**3.4 Biomarkers determined in serum**

Serum in humans is a matrix commonly used in clinical and biological studies. Many authors recommend using the correct matrix. Both plasma and serum are derived from whole blood that has undergone different biochemical processes after blood extraction. The serum is obtained from the blood that has been clotted [84].

### *3.4.1 Proteins (cytokines) involved in the inflammatory process of periodontitis*

In a study conducted by Nile et al. [85], interleukin IL-17 was identified as a reliable biomarker in 40 patients with chronic periodontitis, later these subjects underwent periodontal therapy, and the values of this interleukin decreased; thus, they consider that IL-17 can be a valuable protein to monitor healing processes after a periodontal intervention.

Another protein related to the immune response MCP-1 was studied by Boström et al. [86]. They studied healthy patients with periodontitis and detected that the MCP-1 protein was increased in the serum and inflamed connective tissue comparing it with healthy patients. In this way, they considered that MCP-1 can help identify patients with periodontitis.

#### *3.4.2 Metalloproteinases*

Within the metalloproteinases studied in serum, Lira et al. [65] reported that MMP-8 is elevated in patients with aggressive generalized periodontitis compared with the rest of the patients.

#### *3.4.3 Oxidative stress*

Sreeram et al. [87] studied the transpeptidase biomarker (GGT) in healthy subjects and with periodontitis. This biomarker showed elevated levels in patients with periodontitis with respect to healthy. Among the conclusions found in this study was the GGT is useful, economic, and easy to use.

Onder et al [88] studied 4-hydroxynonenal (4-HNE) as a biomarker in serum, concluding that biomarker 4-HNE was at high levels in patients with periodontitis.

In another serum study, other biomarkers of oxidative stress as total antioxidant capacity (TOS) and oxidative stress index (OSI) used in patients with periodontitis and healthy found high levels of TOS and OSI in patients with chronic periodontitis, this suggests that these biomarkers play important roles in periodontitis [89].

**29**

*Use of Biomarkers for the Diagnosis of Periodontitis DOI: http://dx.doi.org/10.5772/intechopen.85394*

**3.5 Biomarkers determined in plasma**

Calprotectin was studied by Lira et al. [65]; this biomarker was analyzed in patients

Blood can be a universal reflection of the state or phenotype. Its cellular components are erythrocytes, thrombocytes, and lymphocytes. The liquid portion is called plasma, when all the components are retained. The concentrations of various plasma components are routinely determined in clinical practice [92]. In this way, it

Among the cytokines evaluated in plasma, we can say that IL-8 and IL-10 were useful to discriminate patients with aggressive periodontitis from healthy ones, and we can add that they were interleukins different from those expressed in the

On the other hand, IFN-γ is a biomarker that has been determined in plasma and was significantly high in patients with aggressive periodontitis, this correlates with the findings in crevicular fluid where this biomarker was elevated in sick patients [78]. Regarding MIP-1α, when it was determined in plasma, it was determined that it could be useful to discriminate patients with aggressive periodontitis from healthy

Periodontitis is one of the most prevalent illnesses in humans [93]. One of the main challenges faced by the periodontics field is to improve the methods for diagnostic and prognostic of periodontitis [94]. Biomarkers, previously described, can be useful in monitoring the current state of the disease, the effectiveness of the treatment, and possibly predict the progression. However, currently the single ideal biomarker displaying high specificity and sensitivity for discriminating and monitoring this disease has not been determined. Thus, the combination of different biomarkers could be more advantageous than single biomarkers [93]. This would provide a more accurate panorama of the state of periodontal disease.

Classic methods for periodontitis diagnosis as the inspection and the palpation by the specialist can be relatively inaccurate. Additionally, the use of periodontal probes, and radiography could only provide information about previous periodontal damage rather than the current state [93]. Thus, biomarkers have been proposed as complementary methods to defeat the mentioned limitations monitoring the

Desirable's characteristics in test using biomarkers in periodontitis are easy to perform, rapid, and low cost, which could allow clinicians to perform early diagno-

*3.5.1 Proteins (cytokines) involved in the inflammatory process of periodontitis*

with aggressive generalized periodontitis, found elevated levels in these patients compared with healthy and with gingivitis. In addition to the previous study, Nizam et al. [90] studied myeloperoxidase (MOP) and found that it increases in patients with generalized periodontitis compared to healthy ones; nevertheless Meschiari et al. [91] reported similar levels in patients with periodontitis and healthy. This discrepancy between both authors is possible due to the demographic variation and probably the

anatomical site where the sample was extracted for analysis.

is not surprising that biomarkers are sought in plasma.

crevicular fluid where IL-2 and IL-6 were relevant [78].

clinical response to an intervention and future risk [95].

sis and more effective personalized treatment.

ones, since it was found to be elevated [78].

**4. Discussion**

*3.4.4 Others*


**Evaluation of the Effect of Extracted Time Conditions on the Phenolic Content of Olive Pastes from** *cv***. Arbequina and Discrimination Using a Lab-Made Potentiometric Electronic Tongue †**

**Ítala M. G. Marx 1,2,\* , Nuno Rodrigues 1 , Ana C. A. Veloso 3,4, José A. Pereira 1 and António M. Peres 1**

**Abstract:** The present study investigated the effect of malaxation times (Mt) (0, 15, 30, 45 and 60 min), during the industrial extraction of *cv*. Arbequina oils at 25 ◦C on total phenolic content of olive pastes. Additionally, the possibility of applying a lab-made potentiometric electronic tongue (E-tongue), comprising 40 lipid/polymer sensor membranes with cross sensitivity, to discriminate the olive pastes according to the Mt, was evaluated. The results pointed out that the olive pastes' total phenolic contents significantly decreased (*p*-value < 0.001, one-way ANOVA) with the increase of the Mt (from 2.21 ± 0.02 to 1.99 ± 0.03 g gallic acid equivalents/kg olive paste), there being observed a linear decreasing trend (*R*-Pearson = −0.910). These findings may be tentatively attributed to the migration of the phenolic compounds from the olive pastes to the extracted oil and water phases, during the malaxation process. Finally, the E-tongue signals, acquired during the analysis of the olive pastes' methanolic extracts (methanol:water, 80:20 *v*/*v*), together with a linear discriminant analysis (LDA), coupled with a simulated annealing (SA) algorithm, allowed us to establish a successful classification model. The E-tongue-LDA-SA model, based on 11 selected non-redundant sensors, allowed us to correctly discriminate all the studied olive pastes according to the Mt (sensitivities of 100% for training and leave-one-out cross-validation). The satisfactory performance of the E-tongue could be tentatively explained by the known capability of lipid/polymeric sensor membranes to interact with phenolic compounds, through electrostatic interactions and/or hydrogen bonds, which total content depended on the Mt.

