RU2781351C1 - Chemiluminescent analysis device - Google Patents

Abstract

FIELD: instrumentation.

SUBSTANCE: invention relates to analytical sensor systems and can be used to detect reactive oxygen species in biological and other samples, as well as during chemiluminescent immunological analysis with increased sensitivity. The device for chemiluminescent analysis of the content of reactive oxygen species in biological and other samples contains a pump for inserting liquid reagents from containers with chemiluminophore and the samples under study into a microfluidic chip, the output of which is connected by a tube to a drain tank, and a photodetector, while the microfluidic chip and photodetector are placed in a light-tight housing, and the microfluidic chip is made at least two- channel and contains a dielectric substrate with a metasurface of laser-treated metal nanoparticles deposited on it, having localized plasmon resonance in the chemiluminescence spectral band of the chemiluminophore used, and the light-tight housing also includes two linear mutually orthogonally oriented polarizers superimposed on various microfluidic chip channels, located one after the other along the radiation propagation path: a polarization rotator and a third linear radiation polarizer installed directly in front of the photodetector input, coinciding in orientation with one of the linear polarizers superimposed on a microfluidic chip.

EFFECT: coding of chemiluminescent radiation by polarization and an increase in its intensity.

2 cl, 8 dwg, 1 tbl

Description

The invention relates to analytical sensor systems and can be used for detecting reactive oxygen species in biological and other samples, as well as for carrying out chemiluminescent immunoassay with increased sensitivity.

Today, the issue of finding high-speed, sensitive and low-cost sensors for the early diagnosis of socially significant diseases, such as diabetes mellitus, myocardial infarction, and oxidative stress of the body, is acute. The state of oxidative stress is characterized by a high concentration of reactive oxygen species (ROS) in the body, which suppress the human antioxidant defense system. It has been established that ROS are toxic to spermatozoa and lead to male infertility, and are also involved in many inflammatory processes. An excessive concentration of ROS released by cells of the immune system can lead to damage to cell membranes and DNA, which ultimately leads to cell death and tissue damage. Thus, the rapid and accurate determination of the concentration of ROS in biological samples is the most important prerequisite for a correct diagnosis and definition of a treatment strategy.

Traditional approaches to the analysis and diagnosis of oxidative stress have a number of disadvantages, which include the need to collect large volumes of the test material (analyte), lengthy sample preparation using fluorescent markers, and the high cost of analytical equipment. Therefore, the development of new highly sensitive, fast and affordable systems for determining the oxidative stress of the body is of particular importance.

The key advantage of chemiluminescent (CL) devices is that they do not require an optical source or fluorescent markers to function. This circumstance significantly simplifies the design of devices, reduces their weight and final cost. However, these advantages cannot be fully exploited due to the fact that chemiluminophores used in biomedicine have low efficiency. The reason for the low efficiency of CL is the competition of non-radiative channels for the deactivation of molecules excited as a result of a chemical reaction with the process of light emission. In this regard, the development of methods for increasing the efficiency of chemiluminophores and optimizing the processes of collection and registration of CL radiation is an urgent task.

For express diagnostics of oxidative stress, as a rule, containers of relatively large volumes (for analytical and diagnostic instruments) are used, for example, cuvettes, Eppendorf-type microcentrifuge tubes or culture well plates. To study the processes of formation of reactive oxygen species in various liquid media, a small-sized device "Device for the analysis of luminescence of solutions" was patented (RF Patent No. 152424 MPK G01N 21/76, priority date 02.20.2015, published 05.27.2015), which is equipped with a dispenser control unit liquids. In this device, the test substance is placed in microcentrifuge tubes with a volume of 1.5 ml, which leads to the need to select large volumes of the test material.

A known device for recording ultra-weak light fluxes "Automatic chemiluminometer device" (RF Patent No. 146739, IPC G01N 21/76, priority date 03/13/2014, published 10/20/2014), which allows for immunochemical studies and analyzes of free radical pathologies. The proposed technical solution allows to increase productivity by using a standard well plate with up to 96 samples, however, the working volume of one well is 0.1-0.2 ml, which also leads to the need to select large volumes of the test material.

