RU2797775C1 - Method for using an integrating sphere for photometric registration of de novo formation of biogenic metal nanoparticles - Google Patents

Abstract

FIELD: optical instrumentation.

SUBSTANCE: invention concerns a method for photometric registration of the formation of biogenic metal nanoparticles using an integrating sphere. The method includes arranging the integrating sphere so that its opening is in a horizontal position from above, placing a coverslip or well plate over the opening, applying the test suspension of living cells or tissue fragments to the coverslip or well plate. The sample under study is illuminated by means of a module for providing light flux modes both in the spectrometry mode in the visible wavelength range and in the fluorescence spectrometry mode, and the spectral characteristics of the sample are measured. Next, a source of metal cations is introduced into the test sample and repeated measurements of the spectral characteristics of the sample are carried out at specified time intervals during the cation reduction reaction.

EFFECT: increasing the accuracy of assessing the metabolic activity of the studied biological objects, as well as providing the possibility of measuring biogenic nanoparticles in a drop of the reaction mixture or in a well plate.

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Description

The invention relates to optical instrumentation and is intended for a quick comparative assessment of the metabolic activity of cells based on the registration of parameters for the formation of metal nanoparticles by them. The objective of the invention is to increase the sensitivity of photometric registration and evaluation of the parameters of biogenic metal nanoparticles formed in situ by living cells upon contact with a salt solution introduced directly into their suspension as a source of cations. To increase the sensitivity when recording the parameters of the emerging biogenic metal nanoparticles, the source of the “white” light flux of the integrating sphere is supplemented with a system of fiber-optic supply of light fluxes and a module for regulating the light flux, which has several fixed wavelengths corresponding to the exciting effect for the fluorescence of metal nanoparticles of a certain composition. The purpose and technical result of using the integrating sphere is to increase the accuracy of assessing the metabolic activity of the studied biological objects based on the registration of the parameters of the dynamics of the formation of metal nanoparticles directly in the samples containing the studied cells. The technical result is also the possibility of measuring biogenic nanoparticles in a drop of the reaction mixture or in a well plate.

In modern experimental science and many areas of biotechnology, the range of applications of metal nanoparticles is constantly expanding. The natural ability of metabolically active cells to reduce cations and form in situ , first, nanoclusters of zero-valent atoms, and then larger nanoparticles of the corresponding metals, is known. The method based on this property of metabolically active cells makes it possible to control the presence of viable microorganisms and even spores or viral particles in various solutions and liquid media, that is, to control their contamination. It has been shown that the rate of de novo formation and coarsening of biogenic metal nanoparticles increases with an increase in the level of physiological activity of microorganism cultures, and also reflects the state of microbial communities and cells of higher organisms. This analytical approach is based on the exact knowledge that the linear dimensions of biogenic metal nanoparticles formed de novo in the presence of cells from reduced cations by self-assembly of null-valent atoms are proportional to the metabolic reducing activity of living cells [Zhou et al., 2013, Sorokin et al. , 2013, Skladnev et al., 2020]. In sterile media, the reduction of introduced cations does not lead to the formation of nanoparticles.

The present invention improves the efficiency of photometric registration and evaluation of the parameters of biogenic metal nanoparticles formed de novo , which makes it possible to assess the level of metabolic activity of cells in practically significant areas of biotechnology, ecology and medicine.

Currently, as a generally accepted contamination control system for sterile solutions of pharmaceutical, medical and biological preparations, substances and other sterile materials, in accordance with the rules of national standards, the method of direct seeding on indicator media is used (GF RF XII OFS 42-0066-07, USP37 - NF 32, R 2014, EP, 8th ed., JP 15th ed.). The sterility of the test liquid, according to these documents, is judged by the absence of growth of microorganisms during control crops on special indicator media after incubation under optimal conditions. The most significant disadvantage of this method of direct seeding on solid media is the duration of its implementation - at least 14-18 days, as well as its laboriousness and the need to use many expensive reagents. In recent years, to detect microbial and viral contamination of solutions and biological fluids, molecular diagnostic methods have been used, based on specific reactions of individual markers of biological objects - nucleic acids, proteins, lipopolysaccharides of bacterial membranes: RT2-PCR Array Handbook 06/2013, Method for determining bacterial contamination of blood - RF patent 2208645, Method for detecting pathogenic strains and isolates of the bacteria Pasteurella multocida - RF patent 2477321. Such methods significantly reduce the time of analysis, but require the use of expensive reagents, even more expensive analytical equipment and highly qualified personnel.

