Effect of ionic strength on intra-protein electron transfer reactions: The case study of charge recombination within the bacterial reaction center.

It is a common believe that intra-protein electron transfer (ET) involving reactants and products that are overall electroneutral are not influenced by the ions of the surrounding solution. The results presented here show an electrostatic coupling between the ionic atmosphere surrounding a membrane protein (the reaction center (RC) from the photosynthetic bacterium Rhodobacter sphaeroides) and two very different intra-protein ET processes taking place within it. Specifically we have studied the effect of salt concentration on: i) the kinetics of the charge recombination between the reduced primary quinone acceptor QA(-) and the primary photoxidized donor P(+); ii) the thermodynamic equilibrium (QA(-)↔QB(-)) for the ET between QA(-) and the secondary quinone acceptor QB. A distinctive point of this investigation is that reactants and products are overall electroneutral. The protein electrostatics has been described adopting the lowest level of complexity sufficient to grasp the experimental phenomenology and the impact of salt on the relative free energy level of reactants and products has been evaluated according to suitable thermodynamic cycles. The ionic strength effect was found to be independent on the ion nature for P(+)QA(-) charge recombination where the leading electrostatic term was the dipole moment. In the case of the QA(-)↔QB(-) equilibrium, the relative stability of QA(-) and QB(-) was found to depend on the salt concentration in a fashion that is different for chaotropic and kosmotropic ions. In such a case both dipole moment and quadrupole moments of the RC must be considered.

General Approach to the Immobilization of Glycoenzyme Chains Inside Calcium Alginate Beads for Bioassay.

A general method to obtain the efficient entrapment of mixtures of glycoenzymes in calcium alginate hydrogel is proposed in this paper. As a proof of principle, three glycoenzymes acting in series (trehalase, glucose oxidase, and horseradish peroxidase) have been coimmobilized in calcium alginate beads. The release of the enzymes from the hydrogel mesh (leakage) is avoided by exploiting the enzyme's aggregation induced by the concanavalin A. The aggregation process has been monitored by dynamic light scattering technique, while both enzyme encapsulation efficiency and leakage have been quantified spectrophotometrically. Obtained data show an encapsulation efficiency above 95% and a negligible leakage from the beads when enzyme aggregates are larger than 300 nm. Operational stability of "as prepared" beads has been largely improved by a coating of alternated shells of polycation poly(diallyldimethylammonium chloride) and of alginate. As a test for the effectiveness of the overall procedure, analytical bioassays exploiting the enzyme-containing beads have been developed for the optical determination of glucose and trehalose, and limit of detection values of 0.2 and of 40 µM, respectively, have been obtained.

Interfacial electronic effects in functional biolayers integrated into organic field-effect transistors.

Biosystems integration into an organic field-effect transistor (OFET) structure is achieved by spin coating phospholipid or protein layers between the gate dielectric and the organic semiconductor. An architecture directly interfacing supported biological layers to the OFET channel is proposed and, strikingly, both the electronic properties and the biointerlayer functionality are fully retained. The platform bench tests involved OFETs integrating phospholipids and bacteriorhodopsin exposed to 1-5% anesthetic doses that reveal drug-induced changes in the lipid membrane. This result challenges the current anesthetic action model relying on the so far provided evidence that doses much higher than clinically relevant ones (2.4%) do not alter lipid bilayers' structure significantly. Furthermore, a streptavidin embedding OFET shows label-free biotin electronic detection at 10 parts-per-trillion concentration level, reaching state-of-the-art fluorescent assay performances. These examples show how the proposed bioelectronic platform, besides resulting in extremely performing biosensors, can open insights into biologically relevant phenomena involving membrane weak interfacial modifications.

Mechanism of surfactant interactions with feline coronavirus: A physical chemistry perspective.