In the Patent "Microwave trigger inducing metal-enhanced chemiluminescence (MT MX) and its spatial and temporal control" (US Patent No. 9075018 B2, IPC G01N 21/763, G01N 21/76, G01N 33/54306, G01N 33/5438 , G01N 33/553, Y10T 436/143333, Y10T 436/144444, Y10T 436/145555, Y10T 436/147777, Y10T 436/201666, Y10T 436/21, Y10T 436/23, priority date published 02/13/07. 2015) presents a CL system designed to visualize structures and elements using the plasmon effect from metal surfaces, the visually observed glow of which is caused by chemical and biological reactions based on the CL effect. The contrast in visualization of CL images is improved by the use of microwave radiation, and additionally by the use of metallic nanostructures. In this patent, the issues of comparing the intensity of CL in the sample and the reference sample have not been worked out, which does not allow using it to determine oxidative stress.

A method for improving the detection limit of analyte molecules is described in the patent "Method and device for chemiluminescent analysis" (US Patent No. 20190195806 A1, IPC G01N 21/76, B01L 3/5027, G01N 21/05, G01N 33/582, G01N 33/587, G01N 33/725, B01L 2300/0663, B01L 2300/0877, B01L 2300/0883, B01L 2300/123, G01N 2021/0346, priority date 02/13/2016, published 07/07/2015), which uses colloidal solutions of silver and gold nanoparticles. To reduce analyte consumption, a polydimethylsiloxane (PDMS) microfluidic chip was used. Despite the possibility of switching from milliliter volumes of the analyte to microliter volumes declared in the patent, it should be noted that in order to maintain the declared increased sensitivity of the CL sensor operating in the flow mode, the constant addition of colloidal noble metal nanoparticles to the chemiluminophore solution is required.

Known patent "Luminometer" (RF Patent No. 180961 U1, IPC G01N 21/01, G01N 21/76, priority date 07/14/2017, published 07/02/2018), containing an articulated body, consisting of an upper and lower parts, in which are located sample compartment and an electronic control system, including a solid-state photomultiplier, a signal amplification unit, a power supply unit, a control unit, a pulse counter, a USB port, a position sensor of the upper part of the case, and a rechargeable battery.

The invention "Device for the analysis of chemi- and bioluminescence of liquid media" (RF Patent No. 2452937 C1, IPC G01N 21/76, priority date 15.02.2011, published 10.06.2012) for a device for the analysis of CL solutions containing a cuvette compartment for the test or a reference sample, a heater, a PMT, an interface, a control unit, an electric drive and a power supply connected via an electrical circuit and connected to a personal computer. Despite the fact that the device includes additional auxiliary objects (temperature sensors, a heater, and dispensers), its obvious disadvantages include the inability to conduct a quick comparative analysis of the content of reactive oxygen species in the samples under study.

Currently, various methods are used to record the CL signal in stationary devices. However, in portable devices, in most cases, photomultiplier tubes (PMTs) are used. As a rule, the CL signal is recorded sequentially for each sample: first, the CL intensity of the reference sample with a predetermined ROS content is determined, then the signals from the studied samples are recorded. The same approach underlies the “Device for express determination of the concentration of reactive oxygen species in aqueous solutions” (RF Patent No. 162866 U1, IPC G01N 21/76, priority date 12/30/2015, published 06/27/2016), where serial measurements are performed using one cuvette. The main disadvantage of this device is the need for constant washing of the cuvette to record the reference CL signal, provided that different test substances and components are used.

