Holder for mounting thin layers of materials in cuvette for fluorimetric measurements

Abstract

The subject-matter of the present invention is a holder (1) for mounting thin layers of test materials in a spectrofluorimetric cuvette (2), characterized in that it has the shape of a prism with the base of a rectangular trapezoid; two adjacent sidewalls, the first sidewall (3) and the second sidewall (4) being at 90º to each other ultimately adjoin the two adjacent walls of the spectrofluorimetric cuvette, the second sidewall (4) being equipped with at least two protrusions (5, 6) adjoining the second wall of the spectrofluorimetric cuvette and the third wall of the spectrofluorimetric cuvette, between the protrusions (5, 6), in the third sidewall (7) of the holder prism (1), an attachment (8) is placed to immobilize the thin test layer, in the form of a frame; the third sidewall (7) of the holder prism (1) is inclined with respect to the first sidewall (3) of the holder prism (1) at an angle of more than 0º to 45º.

Description

[0001] HOLDER FOR MOUNTING THIN LAYERS OF MATERIALS IN CUVETTE FOR FLUORIMETRIC MEASUREMENTS

[0002] The present invention relates to a holder for mounting thin layers of materials, i.e. films, membranes, nanostructured layers, in a cuvette for fluorimetric measurements. With the use of the present invention, samples of test materials are placed in a repeatable and reproducible manner in the cuvette, thus significantly reducing the measurement error. It is possible to repeatedly insert and remove the sample from the cuvette, to compare the spectra obtained for different materials. With the above- mentioned solution, the measurement of fluorescence emission in the cuvette can be carried out both in solution and without solution. The present invention eliminates the necessity to purchase a holder for measurement / testing of solid samples, optionally offered by fluorimeter manufacturers. A significant disadvantage of this type of holder is the need to work in the absence of solution and the unique mounting of the sample(s) in the holder, which can cause a significant change in the spectrum unrelated, for example, to the processes studied in which the material emitting the spectrum is involved, or to the different properties of the test materials. Holders offered on the market are designed and constructed to accommodate relatively rigid solid samples in the device.

[0003] For spectrofluorimetric measurements, the light should fall on the test sample (e.g., a thin film), at the same angle each time, and the sample should be immobile in the solution during the measurement. In addition, the test sample must be in the beam of excitation radiation, if the measurement is repeated (e.g., to monitor the process flow) the excitation light must fall in the same place so that the results obtained are repeatable and comparable. The present invention makes it possible to record the spectrum of a thin layer of solid material (transfer the holder with the material sample to another medium, e.g., for the reaction to take place) and place the test material in the cuvette again in the same way as the first time, i.e., repeatability. This makes it possible to compare the emission spectrum of the test material before and after the reaction. Thin, especially flaccid layers (e.g., polymer films / membranes, nanofiber mats) tend to float / change their position in solution, and as a result, the intensity of the recorded spectra changes significantly while maintaining other measurement parameters. In extreme cases, the sample of the test material can become outside the excitation light beam, making measurement impossible. In general, spontaneous movement of thin layers of materials in solution, leading to random changes in the intensity of recorded spectra, makes it impossible to directly investigate the influence of other processes (e.g., reaction with the dye present in the material) on changes in the fluorescence emission intensity of films, e.g., polymer films, providing measurements in solution. Such measurements can be important, for example, to study the interaction of a thin layer of material (membrane, film, nanofiber mat) with a solution, to study the composition of a solution, to study the properties of a film / mat, or to study the interaction of a film / mat with a solution, such as drug release study.

[0004] So far, no methods have been presented to solve the problems associated with fluorimetric measurements using solid, thin, flaccid layers of polymeric materials, such as layers or nanofiber mats.

[0005] The developed holder makes it possible to mount the sample at a selected angle with respect to the incident excitation beam, and as a result optimize the scattering and reflection of the incident light and maximize the intensity of the emission spectrum. The invention includes holder models with different angles of incidence of the light beam on the sample, of 0° to 45°, with an optical path of, for example, 5.5 mm. For most materials (structures), the preferred value of the angle of incidence of the excitation beam will be 35° for non-reflective samples. In the examples, the effect of changing the value of the incident beam angle for selected angles of 15°, 20°, 25°, 30°, 35°, and 45° is presented.