HYPOTHESIS: Surfactants are inexpensive chemicals with promising applications in virus inactivation, particularly for enveloped viruses. Yet, the detailed mechanisms by which surfactants deactivate coronaviruses remain underexplored. This study delves into the virucidal mechanisms of various surfactants on Feline Coronavirus (FCoV) and their potential applications against more pathogenic coronaviruses. EXPERIMENTS: By integrating virucidal activity assays with fluorescence spectroscopy, dynamic light scattering and laser Doppler electrophoresis, alongside liposome permeability experiments, we have analyzed the effects of non-ionic and ionic surfactants on viral activity. FINDINGS: The non-ionic surfactant octaethylene glycol monodecyl ether (C10EO8) inactivates the virus by disrupting the lipid envelope, whereas ionic surfactants like Sodium Dodecyl Sulfate and Cetylpyridinium Chloride predominantly affect the spike proteins, with their impact on the viral membrane being hampered by kinetic and thermodynamic constraints. FCoV served as a safe model for studying virucidal activity, offering a faster alternative to traditional virucidal assays. The study demonstrates that physicochemical techniques can expedite the screening of virucidal compounds, contributing to the design of effective disinfectant formulations. Our results not only highlight the critical role of surfactant-virus interactions but also contribute to strategic advancements in public health measures for future pandemic containment and the ongoing challenge of antimicrobial resistance.

Part per trillion label-free electronic bioanalytical detection.

A Functional Bio-Interlayer Organic Field-Effect Transistor (FBI-OFET) sensor, embedding a streptavidin protein capturing layer, capable of performing label-free selective electronic detection of biotin at 3 part per trillion (mass fraction) or 15 pM, is proposed here. The response shows a logarithmic dependence spanning over 5 orders of magnitude of analyte concentration. The optimization of the FBI analytical performances is achieved by depositing the capturing layer through a controllable Layer-by-Layer (LbL) assembly, while an easy processable spin-coating deposition is proposed for potential low-cost production of equally highly performing sensors. Furthermore, a Langmuirian adsorption based model allows rationalizing the analyte binding process to the capturing layer. The FBI-OFET device is shown to operate also with an antibody interlayer as well as with an ad hoc designed microfluidic system. These occurrences, along with the proven extremely high sensitivity and selectivity, open to FBI-OFETs consideration as disposable electronic strip-tests for assays in biological fluids requiring very low detection limits.

Plasma Parameters During Nanoparticle-Enhanced Laser-Induced Breakdown Spectroscopy (NELIBS) in the Presence of Nanoparticle-Protein Conjugates.

Nanoparticle-enhanced laser-induced breakdown spectroscopy (NELIBS) is an optical emission technique based on the laser-induced plasma (LIP) on a sample after the deposition of plasmonic nanoparticles (NPs) on its surface. The employment of the NPs allows an enhancement of the signal with respect to the one obtained with the conventional laser-induced breakdown spectroscopy (LIBS) enabling an extremely high sensitivity and very low limits of detection compared with the LIBS performance. Recently, NELIBS was used for monitoring the NP protein corona formation. As a matter of fact, the NPs in the presence of proteins adsorbed on the surface change their surface properties, therefore the sensing of protein corona formation was possible because of the strong dependence of NELIBS effects on the NP organization on the substrate, which in turn is deeply affected by the surface properties of the NPs. A correlation was found between NELIBS enhancement and the structure of the NP-protein conjugate in terms of protein content absorbed on the NP surface. An interesting question that was not yet exploited regards the role of LIP during the NELIBS when the NPs are covered with proteins. Since the presence of organic matter can strongly quench the LIP emission, the study of the LIP properties during protein corona sensing by NELIBS is of interest for two main reasons: (i) to understand whether the plasma parameters can vary in the presence of proteins adsorbed on the NP surface and (ii) to investigate how and if the plasma parameters themselves can influence the NELIBS processes. With this aim, the study of plasma parameters, i.e., electron densities and temperatures, during the sensing of NP protein corona by NELIBS is presented and discussed. The NPs used during these experiments were ultrapure gold NPs (AuNPs) produced by pulsed laser ablation in liquid, which are stable without any stabilizer. The human serum albumin protein is used to form AuNP-protein conjugates further deposited on a titanium target in NELIBS measurements. Dynamic light scattering, surface plasmon resonance spectroscopy, and laser Doppler electrophoresis for ζ-potential determination were employed to monitor the protein coverage of NP surface in the conjugate solutions before the NELIBS measurements.