**Keywords:** electronic tongue; lipid sensor membranes; chemometrics; olive pastes; total phenolic content

#### **1. Introduction**

The worldwide consumption of virgin olive oil (VOO) is associated with its appreciated sensory attributes as well as with the recognized health benefits, namely, the reduced risk of chronic diseases and increased longevity, mainly related to the unsaturated fatty acids and minor components like polyphenols [1]. One strategy to ensure the natural enrichment of olive oils in phenolic compounds is based on the optimization of the extraction conditions, namely, using different malaxation times and/or temperatures [2–4].

Several destructive and nondestructive analytical techniques (e.g., chromatography, electrochemical sensor devices and spectroscopy) have been applied to evaluate the olive oil physicochemical and quality characteristics, including the assessment of total and

**Citation:** Marx, Í.M.G.; Rodrigues, N.; Veloso, A.C.A.; Pereira, J.A.; Peres, A.M. Evaluation of the Effect of Extracted Time Conditions on the Phenolic Content of Olive Pastes from *cv*. Arbequina and Discrimination Using a Lab-Made Potentiometric Electronic Tongue. *Chem. Proc.* **2021**, *5*, 36. https://doi.org/10.3390/ CSAC2021-10556

Academic Editor: Manel del Valle

Published: 1 July 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

individual compositions in phenolics [5,6]. Nevertheless, most of the studies are focused on the olive oil evaluation after being extracted or during the storage period. In those studies, the proposed methodologies were not used as prognostic tools of olive oil quality, i.e., to predict the quality of the olive oil to be processed from measurements on the olive pastes collected during the olive oil extraction process. Actually, a small number of works have been published on the potential prediction of olive oil composition and quality before or during olive oil production [4,7,8]. In this context, this study aimed to evaluate the effect of malaxation time (Mt), during the industrial extraction of oils, on the total phenolic content (TPC) of *cv.* Arbequina pastes. Additionally, the use of a potentiometric lab-made electronic tongue (E-tongue) to estimate the TPC in olive pastes collected at different Mt, was also evaluated. This capability could allow establishing indirect correlations between the composition of olive pastes and the TPC of the *cv*. Arbequina oils industrially extracted. It is important to emphasize that E-tongues comprising lipid sensor membranes have been extensively used to determine the phenolic profile and the sensory sensations of olive oils [3,7], which versatility has been related to the low selectivity and cross-sensitivity of the sensors that mimic the behavior of the human biological gustatory receptors [9].

#### **2. Materials and Methods**

#### *2.1. Olives and Olive Pastes Samples*

Olives from the *cv*. Arbequina were harvested in mid-November 2017 from an orchard located in Trás-os-Montes region (northeast Portugal). Olive pastes were collected at 5 time-periods (0, 15, 30, 45 and 60 min) during the oil extraction at 25 ◦C, in an industrial olive mill (OLIMONTES, Macedo de Cavaleiros, Portugal. Five samples of olive pastes (~100 g) were collected from the malaxers during the extraction, totalizing 25 olive paste sub-samples (5 replicas × 5 time-periods). The TPC and the potentiometric profiles of the olive pastes were determined.

#### *2.2. Olive Pastes*

#### 2.2.1. Analytical Extraction for TPC and Potentiometric Analysis

The methodology applied was previously described by Marx et al. [6]. The polar extract containing the phenolic compounds was collected to assess the TPC and to establish the potentiometric profiles.

#### 2.2.2. TPC of Olive Paste Extracts

The TPC was determined following the methodology proposed by Singleton and Rossi [10] and previous described by Marx et al. [7]. Gallic acid was used as the external standard compound to establish the calibration curve (R2 > 0.999), being the results expressed as g of Gallic acid equivalents (GAE) per kg of olive paste (g GAE/kg olive paste).

#### 2.2.3. E-Tongue Apparatus and Potentiometric Analysis of Olive Paste Extracts

A lab-made potentiometric E-tongue, comprising two cylindrical arrays, was used. Each array contained 20 lipid polymeric cross-sensitive sensor membranes (1st array: S1:1 to S1:20; 2nd array: S2:1 to 2:20). The construction details, as well as the composition of the membranes were previously reported by Marx et al. [3]. The device was connected to an Agilent Data Acquisition unit (model 34970A), which was controlled by an Agilent BenchLink Data Logger software. For the olive pastes analysis, the TPC polar extract was used after a 1:5 (*v*/*v*) dilution in deionized water [7]. The diluted solution was analyzed with the E-tongue during 5 min to allow reaching a pseudo-equilibrium between the non-specific lipid polymeric membranes and the dissolved chemical compounds [7].

#### *2.3. Statistical Analysis*

The TPC of olive pastes were analyzed using the one-way ANOVA followed by the Tukey's post-hoc multi-comparison test. Linear discriminant analysis (LDA) was applied to evaluate the correct discrimination of the studied pastes based on the best subsets of E-tongue sensors selected using the simulated annealing (SA) algorithm. The leave-oneout cross-validation (LOO-CV) variant was used to evaluate the predictive performance of the classification model and the repeated K-fold-CV. The quality of the results was assessed considering the sensitivity (i.e., the percentage of corrected classified samples). The statistical analysis was performed using the Sub-select and MASS packages of the open-source statistical program R (RStudio version Version 1.2.5033), at a 5% significance level, as previous detailed by Marx et al. [3,7].

#### **3. Results and Discussion**

#### *3.1. TPC of Olive Pastes*

The TPC of the olive pastes, collected at five time-periods (0, 15, 30, 45 and 60 min) during the industrial extraction of *cv*. Arbequina oils, were determined following the Folin-Ciocalteau spectrophotometric method and are shown in Table 1. According to the results, the TPC of the pastes linearly decreases with the Mt (*R*-Pearson = −0.910). However, until 30 min of malaxation, the observed decrease is not significant, there being observed a reduction of 0.46% between 15 and 30 min. On the other hand, after 30 min of malaxation, the reduction on the TPC of the studied pastes was more pronounced. Similar trends (negative correlation between the TPC of the olive paste and the Mt) have already been reported in the literature [4]. According to Trapani et al. [4], the decreasing trend was attributed to the enzymatic oxidation of phenolic compounds, probably due to the fact that during the malaxation process the olive paste was exposed to air. The knowledge of the TPC of olive pastes during malaxation could pave the way towards a real-time control of the impact of the Mt on the olive oils being extracted in order to promote the increase of the total phenolics in olive oils.

**Table 1.** Statistical analysis of TPC of olive pastes collected at five different malaxation times, during the industrial extraction of olive oil (average, standard deviation, *p*-value and *R*-Pearson).