Previously, we have declared a method for detecting contamination of liquids by microorganisms and viral particles, based on registration of the process of formation of biogenic metal nanoparticles, which are formed directly in the test samples from cation reduction products artificially introduced into the test samples in the form of sterile solutions of metal salts (RF Patent 2641960, 2016) . During the cation reduction reaction in the presence of metabolically active cells, the proportion of unreduced cations on the surface of biogenic nanoclusters constantly and rapidly decreases. A decrease in surface charge minimizes the repulsive forces of de novo formed nanoclusters, which promotes their self-assembly and the formation of ever larger nanoparticles. Suspensions of living cells are capable of forming in situ silver nanoparticles with a diameter of 20–150 nm in 20 minutes. Under similar conditions, the reaction of reduction of cations of a suspension of resting microbial cells, a suspension of spores or viral particles for the same time form biogenic silver nanoparticles with a diameter of only up to 2 - 10 nm. In sterile samples of growth media and other liquids, only precursors of nanoparticles (nanoclusters of reduced atoms) with a size of about 1–1.5 nm are formed.

Since metal nanoparticles have a crystalline structure, their parameters, properties, and formation dynamics can be recorded by a larger number of analytical methods than are suitable for registering the presence of microorganism cells or other biological objects. For the detection of metal nanoparticles, their specific optical properties are widely used, due to the phenomenon of surface plasmon resonance, highly developed surface, high capacitance of the double electric layer, as well as the ability of nanoparticles to amplify the signal in Raman, light, and fluorescence spectrometry. One of the unique features of nanoclusters of many metals ranging in size from 2-5 nm is their pronounced fluorescent properties. With the size of nanoclusters of reduced metal atoms comparable to the Fermi wavelength for electrons, due to discrete electronic states caused by strong quantum confinement of free electrons, nanoparticles exhibit supramolecular properties. As the size of metal nanocrystalline structures increases (during the self-assembly of nanoclusters into nanoparticles), the ratio of surface and deep atoms changes, which leads to a shift in fluorescence emission and even its complete quenching [Szerencsés et al., 2020]. We propose to use these natural properties of the fluorescence of biogenic nanoclusters and nanoparticles of various sizes for convenient and accurate photometric registration and assessment of the dynamics of the process of their de novo formation directly in living cell suspensions.

When measuring the parameters of preparations of metal nanoparticles, light and fluorescence spectrometry is most widely used. However, when studying biogenic metal nanoparticles obtained in situ with the participation of various biological objects, significant distortions of the results arise due to the presence of numerous cells or spores in the reaction mixture. The greatest distortions are observed in cases where nanoparticles are formed directly on the cell surface (the so-called encrustation of a biological object with nanoparticles). These reasons make the method of transmission spectrometry insufficiently effective and do not allow one to accurately solve the set problems.

Previously, we proposed the use of an integrating sphere as a measuring element of a spectrophotometer in the vis range for studying preparations of biogenic silver nanoparticles formed in situ using the reflected spectrum parameters [Sorokin et al., 2013]. In this invention, we propose to use a special integrating sphere as a structural element of a spectrophotometer for recording biogenic nanoparticles in the vis range, as well as in the fluorescence mode directly in suspensions against the background of the presence of cells, spores, or tissue fragments in the reaction mixture.

Closest to the set of essential features of the proposed invention is a special integrating sphere, which is a hollow ball, the inner surface of which is covered with diffusely scattering materials, which makes it possible to obtain the most accurate spectral characteristics of the samples in the region of λ _{200 - 2600} nm in terms of reflectance and transmittance [RF Patent 2251667 , 2005]. The prototype relates to optical instrumentation and is designed to evaluate the properties of light-diffusing materials. The disadvantage of the prototype is the large pitch of the recesses on the inner surface of the sphere (7 mm), incommensurable with the wavelength of 1-5 μm in the near infrared range, which determines the low degree of diffuseness of the reflection indicatrix of the surface of the Morris sphere. In addition, deviations of the reflection indicatrix from ideally diffuse in the short-wavelength IR region reach 20-60% at large viewing angles.

