In the present work, the performance of ultra-high molecular weight polyethylene (UHMWPE) barrier nets in marine environments is investigated by Fourier transform infrared spectroscopy, thermogravimetry, scanning electron microscopy, and tensile experiments. The chemical, morphological, thermal stability, and strength changes after aging in salt spray, hygrothermal, and ultraviolet (UV) environments are characterized. An environmental spectrum is designed to simulate a real service environment and predict the service life of UHMWPE. The results show that UV energy can activate UHMWPE molecules and lead to chain breaking, which lowers the breaking strength more efficiently than salt spray. In a hygrothermal environment, the UHMPE fibers bond into clumps, which causes a slight increase in breaking strength after the initial rapid decrease with aging time. The acceleration ratio of the environmental spectrum increases with increasing aging time, which may be caused by the cross-linking and degradation of macromolecular chains in the material. The environmental spectrum given by this work can be used to evaluate performance and predict the service life of UHMWPE barrier nets in marine environments. Owing to its high strength, superior wear resistance, and chemical stability, ultra-high molecular weight polyethylene (UHMWPE) has been used as barrier to prevent the clogging of cold source intakes of nuclear power plants by marine organisms ,2,3,4,5.Environmental exposure tests and laboratory-accelerated aging tests such as hygrothermal and ultraviolet irradiation (UV) are used to evaluate the performance deterioration of UHMWPE ,13,14,15In a nuclear power plant, UHMWPE is used as a barrier net to prevent the ingress of plankton and ensure cleaning of the cooling water from the ocean. In this case, the barrier net is exposed to the marine environment and to salt spray aging processes. In this paper, UV irradiation, hygrothermal, and salt spray aging tests are used to simulate the operating environment of UHMWPE in a tropical marine environment in China. Surface morphologies, fracture strength, and chemical variations are determined. Subsequently, an environmental spectrum is established to simulate the real service environment considering all of the factors, and the accelerated ratio of the environmental spectrum is evaluated.The material used in this study was ultra-high molecular weight polyethylene (UHMWPE) with a wire diameter of 0.6 mm and a mesh diameter of 3.5–4.0 mm. The UHMWPE barrier net was woven into a network through knots by several fine fibers, which were twisted into a strand. Samples with a length of 300 mm and a width of 4.5 mesh were taken from an as-received barrier net for acceleration aging tests in a laboratory, as shown in 2. For the UV test, the temperature was set to 60 °C, the samples were irradiated for 24 h per cycle, and a total of 10 cycles were performed. The UV aging test equipment was an UV weather test box with a type 1A (UVA-351) lamp, supplied by Shanghai Forest Instrument Co., LTD. The wavelength of UV radiation was 340 nm, and the intensity was 0.76 W/mThe salt spray test equipment was conducted with the LRHS-412-RFHY type test chamber of Forest Frequency Company. The samples were sprayed by 5 wt.% NaCl at 35 °C according to ISO4611-2010. Each cycle lasted for 10 days and the longest exposure time was 60 days of six cycles. The LHS-100CL type test chamber was used to perform the hygrothermal experiment. The experiment condition was set to 50 °C and 93% humidity, and the conductivity of the water vapor used to maintain the humidity of the test chamber was less than 20 μS/cm. For each cycle, the samples were exposed in the chamber for 10 days. The longest exposure time was 60 days of six cycles.2 for another 12 h at 50 °C.An environmental spectrum was designed to simulate the performance of the UHMWPE barrier net in the actual environment according to the ultraviolet, hygrothermal, and salt spray experimental results. The environmental spectrum was composed of ultraviolet, hygrothermal, and salt spray experiments, as shown in The tensile properties were characterized by a GOTECH tensile testing machine. According to Chinese standard GB/T4925-2008, the breaking strength of UHMWPE after natural aging and artificial aging was derived. As shown in The scanning electron microscope  was used to observe morphology of samples with a length of about 1 cm in the middle part of the mesh. The sample surfaces were coated with a layer of gold with a thickness of 8~10 nm to ensure the conductivity. The samples were attached to the sample stage with conductive adhesive. During the tests, the working distance (WD) of SEM was about 8.5 mm, the working voltage was 10 kV (EHT = 10 kV), and the signal mode was secondary electron mode.−1. It was tested in the wave-number range of 4000~400 cm−1. The changes in the fiber groups were judged according to the main absorption band and characteristic frequency in the spectra.Infrared spectral analysis was carried out in a Nicolet iS50 Fourier transform infrared spectrometer in attenuated total reflectance (ATR) mode because of the opacity of UHMWPE fibers. The laboratory temperature was kept at 20 °C and the relative humidity was below 65%. The number of scans was 32 and the resolution was 4 cmThermogravimetry analysis (TGA) was performed to observe the effect of the aging method on oxidation temperature and to determine the thermal stability with a TGA-Q5000 . The samples were heated from room temperature to 800 °C at a heating rate of 10 °C/min in a nitrogen atmosphere. −1 reflect the CH2 asymmetric stretching and stretching vibration. The peaks at 1470 and 1461 cm−1 are CH2 in-plane bending and wagging vibration of UHMWPE chains. The 730 and 716 cm−1 peaks belong to C-H out-of-plane bending vibration [Aging can lead to long chain breaking and new side group formation of UHMWPE, and the changes in chain configuration and conformation can be reflected in infrared spectroscopy. ibration ,18. The −1, which can be assigned to the C=O stretching vibration, and a band around 1163 cm−1 denoting C–O–C stretching vibration appears on the spectrum of UV irradiation [−1 representing C=C stretching appears on the spectrum of both UV irradiation and salt spray. The three peaks appearing on the spectrum of UV irradiation also appear on the spectrum aging in the environmental spectrum for 19 cycles, as shown in adiation ,20. The 1,a2)), the surface of the fiber is corroded. The existence of chloride ion with a small radius and high permeability in the salt spray environment results in the obvious shedding of the UHMWPE filament fiber. After 60 days of hygrothermal aging ), the surface of the UHMWPE fiber is no longer smooth, but no obvious surface damage is found in the high temperature and humid environment. 1,c2) shows that the fiber has obvious cracks and damage after UV irradiation for 10 days, which affects the mechanical properties of the material.SEM is used to examine the surface morphology of the fibers and analyze the failure mechanism. The environmental spectrum as described in The deterioration in mechanical properties is one of the most evident effects of aging, which significantly affects the service safety and lowers the service life of UHMWPE. 1,a2)), slight adhesion between thin fibers is observed. After hygrothermal aging ), several fine fibers are bonded into clumps, resulting in an increase in cross section, which slows down the loss of mechanical properties to a certain extent. After UV irradiation aging ), the fine fibers basically exist in the form of a single root without adhesion, and the fracture surface shows a neat brittle section, indicating a decrease in toughness [oughness . Figure To analyze the accelerated ratio of the environmental spectrum, the breaking strength of the UHMWPE fiber interception net aging in the environmental spectrum for different cycles and in the real service environment for different times is compared in The environmental factors that affect the service life of the UHMWPE fiber interception net include temperature, humidity, light, chemical, and biological erosion . To obtaAccording to the experimental results of aging in the individual salt spray, hygrothermal, and UV environments, the environmental spectrum is designed considering the influence of multiple factors existing in the real service environment. The UHMWPE fiber interception net is exposed to the interaction of light, heat, water, and salt spray in the actual marine environment, as schematically shown in (1)The breaking strength deterioration in UV is more significant than that in the salt spray environment owing to changes in polymer chemical structure caused by chain breaking under light.(2)After hygrothermal aging, some fine fibers bond into clumps, which lowers the attenuation of breaking strength.(3)The environmental spectrum is designed according to the real environmental factors affecting the aging property. The degradation mechanism after aging in the environmental spectrum is the same to that in the real service environment.(4)The acceleration ratio of the environmental spectrum to the real service environment increases with the elongation of aging time, which may be caused by the cross-linking and degradation of macromolecular chains in the material.To investigate the performance of the UHMWPE fiber interception net in the marine environment, the material is aged in salt spray, hygrothermal, and ultraviolet environments; a designed environmental spectrum; and the real service environment. The conclusions are as follows:


Type 1 diabetes mellitus (T1D) is, next to obesity and asthma, the most common chronic disease in children in Poland. The results of T1D treatment strongly depend on the patient’s compliance with therapeutic recommendations, which entails the use of necessary health services. Based on a retrospective analysis of the data on health services provided in 2016–2020 to over 15.5 thousand patients with T1D in Poland, we assessed the compliance of the actual model of treatment of T1D in children with the current guidelines. It was found that only about 50% of patients received the number of diabetes consultations corresponding to the recognized standards, with about 15% of children with T1D remaining outside the public healthcare system. In the case of many outpatient services , the number of consultations was extremely low—one order of magnitude lower than in general population and dropped even lower in 2020. This shows that the health needs of children with T1D are not being met within the public healthcare system. The COVID-19 pandemic caused significant limitations in access to healthcare in Poland. Compared to the pre-pandemic period there was a significant decrease (−27% compared to 2019) in the number of hospitalizations, and a substantial increase (+22% compared to 2019) in the number of diabetic ketoacidoses (DKA) cases. The proportion of hospitalizations caused by DKA rose to 8.9% compared to 7.3% in 2019. Type 1 diabetes mellitus (T1D) is an immune-mediated disease. It is a disorder characterized by progressive destruction of pancreatic beta cells, leading to the cessation of insulin secretion and consequent hyperglycemia . For morThe main clinical and social problems associated with T1D are the acute (particularly diabetic ketoacidosis (DKA)) and chronic  complications caused by the disease . Their oThere is a consensus that modern T1D therapy should include the use of modern