rus
Issue #3-4/2024
S.V.Zamalutdinova,P.A.Petrova, A.A.Ramonova, D.V.Bagrov
ORIGINALITY WITHOUT PRIVILEGE: COMPARATIVE ANALYSIS OF ELECTROFORMED AND STANDARD NITROCELLULOSE MEMBRANES IN MELATONIN IMMUNOASSAYS
ORIGINALITY WITHOUT PRIVILEGE: COMPARATIVE ANALYSIS OF ELECTROFORMED AND STANDARD NITROCELLULOSE MEMBRANES IN MELATONIN IMMUNOASSAYS
DOI: https://doi.org/10.22184/1993-8578.2024.17.3-4.220.229
Electrospinning enables manufacturing of polymer membranes composed of nanofibres. These membranes find application as filters, wound coatings, and tissue engineering scaffolds. They are also considered promising substrates for immunoassays. Despite the scientific community’s keen interest in the immunoassays based on electrospun membranes, a direct comparison with membranes formed through alternative technologies has not been conducted to date. In this study, we performed such a comparison and demonstrated that the detection of melatonin via enzyme-linked immunosorbent assay (ELISA) is virtually identical on the electrospun membranes and the conventional commercially available membranes.
Electrospinning enables manufacturing of polymer membranes composed of nanofibres. These membranes find application as filters, wound coatings, and tissue engineering scaffolds. They are also considered promising substrates for immunoassays. Despite the scientific community’s keen interest in the immunoassays based on electrospun membranes, a direct comparison with membranes formed through alternative technologies has not been conducted to date. In this study, we performed such a comparison and demonstrated that the detection of melatonin via enzyme-linked immunosorbent assay (ELISA) is virtually identical on the electrospun membranes and the conventional commercially available membranes.
INTRODUCTION
Immunoassay is an integral part of clinical laboratory diagnosis and research. It is based on the specific interaction between antigen and antibody; the dissociation constant of this interaction is usually in the range of ~0.1–1 nM [1]. Any molecule from an antigen-antibody pair can be an analyte - when measuring antibody levels in the blood, they are captured by antigens, and conversely, to detect antigens, they are captured by antibodies.
The nanotechnology development has significantly improved many types of immunoassays. For example, antibody-modified nanoparticles [2] and affinity biosensors created using nanotechnology [3] have entered laboratory practice. Unique record-sensitive immunoanalytical systems based on silicon “nanowells” capable of registering an analyte at a concentration of ~0.1 fM have been developed [4]. In general, nanomaterials have become an integral component of many immunoanalytical systems and have enabled faster, more accurate and cheaper analyses. Based in this background, the use of electroformed membranes as substrates for immunosensors is of particular interest.
Electroformed membranes are polymeric nonwovens consisting of submicron diameter fibres. Most commercially available nonwovens fabricated using conventional approaches consist of fibres with diameters on the order of ~1–100 µm, while for electroformed membranes, fibre diameters are in the range of ~100 nm – 1 µm and sometimes ~10 nm [5]. The small fibre diameter provides the membrane with a high specific surface area (~10 m2/g) and ability to immobilise a large number of receptor layer molecules that will bind the analyte from the sample [6]. A number of works claim that electroformed membranes can be used as a promising element in systems for dot-blotting and similar laboratory analyses. For example, this has been demonstrated in experiments with Dengue virus - it is usually detected by enzyme-linked immunosorbent assay (ELISA), and detection limit can be reduced by about 10 times if the reaction is carried out on the of electroformed membrane fibres surface rather than on the bottom of a polystyrene plate [7, 8].
Electroforming (electrospinning) is based on the idea that a polymer solution jet becomes thinner if there is a strong electric charge on it. Indeed, repulsion between homonymous closely spaced regions of the jet causes it to stretch and reduce its diameter. Laboratory electroforming works as follows (Fig.1). The polymer solution is placed in a syringe and squeezed out through a needle located opposite the conductive element, the collector. A high voltage, on the order of ~10–40 kV, is applied between the collector and the needle. The charged jet of polymer solution moves towards the collector, usually deforming and becoming thinner. The solvent evaporates – and a polymer fibre is formed, the diameter of which is several orders of magnitude smaller than the diameter of the syringe needle. The fibre is deposited chaotically on the collector, where the electroformed membrane is assembled.