[0006] The proposed holder structure can be obtained from any material, as long as at least its surface is protected against reflection and scattering of the incident excitation beam. Examples illustrating the subject of the invention were made and printed from a thermoplastic material (polylactide) with a 3D printer.

[0007] The use of the proposed cuvette holder, for immobilizing thin films, also allows fluorimetric measurements carried out at a controlled temperature using a standard fluorimeter accessory: a thermostated cuvette holder.

[0008] In the state of the art, the existing solutions for fluorescence measurements of solid samples offer the possibility of measurement with the use of a holder, which is an optional (extra paid) equipment of various spectrofluorimeters offered on the market. As a rule, such a device requires working in the absence of solution (dry samples) and each time mounting the sample in the holder and in the incident light beam, which, as a rule, is a source of significant fluctuations in the fluorescence emission intensity values recorded, even for the same sample, under the same measurement conditions. In addition, it is very difficult, if not impossible, to mount a thin — so flaccid, mechanically non-resistant — sample in a holder. The proposed holders for solid samples do not have the ability to adjust the measurement temperature [https: / / www.hitachi- hightech.com / file / global / products / science / appli / ana / fl / solid / fl_120005.pdf; https: / / store.eu.horiba.com / products / solid-sample-holder-5500001934, https: / / www.agilent.com / en / product / molecular-spectroscopy / fluorescence- spectroscopy / fluorescence-accessories / cary-eclipse-solid-sample-holder, as of 13.03.2023],

[0009] Another solution used in practice, although cumbersome experimentally and not providing sufficiently high repeatability of recorded spectra, requires mounting the sample (a thin layer of solid or semi-solid material) in an adaptor, which is then placed in a commercial solid sample holder described above [A. Baranowska-Korczyc, K. Maksymiuk, A. Michalska Electrospun nanofiber supported optodes: scaling down the receptor layer thickness to nanometers - towards 2D optodes, Analyst, 2019, 144, 4667], Such an adaptor can be a fragment of a suitably cut plastic (Eppendorf) vial. Such a solution allows only the registration of spectra in air (in the absence of solution) and does not ensure repeatability between successive measurements — the sample must be placed in the frame each time and mounted in the holder. Even small changes in the position and angle of incidence of the excitation radiation lead to significant changes in the intensity of the recorded spectra. In addition, the described method is very labor-intensive. A thin solid sample mounted in the (home-made) adaptor described above is not supported on a substrate which further increases the risk of changing the positioning of the outer surface of the sample, and thus the angle of incidence of the excitation radiation and, as a result, the intensity of the recorded emission spectra.

[0010] Other possible solutions enabling recording the emission spectrum of a thin layer include the use of a special attachment connected via optical fibers to the spectrofluorimeter — also an optional accessory offered by some manufacturers [https: / / www.perkinelmer.com / pl / product / ls-series-front-surface-accessory-52123130, as of March 28, 2023], Again, this solution requires working in the absence of solvents such as water, and the intensities of the recorded spectra depend on how the tip is placed on the sample, so they are highly non-reproducible.

[0011] Another possible solution is, for example, to “paste” the test sample — a thin layer — into a planar holder made of plastic. The assembly obtained in this way is adhered, for example, using double-sided tape in a cuvette. Such a solution makes it possible to record reproducible spectra in different solutions, but does not make it possible to remove the sample and carry out the reaction outside the cuvette. In addition, the method of mounting the sample in the cuvette on double-sided tape is very labor- intensive, poorly reproducible between different samples. Moreover, it is an instable method: in contact with water or reagents, the tape or sample holder can become damaged, causing the release of the test material or introducing other, undesirable components into the sample as a result of the reaction of, for example, double-sided tape with water (hydrolysis of the adhesive and / or tape material) [Kalisz, J., Zarqbska, J., Kijenska-Gawronska, E., Maksymiuk, K., Michalska, A. Colorimetric and fluorimetric pH sensing using polydiacetylene embedded within PVC / PCL nanofibers. Electroanalysis, e202200497].

