The malaxation step, one of the most important phases of the virgin olive oil (VOO) mechanical extraction process involved in the development of the main quality characteristics of the final product, was carried out at a low temperature (18 °C). The rapid control of malaxer temperature was handled with the same chiller as that of the heat exchanger used in a semi-industrial extraction plant. Low temperature was used during the full olive paste kneading process and also for half of this process, which showed that there was a significant impact on the phenolic and volatile contents of VOO. Trials were conducted on three different cultivars , and their phenolic and volatile concentrations showed different quantitative and qualitative effects due to the prolonged use of low temperature after the crushing phase, as a function of the different genetic origins of the olives. The process of phenolic compound solubilization into the oily phase was negatively influenced by the use of low temperature during the entire malaxation period for all the cultivars, whereas the volatile fraction showed an improvement in VOO flavor mainly due to the oil extracted from Canino olives. In recent years, the thermal conditioning of olive paste during the olive oil extraction process has increased in usage due to the different impact in oil yield and the quality characteristics of the final product. Previous studies focused their attention on the biochemical process, showing that a differentiated activity level of endogenous enzymes occurred due to the application of different processing temperatures ,3,4,5,6.The application of high temperature favors the degradative process of phenolic compounds induced by PPO and POD and promotes enzymatic activities, which increases the oxidation process during the mechanical extraction of VOO. The high temperature of fruits before the extraction process, the increasing temperature of olive paste during crushing and, most of all, the regulation of malaxation temperature are all factors that can strictly influence the activity level of PPO and POD. For this reason, many studies have been performed to optimize the processing temperature in order to partially control the final phenolic content of the product during the different steps of the extraction process ,4,9.5 and C6 compounds [5 and C6 aldehydes, alcohols and esters was influenced not only by the cultivars but also by other factors, such as the ripening stages of the fruit, agricultural practices, growing areas, and climatic and technological aspects [On the other hand, the most important fraction of volatile compounds that guarantees the development of VOO flavor is obtained in the first phases of the technological process, during crushing and immediately thereafter. A minor percentage is also produced during the kneading phase of olive paste, as reported in previous studies ,11. The ompounds . As demoompounds ,16,17. Hompounds ,9,10,11. aspects ,19.Currently, the best way to manage the temperature of extraction plants at an industrial scale is to employ the rapid conditioning of olive paste after the crushing step with a heat exchanger ,20,21,22Probably due to the different temperature values, this group of endogenous enzymes works differently, and an interesting research topic is to examine how the specific activity that characterizes each different cultivar is influenced by the olive oil paste exposure time at low temperature. The research design was carried out on three different cultivars characterized by different concentrations of phenolic and volatile compounds, from a quantitative and qualitative point of view, in order to better evaluate the impact of innovative technology on VOOs obtained by olive fruits of different genetic origin. The main aim of the work concerned the use of temperatures below 20 °C during the malaxation phase to assess the potential effect on the development of volatile compounds and on the phenol content of VOO in relation to the different genetic origins of the raw material.The seco-iridoid derivatives and lignans were extracted from VOO, as described by Selvaggini et al. (2014). The chemical standards, solvents and other compounds used during the analyses of VOO were supplied by Merck , tyrosol was supplied by Cabru s.a.s.  and hydroxy-tyrosol was supplied by Fluka .Control (C-VOO) heat exchanger at 25 °C, malaxer at 25 °C for 30 min;Test 1 (T1-VOO) heat exchanger at 18 °C, malaxer at 25 °C for 30 min;Test 2 (T2-VOO) heat exchanger at 18 °C, malaxer at 18 °C for 30 min;Test 3 (T3-VOO) heat exchanger at 18 °C, malaxer at 18 °C for 15 min + 15 min at 25 °C.The olives belonged to three different Italian cultivars  and were processed within 48 h after the harvesting period and from the middle of October to the first week of November 2019. The olive growing areas of Canino and Peranzana were located in the Lazio and Apulia regions, respectively, whereas the olives of Moraiolo were harvested in the Umbria region. VOOs were extracted from approximately 180 kg of olives using an industrial plant TEM 200 system  equipped with a hammer mill, a malaxer with a gas controller system that had a working capacity of 200 kg of olives, and a two-phase decanter. The final separation step was carried out with a UVPX 305 AGT 14 centrifuge . Rapid control of olive paste after the crushing phase was obtained using an EVO-Line heat exchanger . The same chiller system as the heat exchanger was also used to regulate the temperature of the malaxer during the 30 min kneading phase. The control trials (C-VOO) were conducted at 25 °C, whereas the experimental tests were conducted by settling the temperature of the heat exchanger at 18 °C and changing the temperature of the malaxer as follows:The main legal quality parameter methods  were evaluated according to Regulation (EU) 2015/1830 [The content of the main hydrophilic phenols of VOO controls and extracts at low temperature was analyzed with high-performance liquid chromatography (HPLC) using the same instrument and the same method as described by Taticchi et al. (2021). The concentrations of 3,4-DHPEA-EDA (oleacein), p-HPEA-EDA , 3,4-DHPEA-EA (isomer of the oleuropein aglycon), p-HPEA-EA (ligstroside aglycon), 3,4-DHPEA (hydroxy-tyrosol), p-HPEA (tyrosol), vanillic acid, p-cumaric acid, (+)-1-acetoxypinoresinol and (+)-pino-resinol were expressed in mg/kg of oil.The volatile fractions of the VOO control and extracts at low temperature were analyzed with headspace solid-phase microextraction followed by gas chromatography–mass spectrometry (HS-SPME/GC-MS) using an Agilent Technologies GC 7890B instrument equipped with a “Multimode Injector” (MMI) 7693A  and followed the method described by Taticchi et al. . The conAll the phenolic and volatile compound data of the VOO controls and experiments were analyzed by SigmaPlot Software 12.3  with one-way analysis of variance (ANOVA).Based on the results, in the mechanical extraction process of VOO, the use of low temperature during the first phases, such as the crushing and malaxation of olive paste phases, showed that the temperature had a significant role in controlling the activities of the main endogenous enzymes  that regulate both the neoformation of volatile compounds and the oxidation phenomenon of the phenolic fraction ,8,16,17.The use of low temperature during the malaxation phase did not modify the oil yield, which confirmed the data reported in a previous study that did not observe obstacles to the coalescence phenomena of oil droplets and VOO extractability .232, K270 and ΔK in line with the legal limit [Compared with the control test results, the assessment of the quality indices of T1, T2 and T3-VOOs did not show significant differences for all three cultivars, with values of free fatty acids, peroxide, KThe use of flash thermal conditioning of olive paste after the crushing phase (T1-VOO), induced by a heat exchanger, allowed for the positive variation in the phenolic fraction to be determined, which was a function of the genetic origin of the olives. The settling of the heat exchanger at 18 °C confirmed data that indicated a significant improvement in the phenolic composition for the T1-VOOs extracted from the three cultivars in comparison with the C-VOOs [The high enhancement of Canino was very interesting because the control content of phenolic compounds was very low and the use of a heat exchanger highly improved the sensory and health properties of the product.The rapid reduction in the temperature of crushed olive paste (T1-VOO) also confirmed a significant influence of the cultivar on volatile compounds. The effects were strictly related to the different levels of the activity of the lipoxygenase (LOX) pathway enzymes that regulate the development of the VOO volatile fraction ,25.5 and C6 aldehydes when compared to C-VOO, which was a difference that was not detected for the other two cultivars  also prolonged the use of low temperature during the malaxation phase to evaluate the eventual positive effects on the formation of volatile compounds and on the hydrophilic phenol content of VOOs. The data obtained from Test 2 and Test 3 showed a differentiated impact on VOO quality parameters due to the different cultivars, probably as a consequence of the different activities of endogenous enzymes that regulate volatile and phenolic compounds. In fact, the LOX pathway is characterized by different levels of enzymatic activity, which are influenced by the genetic origin of the fruits, and this explains the variable development of the volatile aldehydes, alcohols and ester compounds that determine the large aromatic variability of VOO, as explained by other authors ,27.5- and C6-saturated and unsaturated alcohols of 24.5% compared to the C-VOO.The different LOX activities are influenced not only by agronomic practices, the growing area, and the ripening index but also by the temperature of the extraction process ,8,11,28.6 aldehydes. In contrast, the application of low temperature during the kneading of olive paste showed a significant impact on the volatile sensory note of Canino VOO with an increase in green and leaf-like aroma due to (E)-2-exenal [5 and C6 volatile compounds was also recently proposed by other authors [Although significant differences have been pointed out among the different tests, the concentration of esters of Canino VOO was probably too low to recognize a variability in the sensory properties of the product ,31. When2-exenal  and a re2-exenal ,32. Simi authors ,34.On the other hand, the use of low temperature during the malaxation phase reduced the phenolic content of Canino T2-VOO by 34% compared to T1-VOO, whereas the value rose again in Test 3 when the low temperature was maintained for only half of the kneading process. The data highlighted that the inhibitory effect of low temperature on PPO and POD activity  was minoAnother interesting result, which was also confirmed by the Moraiolo and Peranzana cultivars, concerned the evolution of VOO phenolic compounds during Test 3, in which increasing the temperature at 25 °C for the last 15 min of malaxation time was enough again to improve the levels of solubilization of the phenolic fraction into the oily phase of olive paste. The T3-VOOs enhanced their concentration of 8% and 12.4% compared with the trials conducted at 18 °C for the entire malaxation time for Moraiolo and Peranzana cultivars .In contrast, the genetic origin of the fruits had a major influence on the evolution of VOO volatile compounds both in quantitative and qualitative terms. In fact, the impact of different temperatures used after the crushing phase or during malaxation highlighted different evolution trends for the other two cultivars compared with the concentrations of the volatile fraction of Canino VOOs. The sum of aldehydes in Test 2 slightly increased, and above all in the Moraiolo VOO, even if the data were not statistically significant. Concerning the sum of alcohols and esters, the results showed a reduction and an increase in their concentration, respectively, compared to the C-VOO of Peranzana. The alcohols of Moraiolo showed the same evolution trend even if the data were not statistically significant, whereas the ester content, as analyzed for Canino VOOs, was too low to recognize the effect of a significant increase . During The first result of this study confirmed the positive role of rapid thermal conditioning on the olive paste of different cultivars after the crushing phase, which was induced by the reduction of temperature using a heat exchanger. The impact was mainly related to the improvement of the hydrophilic phenol content of VOOs in all the cultivars processed with different quantitative increases. In contrast, the low temperature, also applied during the malaxation phase, determined an overall reduction in the phenolic fraction. The data highlighted how the solubilization phenomenon of phenolic compounds into the oily phase, promoted at 25 °C of extraction temperature, played a higher role in the determination of the final VOO phenolic concentration than the impact of a probable reduction in the enzymatic oxidation induced by the use of lower temperature during the kneading of olive paste.The impact on the volatile fraction, which influenced the VOO flavor, was strongly conditioned by the genetic origin of the olives, which determined different responses to the application of low temperature during the malaxation phase. The VOO of the Canino cultivar was the only cultivar that showed a significant evolution of the volatile fraction, with a higher content of the sum of aldehydes and a reduction of the sum of alcohols that contributed to an enhancement of the “green” sensory note of VOOs. The use of low temperature for half of the malaxation phase could represent, for the Canino cultivar, a middle course to optimize the reduction effect of phenolic compounds and the increase of aldehydes, whereas for the other cultivars, which did not show significant improvement in volatile concentration, application of 18 °C temperature during the kneading step would not be recommended.Knowledge of new technologies and the settling of different technological parameters, such as temperature and time of treatment, should be implemented to optimize the mechanical extraction process as a function of the different cultivars processed, with the opportunity of obtaining VOO of higher quality standards. The high variability was also apparent in this study and mainly related to the volatile content evolution. This variability suggests the importance of having a range of solutions to extract from each different cultivar, different VOOs characterized by different levels of volatile and phenolic compounds that can characterize the sensory and health properties of the product and intercept with different groups of consumers.