Interaction of surfactants with phospholipid vesicles in the low concentration regime.

The interactions between diluted phospholipid vesicles (0.3 µM - 40 µM) and surfactants (around their cmc) have been investigated as model of the phenomena taking place when enveloped viruses are challenged by detergent formulations such as mouthwashes or dishwashing liquids. We have used negatively charged Small Unilamellar Vesicles (SUVs) to simulate the negatively charged viral envelope and surfactants with different charges: the anionic Sodium Dodecyl Sulphate (SDS), the cationic Cetylpyridinium Chloride (CPC) and the non-ionic Octaethylene glycol monodecyl ether (C10E8). Dynamic and Electrophoretic Light Scattering have been used to probe variations in size and surface charge of the vesicles. The surfactants effect on the membrane permeability was investigated by measuring the fluorescence of SUVs secluding the fluorophore calcein. All the surfactants perturb the bilayer inducing graded dye leakage. Irrespective of the chemical nature of the surfactant, the membrane leakage follows the same sigmoidal master curve when it is plotted against the ratio surfactant concentration/cmc. The membrane leakage is negligible below cmc/2 and above such a value increases up to the cmc where all the dye has been fully released. For ionic SDS and CPC the dependence of leakage halftime on such a scaled concentration is the same irrespective of the charge of the surfactant and the vesicles. The nonionic surfactant C10E8 induces the dye release from the SUV two orders-of-magnitude faster than the ionic surfactants. These results show that the rate-determining parameter for the permeabilization of the lipid bilayers is the electrostatic penalty to the flip-flop required to transport the surfactant inside the vesicle.

Effect of detergent concentration on the thermal stability of a membrane protein: The case study of bacterial reaction center solubilized by N,N-dimethyldodecylamine-N-oxide.

We report on the response of reaction center (RC) from Rhodobacter sphaeroides (an archetype of membrane proteins) to the exposure at high temperature. The RCs have been solubilized in aqueous solution of the detergent N,N-dimethyldodecylamine-N-oxide (LDAO). Changes in the protein conformation have been probed by monitoring the variation in the absorbance of the bacteriochlorine cofactors and modification in the efficiency of energy transfer from tryptophans to cofactors and among the cofactors (through fluorescence measurements). The RC aggregation taking place at high temperature has been investigated by means of dynamic light scattering. Two experimental protocols have been used: (i) isothermal kinetics, in which the time evolution of RC after a sudden increase of the temperature is probed, and (ii) T-scans, in which the RCs are heated at constant rate. The analysis of the results coming from both the experiments indicates that the minimal kinetic scheme requires an equilibrium step and an irreversible process. The irreversible step is characterized by a activation energy of 205+/-14 kJ/mol and is independent from the detergent concentration. Since the temperature dependence of the aggregation rate was found to obey to the same law, the aggregation process is unfolding-limited. On the other hand, the equilibrium process between the native and a partially unfolded conformations was found to be strongly dependent on the detergent concentration. Increasing the LDAO content from 0.025 to 0.5 wt.% decreases the melting temperature from 49 to 42 degrees C. This corresponds to a sizeable (22 kJ/mol at 25 degrees C) destabilization of the native conformation induced by the detergent. The nature of the aggregates formed by the denatured RCs depends on the temperature. For temperature below 60 degrees C compact aggregates are formed while at 60 degrees C the clusters are less dense with a scaling relation between mass and size close to that expected for diffusion-limited aggregation. The aggregate final sizes formed at different temperatures indicate the presence of an even number of proteins suggesting that these clusters are formed by aggregation of dimers.

Effects of the measuring light on the photochemistry of the bacterial photosynthetic reaction center from Rhodobacter sphaeroides.