[0012] In fluorimetric measurements carried out for solids, several conditions must be met for reliable measurement results that are repeatable and reproducible: the test object must be stationary with respect to the optical path of the excitation light (the position of the sample must be constant). The solution, if used, must not affect the position of the sample (it must not get under the sample and lift it or otherwise move it). Air bubbles must not form under the sample and change the position of the sample surface in relation to the incident excitation light. If measurements are made several times for the same sample — changes over time, as a function of changes in the concentration of selected reactants, etc. are recorded — the excitation light should fall on the solid sample each time in the same place, at the same angle, so that the results obtained are repeatable and comparable.

[0013] In fluorimetric studies, nanomaterials in the form of particles suspended in solution (micro- and nanoparticles) are often used. The study of emission changes of such objects (stable suspensions in a selected liquid medium) as a result of reaction, for example, with reagents dissolved in the liquid phase, or the study of emission changes over time, due to other processes, does not pose technical problems. For the purpose of recording emission spectra, as for homogeneous solutions, a sample of this type can be placed together with the solution in a cuvette and obtain favorable, repeatable and reproducible results i.e. spectra [A. Michalska, K. Maksymiuk, Jonoselektywne sensory potencjometryczne i optyczne: podobne materiafy konstrukcyjne, rozne mozliwosci analityczne (Ion-selective potentiometric and optical sensors: similar construction materials, different analytical possibilities). Wiadomosci Chemiczne, 2015, 69, 9-10: 687-699], Solid samples require proper positioning in the cuvette and immobilization, so that they can be illuminated at the right angle, i.e. not causing reflection, with excitation light. In the case of testing powders, crystals, thick layers, suitable holders offered by manufacturers of measuring equipment can be used. As a rule, these are metal pieces, in which the sample is placed by sliding it between the substrate and metal clamps (springs). However, it should be emphasized that, as a rule, these devices are adapted to work in the absence of solution. While the same sample can be placed in them many times (taking into account its material strength), this process does not allow precise control of the location from which the spectrum is collected, significantly contributing to measurement discrepancies. It should be emphasized that in order to, for example, monitor the leaching of the color reagent from the sample, or the reaction of the sample material with the solution, it is necessary to remove the sample from the holder and place it again after the reaction / process if the aforementioned solution is used.

[0014] Recording spectra from thin, flaccid, solid or semi-solid materials with limited or low mechanical strength is extremely challenging when recording spectra in the absence of liquids. Monitoring possible reactions / processes taking place, involving such materials, in liquid media, such as water, is virtually impossible. It should be emphasized that this is generally related to the low mechanical strength of thin layers of materials, especially nanostructured ones, as well as their high buoyancy. Thin polymer layers, such as nanofiber mats, tend to float in solution, which causes a change in the angle of incidence of excitation light. In extreme cases, the excitation light may not illuminate the solid test sample (the sample will become outside the optical path). As a result, the recorded spectra can vary significantly in intensity even for the same material, in the absence of other reactions / processes. Technical difficulties in registering spectra for thin materials make it impossible in practice to fluorimetrically study these objects, to use them, for example, as fluorimetric sensors, to fluorimetrically study the release of substances from them (e.g., drugs), or to track changes in material emission due to other processes / reactions. To date, no long-term proven, and reproducibly working, methods have been presented to solve the problems associated with spectrofluorimetric measurements of solid thin films, e.g., polymer films, carried out in solution, especially solutions to record spectra for the same sample over time / in different conditions, requiring reactions to be carried out outside the cuvette or in a cuvette in solution, or to compare emission spectra of different nominally identical samples.

[0015] The purpose of the present invention was to develop a holder for mounting thin layers of materials, i.e. films, membranes, nanostructured layers, in a cuvette for fluorimetric measurements, which will enable the immobilization of samples of solids in the cuvette in a repeatable and reproducible manner, in or in absence of solution, allowing the sample to be repeatedly removed and placed in the cuvette along with the holder.