Aars was the most stably expressed HKG in the mouse brain as a whole. However, different genes were more stably expressed in different parts of the brain. Polr2f was the most stably expressed in the cortex, Psmd6 was the most stably expressed in the cerebellum, and Psmd7 was the most stably expressed in the striatum and substantia nigra. HKGs were different in similar tissues of the studied organisms. Polr2f was the most stably expressed HKG in the peripheral blood of mice, whereas PSMD6 was the most stably expressed gene in humans. Thus, there is no universal HKG both for different brain tissues of one organism and for similar tissues of different organisms. Furthermore, the identified most stably expressed HKGs can be considered as such only under conditions in PD.A critical aspect of real-time PCR is the presence of housekeeping genes (HKGs) as an internal control for the normalization of expression data for genes of interest. It is necessary to select correct HKGs in the investigation of various pathologies. Thereby, we analyzed the stability of expression of the HKGs in Parkinson’s disease (PD). The work was carried out in the peripheral blood of patients with PD and in the brain tissues and peripheral blood of mice with MPTP-induced PD. As a result,  Currently, active investigations of gene expression are being carried out. The simplest and most widely used method for analysis of gene expression at the mRNA level is a semiquantitative approach based on the reverse transcription (RT) reaction and real-time quantitative polymerase chain reaction (qPCR). This method is considered the gold standard for the study of changes in gene expression at the mRNA level in biomedical investigations ,2.A critical aspect of this method, as for other semiquantitative methods, is the presence of reference genes as an internal control for the normalization of expression data for the genes of interest. Based on this, we can conclude that the precise determination of changes in the expression of candidate genes depends on the correct choice of internal reference genes. These genes are called housekeeping genes (HKGs).Genes with stable expression in all biological samples and whose expression does not change under experimental conditions or under conditions of disease are most often considered as HKGs . HKGs arIt is believed that the expression of these genes should not depend on the influence of various factors acting on the cell, tissue, and organism, or these factors should have minimal effect. However, it is now becoming clear that the expression of the most common HKGs can depend on external conditions and changes in pathologies ,6. The sBased on this observation, a search and selection of the most stably expressed HKGs for the analysis of gene expression in Parkinson’s disease (PD) were carried out. PD is one of the most common neurological diseases and is characterized predominantly by the death of dopaminergic neurons in the substantia nigra. Degeneration of dopaminergic neurons leads to disorganization of the function of almost all neurotransmitter systems of the brain. In this regard, the etiopathogenesis of PD has been widely studied, and active analysis of changes in gene expression under conditions of this pathology is being carried out. However, experimental results are often not reproducible or do not match. Perhaps this is due to differences in the HKGs used.In this regard, we analyzed the reference genes in the peripheral blood of patients with PD, as well as in the tissues of the brain and peripheral blood of mice (Mus musculus), as the most widespread object for modeling PD .l-dopa, piribedil, pramipexole, or amantadine) in various combinations.In this study, 35 untreated and 12 treated patients with the sporadic form of PD  were analyzed. The mean age ± standard deviation (SD) at disease onset was 58.6 ± 7.4 years (range 46–71), and the mean age at enrollment was 59.2 ± 7.1 years (range 47–73). The PD patients with treatment received different medications with dopamine receptor agonists . They were selected and studied according to the International Movement Disorder Society Unified Parkinson’s Disease Rating Scale  and Hoeh44 neurologically normal healthy individuals were studied as controls. These participants were matched to the PD patients regarding gender, age, and ethnicity.Patients and neurologically healthy volunteers (of Russian ethnic origin residing in European Russia) were selected at the Research Center of Neurology . All participants were examined for the presence of frequent mutations in all genes responsible for monogenic forms of PD and did not have any family history of PD. All participants had no severe concomitant diseases, such as severe cardiovascular disease, cancer, or diabetes mellitus.The study was conducted in accordance with the World Medical Assembly Declaration of Helsinki—Ethical Principles for Medical Research Involving Human Subjects. All blood samples were collected with the informed consent of the investigated subjects. The study was approved by the Ethics Committee of the Research Center of Neurology , protocol 18/4.Twenty C57BL/6 mice were purchased from the specific pathogen-free licensed nursery of the Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences. Eight-week-old male mice weighing 22–25 g were randomly divided into two groups for analysis: one control group and one experimental group. Each group of mice contained 10 individual animals. The mice were housed 3–4 to a cage under standard conditions with free access to food and water with a 12:12 h light:dark regime.A mouse model of the early symptomatic stage of PD was used in this study. Experimental work with laboratory animals was carried out in accordance with The Guide for the Care of Animals  as approAll efforts were made to minimize the potential suffering and discomfort of animals, and care was taken according to the 3R rule .The isolation of total RNA from brain tissues was carried out using the RNAeasy Mini Kit  and Quick-RNA MiniPrep Kit .The isolation of total RNA from 250 µL of each sample of human and mouse peripheral blood was performed using Whole Blood RNA MiniPrep Kit . The concentration of total isolated RNA from all samples was measured using a Quant-iT RNA BR Assay Kit and a Qubit 3.0 fluorimeter . RNA quality was monitored using an Experion automated electrophoresis system . The RNA quality index was higher than 8.5 in all samples.All procedures were carried out according to the manufacturers’ recommendations.Gene expression analysis was conducted using RT-qPCR with TaqMan probes. All samples were handled as equally as possible in order to minimize any random biases. Single-stranded DNA was synthesized using 120 ng of total RNA, 100 ng of Escherichia coli tRNA as a carrier , specifiPrimers and probes for qPCR were selected using the Beacon Designer 7.0.2 program and the nucleotide sequences of the genes of humans, mice, and rats. The sequences of gene-specific primers and probes are listed in cDNA obtained in the reverse transcription reaction was used as a template for qPCR. This was diluted 50 times in an aqueous solution of tRNA from Escherichia coli (0.02 ng/μL) . qPCR waPrimers and probes for analysis were selected using Beacon Designer 7.0  and corresponding sequences from the NCBI database . The speThe RefFinder program calculates the score of reference genes using the algorithms of geNorm , NormfinBased on these scores, the program assigns an appropriate score to each gene and calculates the geometric mean of their weights for the overall final ranking. The program chooses the lowest score among the ranks assigned by each algorithm separately and then assigns the final comprehensive score. The closer the final score is to one, the more stably expressed the HKG is considered to be.For the selection of candidate genes in the HKGs database, screening was conducted in PubMed. A search for the key MeSH term “housekeeping gene” (filters applied: humans) yielded 26,399 publications since 2000. A search for the keyword “housekeeping PCR” (filters applied: humans) returned 880 results since 2000. Searching for the keyword “housekeeping real-time PCR” (filters applied: humans) returned 562 results since 2000. The search term “housekeeping real-time PCR brain” (filters applied: humans) returned only 29 results since 2001. Of these 29 studies, only 4 were directly related to the analysis of HKGs in nontumor samples ,23,24,25In addition, data on the analysis of reference genes in brain tissues , a neuroNext, we carried out an additional screening of publications on the query “gene expression Parkinson’s disease RNA” (filter: human) in the PubMed database, and 715 publications were identified. The TBP [ACTB, RPL30 [GAPDH [YWHAZ [PPIA [B2M [HPRT [GUSB [SARS1, AARS1 [PSMD6, PSMD7 [POLR2F [POLR2A, and PSMA5 [Then, we compared the two generated lists and selected the genes included in both lists to study the stability of their expression in our work. As a result of the analysis, HKGs TBP ,26, ACTBB, RPL30 ,29, GAPD0 [GAPDH ,29, YWHAH [YWHAZ , PPIA [2AZ [PPIA , B2M [262M [HPRT ,27, GUSBRT [GUSB ,27, SARS1, AARS1 , PSMD6, 6, PSMD7 ,29, POLRnd PSMA5 ,31 were ACTB [GUSB [PPIA [YWHAZ [RPL30 [GAPDH [All potential HKGs must satisfy certain conditions. First, the genome should not contain pseudogenes of these genes. It is known that processed pseudogenes lacking introns and being almost complete generate copies of mature mRNA of the corresponding genes making up 70% of all pseudogenes . In thisACTB , GUSB [3TB [GUSB , PPIA [3SB [PPIA , YWHAZ [A [YWHAZ , RPL30 [Z [RPL30 , and GAP0 [GAPDH  were excB2M [Second, a reference gene means a gene whose expression does not change under the conditions of the studied pathology and is also approximately the same in all studied tissues. In this regard, expression of all selected HKGs was analyzed using BioGPS, and HKG B2M  was exclPOLR2F, POLR2A, PSMD6, PSMD7, HPRT1, PSMA5, SARS1, AARS1, TBP, and BCAT2 (the names are given in the nomenclature for humans), TaqMan primer systems were selected, and an analysis of changes in their relative mRNA levels was performed in brains of mice and mouse and human blood samples. It should be noted that at the stage of selection of primer systems and their validation for analysis, the TBP, POLR2F, and BCAT2 genes in humans were eliminated because their levels of transcripts were below the limit of detection. After obtaining the primary data, the Ct genes were analyzed using the RefFinder program.For the remaining HKGs An example of data processing for the analysis of relative mRNA levels in mouse peripheral blood is shown in general in Psmd7 is the most optimal and stably expressed HKG in the brain as a whole, as well as in three separate brain tissues  in the control group of mice; however, this gene is the second in the cortex, where Aars1 is in the first place. At the same time, Psmd6 is the most stably expressed in the brain, as a whole, of experimental animals with the MPTP-induced PD model; however, it occupies only fourth place in the cortex and striatum and third place in the cerebellum and substantia nigra.The cumulative results of the analysis are shown in In general, as can be seen from the data presented, the stability of expression of the HKGs studied differs depending on the presence or absence of pathological processes.Comprehensive scores and gene ranks were obtained for each gene in each tissue examined.Based on the fact that the stability in different tissues and in the presence and absence of pathology is different, when carrying out work on expression analysis, it is necessary to use HKGs whose