EA034343B1 - Method for luminescent analysis on atomic-force microscope using fluorophore-fluorescent probes - Google Patents

Abstract

The invention relates to the field of technique of probe microscopy. The technical result of the supposed invention is that the used experimental technique is simplified on the one hand due to simplification of optical circuit of photon excitation and registration and the methods of classical luminescent analysis are transferred to the nanometer level on the other hand. Such transfer allows to accurately position a fluorescent probe above the test sample; consequently, it becomes possible to perform a local chemical analysis and also to study the resonance energy transfer during luminescence in interrelation with the peculiarities of the surface structure of the test sample.

Description

The invention relates to the field of instrumentation, mainly to measuring equipment. It can be used, for example, in molecular biology, to study the mechanisms of energy migration in the sample under study, to study the chemical composition of samples with nanometer spatial resolution, to study the viscoelastic and electrophysical properties of the studied surface depending on the chemical composition.

There is such a method of researching objects as luminescent analysis [M.A. Konstantinova Schlesinger. Luminescent analysis. M., State. Ed. Phys.-mat. Lit., 1961, 400 pp.]. The most commonly used fluorescence. With this research method, special substances called fluorescent indicators or fluorophores are added to the solution of the studied substance. In turn, fluorophores are subdivided into fluorescent labels or fluorescent probes depending on how they bind to the target molecule, covalent bond or non-covalent one. After this, fluorophores are excited using external radiation sources and then the properties of the emitted fluorescence radiation are studied. Among the studied features of fluorescence radiation are fluorescence time, anisotropy, spectra, fluorescence kinetics, radiation polarization, and other characteristics. Studying the fluorescence spectra allows us to study the chemical composition of the samples, both qualitative and quantitative, as well as the mechanisms of intermolecular and intramolecular energy migration and other issues.

The disadvantages of luminescent analysis include low spatial resolution;

the inability to relate the results to any characteristic structural features of the structure of the test sample without additional studies.

There is also a method of researching objects, called fluorescence microscopy. In the framework of this method, some elements of interest to the researcher (molecules, cells, etc.) are marked with specific fluorescent labels or fluorescent probes. Next, the object is scanned by a laser beam, causing these labels to fluoresce. After that, an image of the studied object containing fluorescent labels can be constructed that shows the spatial distribution of the elements of interest to the researcher and how this distribution changes over time. Fluorescence time, anisotropy, spectra and fluorescence kinetics can also be investigated, and more sophisticated methods of analysis using fluorescence can be used (Optical Fluorescence Microscopy: From the Spectral to the Nano Dimension Summary, ed. By A. Diaspro, Springer, 2011; Lakovich J. Fundamentals of fluorescence spectroscopy. 1986, 488).

The disadvantages of this method of research using fluorescence include low resolution without the use of superresolution;

the inability to control the position of the fluorescent label when using superresolution.

The closest analogue in technical essence and the achieved result is the method of excitation and registration of optical phonons in the cantilever needle of an atomic force microscope (AFM) [Petrov AB, Bakhtizin RZ, Gots S.S. ]], Which allows you to excite and register optical phonons on the tip of the AFM cantilever needle directly near the test surface, and due to the close mechanical connection of the tip of the cantilever needle with the test surface to study the spectra of optical phonons of the test sample.

The disadvantages of this method of excitation and registration of optical phonons in the AFM cantilever needle include a rather complex experimental technique involving the use of a femtosecond laser;

limited functionality that allows the experimenter to work only with optical phonons.

The technical result of the invention consists in simplifying the experimental technique used, on the one hand, by simplifying the optical scheme of excitation and registration of photons and in transferring classical luminescent analysis methods to the nanometer level, on the other hand. Such a transfer makes it possible to accurately position the position of the fluorescent probe over the test sample; accordingly, it becomes possible to make a local chemical analysis, as well as to study the resonant energy transfer during luminescence in conjunction with the structural features of the surface of the studied sample.

The specified technical result is achieved by the fact that the method of luminescence analysis using an atomic force microscope using fluorophores-fluorescent probes, including laser irradiation of the active layer of the material deposited on the tip of a cantilever needle pressed to the sample, excitation and registration of optical phonons in it, processing the obtained information, characterized in that as a layer of material deposited on the tip of the cantilever needle is a fluorophore-fluorescent probe interacting with the investigated awn to form a non-covalent chemical bonding, wherein electrons are excited by an optical radiation source, hereinafter emitted fluorescent cure, which is detected via fotopri

- 1 034343 of the removable device focused on the tip of the cantilever needle, the obtained information is processed, and based on the characteristics of the fluorescence radiation, a conclusion is made about the properties of the test sample at the point of contact of the cantilever needle with it.