[0016] This purpose has been achieved by a holder for mounting thin layers of materials in a cuvette for fluorimetric measurements according to the present invention.

[0017] Thus, the subject-matter of the present invention is a holder for mounting thin layers of materials in a cuvette for fluorimetric measurements, which:

[0018] - has the shape of a prism with the base of a rectangular trapezoid;

[0019] - two adjacent sidewalls, the first sidewall and the second sidewall being at a 90° angle to each other ultimately adjoining two adjacent walls of the spectrofluorimetric cuvette through which neither excitation nor emitted light passes, the first wall of the cuvette and the second wall of the cuvette, wherein the first sidewall should adjoin the first wall of the spectrofluorimetric cuvette over its entire surface, while the second sidewall has a width smaller than the width of the second wall of the spectrofluorimetric cuvette and is equipped with at least two protrusions adjoining the second wall of the spectrofluorimetric cuvette and the third wall of the spectrofluorimetric cuvette, through which the excitation radiation enters;

[0020] - between the protrusions, in the third sidewall of the holder prism, in the optical path of the excitation light, an attachment for immobilizing the test thin layer, in the form of a frame, preferably rectangular, is placed, wherein the immobilizing attachment being mounted on the third sidewall of the holder prism or in a rectangular recess in the third sidewall of the holder prism after the application of the test sample, in order to immobilize it;

[0021] - the third sidewall of the holder prism is inclined relative to the first sidewall of the holder prism at an angle of more than 0° to 45°.

[0022] Preferably, the protrusions have the shape of a prism with the base of a rectangular trapezoid, which first sidewall with the smallest area has to adjoin the second wall of the spectrofluorimetric cuvette, and the second sidewall located at a 90° angle to the first sidewall of the protrusion has to adjoin the third wall of the spectrofluorimetric cuvette.

[0023] Preferably, the holder has the shape of a prism with the base of a rectangular trapezoid with a height higher than the height of the spectrofluorimetric cuvette.

[0024] Preferably, the surface of the holder may be coated with a layer preventing scattering and / or reflection of light.

[0025] Preferably, the third sidewall of the holder prism is inclined with respect to the first sidewall of the holder prism at an angle of 15° to 45°, preferably 15°, 20°, 25°, 30°, 35°, 40°, and 45°.

[0026] Preferably, the holder can be made from various materials, preferably using a 3D printer.

[0027] Preferably, the polymer used to obtain the holder is selected from the group of thermoplastic polymers.

[0028] Preferably, the polymer used to obtain the holder is matte, preferably of a dark color, most preferably black.

[0029] Unexpectedly, it turned out that the proposed holders for thin films, e.g., such as thin polymer layers, nanofiber mats, allow immobilization of thin samples of solids in a repeatable and reproducible manner in the cuvette. This makes it possible to monitor the reaction between the solid and the solution, or to repeatedly remove the sample along with the holder to perform a reaction / other process and place it back in the cuvette to record the spectrum.

[0030] The present invention can be applied to thin layers of various materials, regardless of their chemical composition or nature. The present invention is preferably useful for recording emission spectra of polymer nanofiber mats. Polymer fiber mats are obtained by an electrospinning method, in an electric field of high intensity. A polymer solution, in a suitable solvent, is delivered using a syringe pump at a constant rate, and a high voltage is applied between a needle connected to the syringe and a collector (a material with good electrical conductivity, such as aluminum foil, conductive glass (ITO)). When the voltage is applied, strands of polymer solution are pulled from the needle. After evaporation of the solvent, polymer fibers on the order of nanometers or thicker are formed as the strands fall on the substrate i.e. the collector. The thickness of the obtained polymer mat depends on the length of conducting the electrospinning process, and the finished mat is removed from the collector (conductive substrate), such as aluminum foil. The resulting mats, depending on the polymer material used or the possible modification of the mat after electrospinning, may show fluorescence emission. The typical thickness of a polymer mat is a few to several hundred micrometers.