The closest in technical essence to the claimed object is a portable device described in the patent "Microfluidic device for detecting chemiluminescence" (Patent CN No. 212780531 U, IPC G01N 21/76, B01L 3/00, priority date 09/01/2020, published 03/23/2021 ). The device is designed for quantitative determination of biological oxygen demand (BOD) in a water sample depending on the concentration of polluting organic substances. The operating principle of the device is based on the optical effect of CL of luminol molecules, which appears in the channels of a microfluidic chip. The device includes a microfluidic system, a pump for introducing liquid from a container with reagents in the form of a chemiluminophore, in particular luminol, and test samples in the form of water samples into a microfluidic chip, the outputs of which are connected by tubes to a container for draining, a photodetector for registration of CL and a microdevice for temperature control. The described device is distinguished by the division of a microfluidic chip into a reaction zone with a Tesla valve channel for mixing yeast suspension and water sample, as well as a CL zone with a spiral single microchannel, designed for rapid mixing of sample solutions with luminol and further observation of CL generation in the presence of a water sample. The input of the water sample occurs simultaneously with the input of the yeast suspension in the reaction channel using a peristaltic pump with a given flow rate. The CL of luminol upon activation by a mixed solution of a water sample with yeast is recorded in real time using a photodetector in the form of a PMT located under the microfluidic chip. However, the disadvantages of this device are the length of time for comparative analysis, which, in turn, does not exclude the possibility of contamination of the microchannels with the reaction products of the control sample, as well as the absence of an opaque housing, which necessitates the registration of CL luminescence in a dark room. This circumstance does not allow one to obtain reliable data on the CL intensity of the studied samples.

The task to be solved by the claimed invention is to increase the speed and increase the sensitivity of detection, as well as reduce the volume consumption of biological and other samples containing ROS. The problem is solved by achieving a technical result, which consists in encoding the CL radiation coming from different channels of the microfluidic chip by polarizing it and increasing its intensity. The technical result is achieved due to the fact that the device for chemiluminescent analysis of the content of reactive oxygen species in biological and other samples contains a pump for introducing liquid reagents from containers with chemiluminophore and test samples into a microfluidic chip using tubes, the output of which is connected by a tube to a container for draining, and a photodetector, characterized in that the microfluidic chip and the photodetector are placed in an opaque housing, the microfluidic chip is made at least two-channel and includes a dielectric substrate with a metasurface deposited on it from laser-treated metal nanoparticles having a localized plasmon resonance in the chemiluminescence spectral band of the chemiluminophore used , and the opaque housing also includes two linear mutually orthogonally oriented polarizers superimposed on different channels of the microfluidic chip and located one after the other along the propagation of the rotator radiation. l polarization, made, for example, in the form of a liquid crystal twist cell, the electrical inputs of which are connected to the outputs of the generator of control voltage pulses, the third linear polarizer of radiation, coinciding in orientation with one of the linear polarizers superimposed on a microfluidic chip, a photodetector made with a counting function photons.

The essence of the invention is illustrated by figures 1-8 and their brief description below.

Fig. 1. Schematic diagram of a device for chemiluminescent analysis.

Fig. 2. Microfluidic chip. A flexible transparent PDMS polymer part with two microchannels (a) and a dielectric substrate with a metal coating of nanoparticles (b). The inset shows a picture of silver nanoparticles taken from a scanning electron microscope.

Fig. Fig. 3. Spectral overlap of the absorption band of the metasurface of silver (Ag) nanoparticles (a) and the chemiluminescence band of luminol molecules (b).

Fig. Fig. 4. Kinetics of the chemiluminescent reaction of luminol upon activation with hydrogen peroxide (H ₂ O ₂ ): (a) a microfluidic channel without a metasurface of silver nanoparticles and (b) a microfluidic channel with a metasurface of silver nanoparticles.

Fig. Fig. 5. Kinetics of the chemiluminescent reaction of luminol in the presence of a plasmonic metasurface of silver nanoparticles upon activation with hydrogen peroxide (H ₂ O ₂ ) of various concentrations: accumulation time 100 ms (a) and 10000 ms (b).

Fig. Fig. 6. Kinetics of the chemiluminescent reaction of luminol in the presence of a plasmonic metasurface of silver nanoparticles upon activation with sodium hypochlorite (NaOCl) of various concentrations.

Fig. 7. Demonstration of alternate registration of the kinetics of the chemiluminescent reaction of luminol, using a polarization rotator.