1 *p*-values for the one-way ANOVA. Different letters in the same row show statistically differences from the given mean (*p* < 0.05). n = 5.

#### *3.2. Estimating TPC of Olive Pastes Based on the Potentiometric E-Tongue Analysis of Olive Paste Extracts*

Among the 40 lipid polymeric sensors, it was possible to establish linear correlations (positive or negative, i.e., signal-on or signal-off) between the potentiometric signals recorded by the E-tongue sensor membranes and the decimal logarithm of TPC, for 75% of the sensors (0.836 ≤ R2 ≤ 0.998). The sensors mean sensitivities varied from +3.7 to +376 mV/decade, or between −185 to −42 mV/decade. The linear correlations were obtained for 30 E-tongue sensors (1st array: S1:1, S1:2, S1:3, S4, S1:7, S1:8, S1:10, S1:12, S1:13, S1:15, S1:16, S1:17, S1:18, S1:19 and S1:20; 2nd array: S2:2, S2:3, S2:4, S2:5, S2:7, S2:8, S2:9, S2:10, S2:12, S2:13, S2:15, S2:17, S2:18, S2:19 and S2:20), and the mean TPC calculated by applying the referred linear correlations are shown in Table 2.

**Table 2.** TPC (mean ± standard deviation) of olive pastes estimated using the correlations established between the E-tongue signals and the decimal logarithm of TPC, for the five different malaxation times studied (minimum and maximum contents in brackets).

The agreement between the experimental TPC (Table 1) and those estimated by the device (Table 2), pointed out that the E-tongue could be applied as a real-time analytical tool to estimate the TPC in olive pastes collected during the oil extraction, allowing establishing the best Mt of the olive pastes that would ensure the extraction of an olive oil rich in phenolic compounds.

Finally, the E-tongue signals acquired during the analysis of the olive pastes' methanolic extracts (methanol: water, 80:20 *v*/*v*), allowed the establishing of a successful classification LDA-SA model. The E-tongue-LDA-SA model (Figure 1), based on 11 selected non-redundant sensors, correctly discriminated all the studied olive pastes according to the Mt (sensitivities of 100% for training and LOO-CV) and 91 ± 12% for repeated K-fold-CV. The satisfactory performance of E-tongue could be tentatively attributed by the known capability of the lipid sensor membranes to interact with phenolic compounds, through electrostatic interactions and/or hydrogen bonds, which total content depended on the Mt [7].

**Figure 1.** E-tongue-LDA-SA model performance regarding the supervising classification of *cv.* Arbequina olive pastes extracted at 0 min (-); 15 min (); 30 min (•); 45 min () and 60 min () based on the potentiometric signals gathered by eleven lipid sensor membranes (1st array: S1:1, S1:8, S1:14, S1:17, S1:18, S1:20; 2nd array: S2:2, S2:3, S2:4, S2:5 and S2:18), selected using the SA algorithm from a set of 40 sensors.

#### **4. Conclusions**

The spectrophotometric evaluation of the olive pastes showed that until 30 min of malaxation, the TPC of the olive pastes were not significantly different. Oppositely, after 30 min of malaxation, the TPC of the pastes decreased, being the lowest contents determined for pastes after 60 min of malaxation. However, monitoring the TPC of olive pastes by spectrophotometry is a time-consuming task that requires several sample pre-treatments. Furthermore, this conventional spectrophotometric technique has some practical limitations, like the difficult regarding its implementation as an in-situ and *online* tool, besides being an invasive/destructive technique.

The present study showed that the potentiometric E-tongue analysis of extracts of olive pastes, collected during the industrial extraction of *cv*. Arbequina oils, coupled with chemometric tools, allowed estimating of the TPC. In addition, the E-tongue was capable to correctly discriminate all olive pastes studied according to the malaxation time.

Taking into account its portability, the lab-made E-tongue could be easily implemented in an industrial olive mill allowing estimating of the TPC of the olive pastes and, indirectly, establishing of the optimal malaxation time of the olive pastes to obtain a high-quality oil.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/CSAC2021-10556/s1.

**Author Contributions:** Conceptualization, Í.M.G.M., N.R., J.A.P. and A.M.P.; methodology, Í.M.G.M. and N.R.; software, A.M.P., and A.C.A.V.; validation, J.A.P. and A.M.P.; investigation, Í.M.G.M.; resources, Í.M.G.M. and N.R.; writing—original draft preparation, Í.M.G.M.; writing—review and editing, A.M.P. and J.A.P.; supervision, A.M.P.; funding acquisition, Í.M.G.M. and N.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** Foundation for Science and Technology (FCT, Portugal) FCT/MCTES; CIMO (UIDB/00690/ 2020); CEB (UIDB/04469/2020); REQUIMTE-LAQV (UIDB/50006/2020); BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte. Ph.D. research grant (SFRH/BD/ 137283/2018) provided by FCT.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support by national funds FCT/MCTES to CIMO (UIDB/00690/2020), to CEB (UIDB/04469/2020), to REQUIMTE-LAQV (UIDB/50006/2020) and to BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte. Ítala Marx acknowledges the Ph.D. research grant (SFRH/BD/137283/2018) provided by FCT. Nuno Rodrigues thanks the National funding by FCT-Foundation for Science and Technology, P.I., through the institutional scientific employment program contract.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

### *Abstract* **Conductive Electrospun Nanofibers for Multifunctional Portable Devices †**

**Antonio Fotia 1 , Patrizia Frontera 2 , Lucio Bonaccorsi 2 and Angela Malara 2,\***

**Abstract:** The need to perform in situ sensing measurements lead to the development of innovative and smart field-portable devices. The advantages of such systems are remarkable since they are mainly battery-powered, lightweight and easy to carry and keep. Moreover, field-portable devices are easy to use and are able to give fast sensing responses. In the last few years, many efforts have been made in the development of new performing systems and the advantageous use of nanofibrous materials was assessed. To this purpose, the electrospinning has been recognized as the most powerful and facile technique for generating uniform nanofibers with controlled dimension and morphology. When conductive polymers are electrospun, very interesting electrical properties can be obtained along with the well-known ones that are typical of nanofibers. Among these polymers, polyaniline has been extensively used. In this work, an innovative hybrid material based on polyaniline/polyvinyl acetate/graphene oxide nanofibers was developed and tested as a sensor toward the detection of contaminants in aqueous media. Nanofibers, in the form of a compact mat, were deposited onto a support with suitable electrical contacts. Measurements were performed exploiting the excellent electrical properties of the realized nanofibers in both direct and alternating currents. When a direct current was used, the change in the nanofibers' resistance value was registered upon exposure to contaminated aqueous solutions and used to determine the presence or absence of contaminants, whereas when tests were performed with an alternating current, the quantitative determination of single species in contaminated solutions was also possible. In this way, by integrating the two different measurement methodologies, an opportunely designed multifunctional portable device will be developed for both qualitative and quantitative contaminants determinations.