[0031] The invention relates to a method of immobilizing a thin film, such as a polymer film, in a cuvette for spectrofluorimetric measurements. The invention allows spectrofluorimetric measurement of thin films in a cuvette with an optical length of less than 1 cm, preferably 5.5 mm. The invention makes it possible to repeatedly remove a sample of the solid material from the cuvette (for example, for modification, reaction, etc.) and place it back in the cuvette in an identical position. The invention makes it possible to record spectra in solution as well as in the absence of solution. The invention allows measurement at various angles of incidence, of 0° to 45°, preferably 35° for matte samples and 15° for reflective samples. The holder for immobilizing thin layers of solid samples can be made of any material; optionally its surface can be coated with a layer preventing light scattering / reflection. Preferably, the holder can be prepared by 3D printing technique.

[0032] The holder makes it possible to mount a solid sample in the cuvette i.e. immobilizing it at a selected angle to the incident radiation. The holder uses the typical geometry of a cuvette — its back walls, which meet at an angle of 90° when inserted into the cuvette, adjoin its walls, through which neither excitation nor emitted light passes. In 1 / 3 of the height of the holder (counting from the top) and at the bottom of the holder there are elements that are in contact with the wall of the cuvette, through which the excitation radiation enters; the role of these elements is to prevent the holder from deviating from the vertical and changing the position of the test layer. The front part of the holder is a cross-sectional plane directed at a selected angle (0 to 45°) to the rear edge of the holder, which is in contact with the cuvette wall, parallel to the one through which the excitation radiation enters. This allows mounting the sample at the desired angle to the excitation radiation. On the cross-sectional surface of the holder there is a rectangular recess, with a centrally located, rectangular-shaped space for the sample. A thin film, a layer of the test material, is applied to the surface of the holder at the place intended for the sample. The place is positioned in the holder so that it is in the optical path of the excitation light. The test material is then immobilized with the help of an immobilization attachment (a frame). The sample thus mounted in the holder can be placed in the cuvette, also filled with solution, and the spectrum can be recorded in any spectrofluorimeter and mode of operation. The sample can also be removed along with the holder and placed back in the cuvette. Thanks to the shape of the holder, the adjoining of its rear walls, and the two protrusions located in front and adjoining the walls of the cuvette, as well as the immobilization of the sample with the frame in the selected place, the test material will always be in the same position relative to the excitation light. At the same time, the shape of the holder allows the presence of the solution in the cuvette, i.e. recording spectra in solution, or at the selected temperature, if the cuvette holder in the spectrofluorimeter is equipped with a thermostatic device. The solution can be stirred without causing the sample to move.

[0033] The example presents holders with 5 different angles of incidence of the excitation beam on the sample: 15°, 20°, 25°, 30°, 35° using the same optical path of 5.5 mm.

[0034] Preferably, the proposed holder can be used once (similar to polyacrylate cuvettes), but it can also be used repeatedly. The holder can be inserted into a disposable polyacrylate cuvette, but also into a cuvette such as quartz cuvette.

[0035] In an example showing the performance of the invention, a thin film, a mat of electrospun nanofibers made of poly(vinyl chloride) (PVC) and polycaprolactone (PCL), containing the fluorescent dye Nile Red (NR), was used.

[0036] In the embodiment, the environment in which the usefulness of the invention was tested was double-distilled water, containing no interfering substances.

[0037] In a preferable embodiment of the invention, in an aqueous solution, results were obtained showing the stability of the spectrum recorded using a PVC / PCL mat between 20 and 50 pm thick, with NR added, placed in a holder according to the present invention, after transferring it to other cuvettes. The recorded emission spectra for the mats placed in the holder, being the subject-matter of the invention, have significantly higher intensities, which is due to the more preferable positioning of the sample in relation to the excitation beam. In addition, transferring the holder, along with the sample placed in it, between different cuvettes makes it possible to obtain similar intensities at maximum. In the case of a sample not placed in the holder, significant changes in the recorded values of fluorescence emission intensities are observed. The results obtained for the thin polymer layers, PVC / PCL mats with NR, using the holder that is the subject-matter of the invention are characterized by preferable repeatability and reproducibility.