medications (rapid-acting insulin), advanced glycemic monitoring technologies , and care by various providers  . CurrentAvailable studies indicate a significant association of T1D treatment outcome with adherence to established treatment recommendations , includiThe outbreak of the SARS-CoV-2 pandemic resulted in limited access to diagnostics and treatment of many diseases. This phenomenon occurred in many countries and led to inter alia the so-called health debt . In the Poland is the eighth most populous country in Europe (fifth in the EU). It is also the largest country in Central and Eastern Europe, representative of the former states of the Eastern Bloc. Therefore, epidemiological data from the period before, during, and after the pandemic can show the scale of the challenges in diabetes mellitus management in the region and serve as the basis for planning health policies in this area.The objectives of this study were (1) to analyze the use of healthcare services by children and adolescents with T1D in Poland, (2) to assess the consistency of the actual frequency of use of specific services with recognized standards and guidelines, and (3) to determine the impact of restrictions resulting from the COVID-19 pandemic on the use of healthcare services by children and adolescents with T1D.In Poland, T1D is, next to obesity and asthma, the most common chronic disease in children . AccordiPeople with diabetes are cared for by diabetologists—doctors who specialize in the diagnosis and treatment of diabetes. In the case of children and adolescents, pediatric diabetologists work in diabetes clinics for the children. In addition to the doctors, diabetes nurses and dieticians also care for the children in these clinics. As part of the medical advice, glycated hemoglobin (HbAc1) levels and other laboratory tests are performed in these clinics . ChildreThe Polish Diabetes Association (PTD) guidelines for the therapeutic management of diabetes in children and adolescents are consistent with current standards in other developed countries . Unlike The Polish healthcare system is experiencing many difficulties. Due to the lack of sufficient financial resources and personnel problems , there are limitations on the availability of certain services  resulting in extended waiting periods  .In the literature, there are numerous examples of studies assessing general health status, metabolic compensation, psychological well-being, and social functioning of children and adolescents with T1D in Poland. However, there is no information on the actual use of healthcare services by children and adolescents.The first case of COVID-19 in Poland was diagnosed on 4 March 2020. The increase in the number of cases, although delayed and slower than in other European countries, resulted in the declaration of an epidemic state by the national authorities and the subsequent introduction of many restrictions and organizational changes in the healthcare system in the second half of March 2020.During the pandemic period, scheduled admissions were suspended at many hospitals and outpatient (ambulatory care) clinic operations were limited. The restrictions were strictly enforced until June 2020. In the meantime, the possibility of remote medical assistance was slowly implemented. However, telephone consultations were predominantly used, which significantly limited the physician’s ability to accurately diagnose the problem and appropriately guide treatment ,20.For patients with T1D, the restrictions made it difficult to have regular follow-up appointments. There was also the reluctance to use healthcare services due to the fear of SARS-CoV-2 infection, which raised serious concerns about possible health consequences . PatientThe research utilizes the epidemiological registry-based study approach to determine the number and frequency of various health services used by patients with T1D in Poland. Results were contrasted with established standards and guidelines for T1D treatment in children and adolescents. The data from the pre-pandemic period (years 2016–2019) were compared with the data from the first year of the COVID-19 pandemic.The research material consisted of data collected by the National Health Fund (NHF). NHF is the state-owned entity responsible for settling the costs of benefits provided under the public health insurance system . For this purpose, the NHF collects and processes the data on the number and type of services provided to individual insured persons. These data include, among others, information on clinical diagnoses (following the classification contained in ICD-10). However, the NHF does not possess information about the tests performed  or their results . All children and adolescents (<19 years old) are entitled to health services financed by NHF.The structure of the data collected by the NHF enables the determination of the number of individual healthcare services provided in each of the years of the analyzed period, as well as the determination of the number of persons  to whom they were provided.For the purposes of population comparisons, the official data on the population of Poland aged under 19 were used. These data are made available to the public by the Central Statistical Office (CSO).The number of patients at the end of the accounting/reporting period.The number of new patients/diagnoses in the accounting/reporting period.The number of patients (persons under 19 years of age with diagnosis E.10 according to ICD-10) including (NHF data):In the general population (CSO data);Beneficiaries of healthcare services provided under the universal health insurance scheme (NHF data).The number of persons under 19 years of age:One diabetes consultation;Two diabetes consultations;Three diabetes consultations;Four diabetes consultations;Healthcare services associated with hospitalization in the diabetic ward.The number of patients from pt. 1 above who during the accounting/reporting year were provided with (NHF data):Ophthalmological consultation (examination for retinopathy);Neurological consultation (diagnosis of neuropathy);Mental health advice .The number of persons (separately from points 1 and 2 above) who during the accounting/reporting year were provided with specific healthcare services (NHF data):The subject of the analysis was information concerning children and adolescents—defined as beneficiaries under 19 years of age (about 6.5–7 million people). T1D patients (ca. 14–16 thousand persons) were distinguished from this group—the identification criterion was a diagnosis E10 according to ICD-10. DKA cases  were identified based on diagnosis E10.1 according to ICD-10. The search involved the identification of:Due to the sensitive nature of the collected information, following the regulations on the protection of personal data in force in Poland, the NHF does not provide data allowing aggregation of information on services provided to a specific person . For this reason, the analysis of the collected information was performed using descriptive statistics tools.According to the procedures for verification and control of settlements, data in the NHF system may be corrected by both providers and the NHF. This causes a delay in data availability. It is assumed that after 2 quarters the modification of information in the system may take place only in extraordinary circumstances. In this study, information for the period 2016–2020, as of the end of June 2021, was the subject of analysis.The anonymous character of the analyzed data resulted in the lack of necessity to obtain consent for participation in the study. Nevertheless, the research program under which the study was conducted received a positive assessment from the IRB of the Centre of Postgraduate Medical Education (no. 501-4-44-28-18).Poland’s population decreased during the period under analysis from 38.437 million at the end of 2015 to 38.265 million at the end of 2020. In contrast, the number of people under 19 years of age showed a slight upward trend. A vast majority of children and adolescents (about 96–97%) have used health services provided under the universal health insurance scheme. The only exception was in 2020 when almost 11% of people under 19 years of age did not use any health benefit under the public system. This represented a several-fold increase from previous years. Children and adolescents with T1D were consistently about 0.2% of the total number of beneficiaries .The number of children and adolescents with T1D who received healthcare services under the universal health insurance system in Poland in particular years showed a steady upward trend throughout the analyzed period . It incrThe distribution of the incidence of T1D in the population under 19 years of age benefiting from the public health system is shown in During the study period, children with T1D most frequently received diabetes consultations provided on an outpatient basis . These sThe number of services provided in the inpatient setting  in 2016–2019 oscillated between 6.9–7.5 thousand cases (corresponding to about 47–50% of the population of children with T1D in a given year) and decreased to about 5.5 thousand cases (35.4% of the population of children with T1D) in 2020. . Most hoThe collected data show a large time variability in the number of DKA cases diagnosed in children under 19 years of age by a quarter from 2017 to 2020 . During Data collected by the NHF indicate an extremely low number of outpatient ophthalmic, neurological, and mental health services provided to patients with T1D. In patients with T1D, such consultations occur ten times less frequently than in the general population. The data in Epidemiology of T1D in Poland is characterized by comparable incidence and slightly lower prevalence as in other European countries . The chaIn accordance with the first objective of this study, we found that the vast majority (approximately 85%) of patients with T1D annually receive diabetes counselling provided in outpatient care units under the universal health insurance scheme. The number of outpatient consultations is one of the primary components of assessing patient adherence to treatment recommendations , as wellIn the case of over a half of children and adolescents with T1D in Poland (51.7%), the frequency of these visits corresponds to or even exceeds the minimum PTD recommendations in this respect (at least 4 visits per year). This indicates a high level of patients’ (their guardians) involvement in the therapeutic process . This isOf concern is the suboptimal use of diabetes care by the rest of the population of children and adolescents with T1D, including a relatively high number of patients receiving two or fewer consultations per year (32.5%). This value is higher than that obtained in other studies . The avaFor the remaining outpatient services that should be provided to children and adolescents with T1D according to current treatment standards, the frequency of use of those examinations and consultations that do not fall under diabetes counselling was assessed. These included ophthalmological advice (including funduscopic examination), neurological advice, and mental health services . These services are important in the prevention of T1D complications ,36.The comparison of the obtained results with the current recommendations of PTD  and the available results of analyses of the functioning of the care of children and adolescents with T1D in other countries , indicatThere are many possible reasons for such a low degree