expression is most stable in a certain tissue both in the presence and in the absence of pathology. Accordingly, a table was compiled for the final ranking of HKGs, based on the calculation of the summarized comprehensive score .The summarized comprehensive score of each HKG in each tissue was summarized from the comprehensive score obtained from the control mice and healthy volunteers  and miceAars1 is the most stably expressed and takes the first place (gene rank = 1) with a total comprehensive score of 4.21 = 2.21 + 2.20, where 2.21 is a comprehensive score from the column “whole brain” of For example, in the brain as a whole, regardless of the presence or absence of pathology, The final rank of each HKG in tissues of mice was summarized from the comprehensive score obtained from the control group  and the Aars has the most stable expression among all HKGs studied in the brain. The same gene ranks second in terms of stability of expression in various parts of the brain. Ranking second in the whole brain, Psmd7 was most stably expressed in the striatum and substantia nigra. In the cortex, the most stably expressed HKG was Polr2f, and in the cerebellum, the most stably expressed HKG was Psmd6. It is noteworthy that the first of them was also the most stably expressed in the peripheral blood of mice, and the second was the most stably expressed in the peripheral blood of humans.In general, based on the data obtained, the HKG Summarized comprehensive scores and gene ranks were obtained for each gene in each tissue studied.According to the widespread opinion of the scientific community that when analyzing changes in gene expression, it is necessary to take into account at least two HKGs, one should take into account the genes located at the first, second, and, at the most, at the third place in the stability rating of HKGs given in Polr2f is the most stably expressed HKG in the peripheral blood of mice, whereas PSMD6 is the most stably expressed gene in humans  was chosen. Models based on the introduction of this toxin are currently considered classical . It has ACTB, GUSB, PPIA, YWHAZ, and GAPDH, widely used as HKGs, were removed from further study. For example, ACTB and GAPDH are the most frequently used housekeeping systems. ACTB has 22 pseudogenes and GAPDH has 47 [Based on a bioinformatic analysis searching for the presence of pseudogenes, the genes H has 47 . PseudogH has 47 ,46. In tPSMD7 (NM_002811.5) and HPRT1 (NM_000194.3) genes. However, there is currently no experimental confirmation of their existence, so we selected these genes for further analysis. In addition, primers were selected so that they did not anneal to the region of putative pseudogenes in the genome.The rest of the HKGs selected based on the results of the literature analysis passed the bioinformatic analysis. Coincidentally, in silico pseudogenes were found for the One of the fundamentally important conditions in the selection of HKGs is the stability of their expression level, regardless of the presence or absence of the development of a pathological process. In our work, it was shown that the presence of a pathological process affects the stability of the expression of HKGs. As can be seen from Aars, which encodes an alanyl-tRNA synthetase. This gene is not directly involved in the processes associated with the functioning of the nervous system and processes involved in the pathogenesis of PD. In addition, this gene is the second most stably expressed gene in some of the brain tissues we studied. Thus, this gene is the most stably expressed and attractive HKG for expression analysis.Overall, the most stably expressed HKG in the mouse brain  is Aars,Psmd7 gene in mice turned out to be one of the most stably expressed in the brain as a whole .The gene encoding polymerase 2 subunits in mice, The data obtained indicate that there is no universal HKG both for different brain tissues of one organism and for similar tissues of different organisms. This conclusion is supported by other studies: researchers must select and validate a set of reference genes for each specific cell line . In addition, we have shown that the presence of a pathological process may affect the expression of HKGs. This fact must be taken into account when choosing the optimal HKG for expression analysis in pathological conditions. In this case, it is necessary to choose those HKGs that are most stably expressed in both experimental and control groups, in other words, regardless of experimental conditions.In conclusion, it should be noted that there are certain limitations of the use of the HKGs selected in this study as the most stably expressed. Thus, these HKGs can be most confidently used to study the pathological process caused by the pathogenesis of PD in young mice, as well as for PD patients at relatively early stages of the pathological process.


Advanced pore morphology (APM) foam elements are almost spherical foam elements with a solid outer shell and a porous internal structure mainly used in applications with compressive loading. To determine how the deformation of the internal structure and its changes during compression are related to its mechanical response, in-situ time-resolved X-ray computed microtomography experiments were performed, where the APM foam elements were 3D scanned during a loading procedure. Simultaneously applying mechanical loading and radiographical imaging enabled new insights into the deformation behaviour of the APM foam samples when the mechanical response was correlated with the internal deformation of the samples. It was found that the highest stiffness of the APM elements is reached before the appearance of the first shear band. After this point, the stiffness of the APM element reduces up to the point of the first self-contact between the internal pore walls, increasing the sample stiffness towards the densification region. APM foa3 ,5, where3 ,7,8. Whe3 ,10.Due to their specific properties ,12 and tIn general, APM foam elements have a characteristic stress–strain response in compression. Their mechanical properties have been analysed in detail in many studies using experimental testing and advanced computer simulations ,12,15,20The existing studies focus primarily on the mechanical response or the internal structural changes in the APM foam elements, adding numerical simulations to better understand the mechanical behaviour of APM foam elements. Therefore, such studies give no direct answer to how the deformation of the internal structure and its changes are related to its mechanical response. With the advancements achieved in the field of porous solids for structural applications ,30,31,32Numerous ex-situ compression experiments on metal foams ,37,44 weMechanical experiments were performed using an in-house designed table-top loading device allowing in-situ 4D XCT experiments  to be peIn this study, the APM foam elements produced from the AlSi7 alloy powder and foamed using TiH2 foaming agent were used. The foam elements were not coated with a bonding material and had a nominal diameter of 10 mm. More details about the manufacturing process can be found in ,6. PreviA set of nine APM samples having a quasi-spherical shape with an outer diameter of 10.4 ± 0.4 mm, a meridian diameter of 10.1 ± 0.2 mm , and a weight of 387.2 ± 1.1 mg were subjected to uni-axial compression to investigate their deformation behaviour and microstructural characteristics. Three APM foam samples were compressed and scanned in-situ, while the rest of the samples were only investigated for their mechanical response. All the mechanical tests  were performed using a loading rate of 5 µm/s. During the radiographical imaging, XCT scans were performed in 200 µm displacement increments corresponding to approximately 2% of the overall compressive strain. The experiments were terminated by reaching approximately 50% strain corresponding to the end of the plateau region and the start of the densification.The compressive response of the samples was measured using a load-cell signal with 0.1 N precision and a position encoder signal with 0.25 µm precision. A sensor readout rate of 100 Hz was selected and the experiments were controlled using in-house developed control software .2 and a native resolution of 1944 × 1536 px was used for the image acquisition. Each tomographical scan of the time-lapse measurement consisted of 800 equiangular projections with a 90 ms acquisition time. The overall scanning time per one tomography was 6 min including the image acquisition time, time of detector readout, and rotary stage movement. In total, the in-situ experiments consisted of 26 or 27 scans per each sample. The reconstruction of the 3D images was performed using an FDK filtered backprojection reconstruction algorithm [For the radiographical imaging, an XWT-240-SE  transmission-type X-ray tube operated at an acceleration voltage of 90 kV and a target current of 333 µA was used to irradiate the sample. A Dexela 1512 NDT  flat panel X-ray detector based on a GOS scintillator and CMOS technology with an active area of 145.4 × 114.9 mmlgorithm  implemenThe threshold value was determined based on a histogram evaluation in VG Studio MAX 3.4 by identification of a local minimum in the histogram of the reconstructed 3D image, since this minimum is located between the histogram peaks corresponding to the background intensities and APM material intensities and is, therefore, a good threshold value for binarisation. This enabled one to suppress the artifact of the cone-beam irradiated loading platens at the cost of loss of information near the loading platens , which wNevertheless, due to the sufficient contrast to noise ratio in the reconstructed 3D images, it was possible to treat the photon scatter artefact in the vicinity of the loading platens during the binarisation procedure without further measures. However, an error resulting from such a binarisation procedure with a magnitude of few percent can still be expected.Furthermore, to quantify the effect of the relatively low number of projections (800) per tomography used in the time-lapse measurement for the scan speed increase, each sample was subjected to a high-quality reference CT scan with 2400 projections prior to the loading procedure. 