Biomolecular Binding under Confinement: Statistical Predictions of Steric Influence in Absence of Long-Distance Interactions.

We propose a theoretical model for the influence of confinement on biomolecular binding at the single-molecule scale at equilibrium, based on the change of the number of microstates (localization and orientation) upon reaction. Three cases are discussed: DNA sequences shorter and longer than the single strain DNA Kuhn length and spherical proteins, confined into a spherical container (liposome, droplet, etc.). The influence of confinement is found to be highly dependent on the molecular structure and significant for large molecules (relative to container size).

Statistical predictions on the encapsulation of single molecule binding pairs into sized-dispersed nanocontainers.

Single molecule experiments have recently attracted enormous interest. Many of these studies involve the encapsulation of a single molecule into nanoscale containers (such as vesicles, droplets and nanowells). In such cases, the single molecule encapsulation efficiency is a key parameter to consider in order to get a statistically significant quantitative information. It has been shown that such encapsulation typically follows a Poisson distribution and such theory of encapsulation has only been applied to the encapsulation of single molecules into perfectly sized monodispersed containers. However, experimentally nanocontainers are usually characterized by a size distribution, and often just a single binding pair (rather than a single molecule) is required to be encapsulated. Here the use of Poisson distribution is extended to predict the encapsulation efficiency of two different molecules in an association equilibrium. The Poisson distribution is coupled with a log-normal distribution in order to consider the effect of the container size distribution, and the effect of adsorption to the container is also considered. This theory will allow experimentalists to determine what single molecule encapsulation efficiency can be expected as a function of the experimental conditions. Two case studies, based on experimental data, are given to support the theoretical predictions.

FRET theoretical predictions concerning freely diffusive dyes inside spherical container: how to choose the best pair?

FRET has been massively used to see if biomolecules were bounded or not by labelling both biomolecules by one dye of a FRET pair. This should give a digital answer to the question (fluorescence of the acceptor: high FRET efficency: molecules associated, fluorescence of the donor: low FRET efficency: molecules dissociated). This has been used, inter alia, at the single-molecule scale in containers, such as liposomes. One perspective of the field is to reduce the container's size to study the effect of confinement on binding. The problem is that if the two dyes are encapsulated inside a small liposome, they could have a significant probability to be close one from the other one (and thus to undergo a high FRET efficiency event without binding). This is why we suggest here a theoretical model which gives mean FRET efficiency as a function of liposome radius (the model applies to any spherical container) and Förster radius to help the experimentalist to choose their experimental set-up. Besides, the influence of side effect on mean FRET efficiency has been studied as well. We show here that if this "background FRET" is most of the time non-quantitative, it can remain significant and which makes data analysis trickier. We could show as well that if this background FRET obviously increases when liposome radius decreases, this variation was lower than the one which could be expected because of side effect. We show as well the FRET efficiency function distribution which let the experimentalist know the probability to get one FRET efficiency value.

Electrocatalytic Mechanism Involving Michaelis-Menten Kinetics at the Preparative Scale: Theory and Applicability to Photocurrents from a Photosynthetic Algae Suspension With Quinones.

In the past years, many strategies have been implemented to benefit from oxygenic photosynthesis to harvest photosynthetic electrons and produce a significant photocurrent. Therefore, electrochemical tools were considered and have globally relied on the electron transfer(s) between the photosynthetic chain and a collecting electrode. In this context, we recently reported the implementation of an electrochemical set-up at the preparative scale to produce photocurrents from a Chlamydomonas reinhardtii algae suspension with an appropriate mediator (2,6-DCBQ) and a carbon gauze as the working electrode. In the present work, we wish to describe a mathematical modeling of the recorded photocurrents to better understand the effects of the experimental conditions on the photosynthetic extraction of electrons. In that way, we established a general model of an electrocatalytic mechanism at the preparative scale (that is, assuming a homogenous bulk solution at any time and a constant diffusion layer, both assumptions being valid under forced convection) in which the chemical step involves a Michaelis-Menten-like behaviour. Dependences of transient and steady-state corresponding currents were analysed as a function of different parameters by means of zone diagrams. This model was tested to our experimental data related to photosynthesis. The corresponding results suggest that competitive pathways beyond photosynthetic harvesting alone should be taken into account.

Investigation of quinone reduction by microalgae using fluorescence - do "lake" and "puddle" mechanisms matter?

Photosynthesis is a fundamental process used by Nature to convert solar energy into chemical energy. For the last twenty years, many solutions have been explored to provide electrical power from the photosynthetic chain. In this context, the coupling between microalgae and exogenous quinones is an encouraging strategy because of the capability of quinones to be reduced by the photosynthetic chain. The ability of a quinone to be a good or bad electron acceptor can be evaluated by fluorescence measurements. Fluorescence analyses are thus a convenient tool helping to define a diverting parameter for some quinones. However, this parameter is implicitly designed on the basis of a particular light capture mechanism by algae. In this paper, we propose to revisit previous fluorescence experimental data by considering the two possible mechanisms (lake vs. puddle) and discussing their implication on the conclusions of the analysis. In particular, we show that the maximum extraction efficiency depends on the mechanism (in the case of 2,6-dichlorobenzoquinone - 2,6-DCBQ, (0.45 ± 0.02) vs (0.61 ± 0.03) for lake and puddle mechanisms respectively) but that the trends for different quinones remain correlated to the redox potentials independently of the mechanism.

Impact of the Coverage of Aptamers on a Nanoparticle on the Binding Equilibrium and Kinetics between Aptamer and Protein.