of adherence by patients (their caregivers) to recommendations for regular eye and neurological examinations. This may result from objective reasons—lack of knowledge about the recommended frequency of examinations and consultations, or subjective ones—underestimation or neglect of the problem by the patient/carer ,39. In tOne of the primary causes of hospitalization of children with T1D is the occurrence of DKA. According to an extensive analysis by Usher-Smith et al., the incidence of DKA varies widely across countries (from 12.8% to 80%) and is inversely associated with gross domestic product, latitude, and background incidence of T1D . In chilThe number of hospitalizations observed in the study was much higher (on average about 6900 cases per year) and corresponded to about 47–50% of the population of children with T1D in a given year. It was therefore much higher throughout the study period than observed in other studies ,41,42,43The development of the COVID-19 pandemic and the changes in healthcare systems in response to it have raised a number of concerns about the impact of these measures on the health of children with T1D. There have been pointed out possible risks from limited access to healthcare , impaireIn the present study, it was possible to observe significant changes in the number and structure of health services provided to children with T1D in the period preceding the outbreak of the COVID-19 pandemic and during the restrictions and organizational changes in the functioning of the healthcare system. The introduction of lockdown in Poland  corresponds with a break in the previously sustained upward trend in the number of DKA cases. After a periodic decline in the number of DKA cases in Q2 2020, there was a sharp increase in their number in Q3 2020, and the total number of DKA in 2020 was almost 22% higher than in the previous year. This may indicate a phenomenon of deferred demand for health services due to fear of SARS-CoV2 infection or because of difficulties in obtaining them, as observed in other health systems . At the The number of hospitalizations due to new T1D diagnoses remained relatively unchanged, the reduction in the total number of hospitalizations resulted from the significantly lower number of hospitalizations due to other causes, mainly planned treatment—hospitalizations for diagnostics and consultations. In the long term, this may cause delays in the diagnosis of T1D complications, as access to such diagnostics and consultations in outpatient care is limited.In terms of the use of outpatient services, it was observed that the average annual number of diabetes consultations has not changed despite the pandemic. The average number (ca. 2.8 consultations/patient/year) was similar to the values observed in the pre-pandemic period. At the same time, the differences between the number of visits used by different groups of patients have widened—the group of patients taking four or more visits per year has increased (to 51.7% of the population of children with T1D), as well as those making the fewest visits (1 or less)—20.8%. In 2020, remote medical visits (telemedicine) were implemented on an unprecedented scale in Poland. Available studies indicate the high usefulness and popularity of this form of service to patients with T1D . HoweverAlthough most of the young T1D patients undergo the recommended number of diabetes consultations per year, there is a significant group with very little or no contact with health professionals. The significant percentage of children who do not receive any diabetes counselling or undergo only one consultation per year must be of concern. Pandemic-related limitations have made this problem even more disquieting. Health care practitioners should be aware of this phenomenon and should try to identify reasons for the nonadherence of some patients.The data collected indicate negligible use of ophthalmological, neurological, and mental health consultations by children with T1D. Some form of compensation is carried out in the form of hospitalizations, as half the population of children with T1D in Poland undergo them each year. However, this is a costly solution, and prone to disturbances, as proved by the current pandemic.Therefore, special attention should be given to the outpatient ophthalmological consultations, as they should be a part of regular check-ups in T1D, while the number of patients that actually use such consultations is marginal.This study was based on data gathered and processed in the NHF’s computer system for primarily financial reasons. Analyzed data were provided (entered into the system) by various health service providers acting under the universal health insurance scheme, therefore it did not reflect health services provided outside the public health system.The results of this registry-based study indicate inadequate utilization of health services by the population of T1D children and adolescents in Poland and a substantial decrease in the number of such services used during the COVID-19 pandemic. The relevance of the study’s results is enhanced by the fact that analyzed data (kept in the NHF’s registry) show the use of healthcare services by over 96% of children and adolescents in Poland (approx. 89% during the pandemic).The degree of adherence to the recommendations regarding the type and frequency of use of health services is relatively high only in the case of the use of periodic diabetes consultations. Half of the patients have such consultations regularly, in the number corresponding to the current PTD recommendations. At the same time, the number of ophthalmological, neurological, and mental health consultations is extremely low and does not correspond with current standards of T1D treatment.The impact of the COVID-19 pandemic on the health status and use of healthcare services by children with T1D is difficult to assess precisely at this stage. However, the observed health deterioration of newly diagnosed T1D cases manifested in higher DKA numbers may be accompanied by the increased prevalence of long-term complications due to cancelled or postponed consultations. This risk should be included in the diabetes mellitus management strategies, health policies, and contingency plans at the national level.


The work considers a combination of three enhancing approaches for immunochromatographic assay (ICA) and the integration of their impacts into changes of the limit of detection (LOD). Human fatty acid binding protein (FABP), an early biomarker of acute myocardial infarction, was the target analyte. Starting from the common ICA protocol with an LOD equal to 11.2 ng/mL, three approaches were realized: (1) replacement of spherical gold nanoparticles with gold nanoflowers having a branched surface (20-fold lowering the LOD); (2) enhanced labeling of immune complexes via nanoparticle aggregates (15-fold lowering); (3) in-situ growth of bound nanoparticles by reduction of gold salts (3-fold lowering). Single and combined implementations of these approaches have been studied. It has been shown that the LOD decrease for combined approaches is close to the multiplied contribution of each of them. The final LOD for FABP was 0.05 ng/mL, which is 220 times lower than the LOD for the common ICA protocol. The efficiency of the enhanced ICA with three combined approaches was confirmed by testing human serum samples for FABP presence and content. The development presents a new efficient technique for rapid sensitive detection of FABP for medical diagnostics. Moreover, the demonstrated multiple enhancements could be applied for various demanded analytes. The modern practice of medical diagnostics, as well as safety control of consumer products and the environment requires easy-to-use and rapid analytical systems that allow for productive and widespread testing. Immunochromatographic assay (ICA) fully satisfies these requirements and, because of this, has been successfully introduced into analytical practice ,2,3. A tHowever, to ensure this simplicity, analytical reactions are far from equilibrium, and the detected signal appears at the moment of immune binding. Therefore, the number of formed immune complexes is limited (it is less than in equilibrium systems), and the recorded signal is determined only by the amount and optical properties of the bound label without any possibility for increase, as regards enzyme labels. Due to these reasons, ICA is less sensitive, often significantly, than laboratory immunoassay techniques, such as microplate enzyme immunoassay. Therefore, new variants of ICA with higher sensitivity are in demand ,5,6. Suc(1)the use of new nanodispersed markers, which are detected at lower concentrations and allow the immobilization of a larger number of receptor molecules ,14;(2)increased quantity of nanoparticles bound to the formed immune complex, by aggregation of different functionalized nanoparticles in the course of their movement to the binding zone and directly at this zone of test strip ;(3)growth of the detected analytical signal by increasing size of nanoparticle labels after their attaching to the binding zone of the test strip .Approaches for lowering the LOD in ICA can be divided into groups based on which components of the analytical system, and what processes in it, they act upon. Accordingly, of the greatest interest is the combination of approaches with different directions of their actions. Regarding such different and actively developing directions, the following can be indicated:(1)the use of gold nanoparticles with branched surfaces, namely, gold nanoflowers (GNFs), instead of traditional spherical gold nanoparticles (sGNPs) ;(2)the use of several kinds of nanoparticles functionalized by a biotin–streptavidin interacting module for their aggregation in the course of ICA ; and(3)in situ growth of bound gold nanoparticles by their catalysis of cationic gold reduction .In our earlier developments of ICA for various analytes, the enhancing approaches corresponding to these three groups have been implemented and characterized, namely:The given study was focused on the integration of these approaches in the same ICA system with an estimation of the achieved multiple enhancement, and the factors that influence the reaching of the low LOD. We started from the common ICA format, then considered and combined the three enhancing approaches named above .The target analyte chosen for this study was cardiac isoform of human fatty acid-binding protein (FABP), a specific biomarker of cardiac muscle tissues that is released into the bloodstream when the cardiac muscles are destroyed. Cardiovascular diseases, and especially acute myocardial infarction, are important problems for healthcare, being the leading causes of death . Among o® pro system  (18.2 MΩ cm). The immunochromatographic test system was made using Mdi Easypack  membrane kits, used to fabricate a multi-membrane composite consisting of a working CNPC nitrocellulose membrane (15 µm pore size), PT-R7 glass fiber membrane and an AP045 adsorption membrane.Bialexa  provided us with mouse monoclonal antibodies against human FABP (F5 and F10) and recombinant human FABP. The specificity of the antibodies was demonstrated in  by the aThe Abs F10 and BSA were biotinylated at ratios of 15:1 and 8:1, respectively. The synthesis was described by Hermanson  and in o4 to Au0, as described by Frens [Spherical gold nanoparticles (sGNPs) with expected average diameters of 10 nm (as nuclei) and 30 nm were obtained by citrate reduction of HAuClby Frens . Gold naby Frens , on the The GNPs conjugates with Stp, BSA-biotin, F10 and F10-biotin were obtained through physical adsorption . BSA wasTo carry out transmission electron microscopy (TEM), GNPs were applied to 300-mesh grids  pre-treatment with a poly film. Then, the film was placed on the glass and exposed to 0.15% v/v solution of formvar in chloroform . The JEMZetasizer Nano  was used for measuring the hydrodynamic size and zeta-potential GNPs; the dynamic light scattering of the nanoparticles was registered at a scattering angle of 173° for 10 s at 25 °C.Biochrom Libra S80 spectrophotometer  used to get the absorption spectra of the GNPs in the range of 350–800 nm.For scanning electron microscopy (SEM) of the GNPs at the nitrocellulose membrane, the Tescan Mira 3  microscope with the AZtecOne X-act system for energy dispersive analysis  was applied. The samples were pre-treated with carbon using the Q150R ES Plus spraying device .Assembly of immunochromatographic test strips was carried out as described in . For theIsoFlow dispenser  was used to form the analytical zone  and the control zone  onto the working membrane. The protein deposition rate was 1.2 µL per cm of membrane length, with further drying at room temperature for at least 20 h .The multimembrane composites were assembled, and the sheets were cut into 3.9 mm-wide strips and stored at room temperature with relative humidity under 30% .2O2 and 0.085% HAuCl4 was prepared for the enhancement. 