Fig. 8. Photograph of the device for chemiluminescent analysis.

The device for chemiluminescent (CL) analysis (Fig. 1) consists of an opaque housing, including cover 1 and base 2, which protects the photodetector of CL radiation from extraneous illumination. The key element of the device is a microfluidic chip consisting of an assembly of a flexible transparent polymer part 3 with microchannels with specified parameters, made by the lithographic method, with a dielectric substrate 4 coated with metal (for example, silver) nanoparticles. To receive solutions, the microfluidic chip is connected through silicone tubes 5 to a four-channel infusion pump 6, which includes syringes with the necessary solutions: chemiluminophore (for example, luminol) 7 with test samples 8 for the comparative channel of the microfluidic chip (Channel 2), and chemiluminophore 9 with reference sample 10 (Channel 1). To position the microfluidic chip inside the base 2 of the opaque housing, cylindrical neodymium magnets are inserted, which are precisely mated with the transparent polymer part 3 and polarizers 11. Two linear polarizers 11 located in the same plane, superimposed on the flexible transparent polymer part 3 so that the angle between their main transmission planes is equal to 90°, provide the conversion of CL radiation of channels with a reference sample and samples under study into a linearly polarized one. Modulation of the CL signal is carried out using a polarization rotator 12, made, for example, in the form of a liquid crystal twist cell. Polarizer 13 is used to determine the encoded polarized CL radiation from the channels of the microfluidic chip. To rotate the plane of polarization, the electrical inputs of the polarization rotator 12 are connected to the outputs of the generator of control voltage pulses 14. Registration of CL radiation is carried out using a photodetector 15 that supports the photon counting function. To control the generator of control voltage pulses 14, photodetector 15 and visualize the recorded data in real time, a personal computer 16 is used. Waste solutions are collected in a special container 17.

The device works as follows.

First, an alkaline solution of luminol with a concentration of 0.1 M with pH ~ 12-13 is prepared and drawn into syringe 7 and syringe 9. The solution with the reference sample is drawn into syringe 10, and the solution with the test sample into syringe 8, after which the syringes are fixed in the carriage of the infusion pump 6. Then, in order to obtain correct data and reduce parasitic noises when registering the kinetics of the CL reaction, it is recommended to keep the photodetector 15 for at least 30 minutes in the dark. Then, on one side of the microfluidic chip, silicone tubes 5 are connected to syringes 7, 8, 9, 10 with the necessary solutions, and on the other side, to a container for draining 17. The control voltage pulse generator 14, which controls the polarization rotator 12, is connected to the AC network. current 220 V with a frequency of 50 Hz using a power cable and to a personal computer 16 via a USB cable. Next, you need to run the software for the photodetector 15, the output of which is connected to the personal computer 16 using a USB cable. For subsequent data analysis, a baseline should be recorded with the required exposure time (permissible from 10 ms). After the baseline is registered, the fluid supply rate is set in the infusion pump 6 (allowable from 5 to 50 μl / min), and the pumping of solutions from syringes 7, 8, 9, 10 into the microfluidic chip 3 through the tubes 6 hermetically fastened to it is started. Then, solutions from syringes with luminol 9 and reference sample 10 enter Channel 1 of the microfluidic chip and mix in the reaction section of the microfluidic chip, thereby inducing CL luminescence. Similarly, CL luminescence should be observed in Channel 2, when solutions with luminol 7 and test samples 9 begin to interact with each other. Then the CL radiation of Channels 1 and 2, passing through the polarizers 11 corresponding to them, is polarized, and as a result, the radiation of Channel 1 will be polarized perpendicular to the radiation of Channel 2. Then the CL radiation of both channels passes through the polarization rotator 12, which in turn rotates the radiation polarization plane by 90° in the absence of voltage at its electrical inputs, or does not change the plane of polarization when the voltage is set to U ~ 1.5 V using the control pulse generator 14. Further, the radiation passes through the polarizer 13 of the channel signals with the reference sample and the samples under study and depending on the voltage at the output of the control pulse generator, the voltage alternately falls on the photodetector. As a result of these operations, the monitor of the personal computer 16 displays the dependence of the number of photons on time, the so-called. kinetics of the CL reaction coming to the photodetector from Channels 1 and 2 of the microfluidic chip. After that, on the basis of the data obtained, both qualitative and quantitative assessment of ROS in solution with the test sample is carried out in comparison with a reference sample with a known concentration. To register a stable CL response, it is advisable to pass through the microfluidic chip about 120-240 µl of mixed solutions with chemiluminophore/test sample and chemiluminophore/reference sample. An analysis of the experimental data showed that the volume of one channel in a microfluidic chip does not exceed 60 µl. The technical characteristics of the photodetector 15 and the polarization rotator 12 make it possible to use the device with other chemiluminophores whose emission bands lie in the spectral range from 400 nm to 700 nm. A Photon Counting Head H11890-210 receiver (Hamamatsu, Japan) can be installed as a photodetector 15 of CL radiation.