**Keywords:** polyaniline; electrospinning; sensors; portable devices

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/CSAC2021-10634/s1.

**Author Contributions:** Conceptualization, P.F. and A.M.; methodology, A.F. and A.M.; formal analysis, P.F. and A.M.; investigation, A.F. and A.M.; data curation, A.F. and A.M.; writing—original draft preparation, A.F. and A.M.; writing—review and editing, P.F., L.B. and A.M.; supervision; P.F. and L.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Citation:** Fotia, A.; Frontera, P.; Bonaccorsi, L.; Malara, A. Conductive Electrospun Nanofibers for Multifunctional Portable Devices. *Chem. Proc.* **2021**, *5*, 37. https:// doi.org/10.3390/CSAC2021-10634

Academic Editor: Elisabetta Comini

Published: 7 July 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

### *Proceeding Paper* **Polysaccharide-Based Organic Frameworks with Embedded Nanoparticles: Advanced SPR Study on the Antiviral Activity of Gold Composites Derived from Glucuronoxylomannan †**

**Praskoviya Boltovets 1,\* , Sergii Kravchenko 1 , Oleksiy Kovalenko 2 and Borys Snopok 1,\***

**Abstract:** The nanosized composites based on the natural polysaccharides and nanoparticles of noble metals are promising candidates for efficient antiviral drugs. However, the complexity of such objects, their diversity and novelty necessitate the development of new analytical methods for investigation of such supramolecular architectures. In this work, which was recently developed for SPR-based instrumentation, the concept of variative refraction (DViFA, density variations in fixed architectures) was used to elucidate the mechanism of the antiviral action of a polysaccharide with gold nanoparticles grown in it. The SPR data were confirmed by direct biological tests: the effect of the native polysaccharide glucuronoxylomannan (GXM) obtained from the fungus *Ganoderma adspersum* and gold nanocomposites thereon on the infection of *Datura stramonium* with tobacco mosaic virus (TMV) was investigated. Both drugs suppress the development of viral infections. However, if for high concentrations the characteristic activity of the composite is somewhat lower than for GXM, then with an increase in dilution, the effectiveness of the composite increases significantly, up to a twofold excess. It has been reasonably suggested that the mechanism of antiviral action is associated with the formation of clusters of viruses that are no longer capable of infecting cells.

**Keywords:** nanocomposites; variative refraction; surface plasmon resonance; antiviral activity

#### **1. Introduction**

Nanosized composites combined organic compounds and inorganic nanoparticles extend our capabilities to form supramolecular architectures of advanced functionality. Complex macromolecules of biological origin, which can act as a matrix for the synthesis of such objects, are of particular interest. In this work, the polysaccharide glucuronoxylomannan with known immunomodulatory activity was chosen as such a matrix [1].

Earlier, we considered the potential of polysaccharide glucuronoxylomannan as an antiphytoviral agent. It was demonstrated that GXM isolated from *Tremella mesenterica* culture can suppress TMV infection in *Nicotiana tabacum* and *Datura stramonium* plants [2]. It was shown that GXM affects both the virus before infection and the processes that are proceeding immediately in the cell. In particular, polysaccharide can suppress virus reproduction and induce plant resistance to pathogens. It was suggested that GXM sterically block the virions, thereby suppressing its ability to infect [3]. In this regard, it is reasonable to evaluate the antiviral effectiveness of GXM nanocomposites with gold and well-known inhibitory agents, the effect of which is often associated with the activity of this metal ions in an aqueous medium [4]. It should be emphasized that the "ionic model" of the antiviral action of nanoparticles finds fewer and fewer supporters among researchers: the authors of

**Citation:** Boltovets, P.; Kravchenko, S.; Kovalenko, O.; Snopok, B. Polysaccharide-Based Organic Frameworks with Embedded Nanoparticles: Advanced SPR Study on the Antiviral Activity of Gold Composites Derived from Glucuronoxylomannan. *Chem. Proc.* **2021**, *5*, 38. https://doi.org/ 10.3390/CSAC2021-10475

Academic Editor: Elisabetta Comini

Published: 15 December 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

many publications increasingly prefer various physical processes in the field of contact of nanoparticles with biological objects when describing one or another effect of nanoobjects on biological systems.

Indeed, the nanosized composites turned out to be a fundamentally new objects with unusual properties—in particular, their toxicity, which does not correlate with the properties of the material in the atomic-molecular state or in a solid. This is well illustrated by the example of the effect of nano-objects on viruses and bacteria, for which a large amount of experimental material has been collected. It is reasonable to conclude that the antiviral effects of nanoparticles are due to some physical phenomenon, rather than a chemical interaction of one type or another. Nanoscale analytes induce the change of the toxicity paradigm: physical effects come to the forefront, not the features of the chemical structure. This means that we need take into account not only the chemical composition of the object, but also its geometric characteristics, such as its size and shape [5].

The aim of this work was to study GXM nanocomposites with gold nanoparticles in order to elucidate their potential as antiviral drugs using the example of the TMV virions.

Since macromolecules of polysaccharides are complex branched structures, it is not possible to uniformly introduce pre-synthesized metal nanoparticles into these structures. However, taking into account the fact that polysaccharides contain a sufficiently large set of functional groups with the characteristics necessary for carrying out the corresponding redox reactions, in this work the synthesis of metal nanoparticles was carried out directly inside the polysaccharide matrix.

This polysaccharide can act both as a reducing agent and as a stabilizer due to the presence of the corresponding functional groups in the molecule. GXM consists of a linear backbone of (1 → 3)-linked α-D-mannose with mainly xylose and glucuronic acid in the side chains [6]. Molecules of glucuronic acid contains carboxylic acid group, which gives acidic properties to GXM (acid polysaccharide). In order to clarify the peculiarities of the interaction of composites with viral particles at the molecular level, an analysis of their interactions with the tobacco mosaic virus was carried out using the SPR method. Antiviral activity of the complex was also tested in vivo at the *Datura stramonium* L.