The bacterial reaction center (RC) has become a reference model in the study of the diverse interactions of quinones with electron transfer complexes. In these studies, the RC functionality was probed through flash-induced absorption changes where the state of the primary donor is probed by means of a continuous measuring beam and the electron transfer is triggered by a short intense light pulse. The single-beam set-up implies the use as reference of the transmittance measured before the light pulse. Implicit in the analysis of these data is the assumption that the measuring beam does not elicit the protein photochemistry. At variance, measuring beam is actinic in nature at almost all the suitable wavelengths. In this contribution, the analytical modelling of the time evolution of neutral and charge-separated RCs has been performed. The ability of measuring light to elicit RC photochemistry induces a first order growth of the charge-separated state up to a steady state that depends on the light intensity and on the occupation of the secondary quinone (Q(B)) site. Then the laser pulse pumps all the RCs in the charge-separated state. The following charge recombination is still affected by the measuring beam. Actually, the kinetics of charge recombination measured in RC preparation with the Q(B) site partially occupied are two-exponential. The rate constant of both fast and slow phases depends linearly on the intensity of the measuring beam while their relative weights depend not only on the fractions of RC with the Q(B) site occupied but also on the measuring light intensity itself.

Sensing nanoparticle-protein corona using nanoparticle enhanced Laser Induced Breakdown Spectroscopy signal enhancement.

Recently nanoparticle enhanced Laser Induced Breakdown Spectroscopy (NELIBS) is getting a growing interest as an effective alternative method for improving the analytical performance of LIBS. On the other hand, the plasmonic effect during laser ablation can be used for a different task rather than elemental analysis. In this paper, the dependence of NELIBS emission signal enhancement on nanoparticle-protein solutions dried on a reference substrate (metallic titanium) was investigated. Two proteins were studied: Human Serum Albumin (HSA) and Cytochrome C (CytC). Both proteins have a strong affinity for the gold nanoparticles (AuNPs) due to the bonding between the single free exterior thiol (associated with a cysteine residue) and the gold surface to form a stable protein corona. Then, since the protein sizes are vastly different, a different number of protein units is needed to cover AuNP surface to form a protein layer. The NP-protein solution was dropped and dried onto the titanium substrate. Then the NELIBS signal enhancement of Ti emission lines was correlated to the solution characteristics as determined with Dynamic Light Scattering (DLS), Surface Plasmon Resonance (SPR) spectroscopy and Laser Doppler Electrophoresis (LDE) for ζ-potential determination. Moreover, the dried solutions were studied with TEM (Transmission Electron Microscopy) for the inspection of the inter-particle distance. The structural effect of the NP-protein conjugates on the NELIBS signal reveals that NELIBS can be used to determine the number of protein units required to form the nanoparticle-protein corona with good accuracy. Although the investigated NP-protein systems are simple cases in biological applications, this work demonstrates, for the first time, a different use of NELIBS that is beyond elemental analysis and it opens the way for sensing the nanoparticle protein corona.

Salting-Out Approach Is Worthy of Comparison with Ultracentrifugation for Extracellular Vesicle Isolation from Tumor and Healthy Models.

The role of extracellular vesicles (EVs) has been completely re-evaluated in the recent decades, and EVs are currently considered to be among the main players in intercellular communication. Beyond their functional aspects, there is strong interest in the development of faster and less expensive isolation protocols that are as reliable for post-isolation characterisations as already-established methods. Therefore, the identification of easy and accessible EV isolation techniques with a low price/performance ratio is of paramount importance. We isolated EVs from a wide spectrum of samples of biological and clinical interest by choosing two isolation techniques, based on their wide use and affordability: ultracentrifugation and salting-out. We collected EVs from human cancer and healthy cell culture media, yeast, bacteria and Drosophila culture media and human fluids (plasma, urine and saliva). The size distribution and concentration of EVs were measured by nanoparticle tracking analysis and dynamic light scattering, and protein depletion was measured by a colorimetric nanoplasmonic assay. Finally, the EVs were characterised by flow cytometry. Our results showed that the salting-out method had a good efficiency in EV separation and was more efficient in protein depletion than ultracentrifugation. Thus, salting-out may represent a good alternative to ultracentrifugation.

A Novel Silicon Platform for Selective Isolation, Quantification, and Molecular Analysis of Small Extracellular Vesicles.