During fluorescence at the point of contact of the cantilever needle with the studied sample, the following physical processes occur. After excitation of the fluorophore-fluorescent probe, which serves as a donor, three possible behaviors of the excited state are possible. In the first version, a resonant energy transfer occurs from the fluorophore acting as a donor to the test sample acting as an acceptor, after which the acceptor emits fluorescence radiation. In the second case, the resonant energy transfer occurs from the fluorophore acting as a donor to the studied sample acting as an acceptor, after which nonradiative dissipation of the acceptor energy occurs, i.e. quenching of fluorescence occurs. And in the third case, resonant energy transfer does not occur, in this case the acceptor itself emits fluorescence radiation.

The use of a set of cantilevers with different fluorescent probes will make it possible to study the chemical structure of the surface even in the absence of a priori information on the chemical composition of the test sample. Currently, there are a large number of fluorescent probes designed for luminescent analysis [I.E. Sukovataya, V.A. Kratasyuk, V.V. Mezhevikin et al. Photobiophysics. Krasnoyarsk: IPK SFU, 2008, p. 438]. Here are some examples. Naphthylamine sulfonic acids can be used to study proteins and lipids. The tyrosine amino acid is capable of photoluminescence in the optical range; accordingly, fluorescent probes attached to the AFM cantilever can be used to quench tyrosine fluorescence. In addition, there are fluorescence probes, the fluorescence of which depends not only on the chemical composition of the environment, but also on pH, on the viscosity of the environment, on the value of the local electric field.

A distinctive feature of the proposed method is the use of fluorescent radiation generated by a fluorescent probe - a donor, mounted on the tip of a cantilever needle, after its excitation by an optical radiation source, or by a test sample - an acceptor, after its excitation due to resonant energy transfer. Due to the fact that the fluorescent probe is placed on the tip of the cantilever needle, it becomes possible to accurately position its placement on the surface under study.

Examples of technical implementation of the proposed method.

A diagram of a device for examining a surface on an AFM using fluorescent probes is shown in the drawing. The device includes an optical radiation source 1, cantilever 2, cantilever needle 3, cantilever needle tip 4, molecular chains 5 with donor 6, which comprise a fluorescent probe and are covalently bonded to the cantilever needle and non-covalently bonded to the surface under study, an acceptor 7, which is part of the studied sample 9, and a registration system for fluorescence radiation 8.

The method of luminescent analysis using an atomic force microscope using fluorophore-fluorescence probes is implemented as follows (see drawing). The optical radiation source 1 generates an optical pulse that excites a fluorescent probe, fixed by means of covalent chemical bonds to the tip of the cantilever needle 4 and pressed to the test sample 9. The excited state of the fluorescent probe is transferred using the resonant energy transfer from the donor center 6, which is part of the fluorescent probe , to the acceptor center 7, which is part of the test sample, and then highlighted in the form of fluorescent radiation, which is recorded by the register system radios 8. Characteristics of fluorescent radiation depend on the properties of the acceptor and its immediate environment on the surface. Thus, by studying the characteristics of fluorescence radiation, one can study the resonant energy transfer in the vicinity of the tip of the cantilever needle. Moving a fluorescent probe from one place to another using a cantilever, we can obtain a surface image for various characteristics of fluorescent radiation. The drawing does not depict situations where quenching of the fluorescent radiation of the probe occurs and when resonant energy transfer does not occur.

Obtaining fluorescence emission spectra (as well as studying its other characteristics, such as fluorescence time, anisotropy, spectra and kinetics, etc.), combined with a 3-D image of a probe microscope, will answer many pressing questions in condensed matter physics, chemistry, biology, medicine.

Claims (2)

CLAIM A method of luminescence analysis using an atomic force microscope, including laser irradiation of a layer of a material deposited on the tip of a cantilever needle pressed against a test sample, excitation and registration of optical phonons in it, processing of the obtained information, characterized in that a molecular layer is used as an active layer of material chains of fluorophores, connected by covalent bonding with a cantilever needle, while the fluorescent probe interacts with - 2 034343 with the followed surface with the formation of a non-covalent chemical bond, in which electrons are excited using an optical radiation source, then the emitted fluorescent radiation is detected using a photodetector focused on the tip of the cantilever needle, and the received information is processed, and based on the characteristics of the fluorescent radiation, a conclusion is drawn about the properties of the test sample at the point of contact of the cantilever needle with it.