5 dissimilar voxels in each of the 1943 slices in the reconstructed 3D image. Still, the majority was distributed around the photon scatter artefact caused by the aluminium loading platens.Then, the reconstructed 3D images from this high-quality CT and undeformed state of the time-lapse measurement of every sample were compared. Here, the difference between the binarised 3D images was selected as a measure of conformity. Thus, the binarised 3D image of the reference scan was subtracted from the binarised 3D image of the zero-loading state and the total number of non-zero elements  was calculated. From the three investigated samples, the average ratio of the dissimilar voxels to the total number of voxels in the 3D reconstructed images was 1.95%. In the worst case, the relative difference was 5.85%, which corresponds on average to 1.75 × 10To determine the changes in the APM foam elements’ outer shape, volume and porosity, the outer shape of the APM element had to be found for each 3D binarised image. In the case of porous materials, this is not a trivial task since some pores are opened towards the outside a and no The filter operates on one transversal slice at a time. For each pixel of the slice, four lines are projected to the image border in the following directions: left, right, up and down. The filtering procedure enables one to determine the filled slice area The compressive behaviour of the samples is shown in the form of force–deformation curves in During the intermittent in-situ loading procedure, the anticipated force decreases with a magnitude of 10–15% of the actual load occurred due to the release of elastic energy stored in the specimen and the loading device itself, causing the measured relaxation effect . In everThe compressive force–deformation curves have a characteristic shape for porous materials even though the samples were spherical and did not have a constant cross-sectional area. At the beginning of the loading procedure, a linear increase in the force up to the local maximum is followed by a decrease to a minimum plateau value. After that, the force increases in the transition to the densification region at deformations of approximately 50% of the overall compressive strain. The force values at the first force peak are equal to 449, 533 and 458 N at the deformations of 18.8%, 31.0% and 23.1% for APM foam samples 1, 2 and 3, respectively. The lowest plateau forces (neglecting the relaxation force drops) are equal to 292, 417 and 357 N at the deformations of 30.4%, 39.4% and 32.9% for APM foam samples 1, 2 and 3, respectively. The values for characteristic points in the force–deformation diagrams are summarised in Table 1The deformation behaviour of the internal foam structure is shown in The deformation of the outer sample shape was analysed using the value of the filled circle radius With the increase in the deformation, the outer shape of APM sample 1 mostly compresses at the top and at the bottom of the sample, which is evident from the fact that the spherical sample shape is flattened at the bottom and the top of the sample. At the same time, the value of the maximum filled radius for each deformation stage increases from 5.35 to 5.51 mm and finally to 6.08 mm at the deformation stages of 0%, 26% and 50%, respectively.The comparison of the outer shape changes between the different samples was carried out by comparing the maximum filled radius and the normalised filled volume. The normalised filled volumes of the APM samples are shown in The internal changes in the APM foam samples during the compressive deformation were examined by analysing the slice of the sample and average porosity. The slice porosity changes for APM foam sample 1 due to the applied deformation in dependence on the sample’s height are shown in 3, the computed average sample porosity is equal to 73.4% and is in good agreement with the porosity measured from the reconstructed 3D images. During the deformation of APM foam sample 1, its porosity decreased from 75.1% to 70.1%, it decreased from 73.6% to 67.7% for sample 2 and it decreased from 75.0% to 70.2% for sample 3. The porosity changes yield 5.0%, 5.9% and 4.8% for samples 1, 2 and 3, respectively. Observing the rate of porosity change per deformation step (curve inclination), a point can be found in all three curves when the rate of porosity change is decreased to a local minimum. That point occurs at the deformations of 20%, 30% and 26% for samples 1, 2 and 3, respectively.The decrease in the average sample porosity is also visible in Using computed microtomography and in-situ compressive testing of APM foam samples enables the simultaneous evaluation of the mechanical and geometrical changes in samples. The overall shape of all three mechanical responses in The compressive force response in Comparing the force response of APM foam sample 1 with its internal deformation shown in The deformation level of the shear plane appearance and the deformation level of the first force peak can also be predicted from the data of the maximum filled radius. The maximum filled radius of APM foam sample 1 starts to increase at the deformation of 22%, which coincides with the appearance of the shear plane at the deformation of 20%. Up to the point of the shear plane appearance, the sample mostly deforms parallel to the loading direction as there is enough internal pore space for the buckling and bending of the pore walls and, therefore, the maximum filled radius remains the same. As shearing deformation appears and parts of the sample start to move in the transversal direction, the maximum filled radius increases. Thus, an increase in the maximum filled radius points to the appearance of the shear deformation. Analysing the results for samples 2 and 3 gives the same conclusions.Analysing the rate of the change in the normalised filled volume and the average sample porosity gives similar results. For APM foam sample 1, a local minimum in the volume change was recorded at a deformation of 22% and a local minimum in the porosity change was recorded at a deformation of 20%, which coincides with the appearance of the shear plane. Up to the appearance of the shear plane, the outer volume of the sample is reduced. After initiation of the transversal shear deformation, the loading is not only converted into compression of the sample, but also in its transversal deformation and the rate of volume change is reduced. Since the average sample porosity is computed using the filled volume, this effect is also observable in the values of the average porosity. The results of samples 2 and 3 confirm these findings.The analysis of the maximum filled radius of all three APM foam samples showed that the maximum filled radius remains almost constant in the first stages of the compression. Up to the deformation of 20% the maximum filled radius changes for less than 1%.In the first stages of the sample compression, the spherical sample shape changes the characteristic compressive response of the porous material because the regions with the smallest cross-sectional area at the surface of the sphere are compressed first and so the plateau region is reached at higher deformation levels.The mechanical response correlated with the internal deformation of the samples. It was found that the highest stiffness of the samples is reached before initiation of the shear plane and the resulting transversal sample deformation. After this point, the stiffness of the sample reduces up to the point of the first contact between the internal pore walls which increases the sample stiffness towards the densification region.The geometrical analysis of the outer shape and porosity of the APM foam samples showed that the occurrence of the shear plane is also noticeable in the values of the maximum filled radius, the filled volume and the average porosity.Observation of the compressive behaviour of APM foam samples using two different methods, micro-computed tomography coupled with in-situ compressive testing, enabled new insights into the deformation behaviour of the APM foam samples to be determined. The most important findings comparing the results of both methods are as follows:


This research is focused on cellulose and starch derivatives, carboxymethyl cellulose (CMC) and carboxymethyl starch (CMS), added to the detergent in washing reference cotton fabric in soft and hard water at 40, 60 and 90 °C. The applied polymers were analyzed through the potential of surface cellulose modification and inhibition of stain transfer from standard stain donors to modified and initial cotton fabrics. The surface modification of the cotton fabrics, characterized by the zeta potential and amounts of deposits, was coupled with the cluster analysis as well as a whiteness assessment. The obtained results of the zeta potential and degree of whiteness of the reference cotton fabrics before and after washing showed differences between CMC and CMS. The appropriateness of the cluster analysis was confirmed in assessing the potential of applied polymers for surface modification of cotton fabrics and greying inhibition. Washing removes dirt from textiles in an aqueous medium, with the synergy of four factors playing an important role: temperature, time, mechanical effect and chemical effect, which were combined into a process–theoretical system by Dr. Herbert Sinner in 1959 . Water-sThe polymer mostly used as a greying inhibitor or anti-redeposition agent (ARA) of cellulose textiles and is a derivative of cellulose—sodium carboxymethyl cellulose. Due to its water solubility, non-toxicity and biodegradability, carboxymethyl cellulose (CMC), as an anionic polyelectrolyte, is widely applied in numerous fields. In applications, most properties of CMCs depend on three parameters, namely, the molecular weight of the polymer, the average number of carboxymethyl substituents per anhydroglucose unit (or degree of substitution) and the distribution of carboxymethyl substituents along the polymer chain ,14,15. TThe mechanism of action of this polymer is a specific adsorption on cellulose, specified as a very complex process determined by many factors and classified into three categories: (1) polymers, (2) cotton cellulose as an adsorbent, and (3) the composition and the properties of the solution .These polymers have a specific orientation towards cellulose within several layers, which increases the number and density of negatively charged carboxyl groups and the negative charge of the textile surface.In the first phase of rinsing, the dirt is removed, and then in the second phase, the connection between the greying inhibitor (ARA) and the fiber is broken. Modified starches with improved physicochemical properties relative to the native starch are also being researched as functional ingredients in numerous applications. Carboxymethyl starch (CMS), an etherified anionic starch derivative, is considered a green polymer with significant performance in the pharmaceutical, medical, cosmetics, food, environmental, and numerous other industries. This polymer has a low temperature of gelling, swelling and solubility in cold water and degree of substitution (DS), i.e., the number of hydroxyl groups replaced by carboxyl groups determines its properties ,21.In contrast to CMC, the properties of carboxymethyl starch (CMS) have not been investigated regarding surface modification and greying inhibition in the washing of cellulosic fibers. This is why this study is focused on comparing these two polymers, and their mixture (CMC + CMS) in surface modification and greying inhibition of cotton fabric. The polymers, CMC, CMS and their mixture, were added to a detergent dry slurry for washing the reference cotton fabrics in hard and soft water at 40, 60 and 90 °C.The surface modification of the cotton fabric was analyzed after 10 washing cycles by determining the surface charge and deposit content relative to the initial one. The greying inhibition of the cotton fabric before and after modification, washed with stain donors, was analyzed by a whiteness measurement. Given the complexity of the washing process and the involvement of a large number of parameters, their impact was analyzed by cluster analysis as an advanced statistical technique that belongs to the field of multivariate statistical analysis (MVA).Reference cotton fabric without fluorescent whitening agents and finishing agents (F), for which technical characteristics are prescribed by ISO 2267: 2001, was modified (MF) by adding the polymer–surface modifier (SM): CMC, CMS and their mixture, into a detergent dry slurry for 10 washing cycles in hard and soft water at temperatures of 40, 60 and 90 °C. The composition of the basic detergent, supplied by Labud d.d., Zagreb, Croatia is shown in The detergent contained anionic surfactant, soap, sodium percarbonate as bleach, silicate, builders (zeolites and sodium carbonate), fluorescent whitening agents and water. To this detergent, applied at a concentration of 5 g/L, surface modifiers CMC, CMS and their