Example 1

A microfluidic chip with embedded metal nanoparticles was used to increase the sensitivity, reduce the consumption of samples, and increase the speed of the analysis of the CL device. The flexible polymer part 3 (Fig. 2a) was fabricated from optically transparent polydimethylsiloxane (PDMS) using standard soft lithography [Bukatin AS, Mukhin IS, Malyshev EI, Kukhtevich IV, Evstrapov AA, Dubina MV Fabrication of high-aspect-ratio microstructuresin polymer microfluid chips for in vitro single-cell analysis//Technical Physics. - 2016. - V. 61, No. 10. - P. 1566-1571]. The assembled microfluidic chip from a flexible polymer part 3 and a dielectric substrate 4 with metal nanoparticles contains two separate rhomboid channels necessary for comparative analysis (Fig. 2a), and four holes for inlet and two holes for output of solutions. To provide the best detection sensitivity for ROS with metal nanoparticles, the channel height should not exceed 10 μm. In turn, the silver nanoparticles (Fig. 2b) on the substrate were obtained by vapor deposition in a PVD-75 vacuum chamber (Kurt J. Lesker) according to the procedure in [Leonov NB et al. Evolution of the optical properties and morphology of thin metal films during growth and annealing //Optics and spectroscopy. - 2015. - T. 119. - No. 3. - S. 450-455.] when using the necessary mask. The subsequent high-temperature annealing of silver nanoparticles led to the self-organization of metal nanoclusters on the substrate surface due to the surface diffusion of silver atoms. An analysis of the image from a scanning electron microscope showed that the nanoparticles have a rounded shape with sizes from 10 nm to 200 nm (Fig. 2b). To significantly improve their resistance to chemicals, according to the procedure described in [Dadadzhanov DR et al. Self-organized plasmonic metasurfaces: The role of the Purcell effect in metal-enhanced chemiluminescence (MEC)//Sensors and Actuators B: Chemical. - 2021. - T. 333. - S. 129453.], the substrates were exposed to pulsed laser radiation with an energy dose of 80 mJ / cm ² at a wavelength of the 3rd harmonic of the Nd: YAG - laser (SOLAR Laser System) - 355 n . Processing with laser radiation avoids the consumption of metal nanoparticles during analysis and allows multiple use of microfluidic chips due to their fixation on the surface of a dielectric substrate. It is also worth noting that one of the necessary conditions for amplifying the CL signal is fulfilled due to the almost complete spectral overlap of the silver absorption band and the luminol CL band, as shown in Fig. 3. The luminol CL spectrum has a maximum at a wavelength of 428 nm, while the absorption maximum plasmon resonance of the metasurface of silver nanoparticles falls at 450 nm. For a comparative demonstration of the effect of enhancing the CL of luminol, a microfluidic chip was used, in which only one channel had inclusions of metal nanoparticles on a dielectric substrate (insert in Fig. 3). Next, a previously prepared 3% hydrogen peroxide solution and an alkaline solution of luminol with pH = 12.7 were simultaneously introduced into the microfluidic chip at a rate of 1.5 μL/min, first into Channel 1 without nanoparticles, and then into Channel 2 with nanoparticles. From the CL kinetics in Fig. It can be seen from Fig. 4 that the luminescence of luminol without nanoparticles (a) sharply increases, after some time it decreases and practically does not change. In the case of using a metasurface of metal nanoparticles with luminol (b), a pronounced increase in the CL intensity is observed, which eventually reaches the saturation level. Comparison of the CL intensity values in Channels 1 and 2 over the time interval in which the signals are stable showed that the CL intensity in the channel with silver nanoparticles is 3 times higher than in the channel without nanoparticles (the so-called CL amplification factor). An increase in the intensity of CL is achieved due to the Purcell effect, in which there is an increase in the rate of radiation decay of excited chemiluminophore molecules located near the nanoparticles [Sauvan C. et al. Theory of the spontaneous optical emission of nanosize photonic and plasmon resonators //Physical Review Letters. - 2013. - T. 110. - No. 23. - S. 237401.]. Thus, the use of a plasmonic metasurface allows increasing the CL intensity without increasing the volume of the analyte and without complicating the measurement procedure, but also eliminating the use of additional chemical CL catalysts [Iranifam M. Chemiluminescence reactions enhanced by silver nanoparticles and silver alloy nanoparticles: applications in analytical chemistry // TrAC Trends in Analytical Chemistry. - 2016. - T. 82. - S. 126-142.].