The electroforming method was proposed in the middle of the XX century by Academician Petryanov-Sokolov, and it was used to produce air filters to remove aerosol particles from the air [9, 10]. At the beginning of the XXI century, interest in electroforming has increased dramatically, and this can apparently be attributed to two factors. Firstly, scanning electron microscopes have become widespread and are a necessary tool for controlling the structure of electroformed membranes and measuring fibre diameters. Secondly, tissue engineering emerged and it became clear that electroformed membranes made of biodegradable polymers could be used to mimic the extracellular matrix structure [5]. Thus, the electroforming technology was adapted for biological applications, and against this background, the applicability of the membranes fabricated with its help in biosensors was hypothesised.
In a recent review [6] it was shown that electroformed membranes can be used in biological and chemical sensors designed for detection of a wide class of analytes - from metal ions and explosives to whole viruses and bacteria. Among biological sensors, a special place is occupied by immunosensors in which the receptor layer is immobilised on the electroformed fibres surface (analyte binds to the molecules of the receptor layer). In some cases, such sensors can drastically reduce analysis time. For example, using an electroformed membrane sensor, antibodies against MERS-CoV can be detected in less than 6 minutes, which is incomparably shorter than the typical ELISA time (~8 hours) [11]. This radical reduction in time is based on the idea of passing the sample through a porous membrane on which affinity binding of the analyte occurs. A similar, although not so drastic, reduction in analysis time is sometimes achieved using dot-blotting [12, 13]. In this method, a porous nitrocellulose membrane is used as a substrate, and most solutions and reagents are applied using a vacuum pump.
This raises the question: is it true that an electroformed membrane consisting of thin nanofibres has advantages over conventional, commercially available porous membranes? To answer this question, it is necessary to compare the results of ELISA performed on the two types of membranes, and they must be made of the same material. In order for the experiment to be most representative, it is necessary to use a membrane clamp with a vacuum pump, which is designed for dot-blotting. Such an experiment was performed with nitrocellulose (NC) membranes; a reagent kit for melatonin detection was used as the ELISA system. It was shown that the electroformed membrane and commercially available porous NC provided almost identical results, with similar sensitivity and dynamic range.
MATERIALS AND METHODS
Membranes. Two types of NC membranes were used in this work - commercially available, mass-produced Bio-rad membranes (number 1620115), and original, electroformed membranes. They were fabricated as follows. The Bio-rad membrane was dissolved in acetone, dimethylformamide (DMFA) or a mixture thereof. The concentration of NC was 100 mg/ml. Electrospinning was carried out on a Nanofiber Electrospinning Unit (China) under the following conditions 38–40 kV voltage, 15 cm collector distance, 0.514 mm needle inner diameter (21g gauge), and 1 ml/h solution feed rate.
Scanning electron microscopy. For scanning electron microscopy (SEM) study, the samples were coated with a thin layer of gold with a thickness of 20 nm using an Eiko IB3 sputterer (Japan). The structure was examined using a TM3000 SEM (Hitachi, Japan) at an accelerating voltage of 15 kV. Images were processed using Fiji software.
Immunoassay. A commercial kit KSA908Gel1 from Cloude-Clone Corp (USA) was used to detect melatonin by ELISA. Reactions were performed in the cells of a 96-well plate or on the surface of NC membranes. In both cases, the assay involved a series of steps that are commonly used in competitive ELISA (Table 1). After each step, the surface on which the reactions were performed was washed.
The analysis is based on the idea that a special reagent, a biotinylated competitor, must be introduced into each solution under investigation. This is a conjugate of albumin with MT and biotin, and it competes with the MT contained in the test solution for binding to antibodies immobilised on the surface. The biotin in this conjugate allows it to be shown using standard reagents such as streptavidin with horseradish peroxidase and substrates for optical detection (colourimetric or chemiluminescent).
Three buffers were used for reagent dilution: phosphate-salt buffer (PBS, pH 7.4), phosphate-salt buffer with 0.075% Tween-20 (PBST), and proprietary buffer with pH 9.5 for antibody dilution (Coating buffer, Cloud-clone). Blocking buffer solution (Cloud-clone) was used to block non-specific binding sites. Between incubations, the solid carrier was washed in PBST buffer to remove unbound molecules.