10 μL of this solution was applied to the test zone of the horizontally located test strips by dropping from an automatic pipette. The test strips were incubated for 1 min at room temperature and then their images after the enhancement were registered. To characterize the errors, the number of ICA repetitions was at least 5. The CanonScan 8800F Photo scanner  and TotalLAB software  were used to obtained the quantitative results.TruLab N serum  was used to prepare the stock and working antigen solutions. The procedure for carrying out the ICA with the assembled test strips and their subsequent quantitative processing is described in . The tesb1—the mean concentration of the blank (C (FABP) = 0 ng/mL), Sb1—standard deviation of the blank [The visual limit of detection (cut off) was defined as the minimum analyte concentration at which the colored line in the analytical zone could be observed visually. Instrumental LOD was defined as the analyte concentration at which the analytical zone coloration intensity exceeds by three times the standard deviation of the analytical zone coloration for zero samples (without FABP):he blank .At the first stage, a common immunochromatographic test system was realized and optimized. sGNPs were synthesized by the citrate technique and characterized by transmission electron microscopy—see The obtained sGNPs were conjugated with specific anti-FABP antibodies, F10. The optimal concentration of the antibodies for the conjugation was chosen based on the flocculation curve  and amouv/v), which makes it possible to reach an LOD equal to 11.2 ng/mL. Lower concentrations of the detergent impeded the movement of GNPs and increased the background coloration. On the other hand, an increase in the Tween-20 concentration led to higher LODs. Other parameters of the common ICA protocol, such as optical density of the applied GNP conjugate, concentration of antibodies applied to the analytical zone, etc. were chosen according to an earlier study [Special consideration was given to the choice of detergent, a common compound in immunoassays that prevents non-specific interactions. Detergents of different chemical nature were tested: non-ionic surface-active compounds—Tween-20, Tween-80 and Triton X-100, which differ in the length of the polymer chain and, as a result, in solubility and micelle formation constant; and anionic surfactant Pluronic 121, with weak alkaline properties. Concentrations of the detergents varied from 0.2 to 5%. As can be seen from er study .Finally, analytical parameters of the common ICA of FABP were determined under the optimized conditions. The given assay was characterized by cut off and LOD equal to 33 ng/mL and 11.2 ng/mL, respectively .The advantages of GNFs are the larger size and significantly more developed surface of nanoparticles due to their complex structures (tips). These factors contribute to the immobilization of a larger amount of antibodies for more efficient antigen interaction, which reduces the detection limit of high molecular weight antigens. The benefits of GNFs are determined by their developed surface, which provides increased binding of antibodies and more efficient interaction with the target antigen, as specified in . To use For the ICA using GNFs, the optimal concentration of antibodies in the conjugate with GNFs, the concentration of specific antibodies in the analytical zone of the test strip, and the concentration of the detecting conjugate were selected.The optimal concentration of the F10/FABP antibodies for their conjugation with GNFs was 10 μg/mL, which coincides with the data for sGNPs. Comparison of the conjugates with different antibody loadings demonstrated that the maximum level of the analytical zone coloration was achieved with the maximum amount of antibodies used for the conjugate synthesis—20 μg/mL. However, the minimum cut off was achieved at 10 µg/mL; it was equal to 1.2 ng/mL.520) of the conjugate of GNFs and sGNPs was 2 opt. units; its use provided the minimum cut off  and 27 times (cut off).Under the chosen conditions, the test system based on a single GNF-F10 conjugate has a cut off and LOD of 1.2 ng/mL and 0.4 ng/mL, respectively . Thus, rEffectiveness of the aggregation method of signal amplification in lowering the LOD is determined by the introduction of multiple nanoparticle labels into the formed immune complexes ,28. TakiAs an additional biotinylated component, bovine serum albumin and soybean trypsin inhibitor were considered as two proteins not involved in specific interactions. Both proteins were biotinylated by standard protocol and conjugated with sGNPs. The analytical parameters of ICA reached for these two cases are summarized in The use of three aggregating conjugates led to an increase in the background signal and uneven movement of the conjugates along the working membrane while maintaining the analytical characteristics of the test system. In this connection, individually adsorbed GNP-Stp conjugate and a mixture of GNP-BSA-biotin and GNP-F10-biotin conjugates were used. This combination minimized the background coloration of the test strip.the use of the aggregation system based on the sGNPs allowed for a reduction of the LOD by a factor of 15 times (LOD) and 14 times (cut off).The aggregating ICA protocol under the chosen optimal conditions was characterized by a cut off and LOD equal to 2.2 ng/mL and 0.7 ng/mL, respectively . Thus, tthe use of an aggregation system based on GNFs allowed for a reduction of the LOD by a factor of more then 100 times (LOD) and 33 times (cut off).Another completion of the aggregation system included a detecting GNF–F10–biotin conjugate and two amplifying sGNP–Stp and GNF–biotin conjugates. This ICA was characterized by a cut off and LOD equal to 1.2 ng/mL and 0.3 ng/mL, respectively . The lowFinally, the aggregation ICA protocol with GNFs as the most efficient double combination was integrated with post-assay growth of gold nanoparticles by reduction of gold salt. This additional technique increases the coloration of the label bound to the formed immune complex, and thus, also causes a lowering of the ICA LOD, as demonstrated in its earlier application for various analytes ,36. The 2O2 and HAuCl4. The incubation time of the test strip with the growing solution varied from 1 to 5 min. The optimal time was 2 min, which made it possible to obtain well-reproducible results. Increasing the incubation time leads to an increase in non-specific interaction and the formation of a high background signal. The assay implemented under optimal conditions was characterized by cut off and LOD values equal to 0.4 ng/mL and 0.05 ng/mL, respectively (the integration of three enhancing approaches made it possible to reduce the LOD bya factor of 220 times (LOD) and 80 times (cut off). The size of the aggregates in the analytical zone increased by a factor of 3–5 times  in a target matrix (serum). Finally, the practical applicability of the proposed triple enhancing assay was additionally evaluated by quantitative characterization of FABP recovery in spiked samples. A high rate of FABP recovery was shown, reaching 98 ± 2% (C(FABP) = 33 ng/mL). For all developed test systems, reproducibility was evaluated by the coefficient of variation in the range from 8 to 12% in the working ranges.Gold nanoparticles are the most widely used labels in LFIA due to the simplicity of their preparation and the variation of properties, the possibility of effective functionalization and the low detectable concentrations caused by plasmonic properties. Replacing spherical nanoparticles with alternative gold nanoparticles, anisotropic or non-oriented, but with a developed surface, provides for a lowering of the ICA LOD ,38. ThusA promising method for lowering the ICA LOD is to increase the amount of colored marker in the assay area of the test strip, which allows for an increase of the intensity of the colorimetric signal. The formation of aggregates or networks of functionalized small nanoparticles makes it possible to avoid non-specific binding or difficulties in the migration of particles along the membrane ,43. For The growth of nanoparticles by reducing gold or silver salts on their surface can significantly lower the LOD . VariousIn contrast to the considered individual application of different enhancing approaches, the presented study demonstrates that their integration in the same test system leads to multiple lowerings of the detection limit and a final improvement of the analytical parameters by more than two orders of magnitude, thereby significantly extending the possibility for on-site immunochromatographic control of various analytes. The data regarding the existing developments of ICA for FABP collected in The work proposes the integration of three approaches for lowering the LOD of immunochromatographic detection of fatty acid binding protein (FABP), namely: (1) replacement of spherical gold nanoparticles with gold nanoflowers (20 times); (2) formation of aggregates in the analytical zone (15 times); and (3) growth of nanoparticles by gold salt reduction (3 times). It is shown that the combination of three methods integrates the impacts of the individual amplification factors into lowering the limit of detection. The final limit of detection of FABP was 0.05 ng/mL, which is 220 times lower than the common test system.The developed test system, in addition to its undeniable advantages, has disadvantages. The main advantage of the proposed approach lies in the very low detection limit of the analysis, which exceeds each of the methods used separately. Combining the three methods slightly increases the assay time, which does not affect its rapidity.Its implementation requires the use of additional reagents, which is an additional stage that increases the analysis time by 1 min. However, the introduced changes slightly affect the total analysis time while significantly improving the analytical performance. The transfer of the proposed test system into a dry format by finding the best protocols of extra reactants to be applied on an additional membrane seems to be a reasonable task for further investigations to overcome this limitation.The triple enhanced test system meets the requirements of AGREE  , as well