#### **2. Methods**

*Preparation of GXM*. The principle of the method of obtaining preparations of β-glucan from the mycelium of the fungus *G. adspersum* was the same as in obtaining β(1 → 3) β(1 → 6)-bound glucan ("ganoderan") from other species of *Ganoderma* sp. Namely: polysaccharide preparations were obtained from lyophilized mycelium of the fungus by sequential aqueous, alkaline and acid extraction [7]. First, to remove low molecular weight compounds to the dry mycelium of the fungus *G. adspersum*, thoroughly ground in a porcelain mortar, add 85% ethanol solution (1:5, *v*/*v*) and boil for 3 h. The alcohol extraction procedure was repeated three times. Each time the precipitate was separated by centrifugation (10,000× *g*, 15–20 min) and was used in further work. To obtain the aqueous fraction of β-glucan, water (1:5) was added to the mycelium purified from low molecular weight impurities and was boiled for 3 h. The procedure was repeated 5 times. The extracts were collected by centrifugation (10,000× *g*, 15–20 min) and combined. The resulting extract was dialyzed against running and distilled water and then evaporated to a minimum volume (1:5) on a rotary evaporator 1/5 by volume of a mixture of isoamyl alcohol and chloroform (1:10) was then added to the concentrate, the mixture was shaken vigorously for 10 min and then centrifuged (10,000× *g*, 20 min) to separate the aqueous and organic phases. Deproteinization of the extract was repeated once more, the organic extracts were removed, the aqueous ones were combined and dried in freeze-drying [8].

*Synthesis of AuNPs*. First, GXM water solution was prepared by dissolution of 3 mg GXM in 2.9 mL H2O, and then aqueous solution of HAuCl4 (0.1 mL, 30 mM) was added to it at violent stirring. The mixture was stirred during 1 min at room temperature and heated to 100 ◦C with boiling for 10 min. The AuNPs formation was confirmed by UV-Vis Spectroscopy and TEM.

*Instrumentation and Measurements.* The morphological, optical, and spectroscopic properties of AuNPs were examined using the following measurements. UV-vis spectra were acquired with Umico UV-Vis spectrophotometer (data not shown). TEM was performed at 100 kV using a JEOL-1011 (JEM, Japan) instrument (data not shown). Scanning spectrometer "BioHelper" (ISP NASU, Kiev, Ukraine) was used for SPR measurements with standard chips (50 nm Au/1.5 nm Cr/glass (n = 1.61)) and the protein A immobilization protocol described in detail in [9]. SPR measurements were carried out in a static mode without a sample flow in an open cell configuration (400 μL).

#### **3. Results and Discussion**

#### *3.1. Molecular Level Analysis: Advanced SPR Study*

Conventional SPR studies typically use "2D" interfacial architectures whose thickness is significantly smaller than the penetration depth of an exponentially decaying evanescent wave in a dielectric medium [10,11]. The use of SPR techniques for the investigation of "bigger" objects (e.g., intact virus particles so called virions, cells etc.) has some limitations, since their characteristic size exceeds tens of nanometers, which is required to ensure an adequacy of the quasi-linear approximation in SPR sensing.

In the conventional approach, the SPR response depends on the effective thickness of the analyte layer that is bound to the receptors on the surface—the density of both layers (receptor and analyte) is uniform—i.e., the variation of the SPR signal is due to the changes of the thickness. However, an SPR shift depends also on the change of the refractive index within the layer. Therefore, variations of the layer density can also affect the response value due to the variations of the refractive index inside the layer ("variative" refraction). One of the possible mechanisms is changing the packing of the objects of different sizes and shapes within interfacial architectures on the surface (e.g., a virus, an antibody, a small molecule etc.) If the thickness of the surface layer is fixed due to the constant form of the biggest interacting components (e.g., virion), the SPR shift is a single-valued function of the molecular assembly compactness. Exactly such an unconventional approach DViFA (density variations in fixed architectures) has been proposed by us early [12–14] for quantitative detection of native virus particles t (V) using SPR techniques.

Typical protocol includes the incubation of a virus-bearing material (V) with a definite concentration of a specific antibody (Ab) followed by the injection of statistically formed V-Ab complexes into SPR instrument with a sensitive surface covered by protein A. A shift of the minimum of SPR signal depended on the compactness of the V-Ab monolayer on the surface. To realize such an approach, we took advantage of (1) specificity of protein A from *Staphylococcus aureus* to the Fc-fragment of immunoglobulins as well as development of (2) statistically stable distribution of complexes with different numbers Abs per V specific for different Ab/V ratios.

To refine the DViFA approach, the investigation of TMV interaction with serum containing specific antibodies was performed, both with and without the presence of GXM-Au composites in the sample during the incubation phase. We investigated how the nanocomposite affects the interaction of the virus with specific antibodies. During the measurements, the concentration of TMV particles was constant (at constant concentration of 100 μg/mL), and the concentration of antibodies was changed by serial dilutions of serum in the range from 1:25 to 1:1600. The results obtained were compared with the data for a virus at the same concentration preincubated with GXM-Au complex (Figure 1):

At dilution levels between 1:25 and 1:200, the antibodies fill the free surface of the V-Ab cylinders. Such complexes experience a mutual orienting effect on the surface modified by protein A and their configuration is typical for the classical "car parking" problem. The general structure of such an ensemble of viral particles on the surface remains practically unchanged up to a dilution of antibodies of about 1:200. The decrease in the SPR response in this range is due to a decrease in the number of antibodies associated with the virus, i.e., density of surface architecture. Some stabilization of the response in the range from 1:100 to 1:200 can be associated with two competing processes: on the one hand, a decrease in the number of surface-bound antibodies decreases the density, and on the other hand, it increases through some convergence of individual viral particles. In the presence of a GXM-Au composite, all the considered effects are less pronounced and are observed at low dilutions, since the GXM composite blocks some of the surface binding sites of Ab on the virion surface. At a dilution level of 1:200 and 1:100 for a virus without and in the presence of a nanocomposite, respectively, the amount of antibodies becomes insufficient to keep the virus "on side" on the surface and some of the viral particles become "at end", fixing pointwise on the surface with a chain of virus–antibody–protein A. In the absence of a GXM-Au composite, this process continues with further dilution (1:400–1:1600), leading to a decrease in the mass (number of attached virus particles) on the surface.

A radically different situation is observed in the case of the presence of a GXM gold nanocomposite: a decrease in the amount of antibodies at dilutions greater than 1:200 leads to an increase and subsequent stabilization of the response for dilutions up to 1:1600. In this case, the stabilization of the response occurs approximately at the same level, which corresponded to the densest structure of viral particles in the position "on the side". Since the diameter of the virus particle coated with antibodies is about 40 nm, and the length of the virus particle is 300 nm, then in the orientation "on side" and "at end" the surface ensemble of viral particles practically completely overlap the region of the highest concentration of the evanescent wave (less then 100 nm due to its exponential decay with distance from the surface). All this suggests that the presence of a polysaccharide nanocomposite stimulates the aggregation of virus particles into large clusters that are attached to the surface by only a few antibody molecules. Summarizing the obtained

results, it can be argued that the nanocomposite stimulates the aggregation of viral particles into clusters, preventing their "independent" functioning.