In the case of experiments in a 96-well plate, all incubations were performed in a thermostat at 37 °C and shaking at 130 rpm. The contents of the wells were removed by decantation. In the final step of the assay, 90 µl of tetramethylbenzidine was added to each well as a substrate for horseradish peroxidase, incubated for 15 min, then the reaction was stopped by adding 50 µl of 0.5 M sulfuric acid. Optical density of the plate wells was measured on a Hidex Chameleon reader (UK) at a wavelength of 450 nm.
In the case of membrane assays, incubations were performed at room temperature without shaking. The contents of the wells were removed by drawing the liquid through the membrane using a pump. At the final stage, the results were visualised using Affinity ECL chemiluminescent substrate (ProteinAntibodies LLC, RF). Chemiluminescence intensity was measured on a ChemiDoc device (BioRad, USA).
RESULTS AND DISCUSSION
Membrane morphology. The electroforming method is applicable to a very wide class of polymers - polyolefins, polyamides, polyesters, and others. At the same time, the number of papers devoted to the electroforming of NCs is relatively small [14,15]. This seems particularly strange in view of the fact that it was in experiments with NCs that the electroforming process was carried out for the first time [9, 10].
For this work, electroforming conditions were chosen to produce membranes consisting of fibres with an average diameter of 280 ± 60 nm (Fig.2a, b). This is generally close to the size of pore boundaries in commercially available membranes (Fig.2, c, d). Optimal results were achieved using a two-component solvent consisting of a mixture of acetone and DMFA in a ratio of 7 : 3 or 8 : 2 by volume. A similar approach was used in [16]. When attempting to electroform NC from a solution in acetone, the syringe needle quickly clogged, and we failed to obtain a nonwoven membrane, although such experiments are described in [14, 15]. When using a solution of NC in DMFA, a different problem arose: DMFA has a relatively high boiling point of 153 °C and belongs to non-volatile solvents, it did not evaporate quickly enough, and a continuous film was formed on the collector. As a result, it turned out that the two-component solvent was optimal and allowed to obtain thin NC fibres.
Membranes of the two types described (Fig.2) were used in dot-blotting experiments.
Immunoassay results. To evaluate applicability of electroformed membranes in dot-blotting, it is necessary to select an analyte and a set of biochemical reagents for its detection. Melatonin (N-acetyl-5-methoxytryptamine, molecular weight 232 g/mol), a hormone that is responsible for regulating the sleep-wake cycle, was chosen as the analyte. Melatonin (MT) is usually measured using high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS), which is the method used by clinical diagnostic laboratories in Russia. At the same time, there are commercially available kits for determination of MT by ELISA. MT is a low molecular weight substance, so the capture antibodies are not directed against it, but against its conjugate with bovine serum albumin, and the same conjugate is used as a standard. The assay is performed in a competitive manner – a biotinylated competitor is introduced into the sample being assayed and the MT in the sample competes with it for binding to the antibody. Biotin can then be detected by conjugating streptavidin with horseradish peroxidase. A typical calibration curve of such an assay obtained in a conventional 96-well plate is shown in Fig.3a – as the concentration of MT increases, the signal decreases.
Reagents from the described kit were used for dot-blotting, using a vacuum clamp (Fig.3b). When a membrane is placed in it, it is similar to a 96-well plate with a permeable bottom. The liquid introduced into the cells does not flow through the porous bottom spontaneously because it is held back by the surface tension force. However, when the pump is switched on, a pressure differential occurs which causes the liquid to seep through the membrane and collect in a reservoir below it. The rate at which liquid flows through the membrane depends not only on the pressure drop created by the pump, but also on the location of the cell within the clamp. It is likely that the pressure drop is higher near the pump connection than away from it. Therefore, in order to compare the two types of membranes correctly, they were placed in adjacent rows in the central part of the clamp, with the row of cells covered by the electroformed membrane placed between the two rows covered by the fabricated membrane (Fig.3c, d). This arrangement of membranes allowed us to minimise variations in reagent application conditions and focus on membrane properties.