Electron microscopy showed Fe2O3 nanoparticles in the tissues of olfactory bulbs but not in the basal ganglia of the brain after their subchronic intranasal administration. We observed an increase in the number of axons with damaged myelin sheaths and in the proportion of pathologically altered mitochondria in the brains of the exposed animals against the background of almost stable blood parameters. We conclude that the central nervous system can be a target for toxicity of low-dose exposure to Fe2O3 nanoparticles.Particulate matter, including iron nanoparticles, is one of the constituents of ambient air pollution. We assessed the effect of iron oxide (Fe Numerous metallurgical processes and electric arc welding generate aerosols of iron condensation, containing submicron and nano-sized particles, the proportion of which can be as high as 40% of PM2.5 . We assu3O4 nanoparticles to induce oxidative stress, owing not only to the small size but also to their chemical identity, has been also demonstrated in vivo [Up-to-date literary sources describe multiple organ toxicity of iron oxide nanoparticles. The impact of iron-rich airborne particles on the cardiovascular system has been proven both in vivo using Danio rerio  and in e in vivo . At the  in vivo .Recent studies have proven that the central nervous system is one of the targets for toxicity of metal and metal oxide nanoparticles ,13,14, w2O3, has been repeatedly evidenced by animal experiments. In their studies on mice exposed to Fe2O3 nanoparticles at doses of 25 and 50 mg/kg body weight for four weeks, Dhakshinamoorthy et al. showed a change in motor behavior and spatial memory, as well as impaired permeability of the blood–brain barrier and accumulation of nanoparticles in the brain. The authors associated the observed health effects with the accumulated nanoparticles, oxidative stress, and apoptosis in the brain of mice [The neurotoxic effect of iron nanoparticles, including those of Fe of mice .Maher B. et al. also associated the exposure to iron compounds (magnetite nanoparticles) with a unique combination of enhanced production of reactive oxygen species, surface charge, and strong magnetism .2O3 nanoparticles and found the ability of the latter to deposit in brain tissues and damage the striatum [Wu J. et al.  conductestriatum . Oral exstriatum .2O3 was evaluated by Garcia-Fernandez J. et al. in in vitro studies by modeling the distribution of ultrasmall Fe2O3 particles in the gastrointestinal tract [3O4 could be captured by cells of tissue macrophages in the liver, spleen, and lungs [The kinetics of Feal tract . Popescund lungs ,24. Longnd lungs . In addind lungs . At the 2O3 nanoparticles. Current occupational exposure doses, however, demonstrate an obvious decreasing trend, e.g., in Canada [2O3 nanoparticles are capable of exerting toxic effects at low doses. Based on published data, we assumed that even low-dose exposure to iron oxide nanoparticles can induce changes at the ultrastructural and, probably, organismal level .The studies that we have found were conducted using relatively high doses of Fen Canada , the Unin Canada . Data arn Canada . Our objThe analysis of the rats’ behavioral activity data revealed some statistically significant changes in different weeks of the exposure period . Total pAnxiety in the exposed animals was measured in the hole-board test by grooming a and defThe reaction time of the rats to a stimulus differed significantly across the exposure period, as evidenced by changes in the summation threshold index shown in Due to the ambiguity of data on changes in behavioral reactions, we decided to analyze average indicators of behavior and summation threshold index, as shown in Despite the pronounced and unambiguous changes in the behavior of the exposed animals, we observed no significant changes in weight or hematological parameters. We can only note a statistically insignificant yet obvious increase in serum myelin basic protein (MBP) .The analysis of the proportions of different cells in rhino-cytological wash specimens a and imp2O3 nanoparticles in tissues of olfactory bulbs of the rats’ brains; their composition was later confirmed by energy-dispersive X-ray spectroscopy , we confirmed the abovementioned differences in the distribution of mitochondrial morphotypes between the exposed animals and the controls  = 1090.6; p ≤ 0.0001; χ2OB  = 1010.0; p ≤ 0.0001).Using Pearson’s goodness-of-fit test  = 283.1; p ≤ 0.0001; χ2Fe2O3 NPs  = 183.3; p ≤ 0.0001).We also established distinctions in the distribution of mitochondrial morphotypes between different brain regions in both groups . The results of the paired Student’s t-test were similar for all the comparisons.However, no significant differences were found between mitochondria of the vesicular morphotype due to a high variability of values  and in neuron cytoplasm. Our results are consistent with the findings of previous studies showing the presence of nanoparticles in intracellular structures, including mitochondria and nuclei, despite the differences in physical (shape and size) and chemical (silver and gold) characteristics of nanoparticles [2O3 nanoparticles to subcellular structures, since the true mechanism of the toxic effect of nanoparticles may be the result of several risk factors acting simultaneously and difficult to differentiate  [It is worth noting that Fearticles ,36. It ike rate) .2O3 nanoparticles caused significant changes in behavior but not in cytological characteristics of nasal wash specimens and imprint smears of the nasal cavity, hematological parameters, or biochemical parameters of blood serum, with the exception of a statistically insignificant increase in the serum level of the myelin basic protein of 22.5%. Our findings are consistent with those reported by Badman et al. [p < 0.05) and olfactory bulbs, respectively  sized 18 ± 4 nm in the total dose of 0.45 mg Fe2O3 NPs per rat, administered intranasally to three- to four-month-old female rats weighing about 200 g, with 12 animals per group. The choice of dose in the study we analyzed was limited by the nanoparticle concentration in the suspension and the maximum allowable volume for intranasal instillation in rats. The design of the experiment was similar to that described elsewhere [2O3 nanoparticles were prepared at the Ural Federal University, Yekaterinburg, Russian Federation, by pulsed-laser ablation of thin sheet targets of 99.9% pure iron in sterile deionized water, as described elsewhere [Within the framework of the present work, we have analyzed data obtained in a subchronic toxicity study of Felsewhere . The samlsewhere . We examlsewhere  and imprlsewhere  for seveEnergy dispersive X-ray spectroscopy (EDS) spectra were analyzed using the AZtec software .2O3 nanoparticles in rat brain structures, 420 ultrathin sections of two parts of the brain of the exposed and control animals were examined.To identify FeWhen analyzing morphological changes in the myelin sheaths of axons and assessing the state of neuron mitochondria, a series of five to six sections was prepared, and the highest quality ultrathin section of the series was divided into 20+ fields of view, evenly distributed over the entire surface, and then examined.When determining the degree of damage to the myelin sheath of axons in sections of two regions of the brain, 1387 unique sites of the myelin sheath of axons (40 to 60 sites × 2 areas of the brain × 7 rats × 2 groups) were ranked by the presence or absence of damage to its structure.n = 7) and experimental (n = 7) groups. Morphotyping was carried out by two independent readers to exclude bias in establishing mitochondrial morphotypes, after which the results of the percentage distribution of morphotypes were averaged for each type of tissue and animal.When analyzing the damage to mitochondria following the exposure to nanoparticles, we assumed five successive stages of transformation of the inner membrane according to the classification of Sun et al. . We rankt-test. In addition, we compared p values estimated using the Student’s t-test and the Mann–Whitney U test, and their general coincidence proved the appropriateness of applying the t-test.The statistical significance of data on behavioral reactions and blood parameters was established using the Student’s 2), while that between the groups was determined using the Mann–Whitney U test and Student’s t-test.Scanning electron microscopy findings were analyzed using the Statistica 12 software. The statistical significance of differences in the distribution of mitochondrial profiles was assessed using Pearson’s goodness-of-fit test (χp ≤ 0.05).The difference between means was considered statistically significant if the probability of its occurrence by chance was equal to or less than 0.05 (2O3 nanoparticles at a total dose of 0.45 mg to have a toxic effect on rats. Administered intranasally, the particles significantly affected behavioral reactions but induced no changes in cytological characteristics of nasal swabs or imprint smears of the nasal cavity, hematological parameters, or biochemical indices of blood serum.Our data analysis showed the ability of Fe2O3 nanoparticles in the brain and to establish the dependence of their distribution therein on physical properties, dose, exposure duration, and route of administration of the nanoparticles. In addition, it is necessary to study correlations between the presence of nanoparticles in tissues, cells, and organelles, and ultrastructural damage to biological objects under study.The examined kinetics demonstrated the ability of nanoparticles to enter the brain structures and to distribute unevenly in different parts of the rat’s brain. This conclusion requires further research to elucidate patterns of accumulation of Fe