mixture (CMC + CMS) were added at a concentration of 0.4%. The supplier of powder CMC was Dencell, Bulgaria, and of the liquid CMS, Likval, Bosnia and Herzegovina.The properties of CMC, CMS and their mixture in an inhibiting stain transfer were analyzed during a separate washing cycle of the reference cotton fabric (F) and the modified cotton fabric (MF), where EMPA stain donors  were added to each individual fabric. The characteristics of these specific stained textile carriers or stain donors from the EMPA supplier  are shown in The analysis of the potential of CMC, CMS polymers and their mixture in modifying cotton fabric through 10 cycles was performed in a laboratory washing machine with temperature variation  and water quality variation (hard (HW) and soft (SW)). The cycles were performed with a bath ratio (1:5), filling ratio (1:12), reversible rotation (40 to 50 rpm), and rinsing and cyclic centrifugation at 1400 rpm.The degree of modification of the cotton fabrics (MF) under the described conditions of 10 washing cycles in relation to the reference cotton fabric (F) was analyzed by the streaming potential method and by determining the zeta potential as an appropriate method for analysis of the polymeric materials’ surface ,23,24.Through the amounts of deposits, the impact of water hardness and washing temperature on the polymer orientation on the surface of the cotton fabric and the degree of its modification (MF) in relation to the reference cotton fabric (F) was investigated.The degree of inhibition of the stain transfer from the donor to reference cotton fabric (F) and modified fabric (MF) was evaluated before and after the 1st washing cycle in hard and soft water at 40, 60 and 90 ° by measuring the whiteness of the cotton fabric.The degree of substitution (DS) affects the properties of polymers, and these must be determined before application . The degThe hardness of soft (SW) and hard water (HW), as an indicator of mineral load was determined by complexometric titration in an alkaline medium with the addition of indicator Eriochrom black T.Deposits as incineration residues were determined directly on the standard reference cotton fabrics before (F) and after modification (MF). This residue comprises only a small fraction of the organic deposits; the amount of total ash depends on the magnitude of the mineral deposits according to ISO 4312: Surface active agents—Evaluation of certain effects of laundering—Methods of analysis and test for unsoiled cotton control cloth. The amount of ash from mineral substances is a more accurate index of the presence of mineral deposits, which originate from salts from the water or from the detergent, or their mutual interaction. The amount of total ash depends on the amount of mineral deposits, since only a small proportion of organic deposits is present in the total ash.The incineration residue (A) of the reference cotton fabrics (F) and modified cotton fabric (MF) was determined according to the standard method (ISO 4312). Samples of reference cotton fabrics were incinerated in a muffle furnace for 2 h at 800 °C . The percentage of ash content was calculated from the masses of ash content and cotton fabric .The impact of surface modifiers  on the properties of cotton fabric (F) washed with base detergent in hard (HW) and soft water (SW) at temperatures of 40, 60 and 90 °C was analyzed via charge surfaces. The characterization of the charge of the cotton fabric surface before (F) and after modification (MF) was performed by the streaming potential method in an electrokinetic analyzer, EKA, A. Paar, Austria, where the cotton fabrics were the solid stationary phase, and the electrolyte solution KCl (1 mmol/l), the mobile phase. From the values of the streaming potential of the cotton fabric embedded in the adjustable gap cell (AGC) and the parameters in the system depending on the pH value of 1 mmol/L of KCl, the zeta potential (ζ) was calculated according to the Helmoltz–Smoluchovsky equation .The deposition of stains removed from the reference donors on polymer modified fabrics (MF) and the initial fabric (F) in the 1st washing cycle was evaluated by the degree of their whiteness. Spectral parameters of reference fabrics, F, MF, F1 and MF1, were determined using the Spectrophotometer Data Color Spectraflash SF 600 Plus-CT, with the aperture size of 20 mm, under standard illumination D65, with the measuring range of 360–700 nm. The whiteness degree (W) was calculated automatically according to ISO 105-J02: 1997 Textiles—Tests for color fastness—Part J02: Instrumental assessment of relative whiteness and expressed as medium values of four individual measurements.A cluster analysis was applied for evaluation of CMC, CMS and their mixture on deposits and the degree of inhibition of the stain transfer from donor to cotton fabrics before and after modification in washing with detergent in hard and soft water at 40, 60 and 90 °C.d, between two points in n -dimensional space with coordinates jx and jy is usually taken as the Euclidean distance defined by Equation (1):This method belongs to the field of multivariate statistical analysis (MVA). Multivariate techniques are mainly empirical, and their task is to present the analysis of complex sets of data and their interrelationships in a simpler way, characterized by a lot of new information contained in the output data. They are applied in many areas of spectral data analysis   and timeA hierarchical cluster analysis is basically an iterative process of merging objects into groups. The method is based on the formation of clusters in a hierarchy, so that at each subsequent level, the number of clusters is decreased by one, with no possibility of transition from one group to another. This method is characterized by an unknown number of clusters determined on the basis of dendrograms. A dendrogram is a graphical presentation of the gradual combination of objects into clusters on which the distances between individual levels can be observed.A single linkage method using TIBCO Statistics software to the incineration residues data, zeta potential data for deposit analysis and whiteness degree data for stain transfer were applied. As the measure of homogeneity, the error sum of squares (ESS) within the cluster was taken.The degree of substitution (DS) of surface modifiers and greying inhibitors, CMC and CMS, was determined. The obtained average values showed that the degree of substitution CMS (0.75) was higher than CMC (0.57).3) = 404.1 ppm and soft water concentration c (CaCO3) = 44.5 ppm.Water hardness was analyzed, where the average value of hard water was expressed as concentration c  and after modification through 10 cycles (MF) in different conditions, the degree of surface loading with residues was determined. Since the obtained surface loading depends on a number of parameters of the washing process, the goal of this paper was to analyze the interaction of parameters that varied during the washing process. For the interrelationship of all parameters, a cluster analysis was performed on all the results of deposits (ash) of washed cotton fabrics (MF) with variations of detergent composition through different ARAs , and washing conditions  and temperature ).A single linkage method to the incineration residues (IR) data was applied. The results are presented as dendrograms in 3) is low and the value of incineration residue can be compared with unwashed fabric, which is 0.2%.The ash content of cotton fabrics modified in washing with detergent, in which the type of polymer was varied in soft water through 10 cycles at all temperatures, is almost identical. The presented dendrogram shows small differences in the stages of linkage of belonging to the group of samples washed in soft water. Washing in soft water shows no additional reactions or salt deposits on the fabric, since the value c  after 10 cycles of washing (MF) with basic detergent was analyzed by titration curves of the zeta potential depending on the pH of 1 mmol/L KCl. The zeta potential of the reference cotton fabric (F) before modification depending on the pH of 1 mmol/L KCl is shown in The zeta potential curve of the reference cotton fabric depending on the pH is characteristic of bleached cotton cellulose with hydroxyl (-OH) groups and a smaller number of carboxyl (-COOH) groups. The range of the curve from pH 9 to pH 6 is unchanged, and at lower pH values, it has a characteristic shape, which indicates the gradual neutralization of the negative charge up to reaching the isoelectric point . The resThe zeta potential values of the modified cotton fabrics (MF) under all the varied conditions in the range of pH 9 to pH 4 are more negative than the zeta potential values of the reference cotton fabric (F). Following the differences between the curves and the values, a behavior pattern cannot be established but generally, polymer-modified cotton fabrics in soft water have a more negative surface charge than polymer-modified fabrics in hard water. Based on the obtained ratios, CMC as a polymer impacts the increase of the negative charge of the cotton fabric surface modified in soft water at 40 and 60 °C. It agrees with the previous findings which showed the responsibility of CMC for the zeta potential and also the importance of electrostatic interactions in the adsorption process of CMC . Cotton The above ratio may also indicate some steric changes of fabric at 90 °C compared to 40 and 60 °C. According to the obtained results of the zeta potential of cotton fabrics modified with CMC at 40, 60 and 90 °C in hard water, and taking into account the surface condition in view of the content of deposits in the modification degree at 40 and 60 °C, the CMC orientation prevails, and at 90 °C the impact of temperature prevails.The zeta potential values of all cotton fabrics modified with CMS are less negative than the values of cotton fabrics with CMC. The zeta potential curves of cotton fabrics modified with CMS in hard and soft water are characterized by grouping (1) soft water at 40 and 60 °C, and (2) hard water at 40 and 90 °C. The surface charge of the CMS-modified fabric in hard water at 60 °C is similar to the surface charge of the reference cotton fabric (F) in the highly alkaline range. Based on the obtained relations, it can be concluded that CMC as a modifier in the detergent leads to an increase in the negative charge of the cotton fabric surface in soft water at 40 and 60 °C. The curves in The zeta potential curves are not grouped and are similar to the curves of fabrics modified by CMC, indicating a dominant impact of CMC over CMS in the CMC/CMS blend, which is shown by the grouping and lower surface charge of the modified cotton fabric. An interesting phenomenon in the modification of cotton fabric at 40 °C in hard water is reflected in the values of the zeta potential. The cotton fabric modified with CMC is more negative than the unmodified one, indicating a proper orientation of the -COOH polymer groups. A similar behavior is also observed by the modification with CMS. The specific orientation of the polymers, CMC and CMS, covered the cotton cellulose and prevented the dissociation of the reactive groups. This can be influenced by the rheological properties of the polymers. The cluster analysis and grouping of objects according to the action of SMs, i.e., the grouping of individual SMs for different values of pH 4, 6 and 9 was performed for the data on zeta potentials. On the dendrograms in To understand the interaction of SMs in the determination of incineration residue (IR) and zeta potential (ZP), a cluster analysis was performed for all