Example 2

To demonstrate the possibility of detecting ROS by the proposed device, the kinetics of the CL reaction of luminol with a metasurface of metal nanoparticles was recorded upon activation by hydrogen peroxide molecules (Fig. 5 a, b) and sodium hypochlorite (Fig. 6) with different concentrations. As can be seen from FIG. 5a, the CL intensity begins to increase sharply with time, and then sharply decreases to a stationary level. To assess the level of oxidative stress caused by the presence of hydrogen peroxide, it is advisable to evaluate this indicator according to three criteria. The first criterion considers the estimate of the ratio of the intensity maxima of the studied and reference samples during the first bright flash, for example, in the region of 40 s on all graphs of the CL kinetics in Fig. 5a. The second criterion considers the estimation of the ratios of the mean values of the CL intensity of the test and reference samples upon reaching the steady state after a sharp flash. The last evaluation criterion involves comparing the average value of the area under the CL reaction curve with the average value of the area under the curve for the reference sample for the same time intervals. From FIG. It is also seen in Fig. 5a that the CL device, with a signal accumulation time of 100 ms, makes it possible to detect the presence of hydrogen peroxide molecules up to 0.3 ng/mL. Of course, the achievement of better CL kinetics can also be achieved by increasing the CL signal accumulation time, which is confirmed at an accumulation time of 10000 ms and at the previously used hydrogen peroxide concentration (0.3 ng/mL) (Fig. 5b). In order to confirm the possibility of detecting other types of reactive oxygen species, an aqueous solution of sodium hypochlorite with different concentrations was used, as indicated in FIG. 6. It has been demonstrated that the CL device is capable of detecting hypochlorite content in a sample up to 2.5 ng/mL. The volume of used mixed solutions in one channel did not exceed 250 µl. Thus, the CL performance of the luminol CL detection device was demonstrated using the example of detection of ROS (hydrogen peroxide and hypochlorite). The advantage of using a metasurface of metal nanoparticles embedded in a microfluidic chip was also demonstrated, which not only increases the sensitivity of ROS detection, but is also characterized by good stability on the surface of a dielectric substrate in contact with an alkaline solution of luminol. Repeated use of a microfluidic chip with a metasurface is confirmed by a stable CL signal for a long time. It is shown that the use of a microfluidic chip can significantly reduce the volume of a sample with hydrogen peroxide (H ₂ O ₂ ) and sodium hypochlorite (NaClO).