The spectral luminescent properties of dipyrromethenates halogenated with bromine on both ends of the long axis and coordinated using boron fluoride, zinc, or cadmium in neutral ethanol and acidified with hydrochloric acid solutions were studied. The constants of the acid–base equilibrium of the complexes in the proton-donor solvents in the ground and excited states was determined. The mechanisms of complex protonation were discussed, depending on the structure of the compounds. The electronic structures of the neutral and protonated compounds were modeled and analyzed based on the quantum-chemical method. The structures and spectral-luminescence properties were calculated using the SMD model of ethanol solvent using the TD-DFT theory with the B3LYP functional and the composite def2-SVP/def2-TZVP/def2-TZVPP_ECP basis sets, depending on the atomic number of the elements. The chemistry of dipyrromethene (dpm) compounds has been widely developed due to the existing variety of their various metal complexes, which have outstanding spectral and photophysical characteristics. Covalent dpm complexes with cations of p-, d-, and f-elements exhibit high chromophore activity in the visible region of the electromagnetic spectrum. Efficient fluorescence yields and high photostability of dpm complexes with boron fluoride BODIPY have been established ,2,3,4. TThe specific spectral-luminescence and photochemical properties allow for predicting and designing the broad avenue of materials based on dpm compounds in their practical use as active components of various optical devices: fluorescent probes ,10,11, oDespite the large number of works describing the synthesis, there is a lack of published studies devoted to a detailed analysis of the influence of electronic, structural, and solvation factors on the stability and photo-induced properties of dpm and their metal complexes. The manifested properties have only recently begun to be analyzed ,23,24. TThus, our research was aimed at studying the protonation processes of bromine substituted dipyrromethene complexes with various complexing agents such as metalloid B vs. transition metals Zn and Cd in regards to the structural stability and photophysical properties of these compounds in neutral and acidic solvents. The information about the features of protonation becomes important in the practical use of these complexes, namely, the incorporation of dyes into solid-state polymer matrices because the polymerization process often proceeds at low pH, i.e., in acidic media, which certainly affects the spectral and luminescent properties of the materials. In order to establish the influence of neutral and acidified environments, the dpm complexes, both in the ground and in the excited state, were investigated for a practical assessment of their stability in proton-donor media. The obtained values of the acid–base interaction constants and the revealed patterns of the influence of structural and solvation factors will allow us to optimize the search for leading compounds with the most successful combination of spectral and photophysical characteristics in dpm and their metal complexes.2-dpm [BF2L] makes the B-N and B-F coordination bonds weaker. The weakening leads to their cleavage, with proton attaching to the pyrrole nitrogen that forms the protonated ligand H2L+ through one isosbestic point. As was noted in these publications, the isosbestic point indicates that the third chromophore, the molecular form of HL, does not accumulate in solution at recorded concentrations. However, it should be borne in mind that this is typical only for boron fluoride dpm complexes. For other dpm metal complexes, a change in the “classical” protonation mechanism, i.e., that the coordination center is not modified in the first place 50 values for the Franck–Condon state S1 were determined, from which it follows that the stability of the excited BODIPY complex is higher than that in the ground state, which is consistent with the high photostability of BODIPY-based laser media.The experiment showed that in acidified ethanol solutions, the studied dpm complexes entered into an exchange reaction with hydrochloric acid. The electronic absorption spectra of the products corresponded to the spectra of the protonated structures formed in the exchange reactions of the ligands, with the presence of an isosbestic point (500 nm) in the patterns of irreversible spectral transformations a. Table 2L+ ligands formed, the fluorescence quantum yield of which does not exceed 0.003.Changes are also observed in the fluorescence spectra b. CompleWhen ethanol solutions are acidified, Zn and Cd dpm complexes also enter into irreversible exchange reactions with hydrochloric acid. The degree of conversion of complexes into intermediate and final products of the proteolytic dissociation processes clearly reflects changes in the electronic absorption and fluorescence spectra . Complet2 in the corresponding complexes with the structurally identical ligands: 488–489 nm for Br2(CH3)4BODIPY, Zn[Br2(CH3)4dpm]2, and Cd[Br2(CH3)4dpm]2. At the same time, the absorption spectra of the corresponding neutral dpm differ significantly (by 22–28 nm)  and BF2–28 nm) .In our case, it should be taken into account that the introduction of bromine atoms increases the stability of the complexes. It can be assumed that, due to the high electronegativity of bromine atoms, there is a decrease in the electron density in the coordinated nitrogen atoms and accordingly, in their basicity, which makes it difficult for the proton to attack the coordinated nitrogen atoms in the initial, limiting process of the proteolytic dissociation of these metal complexes. As a result, the described effect dominates over the opposite effect of the weakening of the Me-N coordination bonds, caused by the same structural factor, i.e., the outflow of electron density to the bromine atoms.50(S1)F-C values for the S1 Franck–Condon states were determined 4BODIPY exhibits an intensive first band at 528 nm, which determines high fluorescence efficiency vs. the Zn and Cd complexes.A high accuracy of the calculated spectral-luminescence properties of the isolated halogen–dpm compounds coordinated with BFrocesses ,29. The The two lowest states show the same symmetry of ED localization on both fragments, forming a dissipative channel of internal conversion between the bright and dark states preferable over emission, leading to very low quantum harvest of fluorescence from the higher state , with th2-based compounds, which exhibit very efficiency fluorescence, but halogenation, particularly with Br, quenches the process due to increasing nonradiative intersystem spin crossing from the lowest excited photoactive singlet to the triplet system 2 , and the dissociation mechanisms are different. The fact that the halogen substituents partially pull the electron density protonation of complexing BF2 remains the main reason for BODIPY dissociation, whereas the case of the Zn and Cd are more complicated because their low stability depends on weaker metal bonds of the transition elements with two dpm fragments, although the protonation of the halogenated ends does not make the bonds stronger.Br2(CH3)4dpm]2 in the ground state because of the nearly C2v symmetry of the compounds (2 was considered in both the ground and excited states of Br2(CH3)4BODIPY because this is the main method of dissociation.The most preferred points of the metalloid complex in the ground and excited states for protonation are near the strongly negatively charged two fluorine atoms  at the gompounds . The symabs = 30 than for the neutral solvent because of slight distortion symmetry 4dpm]2, which is significantly stronger than that of Δλabs = 30 nm in Br2(CH3)4BODIPY. Moreover, the last system demonstrates a clear increase in the dissociated product with an increase in acidity, in comparison with the rest of compounds, where complexing center is protected from proton attack.Acidification scarcely changes the fluorescence wavelengths  of all t2, Zn, and Cd, which are abbreviated in this work as Br2(CH3)4BODIPY, Zn[Br2(CH3)4dpm]2, and Cd[Br2(CH3)4dpm]2, respectively, are shown in The presented dpm complexes were synthesized at the G.A. Krestov Institute of Solution Chemistry of the Russian Academy of Sciences (Ivanovo). The descriptions of the synthesis and control of the purity of the structure using thin-layer chromatography and IR spectroscopy are described in ,32,33. S2, Zn, Cd) and contain heavy Br atoms located on the periphery of the dpm core in the β-position, i.e., all dpm fragments halogenated with Br on both ends of the long axis. Undried ethanol was used as a solvent. The solutions were acidified using a high-purity 36% aqueous HCl (11.6 M).The studied complexes differ in the complexing agents (BF−5–10−6 M.The absorption spectra of the studied compounds were recorded on a Cary5000 spectrometer (Varian) in a quartz cell with an optical path length of 1 cm under standard conditions. The spectra of fluorescence were measured using a Cary Eclipse spectrometer (Varian) at room temperature (298 K). The typical concentration of the investigated compounds was 10To determine the quantitative characteristics of the dye’s stability by spectrophotometric titration, a series of water–ethanol solutions of dpm complexes with a successively changing content of hydrochloric acid were studied. After adding the acid, the solutions were held for up to 180 min to establish equilibrium, which was confirmed by the invariance of the absorption spectra.50. In this case, the concentration of the complexes decreases due to their decomposition by 50% with the formation of a protonated ligand and the release of the complexing agent from the complex. The titration curves were obtained as dependences of the relative change in absorption at the chosen wavelength ΔD/D0 or fluorescence ΔI/I0 on −lg[HCl]50. The lower the −lg[HCl]50 value, the higher the stability of the complexes in the proton-donor media, i.e., the higher the acid concentration required to achieve 50% conversion of the complex to a protonated ligand according to the classical definition ven base . In thisrelation ,41.In order to reveal the spectral-luminescent features of the dpm complexes and their acidified forms, electronic structures were optimized in both the ground and fluorescence states, and the photophysical properties were calculated in the continuum of the solvation model using the electron density (SMD)  of the e2 (Zn[Br2(CH3)4dpm]2, Cd[Br2(CH3)4dpm]2, Br2(CH3)4BODIPY) was carried out in the proton-donor media. The mechanisms of the dissociation of BF2 vs. Zn and Cd complexes in neutral and acidified ethanol solvents were confirmed by analyzing the titration curves and comparing the spectral data for both absorption and fluorescence in different hydrochloric acid concentrations. The theoretical assumptions about the protonation pathways were confirmed using quantum-mechanical calculations.The experimental and theoretical competitive evaluation of the stability and photo-physical properties of 3,3′,5,5′-tetramethyl-4,4′-dibromo-2,2′-dipyrromethene complexes coordinated with Zn, Cd, and BF2(CH3)4BODIPY, Zn[Br2(CH3)4dpm]2 and Cd[Br2(CH3)4dpm]2 were optimized in the ground and fluorescent states to calculate their spectral-luminescence properties in the ethanol solvent SMD model using TD-DFT theory with the B3LYP functional and the hybrid def2-SVP/def2-TZVP/def2-TZVPP_ECP[Cd] basis sets. The calculations exhibited the expected correlation between the measured and theoretical absorption spectra for the neutral, and better correlation for acidified, structures, as well as perfect agreement for fluorescence. The pathways of the protonation attacks were detected, based on ESP analysis. In addition, these results point to differences in the initial stages of proteolytic dissociation, depending on the nature of the complexing agent. In BODIPY complexes, the primary interaction occurs with the complexing agent BF2, but in the transition metal Zn and Cd complexes of dipyrromethenes, the most preferable direction for initial protonation is along the long axis of the dpm fragments between the brominated end and HCl from the solvent. Thus, it has been shown that the introduction of halogen atoms has a significant effect on the protonation process of the dipyrromethene metal complexes, engaging in interaction with the protons of the medium and screening the coordination centers of the molecules.The electronic structures of the neutral and HCl acidified BrThe obtained results show that the highest stability in the ground and excited states is typical for BODIPY. This complex is stable, even in strongly acidified media, while the metal complexes of dpm (Zn and Cd) are more likely to undergo dissociation in the proton donor solvents. In the future, it is necessary to develop approaches to increase the stability of the dpm metal complexes for their successful practical application in various optical devices.