The calibration curves obtained with the conventional and electroformed membrane are compared in Fig.3c. These are decreasing curves similar to that obtained with the conventional ELISA (Fig.3a). The calibration curves obtained with the two types of membranes match well. We were unable to detect any obvious differences in sensitivity or in the width of the dynamic range. Apparently, in this case, the structure of nanofibres and the electrostatic charge present on them have little influence on the analysis results.
CONCLUSIONS
In this study, we performed a competitive ELISA for melatonin detection; the ELISA was performed in a dot-blotting variant using two types of nitrocellulose membranes, standard commercial and electroformed nanofibre membranes. It turned out that the membranes of these two types showed almost identical behaviour, with no obvious difference in sensitivity or dynamic range width. Nanostructured EF membranes may be of interest in other applications such as air purification or tissue engineering, but to our knowledge they have no clear advantage in immunoassays. The possibilities of improving the analytical performance of ELISA achieved by electroformed membranes require further investigation.
ACKNOWLEDGMENTS
This work was supported by the grant of the Russian Science Foundation, project No. 21-74-10042.
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Immunoassay is an integral part of clinical laboratory diagnosis and research. It is based on the specific interaction between antigen and antibody; the dissociation constant of this interaction is usually in the range of ~0.1–1 nM [1]. Any molecule from an antigen-antibody pair can be an analyte - when measuring antibody levels in the blood, they are captured by antigens, and conversely, to detect antigens, they are captured by antibodies.
The nanotechnology development has significantly improved many types of immunoassays. For example, antibody-modified nanoparticles [2] and affinity biosensors created using nanotechnology [3] have entered laboratory practice. Unique record-sensitive immunoanalytical systems based on silicon “nanowells” capable of registering an analyte at a concentration of ~0.1 fM have been developed [4]. In general, nanomaterials have become an integral component of many immunoanalytical systems and have enabled faster, more accurate and cheaper analyses. Based in this background, the use of electroformed membranes as substrates for immunosensors is of particular interest.
Electroformed membranes are polymeric nonwovens consisting of submicron diameter fibres. Most commercially available nonwovens fabricated using conventional approaches consist of fibres with diameters on the order of ~1–100 µm, while for electroformed membranes, fibre diameters are in the range of ~100 nm – 1 µm and sometimes ~10 nm [5]. The small fibre diameter provides the membrane with a high specific surface area (~10 m2/g) and ability to immobilise a large number of receptor layer molecules that will bind the analyte from the sample [6]. A number of works claim that electroformed membranes can be used as a promising element in systems for dot-blotting and similar laboratory analyses. For example, this has been demonstrated in experiments with Dengue virus - it is usually detected by enzyme-linked immunosorbent assay (ELISA), and detection limit can be reduced by about 10 times if the reaction is carried out on the of electroformed membrane fibres surface rather than on the bottom of a polystyrene plate [7, 8].
Electroforming (electrospinning) is based on the idea that a polymer solution jet becomes thinner if there is a strong electric charge on it. Indeed, repulsion between homonymous closely spaced regions of the jet causes it to stretch and reduce its diameter. Laboratory electroforming works as follows (Fig.1). The polymer solution is placed in a syringe and squeezed out through a needle located opposite the conductive element, the collector. A high voltage, on the order of ~10–40 kV, is applied between the collector and the needle. The charged jet of polymer solution moves towards the collector, usually deforming and becoming thinner. The solvent evaporates – and a polymer fibre is formed, the diameter of which is several orders of magnitude smaller than the diameter of the syringe needle. The fibre is deposited chaotically on the collector, where the electroformed membrane is assembled.
The electroforming method was proposed in the middle of the XX century by Academician Petryanov-Sokolov, and it was used to produce air filters to remove aerosol particles from the air [9, 10]. At the beginning of the XXI century, interest in electroforming has increased dramatically, and this can apparently be attributed to two factors. Firstly, scanning electron microscopes have become widespread and are a necessary tool for controlling the structure of electroformed membranes and measuring fibre diameters. Secondly, tissue engineering emerged and it became clear that electroformed membranes made of biodegradable polymers could be used to mimic the extracellular matrix structure [5]. Thus, the electroforming technology was adapted for biological applications, and against this background, the applicability of the membranes fabricated with its help in biosensors was hypothesised.