#### *3.2. Biological Experiments In Vivo*

To verify the antiviral activity of the nanosized composite, the classical biological test has been performed. Aqueous solutions of Au-GXM (in the concentration range 1–500 μg/mL) were added to a suspension of TMV (10 μg/mL) and the mixture was incubated for 30 min at room temperature. Then, the left halves *Datura stramonium* L. were inoculated with mixture, whereas the right halves were infected with the virus at the same concentration without a composite.

The degree of viral infection suppression (in percentage terms) was calculated from the number of necrotic local lesions on the test and control leaves using the following expression [8]:

$$\mathbf{I} = ((\mathbf{C} - \mathbf{P})/\mathbf{C}) \cdot 100\% \tag{1}$$

where I is the degree of viral inhibition in percent; C—local lesions number on the control half; P—number of local lesions on the test half. The results of the calculation of necrosis were subjected to statistical processing by parametric criteria, calculating their average number (M) and the ratio of these data in the experiment and control, as well as the average error of the ratio (m). On the graph, the data of statistical evaluation of the results were expressed as M ± m (Figure 2).

The in-vivo experiment showed that both native GXM and GXM-Au composites inhibit the development of viral infection: this is manifested in a much smaller number of necrosis on the experimental halves of the leaves of the studied plants (see Insert in Figure 2).

In order to compare the effectiveness of potent antiviral drugs, a concentration dependence of the percentage of necrosis on the experimental leaf halves to the necrosis on the control leaf halves on the drug concentration was constructed (i.e., the lower the value, the more effectively the virus is suppressed). For the preparation of native GXM, a clear linear (exponential in linear coordinates) dependence of the degree of viral infection suppression on the concentration was demonstrated. The difference in the concentration range of 0.5 mg/mL–0.001 mg/mL is c.a. 50%. It was shown that at high concentrations (0.5 mg/mL) the activity of the GXM is higher than the activity of the composite. However, the effectiveness of the GXM-Au composite is significantly less dependent on the concentration of the drug. This leads to the fact that the antiviral activity of the composite is much higher at low concentrations. In particular, for a concentration of 0.001 mg/mL, the degree of viral infection suppression was more than twice better than that for Au composites in respect to native glucan.

The results of molecular analysis and in vivo studies suggest that polysaccharide matrices with embedded gold nanoparticles have a stronger antiviral effect at low concentrations in comparison with natural polysaccharides. The mechanism of this action is due to the fact that the metal composite induces the aggregation of viral particles into clusters incapable of subsequent infection of plant cells.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/CSAC2021-10475/s1.

**Author Contributions:** Conceptualization, B.S.; methodology, P.B. and S.K.; software, B.S.; validation, O.K. and P.B.; formal analysis, B.S.; investigation, S.K.; resources, O.K.; data curation, O.K.; writing—original draft preparation, P.B.; writing—review and editing, P.B. and B.S.; visualization, S.K.; supervision, B.S.; project administration, P.B.; funding acquisition, B.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Academy of Sciences and Ministry of Education and Science of Ukraine.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

### *Proceeding Paper* **Metal–Peptide Complexes—A Novel Class of Molecular Receptors for Electrochemical Phosphate Sensing †**

**Aleksandra Tobolska 1,2,\* , Nina E. Wezynfeld 1, Urszula E. Wawrzyniak 1, Wojciech Bal 3 and Wojciech Wróblewski 1**

**Abstract:** Amyloid-β (Aβ) peptides are crucial in the pathology of Alzheimer's disease. On the other hand, their metal complexes possess distinctive coordination properties that could be of great importance in the selective recognition of (bio)analytes, such as anions. Here, we report a novel group of molecular receptors for phosphate anions recognition: metal–peptide complexes of Aβ peptides, which combine features of synthetic inorganic ligands and naturally occurring binding proteins. The influence of the change in the metal ion center on the coordination and redox properties of binary Cu(II)/Ni(II)-Aβ complexes, as well as the affinity of these complexes towards phosphate species, were analyzed. This approach offers the possibility of fine-tuning the receptor affinity for desired applications.

**Keywords:** metal–peptide complexes; voltammetry; molecular receptors

#### **1. Introduction**

The determination of phosphate anions in body fluids provides information about various disorders such as hyperparathyroidism or vitamin D deficiency [1]. Therefore, the monitoring of phosphate levels is of interest for human health. Chemical sensors are an ideal alternative to classic analytical methods, but their construction requires the synthesis of appropriate receptors, selectively binding to the analyte.

Amyloid β peptides (Aβ) related to Alzheimer's disease are well known for their neurotoxic properties [2]. However, metal complexes, with their N-terminally truncated analogs, have unique coordination properties that could be employed in the design of potential receptors for biorelevant anionic species [3,4]. The Aβ5-9 (Arg-His-Asp-Ser-Gly-NH2) peptide possesses a His-2 binding motif, and thus forms stable complexes with transition metal ions such as Cu(II) or Ni(II). At pH 7.4, the Cu(II) or Ni(II) ion is bound by three nitrogen atoms (3N) from the His residue, the N-terminal amine, and the peptide backbone amide [5]. The resulting chelates also exhibit a labile coordination site, enabling ternary interactions. Hence, metal–peptide complexes offer the possibility of fine-tuning their affinity for desired applications by altering the amino acid sequence and the metal ion center.

The present work explores and compares the coordination and redox properties of Cu(II) and Ni(II) complexes of the Aβ5-9 peptide, followed by their ability to interact with biologically relevant phosphate anions and nucleotides.

**Citation:** Tobolska, A.; Wezynfeld, N.E.; Wawrzyniak, U.E.; Bal, W.; Wróblewski, W. Metal–Peptide Complexes—A Novel Class of Molecular Receptors for Electrochemical Phosphate Sensing. *Chem. Proc.* **2021**, *5*, 39. https:// doi.org/10.3390/CSAC2021-10449

Academic Editor: Nicole Jaffrezic-Renault

Published: 30 June 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **2. Materials and Methods**

#### *2.1. Chemicals and Reagents*

All chemicals were purchased from Merck and Sigma Aldrich. All solutions were prepared daily with deionized water (18 MΩ·cm). In order to avoid Cu(II)/Ni(II) contamination, the glassware was raised with 6 M HNO3 followed by deionized water. AMP and ATP stock solutions were adjusted to pH 7.0–7.4 and kept on ice during measurements to prevent nucleotides hydrolysis.