[0038] The holder according to the invention can be used to immobilize solid thin films, preferably polymeric materials, preferably electrospun polymer mats, for spectrofluorometric studies. The invention will now be further illustrated in preferable embodiments with reference to the accompanying drawing, wherein:

[0039] Fig. 1 shows a schematic of the invention with example angles of incidence: 15°, 20°, 25°, 30°, 35°, perspective view;

[0040] Fig. 2 shows a schematic of the invention with an angle of incidence of 35° with a polymer mat immobilized by means of an immobilization attachment in a cuvette, perspective view;

[0041] Fig. 3 shows a schematic of the immobilization attachment with dimensioning, perspective view;

[0042] Fig. 4 shows a schematic of the invention with exemplary angles of incidence: 15°, 20°, 25°, 30°, 35°, together with dimensioning for an angle of incidence equal to 35°, front view;

[0043] Fig. 5 shows a schematic of the invention with exemplary angles of incidence from the right consecutively: 15°, 20°, 25°, 30°, 35°, together with dimensioning for an angle of incidence equal to 35°, top view;

[0044] Fig. 6 shows a schematic of the invention for an angle of incidence equal to 35°, together with dimensioning, top view;

[0045] Fig. 7 shows a schematic of fluorescence excitation in a sample placed at an angle of incidence under the influence of an excitation beam;

[0046] Fig. 8 shows emission spectra for a PVC / PCL film with NR added, recorded using a holder according to the invention with an angle of incidence equal to 35° after being transferred to 5 different cuvettes in air;

[0047] Fig. 9 shows emission spectra for a PVC / PCL film with NR addition, recorded using a holder according to the invention with an angle of incidence equal to 35° after transferring to 5 different cuvettes filled with distilled water;

[0048] Fig. 10 shows emission spectra for a PVC / PCL film with NR addition, recorded using a holder according to the invention with exemplary angles of incidence: 15°, 20°, 25°, 30°, 35°; emission measurements were carried out in a cuvette filled with distilled water;

[0049] Fig. 11 shows emission spectra for a PVC / PCL film with NR addition, recorded using a holder according to the invention with an angle of incidence of 35° for 8 mats (samples) in air; the inset shows the dependence of emission intensity measured at 640 nm for each sample;

[0050] Fig. 12 shows the emission spectra for a PVC / PCL film with NR added, immobilized in a 96-well plate; the measurement was made 5 times for 1 well filled with distilled water with removing and reinserting the plate;

[0051] Fig. 13 shows the emission spectra for a PVC / PCL film with NR added recorded using mat immobilization on ITO after transferring into 5 different cuvettes filled with distilled water;

[0052] Fig. 14 shows a comparison of maximum emission intensities for measurements carried out in distilled water, measured for immobilization of the sample in a holder according to the invention with an angle of incidence of 35°, a well on a 96-well plate, and on ITO; measurements were carried out in 5 different cuvettes (holder according to the invention, ITO) or after the plate was returned to the spectrofluorimeter.

[0053] EXAMPLES

[0054] Reagents and materials used

[0055] Equipment

[0056] The primary technique used to test the invention was fluorimetric spectroscopy. It required the use of a standard acrylic cuvette. PVC / PCL mats with NR addition were obtained by electrospinning. A Prusa Mini 3D printer was used. Cary Eclipse spectrofluorimeter (emission measurement parameters: excitation 540 nm, emission range 560 or 580 to 800 nm, excitation and emission slits: 5 nm, photomultiplier voltage 650 V).

[0057] Reagents Tetrahydrofuran (THF), dimethylformamide (DMF), double-distilled and freshly deionized water (18.2 MQ cm resistance, Milli-Q Plus, Millipore, Austria). Poly(vinyl chloride) (PVC), polycaprolactone (PCL) and Nile Red (NR) were purchased from Sigma Aldrich (Germany), polylactide filament (PLA, BASF).