In a recent review [6] it was shown that electroformed membranes can be used in biological and chemical sensors designed for detection of a wide class of analytes - from metal ions and explosives to whole viruses and bacteria. Among biological sensors, a special place is occupied by immunosensors in which the receptor layer is immobilised on the electroformed fibres surface (analyte binds to the molecules of the receptor layer). In some cases, such sensors can drastically reduce analysis time. For example, using an electroformed membrane sensor, antibodies against MERS-CoV can be detected in less than 6 minutes, which is incomparably shorter than the typical ELISA time (~8 hours) [11]. This radical reduction in time is based on the idea of passing the sample through a porous membrane on which affinity binding of the analyte occurs. A similar, although not so drastic, reduction in analysis time is sometimes achieved using dot-blotting [12, 13]. In this method, a porous nitrocellulose membrane is used as a substrate, and most solutions and reagents are applied using a vacuum pump.
This raises the question: is it true that an electroformed membrane consisting of thin nanofibres has advantages over conventional, commercially available porous membranes? To answer this question, it is necessary to compare the results of ELISA performed on the two types of membranes, and they must be made of the same material. In order for the experiment to be most representative, it is necessary to use a membrane clamp with a vacuum pump, which is designed for dot-blotting. Such an experiment was performed with nitrocellulose (NC) membranes; a reagent kit for melatonin detection was used as the ELISA system. It was shown that the electroformed membrane and commercially available porous NC provided almost identical results, with similar sensitivity and dynamic range.
MATERIALS AND METHODS
Membranes. Two types of NC membranes were used in this work - commercially available, mass-produced Bio-rad membranes (number 1620115), and original, electroformed membranes. They were fabricated as follows. The Bio-rad membrane was dissolved in acetone, dimethylformamide (DMFA) or a mixture thereof. The concentration of NC was 100 mg/ml. Electrospinning was carried out on a Nanofiber Electrospinning Unit (China) under the following conditions 38–40 kV voltage, 15 cm collector distance, 0.514 mm needle inner diameter (21g gauge), and 1 ml/h solution feed rate.
Scanning electron microscopy. For scanning electron microscopy (SEM) study, the samples were coated with a thin layer of gold with a thickness of 20 nm using an Eiko IB3 sputterer (Japan). The structure was examined using a TM3000 SEM (Hitachi, Japan) at an accelerating voltage of 15 kV. Images were processed using Fiji software.
Immunoassay. A commercial kit KSA908Gel1 from Cloude-Clone Corp (USA) was used to detect melatonin by ELISA. Reactions were performed in the cells of a 96-well plate or on the surface of NC membranes. In both cases, the assay involved a series of steps that are commonly used in competitive ELISA (Table 1). After each step, the surface on which the reactions were performed was washed.
The analysis is based on the idea that a special reagent, a biotinylated competitor, must be introduced into each solution under investigation. This is a conjugate of albumin with MT and biotin, and it competes with the MT contained in the test solution for binding to antibodies immobilised on the surface. The biotin in this conjugate allows it to be shown using standard reagents such as streptavidin with horseradish peroxidase and substrates for optical detection (colourimetric or chemiluminescent).
Three buffers were used for reagent dilution: phosphate-salt buffer (PBS, pH 7.4), phosphate-salt buffer with 0.075% Tween-20 (PBST), and proprietary buffer with pH 9.5 for antibody dilution (Coating buffer, Cloud-clone). Blocking buffer solution (Cloud-clone) was used to block non-specific binding sites. Between incubations, the solid carrier was washed in PBST buffer to remove unbound molecules.
In the case of experiments in a 96-well plate, all incubations were performed in a thermostat at 37 °C and shaking at 130 rpm. The contents of the wells were removed by decantation. In the final step of the assay, 90 µl of tetramethylbenzidine was added to each well as a substrate for horseradish peroxidase, incubated for 15 min, then the reaction was stopped by adding 50 µl of 0.5 M sulfuric acid. Optical density of the plate wells was measured on a Hidex Chameleon reader (UK) at a wavelength of 450 nm.
In the case of membrane assays, incubations were performed at room temperature without shaking. The contents of the wells were removed by drawing the liquid through the membrane using a pump. At the final stage, the results were visualised using Affinity ECL chemiluminescent substrate (ProteinAntibodies LLC, RF). Chemiluminescence intensity was measured on a ChemiDoc device (BioRad, USA).
