Daria O. Solovyeva

Thin films and assemblies of photosensitive membrane proteins and colloidal nanocrystals for engineering of hybrid materials with advanced properties

The development and study of nano-bio hybrid materials engineered from membrane proteins (the key functional elements of various biomembranes) and nanoheterostructures (inorganic colloidal nanoparticles, transparent electrodes, and films) is a rapidly growing field at the interface of materials and life sciences. The mainspring of the development of bioinspired materials and devices is the fact that biological evolution has solved many problems similar to those that humans are attempting to solve in the field of light-harvesting and energy-transferring inorganic compounds. Along this way, bioelectronics and biophotonics have shown considerable promise. A number of proteins have been explored in terms of bioelectronic device applications, but bacteriorhodopsin (bR, a photosensitive membrane protein from purple membranes of the bacterium Halobacterium salinarum) and bacterial photosynthetic reaction centres have received the most attention. The energy harvesting in plants has a maximum efficiency of 5%, whereas bR, in the absence of a specific light-harvesting system, allows bacteria to utilize only 0.1-0.5% of the solar light. Recent nano-bioengineering approaches employing colloidal semiconductor and metal nanoparticles conjugated with biosystems permit the enhancement of the light-harvesting capacity of photosensitive proteins, thus providing a strong impetus to protein-based device optimisation. Fabrication of ultrathin and highly oriented films from biological membranes and photosensitive proteins is the key task for prospective bioelectronic and biophotonic applications. In this review, the main advances in techniques of preparation of such films are analyzed. Comparison of the techniques for obtaining thin films leads to the conclusion that the homogeneity and orientation of biomembrane fragments or proteins in these films depend on the method of their fabrication and increase in the following order: electrophoretic sedimentation < Langmuir-Blodgett and Langmuir-Schaefer methods < self-assembly and layer-by-layer methods. The key advances in the techniques of preparation of the assemblies or complexes of colloidal nanocrystals with bR, purple membranes, or photosynthetic reaction centres are also reviewed. Approaches to the fabrication of the prototype photosensitive nano-bio hybrid materials with advanced photovoltaic, energy transfer, and optical switching properties and future prospects in this field are analyzed in the concluding part of the review.

Two-photon-induced Förster resonance energy transfer in a hybrid material engineered from quantum dots and bacteriorhodopsin

Energy transfer from nanostructures to biological supramolecular photosystems is an important fundamental issue related to the possible influence of nanoobjects on biological functions. We demonstrate here two-photon-induced Förster resonance energy transfer (FRET) from fluorescent CdSe/ZnS quantum dots (QDs) to the photosensitive protein bacteriorhodopsin (bR) in a QD-bR hybrid material. The two-photon absorption cross section of QDs has been found to be about two orders of magnitude larger than that of bR. Therefore, highly selective two-photon excitation of QDs in QD-bR complexes is possible. Moreover, the efficiency of FRET from QDs to bR is sufficient to initiate bR photoconversion through two-photon excitation of QDs in the infrared spectral region. The data demonstrate that the effective spectral range in which the bR biological function is excited can be extended beyond the band where the protein itself utilizes light energy, which could open new ways to use this promising biotechnological material.

Supramolecular nanostructures based on bacterial reaction center proteins and quantum dots.

Design of the nanostructures based on membrane proteins (the key functional elements of biomembranes) and colloid nanoparticles is a fascinating field at the interface of biochemistry and colloids, nanotechnology and biomedicine. The review discusses the main achievements in the field of ultrathin films prepared from bacterial reaction center proteins and light-harvesting complexes, as well as these complexes tagged with quantum dots. The principles of preparation of these thin films and their structure and properties at different interfaces are described; as well as their characteristics estimated using a combination of the modern interfacial techniques (absorption and fluorescence spectroscopy, atomic force and Brewster angle microscopy, etc.) are discussed. Further approaches to develop the nanostructures based on the membrane proteins and quantum dots are suggested. These supramolecular nanostructures are promising prototypes of the materials for photovoltaic, optoelectronic and biosensing applications.