#### *2.2. Peptide Synthesis*

Synthesis of the Aβ5-9 peptide was performed according to the Fmoc/tBu strategy [6] on a Prelude™ peptide synthesizer (Protein Technologies, Inc., Tucson, AZ, USA). The crude was purified by HPLC with the UV detection (Waters, Milford, MA, USA) at 220 nm. The purity of the peptide was verified by ESI-MS (Waters, Milford, MA, USA).

#### *2.3. Voltammetry*

Electrochemical measurements (CV, DPV) were performed using the CHI 1030 potentiostat (CH Instrument, Austin, TX, USA) in a three-electrode arrangement with a GCE (BASi, 3 mm diameter) as a working electrode, an Ag/AgCl electrode (Mineral, Warsaw, Poland) as a reference, and a platinum wire as an auxiliary electrode. The GCE was sequentially polished with the alumina powder (1.0 μm and 0.3 μm) on a Buehler polishing cloth. Then, the working electrode was sonicated for 1 min and rinsed thoroughly with deionized water. All voltametric experiments were carried out in 100 mM KNO3 at pH 7.4 under argon. The pH was adjusted with small aliquots of concentrated KOH or HNO3 solutions. The peptide-to-metal(II) ratio was 1.0:0.9.

#### **3. Results and Discussion**

The electrochemical response for the Aβ5-9 metal complexes recorded at pH 7.4 is depicted in Figure 1. The binary Cu(II)-Aβ5-9 complex enabled both the reduction (Figure 1A, blue line) and oxidation of Cu(II) ions (Figure 1B, blue line). The exchange of the metal center complex to Ni(II) caused significant differences in the redox behavior, with a decrease in metal center oxidation by 188 mV compared to Cu(II) complexes (Figure 1B, green line). We did not observe any signals associated with Ni(II) reduction (Figure 1A, green line).

Distinct electrochemical properties are likely caused by the differences in geometry and the stabilities of the Aβ5-9 complexes. Under studied conditions, Cu(II) complex is square-planar, while Ni(II) complexes are mostly octahedral. Additionally, the conditional stability constant of the Cu(II)-Aβ5-9 (5.8 × 1012 M−1) is about five orders magnitude higher than for Ni(II)-Aβ5-9 (1.7 × 106 M−1) [3,5].

Considering the application of the metal complexes of Aβ5-9 as a recognition element, we studied their response to selected anionic species. Observed changes in the oxidation potentials of the metal center upon the addition of 10 mM phosphates were similar (~150–160 mV) for Cu(II) and Ni(II) complexes of Aβ5-9. Comparable sensitivity of chelates is probably related to a similar Lewis acidity of the metal centers. Furthermore, both complexes exhibit a good selectivity towards phosphates in the presence of chlorides and sulfates (Table 1). Aside from phosphates, only acetates, among other tested analytes, interacted with the metal–peptide complexes, causing changes in redox activity. Nevertheless, the voltametric signals for acetates occurred at different potentials than for phosphates.

Since the intercellular level of organic phosphates can be 20 times higher than inorganic phosphates, we decided to investigate the affinities of the studied metal–peptide complexes for selected nucleotides (AMP, ATP). Similar to phosphate anions, the presence of nucleotides shifted the oxidation peak to less positive values. However, in contrast to Cu(II)-Aβ5-9, the signal of the metal center oxidation for Ni(II)-Aβ5-9 occurs at different potentials for mono- and triphosphates (see Table 1). We suggest that this is due to the ability of the octahedral nickel complex to interact with more than one phosphate group of ATP as a result of the chelate effect.

**Table 1.** Comparison of the affinity towards selected anions and nucleotides of the Cu(II)-Aβ5-9 and Ni(II)-Aβ5-9 complexes. ΔEM(II)/M(III) is the difference of the potential values of Cu(II) or Ni(II) oxidation of the respective ternary system and the binary complex. Calculated based on results published previously [3,4].

#### **4. Conclusions**

Metal–peptide complexes of peptides possessing the His-2 motif ensure there are labile coordination sites, enabling ternary interactions with phosphate anions and nucleotides. Such interactions lead to a strong electrochemical response, which could be valuable for designing a promising class of peptide-based molecular receptors with potential applications as recognition elements in electrochemical biosensors and in vitro clinical diagnostics.Our research proved that the change in the metallic center of the Aβ5-9 complex significantly influences its coordination properties and redox activity. Nevertheless, altering the metal center from Cu(II) to Ni(II) does not change the sensitivity of the complex toward phosphate anions.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/CSAC2021-10449/s1.

**Author Contributions:** Conceptualization, W.B., N.E.W. and U.E.W.; methodology, W.W., U.E.W. and N.E.W.; formal analysis, A.T., U.E.W. and N.E.W.; investigation, A.T.; resources, W.W. and W.B.; data curation, A.T., U.E.W. and N.E.W.; writing—original draft preparation, A.T.; writing—review and editing, A.T., N.E.W. and W.W.; visualization, A.T.; supervision, N.E.W., W.W. and W.B.; project administration, N.E.W. and A.T.; funding acquisition, N.E.W. and A.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financially supported by the Warsaw University of Technology under the program Excellence Initiative, Research University (ID-UB), BIOTECHMED-1 project no. PSP 504/04496/1020/45.010407 (N.E.W.) and implemented as a part of the Operational Program Knowledge Education Development 2014–2020 (Project No POWR.03.02.00-00-I007/16-00) co-financed by the European Social Fund (A.T.).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** We gratefully acknowledge the assistance of Dawid Płonka (Institute of Biochemistry and Biophysics, Polish Academy of Sciences) in peptide synthesis.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

### *Proceeding Paper* **Evaluation of Olive Oil Quality Grade Using a Portable Battery-Operated Sensor System †**

**Marco Grossi 1,\* , Enrico Valli 2, Alessandra Bendini 2 , Tullia Gallina Toschi 2 and Bruno Riccò 1**

**Abstract:** Olive oil quality is normally assessed by chemical analysis as well as sensory analysis to detect the presence of organoleptic defects. Two of the most important parameters that define the quality of olive oil are the free acidity and the peroxide index. These chemical parameters are usually determinated by manual titration procedures that must be carried out in a laboratory by trained personnel. In this paper, a portable sensor system to evaluate the quality grade of olive oil is presented. The system is battery operated and characterized by small dimensions, a light weight and quick measurement response. The working principle is based on the measurement of the electrical conductance of an emulsion between a hydro-alcoholic solution and the olive oil sample. Tests have been carried out on a set of 17 olive oil samples. The results have shown how for fresh olive oil samples, the olive oil's free acidity can be estimated from the electrical conductance of the emulsion. In the case of oxidized olive oil, the measured electrical conductance is also the function of the oxidation level, and a conductance threshold can be set to discriminate between extra virgin olive oils and lower-quality grade oils. The proposed system can be a low-cost alternative to standard laboratory analysis to evaluate the quality grade of olive oil.