INTRODUCTION: Small extracellular vesicles (sEVs), thanks to their cargo, are involved in cellular communication and play important roles in cell proliferation, growth, differentiation, apoptosis, stemness and embryo development. Their contribution to human pathology has been widely demonstrated and they are emerging as strategic biomarkers of cancer, neurodegenerative and cardiovascular diseases, and as potential targets for therapeutic intervention. However, the use of sEVs for medical applications is still limited due to the selectivity and sensitivity limits of the commonly applied approaches. METHODS: Novel sensing solutions based on nanomaterials are arising as strategic tools able to surpass traditional sensor limits. Among these, Si nanowires (Si NWs), realized with cost-effective industrially compatible metal-assisted chemical etching, are perfect candidates for sEV detection. RESULTS: In this paper, the realization of a selective sensor able to isolate, concentrate and quantify specific vesicle populations, from minimal volumes of biofluid, is presented. In particular, this Si NW platform has a detection limit of about 2×105 sEVs/mL and was tested with follicular fluid and blastocoel samples. Moreover, the possibility to detach the selectively isolated sEVs allowing further analyses with other approaches was demonstrated by SEM analysis and several PCRs performed on the RNA content of the detached sEVs. DISCUSSION: This platform overcomes the limit of detection of traditional methods and, most importantly, preserves the biological content of sEVs, opening the route toward a reliable liquid biopsy analysis.

Direct Exposure of Dry Enzymes to Atmospheric Pressure Non-Equilibrium Plasmas: The Case of Tyrosinase.

The direct interaction of atmospheric pressure non-equilibrium plasmas with tyrosinase (Tyr) was investigated under typical conditions used in surface processing. Specifically, Tyr dry deposits were exposed to dielectric barrier discharges (DBDs) fed with helium, helium/oxygen, and helium/ethylene mixtures, and effects on enzyme functionality were evaluated. First of all, results show that DBDs have a measurable impact on Tyr only when experiments were carried out using very low enzyme amounts. An appreciable decrease in Tyr activity was observed upon exposure to oxygen-containing DBD. Nevertheless, the combined use of X-ray photoelectron spectroscopy and white-light vertical scanning interferometry revealed that, in this reactive environment, Tyr deposits displayed remarkable etching resistance, reasonably conferred by plasma-induced changes in their surface chemical composition as well as by their coffee-ring structure. Ethylene-containing DBDs were used to coat tyrosinase with a hydrocarbon polymer film, in order to obtain its immobilization. In particular, it was found that Tyr activity can be fully retained by properly adjusting thin film deposition conditions. All these findings enlighten a high stability of dry enzymes in various plasma environments and open new opportunities for the use of atmospheric pressure non-equilibrium plasmas in enzyme immobilization strategies.

The fe2+ site of photosynthetic reaction centers probed by multiple scattering x-ray absorption fine structure spectroscopy: improving structure resolution in dry matrices.

We report on the x-ray absorption fine structure of the Fe(2+) site in photosynthetic reaction centers from Rhodobacter sphaeroides. Crystallographic studies show that Fe(2+) is ligated with four N(epsilon) atoms from four histidine (His) residues and two O(epsilon) atoms from a Glu residue. By considering multiple scattering contributions to the x-ray absorption fine structure function, we improved the structural resolution of the site: His residues were split into two groups, characterized by different Fe-N(epsilon) distances, and two distinct Fe-O(epsilon) bond lengths resolved. The effect of the environment was studied by embedding the reaction centers into a polyvinyl alcohol film and into a dehydrated trehalose matrix. Incorporation into trehalose caused elongation in one of the two Fe-N(epsilon) distances, and in one Fe-O(epsilon) bond length, compared with the polyvinyl alcohol film. The asymmetry detected in the cluster of His residues and its response to incorporation into trehalose are ascribed to the hydrogen bonds between two His residues and the quinone acceptors. The structural distortions observed in the trehalose matrix indicate a strong interaction between the reaction-centers surface and the water-trehalose matrix, which propagates deeply into the interior of the protein. The absence of matrix effects on the Debye-Waller factors is brought back to the static heterogeneity and rigidity of the ligand cluster.

Stabilization of charge separation and cardiolipin confinement in antenna-reaction center complexes purified from Rhodobacter sphaeroides.