[0058] The object of the invention “a” was designed in an image modeling program as a holder for polymer mat printed with a 3D printer. In the image modeling program (e.g., Blender, Fusion), the holder was designed in such a way that the parameters of thickness, length and width were suitable for the cuvette and that the place for immobilization of the mat was located in a place that matched the optical measurements, i.e. that the light fell on the mat always in the same place. The exact dimensions of the designed holder: height 55 mm, width 10 mm, thickness 10 mm. Schematic drawing of the holder with dimensioning is shown in Figs. 1-6. Fig. 7 shows a schematic of fluorescence excitation in a sample placed at an angle of incidence under the influence of an excitation beam. The model of the obtained holder was then printed using a 3D printer (Prusa MINI). For printing, it is necessary to use optically and chemically passive filaments, preferably black-colored thermoplastic filaments, matte, for example, of PLA. For the holder, it is also important to print an immobilization attachment (here in the form of a frame) to put it on the mat to immobilize it. A schematic of the attachment, including dimensioning, is shown in Figs. 2 and 3. The polymer mat is placed on the holder, and then the attachment is applied to immobilize the mat during testing (a schematic of the immobilization of the mat is shown in Fig. 2). The holder with the polymer mat prepared in this way is placed in the cuvette, as illustrated in Fig. 2. The cuvette with the immobilized mat in the holder can be placed in the spectrofluorimeter so that the angle of incidence is, for example: 15°, 20°, 25°, 30°, 35°.

[0059] PVC / PCL polymer mats with NR were obtained by electrospinning with the following parameters: voltage 13 kV, needle distance from the collector 15 cm, spinning time 30 min, a flow rate of 0.8 ml / h from a 15% solution of PVC / PCL (65% PVC, 35% PCL) with the addition of 0.33% NR, dissolved in a THF:DMF polymer mixture in a volume ratio of 1 :1. A rectangular piece of the appropriate size (matching the subject-matter of the invention used) was cut from the finished PVC / PCL mats and such a mat, immobilized with an immobilizing attachment, was placed in a cuvette containing distilled water and the emission spectrum was recorded. The holder with the same PVC / PCL mat was transferred into another cuvette (also containing distilled water) and the emission spectrum was recorded. The operation was repeated 5 times to check the repeatability of the obtained results after transferring the immobilized mat into different cuvettes. The obtained emission spectra are shown in Fig. 7. PVC / PCL polymer mats with NR were also immobilized on sample holders with angles of incidence: 15°, 20°, 25°, 30°, 35°, and emissions were measured in air (Fig. 8) and distilled water (Fig. 9).

[0060] Example 1

[0061] To check the repeatability of the results obtained using the holder according to the present invention with an angle of incidence of 35°, emission measurements were carried out for PVC / PCL mats with NR in air (dry) when transferring the holder with the mat to the successive samples and recording emission spectra (5 repetitions). From the recorded emission spectra (Fig. 8), it is observed that the obtained analytical responses are stable when transferring the same mat, immobilized on the holder, into the next cuvette.

[0062] Example 2

[0063] To check the repeatability of the results obtained using the holder according to the present invention with an angle of incidence of 35°, emission measurements were carried out for PVC / PCL mats with NR in an aqueous environment when transferring the holder with the mat to successive samples and recording the emission spectra (5 repetitions). From the recorded emission spectra (Fig. 9), it is observed that the obtained analytical responses are stable when transferring the same mat, immobilized on the holder, into the next cuvette with the same solution.

[0064] Example 3 To check the effect of the selected angle of incidence on the recorded emission for PVC / PCL mats with NR, in five holders with selected angles of incidence of: 15°, 20°, 25°, 30°, 35° (schematics of the holders are shown in Figs. 1, 4, and 5), the mats were immobilized and the emission measurements for them were carried out in distilled water. The highest emission values are recorded for an angle of incidence of 35°. The emission peak value at a wavelength of about 640 nm increases proportionally as the angle of incidence increases (Fig. 10). However, it is recommended for samples that readily reflect radiation to conduct measurements in a holder with a smaller angle of incidence.