The essential advantages of the proposed invention in comparison with the prototype are as follows:

- the proposed design of the integrating sphere makes it possible to measure biogenic metal nanoparticles formed in situ against the background of cells or spores present in the reaction mixture both in the vis -spectrometry mode and in the fluorescent mode. In the prototype, measurements are possible only in the IR range;

- the proposed design of the integrating sphere makes it more convenient to measure the cell suspension under study, located directly on the cover glass of the opening of the integrating sphere, or in a well plate located opposite this opening. In the prototype, the test sample must be placed inside the sphere;

- the proposed design of the integrating sphere allows the introduction of additional reagents into the test sample to carry out the formation of biogenic nanoparticles in situ directly in the measurement process. In the prototype, this possibility is not provided.

The objective of the present invention is to increase the sensitivity of the photometric determination of the parameters of biogenic metal nanoparticles formed in situ directly in a suspension of living cells. The proposed design of the integrating sphere was developed to increase the sensitivity of the photometric determination of the level of metabolic activity of living cells based on the recording of the parameters of biogenic metal nanoparticles formed de novo from sterile salt solutions introduced directly into the cell suspensions as sources of cations. Since, in the presence of metabolically active cells, the formation of nanoclusters of zero-valent atoms from cations, and then larger nanoparticles, occurs at a high rate, the proposed design of the integrating sphere provides for the possibility of evaluating the dynamics of their formation from the moment the source of cations is introduced.

For many metals, illumination parameters have been established that excite pronounced fluorescent properties of nanoclusters with a size of 2–5 nm. The use of a mixture of solutions of several salts can enhance the fluorescent properties of nanoclusters due to the presence of reduced atoms of different metals in them. Periodic photometric measurements after adding a source of cations to the cell suspension under study make it possible to determine the moment when the diameter of biogenic nanoclusters of reduced atoms formed de novo reaches a size corresponding to the appearance of fluorescence. In the case of high reducing activity of the studied cell suspension, the process of enlargement of nanoclusters by their self-assembly leads to the formation of ever larger biogenic nanoparticles (from 20–50 nm to 200 nm in 20 minutes of the cation reduction reaction). It is shown in detail that for silver nanoclusters, the fluorescence excitation frequency is a flux with a wavelength of λ ₃₃₇ nm, for excitation of fluorescence of gold nanoclusters, a flux with a wavelength of λ ₅₁₆ nm. The enhancement of the fluorescence of nanoclusters can be achieved due to the presence of lanthanide salts in the reaction mixture. Large nanoparticles of some metals are characterized by quenching of fluorescence upon reaching certain sizes. In the case of de novo formation of biogenic nanoparticles, fluorescence quenching of the drug means that all formed nanoparticles exceed this size.

In the proposed design of the integrating sphere, it is possible to continue recording changes in the size of nanoparticles in the cell suspension under study using spectrometry both in the range of vis -spectrometry and in the fluorescence spectrometry mode. To do this, the integrating sphere of the spectrometer is equipped with an additional module for switching the light flux modes (Fig. 1. 5) from “white” with a wide frequency spectrum to separately adjustable specific wavelengths necessary to excite the fluorescence of nanoclusters of reduced atoms of those metals whose cations are used as source during the formation of biogenic nanoparticles. The module of the modes of a light stream allows to regulate the general level of illumination of the studied sample. The use of additional light filters provides the highest sensitivity for measuring the spectral characteristics of biogenic metal nanoparticles formed directly in a suspension of living cells.