The reaction center-light harvesting complex 1 (RC-LH1) purified from the photosynthetic bacterium Rhodobacter sphaeroides has been studied with respect to the kinetics of charge recombination and to the phospholipid and ubiquinone (UQ) complements tightly associated with it. In the antenna-RC complexes, at 6.5 more than three times smaller than that measured in LH1-deprived RCs. At increasing pH values, for which increases, the deceleration observed in RC-LH1 complexes is reduced, vanishing at pH >11.0. In both systems kinetics are described by a continuous rate distribution, which broadens at pH >9.5, revealing a strong kinetic heterogeneity, more pronounced in the RC-LH1 complex. In the presence of the antenna the Q(A)Q(B)(-) state is stabilized by about 40 meV at 6.511. The phospholipid/RC and UQ/RC ratios have been compared in chromatophore membranes, in RC-LH1 complexes and in the isolated peripheral antenna (LH2). The UQ concentration in the lipid phase of the RC-LH1 complexes is about one order of magnitude larger than the average concentration in chromatophores and in LH2 complexes. Following detergent washing RC-LH1 complexes retain 80-90 phospholipid and 10-15 ubiquinone molecules per monomer. The fractional composition of the lipid domain tightly bound to the RC-LH1 (determined by TLC and (31)P-NMR) differs markedly from that of chromatophores and of the peripheral antenna. The content of cardiolipin, close to 10% weight in chromatophores and LH2 complexes, becomes dominant in the RC-LH1 complexes. We propose that the quinone and cardiolipin confinement observed in core complexes reflects the in vivo heterogeneous distributions of these components. Stabilization of the charge separated state in the RC-LH1 complexes is tentatively ascribed to local electrostatic perturbations due to cardiolipin.

Protein-matrix coupling/uncoupling in "dry" systems of photosynthetic reaction center embedded in trehalose/sucrose: the origin of trehalose peculiarity.

Trehalose is a nonreducing disaccharide of glucose found in organisms, which can survive adverse conditions such as extreme drought and high temperatures. Furthermore, isolated structures, as enzymes or liposomes, embedded in trehalose are preserved against stressing conditions [see, e.g., Crowe, L. M. Comp. Biochem. Physiol. A 2002, 131, 505-513]. Among other hypotheses, such protective effect has been suggested to stem, in the case of proteins, from the formation of a water-mediated, hydrogen bond network, which anchors the protein surface to the water-sugar matrix, thus coupling the internal degrees of freedom of the biomolecule to those of the surroundings [Giuffrida, S.; et al. J. Phys. Chem. B 2003, 107, 13211-13217]. Analogous protective effect is also accomplished by other saccharides, although with a lower efficiency. Here, we studied the recombination kinetics of the primary, light-induced charge separated state (P(+)Q(A)(-)) and the thermal stability of the photosynthetic reaction center (RC) of Rhodobacter sphaeroides in trehalose-water and in sucrose-water matrixes of decreasing water content. Our data show that, in sucrose, at variance with trehalose, the system undergoes a "nanophase separation" when the water/sugar mole fraction is lower than the threshold level approximately 0.8. We rationalize this result assuming that the hydrogen bond network, which anchors the RC surface to its surrounding, is formed in trehalose but not in sucrose. We suggest that both the couplings, in the case of trehalose, and the nanophase separation, in the case of sucrose, start at low water content when the components of the system enter in competition for the residual water.

Water Activity Regulates the Q(A)(-) to Q(B) electron transfer in photosynthetic reaction centers from Rhodobacter sphaeroides.

We report on the effects of water activity and surrounding viscosity on electron transfer reactions taking place within a membrane protein: the reaction center (RC) from the photosynthetic bacterium Rhodobacter sphaeroides. We measured the kinetics of charge recombination between the primary photoxidized donor (P(+)) and the reduced quinone acceptors. Water activity (aW) and viscosity (eta) have been tuned by changing the concentration of cosolutes (trehalose, sucrose, glucose, and glycerol) and the temperature. The temperature dependence of the rate of charge recombination between the reduced primary quinone, Q(A)(-), and P(+) was found to be unaffected by the presence of cosolutes. At variance, the kinetics of charge recombination between the reduced secondary quinone (Q(B)(-)) and P(+) was found to be severely influenced by the presence of cosolutes and by the temperature. Results collected over a wide eta-range (2 orders of magnitude) demonstrate that the rate of P(+)Q(B)(-) recombination is uncorrelated to the solution viscosity. The kinetics of P(+)Q(B)(-) recombination depends on the P(+)Q(A)(-)Q(B) <--> P(+)Q(A)Q(B)(-) equilibrium constant. Accordingly, the dependence of the interquinone electron transfer equilibrium constant on T and aW has been explained by assuming that the transfer of one electron from Q(A)(-) to Q(B) is associated with the release of about three water molecules by the RC. This implies that the interquinone electron transfer involves at least two RC substates differing in the stoichiometry of interacting water molecules.