[0065] Example 4

[0066] To check the reproducibility of the results obtained using the holder according to the present invention with an angle of incidence of 35°, emission measurements were carried out for PVC / PCL mats with NR in air (dry) for 8 different samples, in which emission spectra were recorded. From the recorded emission spectra (Fig. 11), it is observed that the analytical responses obtained are similar for the 8 samples, which is better illustrated in the inset of the dependence of the maximum emission intensity measured at 640 nm for each sample.

[0067] Example 5

[0068] For comparison, other methods of immobilizing PVC / PCL mats with NR on a 96-well plate or ITO, on which the mat was directly immobilized during electrospinning, were carried out. For both immobilization methods, 5 emission measurements were carried out for one sample (in air, Figs. 12 and 13). In both cases, the fluorescence intensity was much lower than for the invention with an angle of incidence of 35° (Fig. 8). Also, the stability of the analytical response for the other immobilization methods was worse, as can be seen from the dependence of the emission intensity measured at 640 nm in five different cuvettes or for the multi-well plate shown in Fig. 14. The above examples confirm the preferable use of the subject-matter of the invention for the immobilization of thin films, preferably polymer mats, in applications for optical studies.

Claims

Claims1. A holder (1) for mounting thin layers of test materials in the spectrofluorimetric cuvette (2), characterized in that:- it has the shape of a prism with the base of a rectangular trapezoid;- two adjacent sidewalls, the first sidewall (3) and the second sidewall (4) being at 90° to each other ultimately adjoin two adjacent walls of the spectrofluorimetric cuvette through which neither excitation light nor emitted light passes, the first wall of the cuvette and the second wall of the cuvette, wherein the first sidewall (3) should adjoin the first wall of the spectrofluorimetric cuvette over its entire surface, while the second sidewall (4) has a width smaller than the width of the second wall of the spectrofluorimetric cuvette and is equipped with at least two protrusions (5, 6) adjoining the second wall of the spectrofluorimetric cuvette and the third wall of the spectrofluorimetric cuvette, through which the excitation radiation enters;- between the protrusions (5, 6), in the third sidewall (7) of the holder prism (1), an attachment (8) is placed in the optical path of the excitation light to immobilize the thin test layer, in the form of a frame, preferably rectangular, wherein the immobilizing attachment (8) is mounted on the third sidewall (7) of the holder prism (1) or in a rectangular recess in the third sidewall (7) of the holder prism (1) after the application of the test sample, in order to immobilize it;- the third sidewall (7) of the holder prism (1) is inclined with respect to the first sidewall (3) of the holder prism (1) at an angle of more than 0° to 45°.

2. The holder according to claim 1, characterized in that the protrusions (5, 6) have the shape of a prism with the base of a rectangular trapezoid, which with the first sidewall of the smallest area should adjoin the second wall of the spectrofluorimetric cuvette, and with the second sidewall located at an angle of 90° to the first sidewall of the protrusion should adjoin the third wall of the spectrofluorimetric cuvette.

3. The holder according to claim 1 or 2, characterized in that it has the shape of a prism with the base of a rectangular trapezoid with a height higher than the height of the spectrofluorimetric cuvette.

4. The holder according to claim 1 or 2 or 3, characterized in that the surface of the holder (1) may be coated with a layer preventing scattering and / or reflection of light.

5. The holder according to any of the claims 1-4, characterized in that the third sidewall (7) of the holder prism (1) is inclined with respect to the first sidewall (3) of the holder prism (1) at an angle of 15° to 45°, preferably 15°, 20°, 25°, 30°, 35°, 40°, and 45°.

6. The holder according to any of the claims 1-5, characterized in that it can be made of various materials, preferably with the help of a 3D printer.

7. The holder according to any of the claims 1-6, characterized in that the polymer used to obtain the holder is selected from the group of thermoplastic polymers.

8. The holder according to any of the claims 1-7, characterized in that the polymer used to obtain the holder is matte, preferably of a dark color, preferably black.

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