a promising material for this scope, thanks to its remarkable optical and physicochemical properties. Here, we present a novel microfluidic device realized starting from a lithium niobate crystal, combining engraved microfluidic channels with integrated and self-aligned optical waveguides. Notably, the proposed microfabrication strategy does not compromise the optical coupling between the waveguides and the microchannel, allowing one to measure the transmitted light through the liquid flowing in the channel. In addition, the device shows a high versatility in terms of the optical properties of the light source, such as wavelength and polarization. Finally, the developed opto-microfluidic system is successfully validated as a probe for real-time pH monitoring of the liquid flowing inside the microchannel, showing a high integrability and fast response.

Trifurcated Splitting of Water Droplets on Engineered Lithium Niobate Surfaces.

Controlled splitting of liquid droplets is a key function in many microfluidic applications. In recent years, various methodologies have been used to accomplish this task. Here, we present an optofluidic technique based on an engineered surface formed by coating a z-cut iron-doped lithium niobate crystal with a lubricant-infused layer, which provides a very slippery surface. Illuminating the crystal with a light spot induces surface charges of opposite signs on the two crystal faces because of the photovoltaic effect. If the light spot is sufficiently intense, millimetric water droplets placed near the illuminated spot split into two charged fragments, one fragment being trapped by the bright spot and the other moving away from it. The latter fragment does not move randomly but rather follows one of three well-defined trajectories separated by 120°, which reflect the anisotropic crystalline structure of Fe:LiNbO3. Numerical simulations explain the behavior of water droplets in the framework of the forces induced by the interplay of pyroelectric, piezoelectric, and photovoltaic effects, which originate simultaneously inside the illuminated crystal. Such a synergetic effect can provide a valuable feature in applications that require splitting and coalescence of droplets, such as chemical microreactors and biological encapsulation and screening.

Determination of the Dielectrophoretic Force Induced by the Photovoltaic Effect on Lithium Niobate.

The actuation of droplets on a surface is extremely relevant for microfluidic applications. In recent years, various methodologies have been used. A promising solution relies on iron-doped lithium niobate crystals that, when illuminated, generate an evanescent electric field in the surrounding space due to the photovoltaic effect. This field can be successfully exploited to control the motion of water droplets. Here, we present an experimental method to determine the attractive force exerted by the evanescent field. It consists of the analysis of the elongation of a pendant droplet and its detachment from the suspending syringe needle, caused by the illumination of an iron-doped lithium niobate crystal. We show that this interaction resembles that obtained by applying a voltage between the needle and a metallic substrate, and a quantitative investigation of these two types of actuation yields similar results. Pendant droplet tensiometry is then demonstrated to offer a simple solution for quickly mapping out the force at different distances from the crystal, generated by the photovoltaic effect and its temporal evolution, providing important quantitative data for the design and characterization of optofluidic devices based on lithium niobate crystals.

Force Dependence of Proteins' Transition State Position and the Bell-Evans Model.

Single-molecule force spectroscopy has opened a new field of research in molecular biophysics and biochemistry. Pulling experiments on individual proteins permit us to monitor conformational transitions with high temporal resolution and measure their free energy landscape. The force-extension curves of single proteins often present large hysteresis, with unfolding forces that are higher than refolding ones. Therefore, the high energy of the transition state (TS) in these molecules precludes kinetic rates measurements in equilibrium hopping experiments. In irreversible pulling experiments, force-dependent kinetic rates measurements show a systematic discrepancy between the sum of the folding and unfolding TS distances derived by the kinetic Bell-Evans model and the full molecular extension predicted by elastic models. Here, we show that this discrepancy originates from the force-induced movement of TS. Specifically, we investigate the highly kinetically stable protein barnase, using pulling experiments and the Bell-Evans model to characterize the position of its kinetic barrier. Experimental results show that while the TS stays at a roughly constant distance relative to the native state, it shifts with force relative to the unfolded state. Interestingly, a conversion of the protein extension into amino acid units shows that the TS position follows the Leffler-Hammond postulate: the higher the force, the lower the number of unzipped amino acids relative to the native state. The results are compared with the quasi-reversible unfolding-folding of a short DNA hairpin.

Optofluidic platform using liquid crystals in lithium niobate microchannel.

We demonstrate the all optical control of the molecular orientation of nematic liquid crystals confined in microfluidic channels engraved in lithium niobate. Microchannels are obtained by a novel approach based on femtosecond pulse laser micromachining carried on in controlled atmosphere. The combined effect of photovoltaic and pyroelectric fields generated by light in lithium niobate crystals on the liquid crystal orientation, is reported for the first time. The total space charge field and its dependence on the incident light intensity can be controlled by changing the direction of pump light propagation through the microfluidic chip. The results reported in this manuscript demonstrate that liquid crystals and lithium niobate can efficiently be combined in microfluidic configuration, in order to push forward a novel class of optofluidic devices.