Counting of peripheral extracellular vesicles in Multiple Sclerosis patients by an improved nanoplasmonic assay and dynamic light scattering.

Extracellular vesicles (EVs) are vesicles naturally secreted by the majority of human cells. Being composed by a closed phospholipid bilayer secluding proteins and RNAs they are used to transfer molecular information to other cells, thereby influencing the recipient cell functions. Despite the increasingly recognized relevance of EVs, the clarification of their physiological role is hampered by the lack of suitable analytical tools for their quantification and characterization. In this study, we have implemented a nanoplasmonic assay, previously proposed for the purity of the EV fractions, to achieve a robust analytical protocol in order to quantify the total phospholipid concentration (CPL) and the EVs number. We show how the coupling of the nanoplasmonic assay with serial dilutions of the unknown sample allows, by simple visual inspection, to detect deviations from the physiological EVs content. The use of a response that depends on the absorbance values at three wavelengths permits to reduce the limit of detection of CPL to 5 µM (total) and the limit of quantification to 35 µM. We also propose a method that takes into account the spread in EV size when the concentration of phospholipids is turned into a concentration of vesicles. The proposed analytical protocol is successfully applied to a small cohort of Multiple Sclerosis patients examined in different stages of their clinical diseases.

Functionality of photosynthetic reaction centers in polyelectrolyte multilayers: toward an herbicide biosensor.

The bacterial reaction center (RC), a membrane photosynthetic protein, has been adsorbed onto a glass surface by alternating deposition with the cationic polymer poly(dimethyldiallylammonium chloride) (PDDA) obtaining as an end result an ordinate polyelectrolyte multilayer (PEM) where the protein retains its integrity and photoactivity over a period of several months. Such a system has been characterized from the functional point of view by checking the protein photoactivity at different hydration conditions, from extensive drought to full hydration. The kinetic analysis of charge recombination indicates that incorporation of RCs into dehydrated PEM hinders the conformational dynamics gating QA- to QB electron-transfer leaving unchanged the protein relaxation that stabilizes the primary charge separated state P+QA-. The herbicide-induced inhibition of the QB activity was studied in some detail. By dipping the PEM in herbicide solutions for short times, kinetics of herbicide binding and release have been determined; binding isotherms have been studied using PEM immersed in herbicide solution. QB functionality of RC has been restored by rinsing the PEM with water, thus allowing the reuse of the same sample. This last point has been exploited to design a simple optical biosensor for herbicides. A suitable kinetic model has been proposed to describe the interplay between forward and back electron-transfer processes upon continuous illumination, and the use of the PDDA-RC multilayers in herbicide bioassays was successfully tested.

Confinement of cardiolipin and ubiquinone in reaction-center core complexes purified from the photosynthetic bacterium Rhodobacter sphaeroides.

The core complex formed by the reaction center and the light harvesting complex 1 (RC-LH1) was purified from the photosynthetic bacterium Rhodobacter sphaeroides. We analyzed the lipid and ubiquinone (UQ) complements copurifying with the RC-LH1 complex and with the peripheral antenna (LH2). In RC-LH1 UQ was almost ten times more concentrated than in the LH2 and in the native membranes from which the complexes were extracted. The fractional lipid composition of the RC-LH1 complex also differed from that of the intact membranes, exhibiting a marked increase in cardiolipin concentration. We propose that the confinement of cardiolipin and ubiquinone observed within the RC-LH1 complex, plays a role in vivo in the stabilization of the light-induced charge separation catalyzed by the RC.