Knowledge of the interaction between aptamer and protein is integral to the design and development of aptamer-based biosensors. Nanoparticles functionalized with aptamers are commonly used in these kinds of sensors. As such, studies into how the number of aptamers on the nanoparticle surface influence both kinetics and thermodynamics of the binding interaction are required. In this study, aptamers specific for interferon gamma (IFN-γ) were immobilized on the surface of gold nanoparticles (AuNPs), and the effect of surface coverage of aptamer on the binding interaction with its target was investigated using fluorescence spectroscopy. The number of aptamers were adjusted from an average of 9.6 to 258 per particle. The binding isotherm between AuNPs-aptamer conjugate and protein was modeled with the Hill-Langmuir equation, and the determined equilibrium dissociation constant (K'D) decreased 10-fold when increasing the coverage of aptamer. The kinetics of the reaction as a function of coverage of aptamer were also investigated, including the association rate constant (kon) and the dissociation rate constant (koff). The AuNPs-aptamer conjugate with 258 aptamers per particle had the highest kon, while the koff was similar for AuNPs-aptamer conjugates with different surface coverages. Therefore, the surface coverage of aptamers on AuNPs affects both the thermodynamics and the kinetics of the binding. The AuNPs-aptamer conjugate with the highest surface coverage is the most favorable in biosensors considering the limit of detection, sensitivity, and response time of the assay. These findings deepen our understanding of the interaction between aptamer and target protein on the particle surface, which is important to both improve the scientific design and increase the application of aptamer-nanoparticle based biosensor.

Investigation of photocurrents resulting from a living unicellular algae suspension with quinones over time.

Plants, algae, and some bacteria convert solar energy into chemical energy by using photosynthesis. In light of the current energy environment, many research strategies try to benefit from photosynthesis in order to generate usable photobioelectricity. Among all the strategies developed for transferring electrons from the photosynthetic chain to an outer collecting electrode, we recently implemented a method on a preparative scale (high surface electrode) based on a Chlamydomonas reinhardtii green algae suspension in the presence of exogenous quinones as redox mediators. While giving rise to an interesting performance (10-60 µA cm-2) in the course of one hour, this device appears to cause a slow decrease of the recorded photocurrent. In this paper, we wish to analyze and understand this gradual fall in performance in order to limit this issue in future applications. We thus first show that this kind of degradation could be related to over-irradiation conditions or side-effects of quinones depending on experimental conditions. We therefore built an empirical model involving a kinetic quenching induced by incubation with quinones, which is globally consistent with the experimental data provided by fluorescence measurements achieved after dark incubation of algae in the presence of quinones.

Redesigning the QA binding site of Photosystem II allows reduction of exogenous quinones.

Strategies to harness photosynthesis from living organisms to generate electrical power have long been considered, yet efficiency remains low. Here, we aimed to reroute photosynthetic electron flow in photosynthetic organisms without compromising their phototrophic properties. We show that 2,6-dimethyl-p-benzoquinone (DMBQ) can be used as an electron mediator to assess the efficiency of mutations designed to engineer a novel electron donation pathway downstream of the primary electron acceptor QA of Photosystem (PS) II in the green alga Chlamydomonas reinhardtii. Through the use of structural prediction studies and a screen of site-directed PSII mutants we show that modifying the environment of the QA site increases the reduction rate of DMBQ. Truncating the C-terminus of the PsbT subunit protruding in the stroma provides evidence that shortening the distance between QA and DMBQ leads to sustained electron transfer to DMBQ, as confirmed by chronoamperometry, consistent with a bypass of the natural QA°- to QB pathway.

Protease sensing using nontoxic silicon quantum dots.

Herein is presented a proof-of-concept study of protease sensing that combines nontoxic silicon quantum dots (SiQDs) with Förster resonance energy transfer (FRET). The SiQDs serve as the donor and an organic dye as the acceptor. The dye is covalently attached to the SiQDs using a peptide linker. Enzymatic cleavage of the peptide leads to changes in FRET efficiency. The combination of interfacial design and optical imaging presented in this work opens opportunities for use of nontoxic SiQDs relevant to intracellular sensing and imaging.

Evaluation of photosynthetic electrons derivation by exogenous redox mediators.

Oxygenic photosynthesis is the complex process that occurs in plants or algae by which the energy from the sun is converted into an electrochemical potential that drives the assimilation of carbon dioxide and the synthesis of carbohydrates. Quinones belong to a family of species commonly found in key processes of the Living, like photosynthesis or respiration, in which they act as electron transporters. This makes this class of molecules a popular candidate for biofuel cell and bioenergy applications insofar as they can be used as cargo to ship electrons to an electrode immersed in the cellular suspension. Nevertheless, such electron carriers are mostly selected empirically. This is why we report on a method involving fluorescence measurements to estimate the ability of seven different quinones to accept photosynthetic electrons downstream of photosystem II, the first protein complex in the light-dependent reactions of oxygenic photosynthesis. To this aim we use a mutant of Chlamydomonas reinhardtii, a unicellular green alga, impaired in electron downstream of photosystem II and assess the ability of quinones to restore electron flow by fluorescence. In this work, we defined and extracted a "derivation parameter" D that indicates the derivation efficiency of the exogenous quinones investigated. D then allows electing 2,6-dichlorobenzoquinone, 2,5-dichlorobenzoquinone and p-phenylbenzoquinone as good candidates. More particularly, our investigations suggested that other key parameters like the partition of quinones between different cellular compartments and their propensity to saturate these various compartments should also be taken into account in the process of selecting exogenous quinones for the purpose of deriving photoelectrons from intact algae.