experimental IR and ZP data at pH 9, which represents the pH of the detergent solution. The results of the analysis are presented by dendrograms , which sThe whiteness degree (W) of cotton fabrics after the 10th cycle modification with CMC, CMS and CMC + CMS (W_MF) added to the basic detergent is higher compared to that of standard pre-bleached cotton fabric (W_F), which has a low degree of whiteness, W = 45.90. To the basic detergent, stilbene-type fluorescent whitening agents for cellulosic fabrics and their mixture were added, which cumulatively increased the whiteness of all surface-modified cotton fabrics at all temperatures. Cotton fabrics modified at the temperature of 90 °C have the highest degree of whiteness. In this part of the research, related to the impact of modifiers (SMs), it is also important to examine the interaction of the parameters of the washing process related to temperature and washing in soft and hard water. For performing the cluster analysis, polymers modifiers were analyzed as objects, and temperature and soft and hard water media as variables. The results in the form of dendrograms are shown in In The washing process of reference cotton fabrics (F) and modified fabrics (MF) was performed with stain donors to evaluate the action of greying inhibitors, CMC, CMS and their mixture. The degree of whiteness (W) of cotton fabrics was evaluated before and after modification (F and MF), and after the 1st cycle of washing  of the cotton fabrics with stain donors. The potential of applied polymers in inhibiting soil deposition, i.e., their transfer from the bath to the clean reference and modified fabrics was assessed, The degree of inhibition of stain transfer, or re-deposition, is associated with certain types of stain donors  that areThe degree of whiteness of the reference cotton fabric washed with stain donors (F1) in all variations of washing conditions is higher than the whiteness of the reference cotton fabric . The increase in whiteness of fabrics washed with basic detergent with CMC, CMS and CMC + CMS inhibitors at 40 °C is mild. The increase in the washing temperature from 40 to 60 °C typically increases the degree of whiteness of the washed fabric (F1) by about 20 units, while washing at the temperature of 90 °C significantly increases the whiteness of fabrics, which reaches values from 128.00 to 151.00. An improved whiteness effect is achieved in soft water for all baths. The whiteness of F1 cotton fabric confirms that there was no redeposition of soil from the bath on the F1 fabric. This cycle is dominated by the mechanism of the physical effect of the fluorescent whitening agent, especially at the washing temperature of 90 °C.The degree of whiteness of cotton fabric modified with CMC, CMS, and CMC + CMS in washing with basic detergent at all temperatures is reduced in the subsequent washing cycle. This indicates the soil transfer from the bath to the surface of the cotton fabrics, through which the redeposition is partially inhibited. The results showed that the stain transfer is lower in soft water than in hard water. The reason for this is the more negative charge of the surface of the modified fabric due to the action of the polymer and the low amount of deposits on the surface. The largest decrease in whiteness of the modified cotton fabric (MF1), regardless of water hardness, was obtained in washing at 60 °C. In view of the previously defined significant impact of temperature on the whiteness degree (W) in the implemented protocol of modification and inhibition of stain transfer, a correlation matrix was set for individual modifiers and greying inhibitors for the whiteness of fabrics F1, MF and MF1. The correlation matrix presented in From the obtained results of the whiteness degree of cotton fabrics subjected to cycles of modification (MF) and washing (MF1) with changed parameters—bath composition , water (hard and soft), temperature —a cluster analysis was performed, with the results shown by dendrograms in From the obtained results of the Pearson correlation parameters shown in The paper presents the application of cellulose and starch derivatives, carboxymethyl cellulose and carboxymethyl starch, in the processes of washing reference cotton fabric with detergent at temperatures of 40, 60 and 90 °C in two media: soft and hard water. The application and impact of the polymers were analyzed through the potential of surface modification of cotton cellulose and the greying inhibition of modified and initial cotton fabrics. The results of the surface modification, expressed as deposit and zeta potential, show a significant impact of CMC in the mixture of applied polymers CMC + CMS.The results of greying inhibition expressed as whiteness show a more significant impact of CMS in the polymer mixture.All of the obtained results indicate a substantial impact of temperature and water as a washing medium. Better insight into the individual impacts was gained by the cluster analysis. The cluster analysis has proven to be an effective tool for a better understanding and statistical confirmation of physicochemical interactions in the complex washing process with polymers.Further research activities will be directed towards rheological properties of biopolymers in variation of concentrations and temperatures. Mechanical and advanced energy techniques in dynamic agitation will also be applied.


RSC Advances article due to concerns with the reliability of the data in the published article.The Royal Society of Chemistry hereby wholly retracts this Separation and Purification Technology article to represent Zr-SVTs.1 The TEM in Fig. 3f, which represents ZSTs, was previously published by the authors in a Separation and Purification Technology article to represent Zr-SVTs and in a Journal of Colloid and Interface Science article to represent Zr-SVT-4.1,2 The methods sections of this RSC Advances article and the Separation and Purification Technology and Journal of Colloid and Interface Science articles describe materials synthesised using different protocols.1,2The TEM in Fig. 3b, which represents EC-ZSTs, is a duplicated, translated and scaled version of the TEM in Fig. 3e, which represents ZSTs. The TEM in Fig. 3d, which represents ZSTs, was previously published by the authors in a Journal of Colloid and Interface Science article to represent Zr-SVT-4 with a different corresponding nitrogen adsorption–desorption isotherm.2 In addition, the data points at low and high partial pressures in the isotherm in Fig. 5 in this RSC Advances article appear to be identical to the data points at low and high partial pressures in the isotherm in the Journal of Colloid and Interface Science article.2 The nitrogen adsorption–desorption isotherm in Fig. 5 for ZSTs in this RSC Advances article was also previously published by the authors in a Chemical Engineering Journal article for ZVTs.3The pore size distribution inset in Fig. 5 for ZSTs was previously published by the authors in a Separation and Purification Technology article for Zr-SVTs,1 in a Journal of Colloid and Interface Science article for Zr-SVT-4,2 and in a Chemical Engineering Journal article for ZVTs,3 and was later published by the authors in a Chemical Engineering Journal article for SYFZr-Tis.4 The methods sections of this RSC Advances article and the Separation and Purification Technology, Journal of Colloid and Interface Science and Chemical Engineering Journal articles describe materials synthesised using different protocols.1–4The EDX spectrum in Fig. 7 for EC-ZSTs was previously published by the authors in a Journal of Colloid and Interface Science article for Zr-SVT-4 and SVTs.2The FT-IR spectra in Fig. 8 for EC-ZSTs and STs was previously published by the authors in a 2 in Fig. 9a has two sets of repeating fragments along the baseline. In addition, the XRD patterns in Fig. 9a and b for TiO2 and EC-ZSTs were also previously published by the authors in a Separation and Purification Technology article for TiO2 and Zr-SVTs. The methods sections of this RSC Advances article and the Separation and Purification Technology article describe materials synthesised using different protocols for EC-ZSTs and Zr-SVTs.1The XRD pattern for TiOSeparation and Purification Technology article and for Zr-SVT-4 in a Journal of Colloid and Interface Science article by these authors.1,2 The methods sections of this RSC Advances article and the Separation and Purification Technology and Journal of Colloid and Interface Science articles describe materials synthesised using different protocols.1,2The elemental composition data in Table 1 for EC-ZSTs is the same as reported for Zr-SVTs in a RSC Advances article with other published articles, primarily a Materials & Design article6 and a Chemical Engineering Science article5 by these authors.In addition, there are significant portions of unattributed text overlap throughout this The authors were contacted for comment, but their response was not sufficient to explain the concerns with this article. Raw data was requested, but the authors did not provide it.Given the significance of the concerns about the validity of the data and the lack of raw data, the findings presented in this paper are no longer reliable.Yuqing Zhang and Pinjing Li oppose this retraction.RSC AdvancesSigned: Laura Fisher, Executive Editor, th February 2022Date: 11


Here, we investigate,by means of density functional theory (DFT) calculations, the adsorptionand dissociation of this complex: after an evaluation of the structuraland electronic properties of Ni(acac)2, a comparison betweendifferent dissociation patterns reveals that the most favorable patternfor the complex adsorbed on iron is different from the one suggestedby considering the strength of the bonds in the isolated complex andan attempt to generalize this dissociation model is made in this work.Moreover, the most favorable adsorption configurations turned outto be a long bridge positioning of the nickel atomalong with an on top positioning of the oxygen atomsof Ni(acac)2, while a short bridge positioningis the most favorable for the central metallic unit alone.Amongmetal β-diketonates, nickel acetylacetonate (Ni(acac) Because of their catalyticproperties, these compounds are ideal for several applications.2 adsorbed on ironare still scarcely described in the literature. In 1967, Kishi andco-workers proposed a description of the adsorption of acetylacetonateunits on nickel and iron, based on infrared and ultraviolet spectroscopies.13 The adsorption and the decomposition of Ni(acac)2 on a metallic substrate were never investigated, to the bestof our knowledge. Understanding the atomistic mechanism of the dissociationof this complex on metal can be useful to clarify its functionality.Here, we present a study based on density functional theory (DFT)concerning the adsorption and dissociation of Ni(acac)2 on iron. After presenting structural and electronic properties thatwere never calculated before for isolated Ni(acac)2 bymeans of DFT, we present in this work the adsorption and dissociationenergies of this complex on different sites of the iron surface.However,the properties of Ni(acac)2 complexshown in We focused on the monomeric form of the Ni(acac)2 and its interactionwith an iron surface, the Quantum Espresso package15 was used to perform first-principle calculations within the frameworkof DFT. The Perdew, Burke, and Ernzerhof (PBE) approximation16 was used to describe electronic exchange andcorrelation. The plane wave/pseudopotential approach was employed,with cutoffs of 35 and 280 Ry applied to the kinetic energy of thewave functions and the charge