Surface ligands affect photoinduced modulation of the quantum dots optical performance

Changes of optical properties of the solutions of CdSe/ZnS quantum dots (QDs) covered with the trioctylphosphine oxide (TOPO) ligands under the pulsed ultraviolet (UV) laser irradiation are observed. The fluorescence quantum yield (QY) of QDs decreases by more than an order of magnitude when the radiation dose approaches 2 × 10-15 J per particle. This process is accompanied by a blue shift of both fluorescence and the first excitonic absorption peaks. The fluorescence quenching becomes less pronounced when the overall TOPO content in the solution is increased. When ТОРО ligands are replaced with n-hexadecylamine (HDA), QY and spectral properties are not changed at the same irradiation conditions. We assume that the above changes of the optical properties are associated with photooxidation of TOPO ligands by excited QD. Such process is less probable for the HDA ligand due to its different energy structure.

Resonance energy transfer in nano-bio hybrid structures can be modulated by UV laser irradiation

A method for targeted variation of the radiation properties of quantum dots (QDs) to control the efficiency of resonance energy transfer in nanocrystal assemblies and nano-bio hybrid materials has been developed. The method is based on strong ultraviolet (UV) laser irradiation of QDs and allows the extinction and luminescence spectra to be controlled and the luminescence quantum yield and decay kinetics to be varied. Water-soluble QDs have been synthesized and used for analyzing the effect of energy transfer from semiconductor nanocrystals on the photocycle of the photosensitive protein bacteriorhodopsin (bR) in bR–QD complexes. The UV irradiation mode has been selected in a way permitting the modulation of QD optical parameters without modification of their structure or physico-chemical properties. It is concluded that the QD interaction with bR accelerates its photocycle, but this acceleration is determined by electrostatic interactions, rather than Forster resonance energy transfer from QDs to bR. The method of UV laser irradiation of fluorescent semiconductor QDs has proven to be an efficient technique for variation of nanocrystal optical properties without affecting their structure, as well as for fine modulation of the energy transfer processes in the nanocrystal assemblies and nano-bio hybrid materials.

Novel Precursors of Fluorescent Dyes. 1. Interaction of the Dyes with Model Phospholipid in Monolayers

Photoactivated (“caged”) fluorescent dyes are modern tools for structure and function studies of cell membranes and subcellular organelles. Recently synthesized precursors of rhodamine fluorescent dyes (abbreviations PFD813 and PFD814) important for microscopic probing of biological objects have been studied in solution. In order to characterize the behavior at interfaces, monolayers of PFD813 and PFD814 on water have been formed and investigated. The interactions of these precursors with the biomembrane component dimyristoylphosphatidylethanolamine in monolayers at the air–water interface and after transfer to glass plates have been studied by measuring monolayer parameters and spectroscopic properties before and after photo-chemical formation of the fluorescent rhodamine dyes Rho813 and Rho814, respectively.

The effect of silver nanoparticles on the photocycle of bacteriorhodopsin of purple membranes of Halobacterium salinarum

The effect of silver nanoparticles (AgNPs) that are adsorbed on the surface of the purple membranes of Halobacterium salinarium bacteria on the optical properties and functional peculiarities of the lightsensitive protein bacteriorhodopsin (BR) has been demonstrated for the first time. Two mechanisms of the effect of AgNPs on the protein photocycle have been demonstrated using Raman scattering, giant Raman scattering, flash photolysis, and atomic force microscopy. It has been shown that the nanoparticles in the immediate spatial vicinity of BR fix its photocycle at the stage where it was at the moment of interaction with the nanoparticles. At greater distances, which reach three radii of an AgNPs, they have a weaker effect on BR, under which it retains the ability to be involved in the photocycle, however, has its parameters significantly changed. Thus, in the case of wild-type BR the photocycle accelerates and for the BR-D96N mutant it becomes slower. The data that are obtained could be of significance for creation of such optoelectronic hybrid systems with BR, where the parameters of its photocycle can be controlled using NPs. The results of the study may also be used in the field of nanobioengineering research, which is directed to creation of unique materials with controlled properties for recording and storage of information, energy transformation, and identification and characterization of trace amounts of analytes.