The proposed design provides for convenient placement of the test sample opposite a special hole in the integrating sphere. In the proposed design, the integrating sphere is located in such a way that the hole for the test sample (Fig.1, 1) is on top in a horizontal position. This orientation of the hole for placing the test sample allows you to apply the cell suspension under study on a transparent cover glass that covers this hole (Fig. 1. A). The second option involves carrying out the reaction of cation reduction by living cells in the wells of tablets of one or another standard type (Fig. 1. B). In both cases, the proposed design allows the introduction of sterile solutions of cation sources (to start the reduction reaction) directly into the cell suspension under study, followed by periodic repetition of measurements.

On FIG. Figure 1 shows an integrating sphere device for recording the spectra of biogenic nanoclusters and nanoparticles in the vis range and in fluorescence spectrometry modes. Shown is a scheme for measuring directly in a drop of cell suspension (A), or in a well of a plate (B). A hole in the integrating sphere for placing the test sample 1, a sensor for measuring the characteristics of the reflected light flux 2, a light flux source 3, a sample of the test suspension 4, a module for switching light flux modes 5, a light filter 6.

Example 1

The cell suspension under study is placed on a transparent coverslip covering the opening of the integrating sphere (Fig. 1A). Spectral characteristics are registered in the range of vis spectrometry for the control determination of the parameters of the studied cell suspension at the initial moment of the experiment (to confirm the absence of metal nanoparticles in it). A source of cations in the form of an aliquot of a sterile saline solution as a source of cations is introduced into the cell suspension under study. The spectral characteristics of the cell suspension are re-registered in the range of vis -spectrometry for control confirmation of the absence of metal nanoparticles in the added aliquot of the cation source. Repeat measurements of the spectral characteristics of the sample are continued at the required time intervals in the range near the specific wavelength of the nanoparticles of the metal used to record the parameters of the biogenic nanoparticles formed in situ in the cell suspension under study (Fig. 2).

On FIG. 2. shows the change in the specific spectrum of silver nanoparticles formed in 40 minutes in a sample of bacterial cell suspension.

Example 2

In separate wells of a multiwell plate, cell suspensions are placed, for which it is necessary to determine the level of reducing activity in relation to cations. The multiwell plate is placed opposite the opening of the integrating sphere for the test sample. The spectral characteristics of the samples are recorded (in the range of vis -spectrometry and in the fluorescence spectrometry mode) for the control determination of parameters, confirming the absence of metal nanoparticles at the initial moment of the experiment. Sources of cations are introduced into each of the test samples in the form of a sterile salt solution. The ability to move the plates provides successive cyclic re-measurements of the spectral characteristics of each of the investigated variants of the samples at fixed time intervals. The duration of the reduction reaction of the used cations is determined, which is necessary for the formation of fluorescent biogenic nanoclusters of reduced atoms, as well as the time corresponding to the complete quenching of the fluorescence of the nanoparticles.

Example 3

The studied suspensions of natural aqueous samples are placed in separate wells of a multiwell plate. Place the plate opposite the opening of the integrating sphere for the test sample. The spectral characteristics of the studied cell suspensions of microbial communities are recorded (in the range of vis -spectrometry and in the fluorescence spectrometry mode) for the control determination of parameters, confirming the absence of metal nanoparticles at the initial moment of the experiment. Sources of cations are introduced into each of the test samples in the form of a sterile salt solution. The spectral characteristics of the studied cell suspensions are re-registered for control confirmation of the absence of nanoparticles in the introduced aliquot of the cation source. Repeat measurements of the spectral characteristics of the sample are continued every 3 minutes (in the range of vis spectrometry and in the fluorescence spectrometry mode). The duration of the reduction reaction of the used cations is determined, which is necessary for the formation of fluorescent biogenic nanoclusters of reduced atoms, which corresponds to their size from 5 nm. Based on the obtained results of spectral characteristics at specific wavelengths λ of metal nanoparticles, the level of activity of the compared cell preparations in relation to the used cations is estimated.