density, respectively, as the pseudopotentialswere ultrasoft. The choice for these values of the cutoffs is justifiedin the Supporting Information. Spin polarizationwas used and smearing with Gaussian functions was applied with a widthof 0.02 Ry to better describe electronic occupations around the Fermilevel. For the geometry optimization of the isolated complex, a cubicsupercell of 60 Å was used to avoid interactions between theperiodic replicas. The adsorption of the molecule on iron was studiedconsidering the (110) surface, as it is the most stable surface forthis metal.18 A supercell of iron with a 5× 3√2 in-plane size and a height 6√2 times thelattice parameter of bulk iron was used, containing a slab composedof four layers with 30 atoms each. Integrations were carried out atthe Γ point for the calculations in the cubic cell, while forthe orthorhombic cell, a 2 × 2 × 1 Γ-centered gridwas chosen. An initial guess for the geometry of the complex was obtainedwith the software Avogadro.19 The molecularstructure was then optimized, with the total energy and the forcesconverged under the thresholds of 10–4 and 10–3 Ry, respectively.To study the properties of Ni(acac)2 obtained from the geometry optimizationagrees with the other existing data in the literature.22 Calculated bond lengths and angles, reported in The chemicalstructure of Ni(acac)2 complex werealso evaluated. The ionization energy Ei was calculated asE2+Ni(acac) and E2Ni(acac) are the total energiesof the cationic complex obtained by removing one electron and of theneutral complex, respectively. The calculated value of 6.25 eV iscomparable to the experimental one of 7.41 eV,23 with an underestimation of about 16%, most probably dueto the well-known problem of the fundamental gap in DFT.24Electronic propertiesof the Ni(acac)2 are shownin dxz orbital of Ni, accountingfor almost 70% of the wave function, and one pz orbital for each of the O and C3 atoms,collectively accounting for more than 20% of the wave function. Thelowest unoccupied molecular orbital (LUMO) is composed of almost 60%of the Ni dxy orbitaland of the O py orbitals,which collectively account for almost 25% of the wave function. LUMO+ 1 and LUMO + 2, which are mostly obtained by combining pz orbitals of the C and O atoms of theligand units, are in perfect agreement with the plots proposed inthe literature for the LUMO and LUMO + 1.25 In fact, the high number of nodal planes in the lowest unoccupiedmolecular orbital (LUMO) may suggest that the calculated energy ofthis orbital is underestimated, and it should lie above LUMO + 2.The frontier molecular orbitals of Ni(acac)Efrag was determined asE1frag and E2frag arethetotal energies of the individual fragments obtained after moleculardissociation. This analysis aims at comparing different ways of breakingthe molecules to evaluate the strength of the broken bonds. The absolutevalues of fragmentation energies obtained in To estimate which could be the most favorable dissociationpatternfor the isolated complex, the fragmentation energy The lowest fragmentation energy is observed in the case ofCut2, where two Ni–O bonds are broken. Breaking two C–Cbonds is more difficult, requiring 5.8 (Cut 8) to 7.4 (Cut 1) eV morethan Cut 2. As expected, the fragmentation energies of mixed patternswhere a Ni–O bond and a C–C bond are broken lie in themiddle. Therefore, Cut 2 may be the most likely to occur for isolatedmolecules, as the energy required to separate the two fragments islower with this scheme. However, the effect of a metallic substratecan change the picture provided by these calculations, as the fragmentsof the molecules can be stabilized differently on the surface of themetal, in favor of a dissociative pattern that involves the strongerC–O bonds.One could roughly estimate the strength ofindividual bonds inthe complex by considering half of the fragmentation energies of Cuts1, 2, and 4. This calculation would yield 5.96, 2.25, and 5.46 eVfor C–O, Ni–O, and C–CH bonds, respectively.Cuts 5–9 are indeed the combination of these fragmentationpatterns and summing up the fragmentation energies of the individualbonds would yield 7.71, 8.21, and 11.4 eV for Ni–O + C–CH(Cuts 5 and 6), Ni–O + C–O (Cut 7), and C–O +C–CH (Cuts 8 and 9), respectively. These last values differfrom the energies on the right side of 2, the Ni(acac)2 complex was adsorbed on an iron(110) surface. Three nonequivalent sites of the surface were chosenas the initial position of the Ni atom of the complex: the position on top of an iron atom (OT), the twofold coordination site,called the short bridge (SB), and the fourfold coordinationsite, called the long bridge (LB). Two differentorientations of the complex were considered for all three sites: withthe ligand units parallel to the y-axis of the supercell and with the complex rotated by 45° . During the geometry optimization of the complexes on theiron surfaces, the x and y coordinatesof the Ni atom were kept fixed, while all of the other degrees offreedom were allowed to relax. The final configurations of the adsorbedsystems are shown in To better understandthe effect of a metallic substrate on the chemical properties of Ni(acac)Eads was evaluated asE2Ni(acac)iron and Eiron are the total energies of the system composedby the complex adsorbed on the iron slab and of the iron slab alone,respectively. The adsorption energy for each configuration is includedin When the complex was in the OT, OT*, SB, and LB* configurations,the complex chemisorbed on the iron surface, while in the case ofthe SB* and LB configurations, only physisorption could be established.For all of these systems, the adsorption energy 2 is still the most favorable also on iron, wecalculated the dissociation energies on the substrate Efragiron asEcentraliron and Eligandiron arethe total energies of the fragments corresponding to the central metallicunit and to a single ligand unit, respectively, adsorbed on the ironsurface. The total energy of the iron slab must be subtracted twicein x-axis, rotated by 45°,and parallel to the y-axis, to explore differentadsorption configurations on the surface. For both the fragments,the most favorable configurations turned out to be the ones in whichthe units were rotated by 45°. These adsorption configurations,shown in the bottom panels of The metallic substrate canplay an important role in modifying the mechanisms of molecular dissociation.To verify whether the dissociation mechanism predicted for isolatedNi(acac)2 complexon the iron surface. All of the calculated dissociation energies arenegative, meaning that the dissociation of the complex is favorableon the substrate. Cut 1 is the most favorable way of breaking thecomplex, as the energies obtained from this pattern are higher inabsolute value than the ones of Cut 2. This result shows that thepreferred dissociation pattern changes from Cut 2 for the isolatedmolecule to Cut 1 for the complex adsorbed on the iron substrate.The fragments obtained upon dissociation on the iron surface can bestabilized differently with respect to the isolated molecular fragments,which are unable to relax their strained geometry. In particular,for Cut 1, such favorable values of dissociation energies are alsodue to the detachment of the oxygen atoms of the central metalliccores, leading to significantly higher stability of the system. Forboth dissociation patterns, the most favorable destination for themetallic core is the SB position, providing the lowest  dissociation energy.4 unit on iron is thermodynamically favorable,by calculating the following reaction energyEfrag,centraliron is the energy requiredto fully divide the NiO4 central unit and ENiiron, EOiron, and E4NiOiron are the total energies of the respectiveisolated species, all adsorbed on iron. The values of Efrag,centraliron vary by changing the adsorption site of the Ni atom on the surface;yet, they range from −1.33 (SB) to −2.20 eV (LB), withthe negative sign indicating that the full dissociation of the NiO4 central unit is favorable and that the final product of thedissociation will most probably consist of Ni and O atoms fully separatedon the iron surface, as long as enough energy is provided to the systemto overcome the dissociation barriers.Since only part of the O atoms detached from the Niatom in thecentral units originated by Cut 1, we verified whether the full dissociationof the NiO27 The results of the fragmentation analysis are in perfect agreementwith the ones obtained in this work for Ni(acac)2 and theysuggest the following general rules for small metallorganic complexesinteracting with the iron surface:1.While the molecular dissociation ofthe isolated complexes is energetically unfavorable, the iron substrateis capable of stabilizing the fragments of the dissociation, turningit into a favorable process.2.The dissociation pattern in which theweakest bonds of the isolated complexes are broken is not the mostprobable when the complexes are on the iron surface. Alternative pathscan be observed in which stronger bonds are broken, due to the stabilizationof the relative fragments by the substrate.In fact, Ni(acac)2 and most of the MoDTC structureson iron can be broken more preferably between the chalcogen atomsand the adjacent carbon atoms of the ligand units. These chalcogenatoms remain near the central metallic units, completing the coordinationof the metal, and possibly detach in a subsequent step, while thecarbon atoms with broken bonds strongly bind to the iron surface.In a previous study regardingmolybdenum dithiocarbamates, we discoveredthat the most probable fragmentation pattern for these complexes oniron differs from the one for the isolated complexes, predicted byanalyzing the strength of the bonds that take part in the dissociation.2 with an iron surface by means of DFT. Afterconfirming thegood agreement between the optimized geometry of the complex withthe existing structural data available, we calculated the frontierorbitals and the ionization energy of this complex for the first timewith DFT. A fragmentation analysis both for the isolated complex andfor the complex adsorbed on iron was performed and different adsorptionsites were considered to understand which is the most preferable configurationof Ni(acac)2 on the iron surface. The main results aresummarized as follows:2, the most favorablefragmentation pattern is Cut 2, corresponding tothe dissociation of the acetylacetonate ligand units from the nickelatom.For isolatedNi(acac)2 on the iron surface is LB*, requiring thenickel atom tobe in the long bridge position and the oxygen atomsto be on top of the nearest iron atoms. After dissociation, the centralmetallic unit of the complex is more stabilized in the shortbridge position.The most favorable adsorption configurationfor Ni(acac)Both dissociationpatterns, which are not favorablefor the isolated complex, become favorable on iron. The most favorablefragmentation pattern for the complex adsorbed on iron becomes Cut1 due to the stabilization effect of the substrate. These observations,in agreement with the ones drawn for MoDTC complexes, suggest a possiblegeneralization of the dissociation mechanism on the iron surface forsmall metallorganic complexes where the ligand units are connectedto the metallic center through chalcogen atoms.We presented a study concerning the interactionof Ni(acac)The reaction energies for the fragmentation calculated inthiswork do not take into account the energy barriers associated withthe dissociation. A further step of this study could include the estimationof such barriers and the dynamic simulations of the dissociation,to better understand the kinetics of the different mechanisms.