Photoinduced modification of quantum dot optical properties affects bacteriorhodopsin photocycle in a (quantum dot)- bacteriorhodopsin hybrid material

A method for controlled changes in the radiative properties of quantum dots (QDs) in order to modulate the Forster resonance energy transfer (FRET) rate in nano-hybrid materials is proposed. The mechanism underlying the effect of QDs with optical properties modulated by UV laser irradiation on the photocycle of the photosensitive protein bacteriorhodopsin (bR) in its native purple membranes (PM) isolated from Halobacterium salinarum has been studied. The irradiation leads to a twofold decrease in the QD fluorescence quantum yield without changes in the extinction spectrum or the position or shape of the fluorescence spectrum. The bR photocycle is accelerated, which has been shown to be related to the changes of the surface potential of PM upon formation of their complexes with QDs.

An instrumental approach to combining confocal microspectroscopy and 3D scanning probe nanotomography

In the past decade correlative microscopy, which combines the potentials of different types of high-resolution microscopies with a variety of optical microspectroscopy techniques, has been attracting increasing attention in material science and biological research. One of outstanding solutions in this area is the combination of scanning probe microscopy (SPM), which provides data on not only the topography, but also the spatial distribution of a wide range of physical properties (elasticity, conductivity, etc.), with ultramicrotomy, allowing 3D multiparametric examination of materials. The combination of SPM and ultramicrotomy (scanning probe nanotomography) is very appropriate for characterization of soft multicompound nanostructurized materials, such as polymer matrices and microstructures doped with different types of nanoparticles (magnetic nanoparticles, quantum dots, nanotubes, etc.), and biological materials. A serious problem of this technique is a lack of chemical and optical characterization tools, which may be solved by using optical microspectroscopy. Here, we report the development of an instrumental approach to combining confocal microspectroscopy and 3D scanning probe nanotomography in a single apparatus. This approach retains all the advantages of SPM and upright optical microspectroscopy and allows 3D multiparametric characterization using both techniques. As the first test of the system developed, we have performed correlative characterization of the morphology and the magnetic and fluorescent properties of fluorescent magnetic microspheres doped with a fluorescent dye and magnetic nanoparticles. The results of this study can be used to obtain 3D volume images of a specimen for most high-resolution near-field scanning probe microscopies: SNOM, TERS, AFM-IR, etc. This approach will result in development of unique techniques combining the advantages of SPM (nanoscale morphology and a wide range of physical parameters) and high-resolution optical microspectroscopy (nanoscale chemical mapping and optical properties) and allowing simultaneous 3D measurements.

The effect of plasmon silver and exiton semiconductor nanoparticles on the bacteriorhodopsin photocycle in Halobacterium salinarum membranes

The interaction of semiconductor quantum dots and silver nanoparticles (AgNPs) with bacteriorhodopsin (BR), a membrane protein contained in the purple membrane (PM) of Halobacterium salinarum, is studied. It is shown that both types of nanoparticles are adsorbed efficiently on the surface of the purple membranes, modulating the parameters of the bacteriorhodopsin photocycle. Electrostatic interactions are found to be the main cause of the effect of nanoparticles on the bacteriorhodopsin photocycle. These results explain our earlier data on the “fixation” of the bacteriorhodopsin photocycle for protein molecules trapped after incubation of the purple membranes with silver nanoparticles near the location of the “hot spots” of the effect of surface-enhanced Raman scattering (SERS). It is demonstrated that exposure of silver nanoparticles with bacteriorhodopsin in SERS-active regions lowers the amount of bacteriorhodopsin molecules involved in phototransformations.