Non-histone nuclear proteins HMGB1 and HMGB2 (High Mobility Group) are involved in many biological processes, such as replication, transcription, and repair. The HMGB1 and HMGB2 proteins consist of a short N-terminal region, two DNA-binding domains, A and B, and a C-terminal sequence of glutamic and aspartic acids. In this work, the structural organization of calf thymus HMGB1 and HMGB2 proteins and their complexes with DNA were studied using UV circular dichroism (CD) spectroscopy. Post-translational modifications (PTM) of HMGB1 and HMGB2 proteins were determined with MALDI mass spectrometry. We have shown that despite the similar primary structures of the HMGB1 and HMGB2 proteins, their post-translational modifications (PTMs) demonstrate quite different patterns. The HMGB1 PTMs are located predominantly in the DNA-binding A-domain and linker region connecting the A and B domains. On the contrary, HMGB2 PTMs are found mostly in the B-domain and within the linker region. It was also shown that, despite the high degree of homology between HMGB1 and HMGB2, the secondary structure of these proteins is also slightly different. We believe that the revealed structural properties might determine the difference in the functioning of the HMGB1 and HMGB2 as well as their protein partners. Proteins HMGB1 and HMGB2 belong to a large family of HMG proteins characterized by high electrophoretic mobility (High Mobility Group). HMG proteins are the largest group of nuclear proteins after histones. According to their structure and functions, these proteins are divided into three families: HMGA (formerly known as HMGI/Y), HMGN (formerly known as HMG14/17), and HMGB domain proteins  ,4. HMGA The HMGB1 and HMGB2 proteins are very similar in structure and amino acid sequence. Both consist of a short N-terminal region, two DNA-binding domains, A and B, and a C-terminal sequence of glutamic and aspartic acids. These proteins are evolutionarily conserved . For exaThe variety of functions performed by proteins is associated with their localization in the cellular/intercellular space ,26,27,28Having highly homologous amino acid sequences, the HMGB1 and HMGB2 proteins should have other features that determine their functional differences. Such features can include, for example, the nature/pattern of their PTMs and the structural organization of their polypeptide chains. However, currently, there is a lack of information about the differences between their PTMs and secondary structures. Here, we report new potential sites of PTMs, which have been revealed in HMGB1 and HMGB2. In this work, we also performed a comparative analysis of PTM patterns and secondary structures of the HMGB1 and HMGB2 proteins.For the identification of structural characteristics of proteins and their complexes with DNA and also PTMs, nuclear HMGB1 and HMGB2 were prepared according to the approach described in Materials and Methods. Proteins were isolated from calf thymus by extraction with 5% perchloric acid followed by precipitation with 6 volumes of acidified acetone at −20 °C. Separation of the protein fraction into individual components was performed using ion exchange chromatography on an FPLC system. Results are presented in The secondary structure of a protein is one of the basic characteristics that determine its biological functioning and physicochemical properties. HMG proteins are often compared with natively unfolded proteins due to their high structural flexibility ,46,47,48Increasing the ionic strength of the solution to 1.5 M NaCl leads to the electrostatic screening of charged groups, resulting in suppression of their Coulombic attraction/repulsion, rearrangements of bound counterions, and inducing structural transitions in proteins . Indeed,As shown earlier ,52, the The above differences in the secondary structure of the HMGB1 and HMGB2 chromosomal proteins can be due to at least two reasons: different lengths of the disordered negatively charged C-terminal regions or different nature of PTMs in these proteins, which can significantly modify the physicochemical properties of the polypeptide chain. To test both hypotheses, we conducted a comparative analysis of the PTMs of the HMGB1 and HMGB2 proteins and studied their interaction with DNA, which is the main binding target in the cell nucleus.The HMGB1 and HMGB2 PTMs were analyzed using MALDI mass spectrometry. Since there are very little data on HMGB2 modifications, we set ourselves a more general task—to reveal the modifications possible for these proteins and the regions of their location in the HMGB1 and HMGB2 proteins and compare them with each other. If there are several identified potential modification positions in a certain site, the MALDI-FT-ICR-MS analysis we used does not allow us to accurately identify the site where this modification occurred. The experimental results presented in this table reflect the areas of the amino acid sequence in which these modifications are present. Since we can only indicate the area, we did not highlight specific modification sites in the table. Based on the comparison of literature data with our experimental data, we constructed an approximate map of the position of potential modifications . Further2+-regulated secretory organelles involved in inflammatory and immune responses, secreting their contents into the external environment in response to triggering signals  for protein solutions. The values of the molar ellipticity of proteins were calculated based on the average weight of the amino acid residue, which is equal to 102.31 and 100.39 for HMGB1 and HMGB2, respectively.The obtained CD spectra are presented as the difference between the absorptions of left- and right-polarized light ∆A (∆A = Aθ]) is not quite convenient. This is due to the fact that there may be regions in the spectra where the obtained signal is a superposition of contributions from individual components in the mixture. In our case, there is an overlap of the spectral bands of protein and DNA at wavelengths less than 250 nm. The degree of α-helicity of the proteins was estimated based on two independent approaches. First, using the K2D3 algorithm [θ222]:In cases where it is necessary to analyze the spectra of a multicomponent mixture, the use of specific values  and UV absorption spectroscopy. This approach makes it possible to detect differences in the secondary structure of protein molecules and changes in the structural organization of DNA/protein complexes. As mentioned above, HMGB-domain proteins are often compared with natively unfolded proteins ,9,19, anThe PTMs of HMGB1, which we have identified, are located mainly in the DNA-binding A-domain and in the linker region between two HMGB domains of the protein. On the contrary, the majority of HMGB2 PTMs are found within the B-domain of the protein. There are also some modifications in the linker region of HMGB2; however, they are different compared to HMGB1. It can be expected that the number of modifications is higher in the functionally significant regions of the protein. Hence, it can be assumed that a greater number and variety of modifications within the B-domain of HMGB2 reflects its greater functional activity as compared to the B-domain of HMGB1. HMGB2 has greater conformational flexibility; it demonstrates more effective DNA compaction and condensation compared to HMGB1; it has a greater number of PTMs identified in the B-domain. Altogether, the above results indicate that the B-domain of HMGB2 is involved in protein functioning to a much greater extent than the B-domain of HMGB1.Both proteins are involved in various processes: replication, repair, etc. However, the presence in the cell of both proteins so close in their primary and secondary structures cannot be accidental. Combining the results of CD spectroscopy and mass spectrometry, one can conclude that, despite the high homology of the primary structure of the HMGB1 and HMGB2 proteins, the mechanisms of their binding to DNA and other proteins can differ significantly. Interacting with DNA, both HMGB1 and HMGB2 significantly bend the double helix. However, according to the earlier published data, a single DNA-binding domain of HMGB2 induces a bend of about 100°, while two HMGB1 DNA-binding domains together induce bends of less than 80° . This miChromatin is a complex, highly dynamic system that includes a huge number of different factors. The role of proteins in a living cell is a set of manifestations of their various contacts with many partners. The DNA–protein systems studied in this work are an in vitro model system, which makes it possible to study the interaction of a particular protein with DNA using spectral methods in the absence of other partners. The differences that we have identified may indicate a difference in the functioning of these proteins in chromatin, which manifests itself in the difference in the spectral characteristics of not only the HMGB1 and HMGB2 proteins themselves but also their complexes with DNA. These features of proteins can affect the functioning of the chromatin-remodeling complex since it is the DNA loop formed during the interaction of DNA with HMGB-domain proteins that initiate the recruitment of this complex. This, in turn, can affect basic cellular processes, such as transcription.We believe that it is important to understand the subtle structural differences between the HMGB1 and HMGB2 proteins, which undoubtedly affect the mechanisms of their interaction with DNA.