MATLAB and COMSOL software were used for simulation. Meanwhile, the electrode materials were selected. Then, the performance of the designed annular 2-2-2 ultrasonic transducer was tested. The simulation results show that when the polymer phase material of the piezoelectric ultrasonic transducer is doped PDMS, the piezoelectric phase and the ceramic substrate account for 70% of the total volume, the polymer phase accounts for 30% of the total volume, and the maximum frequency band width can reach 90 kHz. The experimental results show that the maximum bandwidth of −3 dB can reach 104 kHz when the frequency is 160 kHz. The results of the electrode test show that the use of Cu/Ti electrode improves the electrical conductivity of the single electrode. In this paper, the annular 2-2-2 transducer designed in the case of small volume had the characteristics of a wide frequency band, which was conducive to the miniaturization and integration of the transducer. Therefore, we believe that the annular 2-2-2 piezoelectric composite has broad application prospects.The performance of the ultrasonic transducer will directly affect the accuracy of ultrasonic experimental measurement. Therefore, in order to meet the requirements of a wide band, a kind of annular 2-2-2 piezoelectric composite is proposed based on doped PDMS. In this paper, the transducer structure consisted of PZT-5A piezoelectric ceramics and PDMS doped with 3 wt.% Al Ultrasound detection technology has been widely used in the fields of biomedicine ,2, underFor this reason, in the past few decades, researchers have made some attempts to improve the performance of piezoelectric transducers. One way is to change the connection mode of the two-phase composite material. Among them, the 1-3 series and 2-2 series piezoelectric composite materials are representative. The cylindrical piezoelectric composite material designed by Li et al.  based onAnother way to improve the performance of the piezoelectric transducer is to change the material of the polymer phase. Zhong et al.  demonstrIn order to improve the high attenuation of PDMS, fillers are doped in PDMS. The study by Wang et al.  showed tTo sum up, this paper proposes a miniature annular 2-2-2 piezoelectric composite based on doped PDMS. We deduced the equivalent parameter formula and selected the piezoelectric phase and polymer phase materials. Meanwhile, we used MATLAB and COMSOL Multiphysics software for simulation. In terms of the manufacturing process, the magnetron sputtering method was used to sputter the electrode, and the electrode material was selected. In this study, we compared the performance advantages and disadvantages of the transducer polymer phase material doped PDMS and traditional epoxy resin, and the material properties of the polymer phase of annular piezoelectric composite were optimized. In addition, compared to the original piezoelectric composite, the frequency band was broadened. At the same time, more possibilities are provided for the further study of piezoelectric composites.As shown in The second type of piezoelectric equation, whose boundary conditions are mechanical clamping Z direction is set as the polarization direction, so when the 2-2 piezoelectric composite material is stretched along the polarization direction, it will not cause the coupling effect of shear deformation. Therefore, The X, Y, and Z directions are obtained. In the formula, C represents the piezoelectric phase, the right superscript P represents the polymer phase, and the right superscript CP represents the 2-2 type piezoelectric composites as a whole.According to Newnham’s series and parallel model, Smith’s model, and Khan’s derivation theory ,31,32,33X direction, the piezoelectric phase and the polymer are connected in series, each stress is equal, and the strain is the sum of each corresponding variation:In the Y direction, the piezoelectric phase and the polymer are connected in series, each stress is equal, and the strain is the sum of each corresponding variation:In the Z direction, the piezoelectric phase is parallel to the polymer, the strain is equal, the stress is the sum of the corresponding forces, the electric field intensity is equal, and the electric displacement component is the sum of the electric displacement components of each phase.For the After equivalent transformation, the equivalent equation with  and electrical short circuit , are applied. The equivalent properties of 2-2 piezoelectric composites can be calculated as follows:In the same way, the piezoelectric equations of the first kind, whose boundary conditions are mechanical freedom 3 Kg/m3), piezoelectric voltage constant, and sound velocity (m/s).In order to achieve the best electrical and acoustic properties of annular composites, the choice of materials is very important. PZT was selected as the piezoelectric phase and ceramic substrate. The common results are shown in From the parameters in The piezoelectric ultrasonic transducer requires the wafer to have a larger electromechanical coupling coefficient and a wider bandwidth in order to obtain a higher conversion coefficient. However, the electromechanical coupling coefficient and bandwidth of PZT-5A are small, so the choice of polymer phase materials is particularly important. At present, the common material on the market that can be used for the polymer phase is epoxy resin. However, many experiments have shown that when the polymer phase is epoxy resin, the thickness electromechanical coupling coefficient is small, only about 0.6. At the same time, the bandwidth of piezoelectric ultrasonic transducers using epoxy resin as the polymer phase material is relatively small. In order to improve the performance of piezoelectric transducers, many scholars have changed the material of the polymer phase. Among them, the research of Sharma et al.  and Romo2O3:SiO2 (1:6) powder, with the hope of reduce the attenuation, increasing the adhesion of the electrode, and reducing the loss tangent. Experiments have proved that when doped with 3 wt.% Al2O3:SiO2 (1:6) powder, the loss tangent of the polymer phase is about 0.01~0.03, and the adsorption force to the electrode is the best (see the following comparison). However, PDMS is also a high-attenuation acoustic material. In addition, according to The annular 2-2-2 piezoelectric composite is composed of type 2-2 piezoelectric composite and ceramic substrate in series. PZT-5A was chosen as the piezoelectric phase material, and PDMS doped with 3 wt.% Al2O3:SiO2 (1:6) ,27 powdeWe use MATLAB to calculate the equivalent performance parameters of piezoelectric composites. We preliminarily determined the range of the optimal volume percentage and obtained the curves of equivalent density From t (mm), piezoelectric ring heights h (mm) and piezoelectric ceramic sheet diameters r (mm). The results are shown in We used COMSOL Multiphysics software to simulate the structure of the annular 2-2-2 piezoelectric composite. Simultaneously, simulations were performed under different thicknesses Electrodes prepared by magnetron sputtering can be optimized by controlling the parameters of target material, composition ratio, sputtering power, and sputtering time. At the same time, the purity and consistency of the electrode obtained by the sputtering coating method are high. Therefore, this experiment used magnetron sputtering to prepare the electrodes. Before magnetron sputtering, the rougher annular 2-2-2 composite material obtained in the previous step was thinned and polished by a chemical mechanical thinning polishing machine. The thinned and polished material was then etched with oxygen ions in order to make the electrode adhere better to the material. Finally, the composite material was put into acetone or alcohol solvent by ultrasonic shock cleaning machine again to remove all kinds of impurities and surface stains. The composite was blow dried with nitrogen and then we waited for the sputtering electrode.We used COMSOL simulation software to simulate the vibration mode of the annular 2-2-2 transducer of PDMS doped with 3 wt.% Al2O3: SiO2 (1:6) powder in air. An infinite air domain was added to the annular transducer, a perfect matching layer (PML) was added to the outermost layer to simulate the absorption of sound waves in the process of propagation away from the sound source, and an outfield calculation was established at the interface boundary between the air domain and the PML domain. The transmission voltage response TVR and directivity index DI were calculated by Formulas (19) and (20), respectively. Among them, the transmission voltage response refers to the free field sound pressure generated by the transmission transducer in the specified direction, 1 m away from its effective sound center. In fact, the directivity index is the number of decibels higher the sound level on the directivity transmitting sound axis is than the sound level of the non-directivity transmitting sound field at the same distance. The simulation results are shown in In Formula (20), 2O3: SiO2 (1:6) powder varied with frequency hen the piezoelectric phase and the ceramic matrix accounted for 70% of the total volume and the polymer phase accounted for 30% of the total volume. It can be seen from the figure that when the frequency was 160 kHz, the bandwidth of the sensor −3 dB was about 90 kHz.The simulation results in The simulation result of The reason for the difference is that the acoustic impedance of PDMS is lower than that of epoxy resin. Therefore, the acoustic impedance matching between the transducer and the air is improved to obtain a larger frequency band width.We can see from Due to the different thermal expansion coefficient of different metal targets, in order to enhance the adhesion of the electrode, a four-probe tester was used. Under the condition of sputtering with the same thickness, samples with different wt.% were tested by sputtering with different metal targets; that is, the electrical conductivity of the electrodes sputtered by Ag,Cu and Ag/Ti,Cu/Ti was tested, where the transition layer was Ti. Six different points were selected for measurement, and then the test results of the same sputtered layer were averaged. The measured sheet resistivity and sheet resistance are shown in At the same time, the adhesion of the interface between the electrode and the composite material was tested by the direct pull method. The lead wire was soldered onto the electrode film with a diameter of about 2 mm and an area of about 3 mm square. The test results of the electrode are shown in the figure. It can be seen from the data in To sum up, we chose Cu/Ti as the electrode of annular transducer.The annular piezoelectric composite material was prepared by the method described in , longitudinal wave velocity μ (m/s), density ρ (Kg/m3), characteristic impedance Z (MRayl), and relative dielectric constant The following electrical parameters of the sample were tested: primary resonance frequency μ%, ρ%, and Z% are the percentages of the error values of thickness electromechanical coupling coefficient, longitudinal wave sound velocity, equivalent density, and characteristic impedance, respectively. The calculation method of all error values was the ratio of the actual value and the theoretical value, and the results were positive and retained two decimal places. Therefore, it can be considered that the above theoretical parameter analysis and simulation results are close to the actual calculated values.The test results were relatively close to the data calculated by the data analysis software and COMSOL Multiphysics software. The error between the experimental value and the theoretical value is shown in rel (dB) was calculated by Formula (23). The uf and f1 are the high and low frequencies at the echo amplitude 50% (−6 dB) lower than the maximum echo amplitude. VP-P is the peak-to-peak voltage displayed by the oscilloscope from the sample to be measured.The pulse echo method of ultrasonic nondestructive testing was used to test the bandwidth. The experiment needed a pulse signal generator , an oscilloscope with FFT function , and four order probe calibration blocks with thicknesses of 2.5, 5, 10, and 20 mm. The length of the near-field region [D is the diameter of the transducer m, A is the effective area of the transducer m2, and λ is the wavelength of the ultrasonic wave propagating in the medium, which can be calculated using Formula (22):c is the wave speed of ultrasonic wave propagation in the medium m/s, and f is the frequency of ultrasonic wave Hz.eU and aU are the peak-to-peak voltage (V) of the return wave from the specified reflector and the peak-to-peak voltage (V) applied to the tested sample, respectively.In Formula (21), 2O3:SiO2 (1:6) powder, which broadened the frequency band. The experimental results show that the maximum bandwidth of −3 dB could reach 104 kHz when the frequency was 160 kHz. The results of the electrode test show that the use of Cu/Ti electrode improved the electrical conductivity of the single electrode. At the same time, due to the unique copolymerization and adhesion of PDMS, it could be flexibly doped with materials of different properties to change the performance of the polymer phase. Thus, it can be argued that the annular 2-2-2 piezoelectric composite has broad application prospects.In summary, a piezoelectric ultrasonic transducer based on doped PDMS as a polymer phase material was designed. First, the formula for equivalent parameters was derived, and then the optimal size and simulation results were determined with MATLAB software and COMSOL Multiphysics software. For the polymer phase, PDMS was used to replace the common epoxy resin available in the market, and the original PDMS was doped with 3 wt.% Al