RESULTS AND DISCUSSION
Membrane morphology. The electroforming method is applicable to a very wide class of polymers - polyolefins, polyamides, polyesters, and others. At the same time, the number of papers devoted to the electroforming of NCs is relatively small [14,15]. This seems particularly strange in view of the fact that it was in experiments with NCs that the electroforming process was carried out for the first time [9, 10].
For this work, electroforming conditions were chosen to produce membranes consisting of fibres with an average diameter of 280 ± 60 nm (Fig.2a, b). This is generally close to the size of pore boundaries in commercially available membranes (Fig.2, c, d). Optimal results were achieved using a two-component solvent consisting of a mixture of acetone and DMFA in a ratio of 7 : 3 or 8 : 2 by volume. A similar approach was used in [16]. When attempting to electroform NC from a solution in acetone, the syringe needle quickly clogged, and we failed to obtain a nonwoven membrane, although such experiments are described in [14, 15]. When using a solution of NC in DMFA, a different problem arose: DMFA has a relatively high boiling point of 153 °C and belongs to non-volatile solvents, it did not evaporate quickly enough, and a continuous film was formed on the collector. As a result, it turned out that the two-component solvent was optimal and allowed to obtain thin NC fibres.
Membranes of the two types described (Fig.2) were used in dot-blotting experiments.
Immunoassay results. To evaluate applicability of electroformed membranes in dot-blotting, it is necessary to select an analyte and a set of biochemical reagents for its detection. Melatonin (N-acetyl-5-methoxytryptamine, molecular weight 232 g/mol), a hormone that is responsible for regulating the sleep-wake cycle, was chosen as the analyte. Melatonin (MT) is usually measured using high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS), which is the method used by clinical diagnostic laboratories in Russia. At the same time, there are commercially available kits for determination of MT by ELISA. MT is a low molecular weight substance, so the capture antibodies are not directed against it, but against its conjugate with bovine serum albumin, and the same conjugate is used as a standard. The assay is performed in a competitive manner – a biotinylated competitor is introduced into the sample being assayed and the MT in the sample competes with it for binding to the antibody. Biotin can then be detected by conjugating streptavidin with horseradish peroxidase. A typical calibration curve of such an assay obtained in a conventional 96-well plate is shown in Fig.3a – as the concentration of MT increases, the signal decreases.
Reagents from the described kit were used for dot-blotting, using a vacuum clamp (Fig.3b). When a membrane is placed in it, it is similar to a 96-well plate with a permeable bottom. The liquid introduced into the cells does not flow through the porous bottom spontaneously because it is held back by the surface tension force. However, when the pump is switched on, a pressure differential occurs which causes the liquid to seep through the membrane and collect in a reservoir below it. The rate at which liquid flows through the membrane depends not only on the pressure drop created by the pump, but also on the location of the cell within the clamp. It is likely that the pressure drop is higher near the pump connection than away from it. Therefore, in order to compare the two types of membranes correctly, they were placed in adjacent rows in the central part of the clamp, with the row of cells covered by the electroformed membrane placed between the two rows covered by the fabricated membrane (Fig.3c, d). This arrangement of membranes allowed us to minimise variations in reagent application conditions and focus on membrane properties.
The calibration curves obtained with the conventional and electroformed membrane are compared in Fig.3c. These are decreasing curves similar to that obtained with the conventional ELISA (Fig.3a). The calibration curves obtained with the two types of membranes match well. We were unable to detect any obvious differences in sensitivity or in the width of the dynamic range. Apparently, in this case, the structure of nanofibres and the electrostatic charge present on them have little influence on the analysis results.
CONCLUSIONS
In this study, we performed a competitive ELISA for melatonin detection; the ELISA was performed in a dot-blotting variant using two types of nitrocellulose membranes, standard commercial and electroformed nanofibre membranes. It turned out that the membranes of these two types showed almost identical behaviour, with no obvious difference in sensitivity or dynamic range width. Nanostructured EF membranes may be of interest in other applications such as air purification or tissue engineering, but to our knowledge they have no clear advantage in immunoassays. The possibilities of improving the analytical performance of ELISA achieved by electroformed membranes require further investigation.
ACKNOWLEDGMENTS
This work was supported by the grant of the Russian Science Foundation, project No. 21-74-10042.
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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