Example 3

The result of the proposed device is shown in Fig. 7, which shows three graphs showing the CL kinetics of the reaction of luminol with silver nanoparticles upon activation of NaClO of various concentrations (see table 1).

Table 1. Concentrations of sodium hypochlorite (NaClO) in solution. Channel NaClO concentration C1 5 µg/ml C2 1 µg/ml C3 0.5 µg/ml C4 25 ng/ml C5 50 ng/ml

In the absence of voltage on the polarization rotator 12, the plane of polarization of the incident CL radiation rotated by 90°, which ensured the passage of the CL radiation from Channel 1 to the photodetector 15 and, at the same time, blocked the radiation from Channel 2 with the NaClO concentration different from the concentration in Channel 1. At voltage U ~ 1.5 V is applied to the electrical inputs of the polarization rotator 12 in the form of a liquid crystal twist cell, where the liquid crystal molecules are aligned along the direction of light propagation, the rotation of the polarization plane of the radiation does not occur and, as a result, the radiation of Channel 2 falls on the photodetector 15, and the radiation Channel 1 is blocked. Quantitative and qualitative analysis of the content of ROS in samples can be performed by comparing the obtained data from Channel 1 and Channel 2. When using a microfluidic chip with a large number of microchannels, the polarization states of the CL radiation of the studied channels should be the same, but different from the polarization of the radiation of the reference channel. Numerous experiments have shown that switching of the CL response radiation from the reference and analyzed samples can be carried out with a frequency from 0.5 Hz to 500 Hz. In this configuration, when the accumulation time is set to 10 ms, as seen in FIG. 7 notice a stable variable CL signal varying with different frequencies. If the device is used in real conditions, the time to obtain the primary result / diagnosis of the degree of ROS content will be significantly reduced. Based on the experimental data on the registration time of the CL kinetics of the reaction of two samples with different concentrations of NaClO, which was about 70 seconds. Unlike traditional devices for CL analysis, the device is extremely compact and mobile, which is confirmed by its dimensions Fig.8, where in the center there is an opaque housing with the key components of the device described earlier, and on opposite sides there is an infusion pump, a generator of control voltage pulses and a container for collecting waste materials.

Thus, the registration of CL responses from various types of ROS demonstrates the operability of the CL device when registering a CL signal alternately from two channels due to the polarization of their radiation by switching the photodetector to registration of radiation from a reference or analyzed sample at a given frequency. The use of encoding the signals of the reference and analyzed samples using radiation polarization allows not only to reduce the measurement time, but also to get rid of moving parts (for example, optomechanical systems of movement and rotation), which simplifies the use of the device and increases the reliability of its operation. The optical scheme makes it possible to switch the radiation photodetector between measurements of the CL intensity and reaction kinetics in the reference and analyzed samples with a variable frequency, which makes it possible to get rid of low-frequency noise and highlight the useful signal against their background.

Claims (2)

1. A device for chemiluminescent analysis of the content of reactive oxygen species in biological and other samples, containing a pump for introducing liquid reagents from containers with a chemiluminophore and test samples into a microfluidic chip using tubes, the output of which is connected by a tube to a drain container, and a photodetector, characterized in that that the microfluidic chip and the photodetector are placed in an opaque housing, and the microfluidic chip is made at least two-channel and contains a dielectric substrate with a metasurface deposited on it from laser-treated metal nanoparticles having a localized plasmon resonance in the chemiluminescence spectral band of the chemiluminophor used, and in an opaque housing two linear mutually orthogonally oriented polarizers are also included, superimposed on different channels of the microfluidic chip, located one after another along the radiation propagation direction, and a polarization rotator installed directly but before the input of the photodetector there is a third linear polarizer of radiation, coinciding in orientation with one of the linear polarizers superimposed on the microfluidic chip. 2. A device for chemiluminescent analysis of the content of reactive oxygen species in biological and other samples according to claim 1, characterized in that the polarization rotator is made in the form of a liquid crystal twist cell, the electrical inputs of which are connected to the outputs of the generator of control voltage pulses, and the photodetector is made with the function photon counts.

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