A wireless multi-layered sensor that allows electromyography (EMG), mechanomyography (MMG) and near-infrared spectroscopy (NIRS) measurements to be carried out simultaneously is presented. The multi-layered sensor comprises a thin silver electrode, transparent piezo-film and photosensor. EMG and MMG measurements are performed using the electrode and piezo-film, respectively. NIRS measurements are performed using the photosensor. Muscular activity is then analyzed in detail using the three types of data obtained. In experiments, the EMG, MMG and NIRS signals were measured for isometric ramp contraction at the forearm and cycling exercise of the lateral vastus muscle with stepped increments of the load using the layered sensor. The results showed that it was possible to perform simultaneous EMG, MMG and NIRS measurements at a local position using the proposed sensor. It is suggested that the proposed sensor has the potential to evaluate muscular activity during exercise, although the detection of the anaerobic threshold has not been clearly addressed. There is a need to develop a system for monitoring muscular activity and estimating muscular fatigue in the fields of sports science and physical rehabilitation. Many researchers have reported work in this field, and electromyography (EMG) is adopted as the primary method of evaluating muscular activity involving muscular fatigue ,2,3,4.EMG measures the electrical voltage induced by muscular contractions using dish electrodes that are pasted onto the surface of the skin. Additionally, mechanomyography (MMG) ,7,8,9,10Multi-modal evaluation techniques based on two types of sensing methods, including EMG and MMG ,17,18,19The purpose of our research is the development of a portable wireless sensor that allows the evaluation of muscular activity and involves the prediction of muscular fatigue, i.e., the detection of the anaerobic threshold (AT). A new multi-layered sensor that allows the measurement of EMG, MMG and NIRS signals simultaneously at the same muscular motor unit has been proposed . The useN (400) is the number of measured voltages. In NIRS, three types of LED were sequentially switched, and (2) was used because of the measurement error generated by the switching. Data were thus obtained at intervals of 160 ms (6.25 S/s).A micro-computer  having a 12-bit, five-channel analog-to-digital converter for data acquisition and control was used. ZigBee modules  were used for wireless communication. In this system, two types of interrupt handlers were used for voltage measurement and transmission. Voltages in EMG, MMG (LED and PD) and NIRS were sequentially measured through interrupt processing at intervals of 100 μs. Data were thus acquired at intervals of 400 μs (2.5 kS/s). The averages of absolute values in EMG and MMG (LED and PD) derived using (1) and the averages of absolute values in NIRS derived using (2) were sequentially transmitted to a personal computer (PC) through interrupt processing at intervals of 40 ms and a serial baud rate of 38,400.2 concentrations are derived from (3).I(λ) and I0(λ) are, respectively, the quantities of detected and emitted light at a wavelength of λ. εoxy and εdeoxy are, respectively, the absorption coefficients of HbO2 and Hb [Coxy and Cdeoxy are, respectively, the concentrations of HbO2 and Hb. Additionally, d and S are, respectively, the average light path length and the dispersion. In this paper, the unknown value of d is calculated to be 12.5 with reference to the literature [2 were obtained at intervals of 480 ms.The Hb and HbO2 and Hb  and Coxyterature . Using tThe interval for transmission was optimized in a preliminary experiment. The transmission error was greater when the transmission interval was shorter. The voltage source was a 7.4-V lithium battery , and a voltage of 3.3 V was produced for wireless communication and a voltage of 5.0 V for circuit operation. The voltage stability of the sensor system was measured after the full charging of the battery in the preliminary experiment. As a result, for this system, it was found that the voltage of 5.0 V was stable with fluctuation of ±0.05% and attenuation of 0.2% over a period of 8 h. The overall mass of the signal processing circuit in Three healthy male participants, two in their twenties and one in his forties, participated in the experiments. The experiments were conducted according to the Declaration of Helsinki. All experimental procedures were approved by an ethics committee at Saga University. The procedures and risks were explained to each participant and informed written consent was obtained from all participants. Two experiments were conducted, one involving isometric ramp contraction at the forearm and the other cycling exercise of the lateral vastus muscle.In the experiment, the relaxed condition of the forearm was first maintained for 30 s (Rest). Next, the condition that a vessel with a load of 400 g was placed on the participant’s palm was maintained for 10 s (Work). The isometric ramp contraction was then performed until 60% MVC was reached. The load on the palm was changed, flowing water into the vessel at a constant rate of loading. Finally, the rest condition was maintained for 30 s. EMG, MMG and NIRS signals were measured using the layered sensor.2 and CO2 in the respiration gases. The AT was estimated using the V-slope method based on concentrations of O2 and CO2 [ and CO2  and comp and CO2 .2 and CO2 were measured by the aero monitor. In the cycling exercise, a speed of 60 rpm was maintained through the use of a metronome.In the experiment, the relaxed condition in which both feet were placed on the pedals and kept at the same height with the left leg ahead was first maintained for 1 min (rest). Next, the pedals were pushed at a speed of 60 rpm while a work load was added from 15 Watts (W) in increments of 15 W every 1 min (cycling). When the participant could no longer maintain the cycling speed, the cycling exercise was deemed complete, and, finally, the rest condition was maintained for 5 min (rest). During the experiment, EMG, MMG and NIRS signals were measured using the proposed sensor. Additionally, respiration gases such as OThe first experiment was conducted three times for each participant, and the second experiment once for each participant. The temperature in the room where both experiments were performed was controlled at 25 °C.2 and ΔHb decreased and increased during the ramp stage. These quantities then increased and decreased in the rest period after the ramp stage. It is seen that ΔHbO2 and ΔHb changed because the condition of the blood changed, even though there were no corresponding changes in the EMG and MMG signals. The results were in agreement with the results of muscular activity previously reported [reported ,14,20,24reported . The sim2 and a reduction in ΔHb under the load of 15 W. ΔHbO2 and ΔHb, respectively, decreased and increased with step increments of the load from 2 min (30 W). ΔHbO2 and ΔHb increased and decreased during the rest period after the cycling exercise, whereas the EMG and MMG voltages during the rest period were both zero. Both the slopes of ΔHbO2 and ΔHb slightly decreased at around 9 min. These results relate to the AT and are similar to the change in the slope of the EMG voltage [ voltage . It is tHb to VEMG and the approximated quintic curve based on data between 2 and 14 min (red line) to enable the detection of the threshold for the evaluation of fatigue (AT) based on the literature [Hb to VEMG had an extremal value (red dotted line) at 7 min 43 s. Meanwhile, the intersection point of the slopes of VO2 and VCO2 was at 7 min 54 s. The timing of the AT obtained from the ratio of ΔHb to VEMG was thus similar to that obtained from VO2 and VCO2.terature  and the Hb to VEMG and that obtained from VO2 and VCO2 for the three participants. It is seen that the ratio of ΔHb to VEMG can be used for determining the timing of the AT because the times are similar to those determined using VO2 and VCO2. The proposed sensor thus offers the potential to estimate the AT.A wireless multi-layered sensor was developed, and simultaneous EMG, MMG and NIRS measurements were performed for an isometric ramp contraction at the participant’s forearm and cycling exercise of the lateral vastus muscle with step increments in the load. The results of the isometric ramp and cycling exercises showed that simultaneous EMG, MMG and NIRS measurements of local muscular activity were possible using the proposed sensor. Additionally, a comparison of the extremal value (the ratio of ΔHb to EMG) obtained using the proposed sensor and AT obtained using a commercial respiration gas sensor showed that the proposed sensor could be used to predict muscular fatigue.In future work, the relationship between the extremal value, the inflection point of the MMG voltage obtained by the proposed sensor, and the AT obtained by a commercial sensor involved in the prediction of muscular fatigue will be derived for several participants and different exercises. This work will include measurements for large step increments of the load and constant loads. In the present study, the mean power frequency could not be calculated in frequency analysis because the data transmission interval was insufficient even though the sampling frequency was sufficient. Therefore, an improved system with a data logger or combined data transmission that allows frequency analysis will be developed and evaluated as one of the parameters. Additionally, the processing circuit will be smaller because it is possible to halve its size by using a smaller microcomputer and improving the circuit configuration and elements.