Example 4

100-150 μl of sterile salt solutions are added to the wells of a multiwell plate as sources of cations - substrates for the formation of nanoparticles. The multiwell plate is placed opposite the opening of the integrating sphere for the test sample with the possibility of moving it for successive registration of spectral characteristics in each of the wells. The spectral characteristics of each of the samples are recorded (in the range of vis spectrometry and in the modes of fluorescence spectrometry corresponding to the used cation sources) for the control determination of parameters, confirming the absence of metal nanoparticles at the initial moment of the experiment. Preparations of tumor or normal (control) tissues are placed in separate wells, for which it is necessary to determine the level of reducing activity in relation to the cations used in the experiment (Fig. 3). Repeated measurements of the spectral characteristics of each of the studied variants of the samples are continued. Based on the obtained results of the spectral characteristics of the formed nanoparticles, the level of reducing activity of the compared cell preparations in relation to the used cations is estimated.

On FIG. 3. Measurement using an integrating sphere of absorption of silver nanoparticles (at λ ₄₀₀ nm) formed in reaction mixtures containing tumor or normal cells is presented.

Information sources

1. Ilyasov S.G. Integrating sphere for the infrared region of the spectrum. Patent RF 2251667, 2005.

2. Skladnev D.A., Sorokin V.V., Kurakov V.V. Method for detecting microbial and viral contamination of solutions. RF patent No. 2641960, 2016.

3. Morris J.C., Integrating Sphere to the Infrared. Applied Optics, 1966, 5(6), 1035-1037.

4. Zhou, Y., Wang, H., Lin, W., Lin, L., Gao, Y., Yang, F., Du, M., Fang, W., Huang, J., Sun, D ., Li, Q. (2013). Quantitative nucleation and growth kinetics of gold nanoparticles via model-assisted dynamic spectroscopic approach. J. Colloid Interface Sci., 407, 8-16.

5. Sorokin, VV, Skladnev, DA, Volkov, VV, Tereshchenko, EY, Mulyukin, AL, Gal'chenko, VF The pathways of silver nanoparticles formation by Mycobacterium smegmatis . Dokl. Biol. Sci., 2013, 452, 325-328.

6. Skladnev D.A., Vasilyeva L.V., Berestovskaya Yu.Yu., Kotsyurbenko O.R., Kalenov S.V., Sorokin V.V. Detection of microorganisms in low-temperature water environments by in situ generation of biogenic nanoparticles. front. Astron. Space Science, 2020, 7:59.

7. Szerencsés B., Igaz N., Tóbiás Á., Prucsi Z., Rónavári A., Belteky P., Dániel, Papp C., Makra I., Vágvölgyi C., Kónya Z., Pfeiffer I., Kiricsi M Size-dependent activity of silver nanoparticles on the morphological switch and biofilm formation of opportunistic pathogenic yeasts. BMC Microbiol., 2020, 20, 176. Published online 2020 Jun 22. doi: 10.1186/s12866-020-01858-9.

Claims (5)

1. A method for photometric registration of the formation of biogenic metal nanoparticles using an integrating sphere, which includes arranging the integrating sphere in such a way that the opening of the integrating sphere is on top of the integrating sphere in a horizontal position, placing a cover glass covering this opening, or placing a well plate opposite the opening, applying the test suspension of living cells or tissue fragments on a cover glass or well plate, illumination of the test sample by means of a module for providing light flux modes both in the spectrometry mode in the visible wavelength range and in the fluorescence spectrometry mode, measurement of the spectral characteristics of the sample, introducing sources into the test sample metal cations and carrying out repeated measurements of the spectral characteristics of the sample at specified time intervals during the cation reduction reaction. 2. The method according to claim 1, characterized in that the studied suspensions of living cells or tissue fragments are placed in the wells of a multiwell plate, which is placed opposite the opening of the integrating sphere for the sample with the possibility of movement for measurements. 3. The method according to claim 1, characterized in that the cation reduction reaction is carried out directly in the test samples of the cell suspension. 4. The method according to claim 1, characterized in that the dynamics of the formation of biogenic nanoclusters and metal nanoparticles is recorded by sequential measurements of the spectral characteristics of the reaction mixture in the spectrometry mode in the visible wavelength range and in the fluorescence spectrometry mode. 5. The method according to claim 1, characterized in that as a source of cations, a sterile solution of salts is introduced into the cell suspensions under study, providing maximum fluorescence of the forming metal nanoclusters and nanoparticles.

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