**Keywords:** olive oil; free acidity; peroxide index; electrochemical sensors; portable systems; electrical conductance; in situ measurements

#### **1. Introduction**

Olive oil is a vegetable lipid highly appreciated for its beneficial effects on human health [1]. Olive oil quality is normally assessed by chemical analysis as well as sensory analysis to detect the presence of organoleptic defects. Two of the most important parameters that define the quality of olive oil are free acidity, defined as the amount of fatty acids no longer linked to their parent triglyceride molecules, which is affected by the quality of the olives used to produce the oil as well as the production process, and the peroxide index, expressed as milliequivalents of active oxigen for a kg of oil, which is an indicator of the oil primary oxidation and is affected by the storage conditions [2]. The official techniques for measuring these chemical parameters are manual titration procedures that must be carried out in a laboratory by trained personnel [3].

In the case of small industrial environments, such as olive oil mills and small packaging centers, which cannot afford an internal laboratory for quality analysis, the olive oil samples to be tested must be shipped to an external laboratory, and this results in high costs for the analysis and long response times. Thus, the development of simple and quick techniques for the analysis of quality grade of olive oil is important to allow in situ measurements directly in the industrial environment. A substantial research has been carried out in recent years towards the development of portable and low-cost sensor systems

**Citation:** Grossi, M.; Valli, E.; Bendini, A.; Toschi, T.G.; Riccò, B. Evaluation of Olive Oil Quality Grade Using a Portable Battery-Operated Sensor System. *Chem. Proc.* **2021**, *5*, 40. https://doi.org/10.3390/CSAC2021- 10614

Academic Editor: Tobias Placke

Published: 6 July 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

for the quality analysis of food products. Some examples include a portable biosensor system for bacterial concentration detection in raw milk [4], a system for the detection of chicken meat freshness [5], a low-cost handheld system for rapid non-destructive testing of fruit firmness [6], a system for the characterization of ice-cream properties with electrical impedance [7], a system for the determination of solid fat content in fats and oils [8] and an optical system for the assessment of lycopene content in tomatoes [9]. Many portable sensor systems presented in the literature are designed using a microcontroller as the core device of the system as well as commercial electronic chips to realize the analog measurement system and the communication system with an external PC. More recently, substantial research has been carried out for the development of smartphone-based sensor systems, since modern mobile phones integrate powerful processors for data analysis, a rich sensor set (camera, accelerometer, gyroscope, light sensor, etc.) as well as peripherals for wireless and wired communication [10].

In this paper, a battery-operated portable sensor system for the quality analysis of olive oil is presented. The system working principle is based on the measurement of the electrical characteristics of an emulsion between a hydro-alcoholic solution and the olive oil sample [11]. Tests on a set of 17 olive oils have shown how the system can discriminate between extra virgin olive oils (EVOOs) lower-quality grade olive oils and thus represents a low-cost and accurate alternative to standard laboratory analysis for in situ olive oil quality assessment in a real production environment.

#### **2. Materials and Methods**

A portable sensor system to evaluate the quality grade of olive oil samples has been designed and built. The system, shown in Figure 1a, is characterized by small size (11 × 15 × 5 cm), light weight (350 g), quick measurement response (30 s) and can be powered by a USB port or batteries (3 AAA alkaline batteries).

**Figure 1.** Photograph of the portable sensor system for olive oil quality grade detection (**a**) and designed electronic board (**b**).

The system working principle is based on electrical impedance spectroscopy (EIS) measurements [12] on an emulsion between a hydro-alcoholic solution and the olive oil tested. The emulsion electrical properties were measured using a 50 mL Falcon vial modified with a couple of cap-shaped stainless-steel electrodes (hereafter the sensor). In more detail, the following steps were carried out:

• The emulsion electrical conductance and the environmental temperature were measured using the portable system, and these values were used to estimate the olive oil quality grade.

The system's primary function is the measurement of the olive oil free acidity. In fact, in presence of the hydro-alcoholic solution, the free fatty acid molecules dissociate and generate ions that contribute to the increase in the emulsion electrical conductance which, once compensated for by variations of the environmental temperature, can be used to estimate the olive oil's free acidity. In the case of fresh olive oil samples, which are characterized by low values of peroxide index, a very good correlation exists between the emulsion electrical conductance and the oil free acidity measured with the standard titration technique. However, when olive oil storage conditions are not adequate, that is, if the oil is exposed to heath or light, oxidation takes place, and this results in the generation of non-volatile compounds (such as aldehydes, ketones and hydrocarbons) that also contribute to the increase in the emulsion electrical conductance. Thus, by setting a threshold for the emulsion electrical conductance, olive oil top quality grade (EVOO) can be distinguished from lower-quality grades, virgin olive oils (VOOs) and lampante olive oils (LOOs).

All electrical measurements, data processing and data filing are carried were using the electronic board that is shown in Figure 1b. The electronic board integrates a LCD screen to display the measurement results, four buttons for user interaction and a USB port that can be used to power the sensor system as well as to transfer the measured data to a PC for further data analysis. The core device of the electronic board is a microcontroller produced by ST Microelectronics (STM32L152RCT6A) that is responsible for the generation of the test signal, the signals acquisitions, the signals processing and the control of all the electronic components of the board. Different commercial electronic chips are integrated on the electronic board to design the analog circuits for the measurement of the emulsion electrical conductance Gm. A schematic diagram of the measurement circuit is shown in Figure 2. A sinewave test signal VIN(t) is generated with the 12-bit DAC integrated in the microcontroller and applied to the sensor vial electrodes. The current through the electrodes is converted to a voltage signal VOUT(t) using a trans-impedance amplifier. The sinewave voltage signals VIN(t) and VOUT(t) are acquired with the 12-bit ADC integrated in the microcontroller, processed to calculate the sinewave parameters and the emulsion electrical conductance Gm. A temperature sensor (MCP9700A) is integrated in the electronic board to make measurements of environmental temperature to compensate variations of Gm with temperature.

**Figure 2.** Schematic of the circuit for the measurement of the emulsion electrical conductance [13].

The estimation of the olive oil free acidity is carried out with the following steps:

#### **3. Results and Discussion**

The portable sensor system was used to evaluate the quality grade of a set of 17 olive oil samples (11 fresh olive oil samples characterized by a peroxide index < 20 and 6 oxidized olive oil samples characterized by a peroxide index > 20). All samples were tested with the portable sensor system, the quality parameters (free acidity and peroxide index) were determined using the reference manual titration techniques, and the oil quality category was defined as suggested by the EU Reg. 2019-1604.

#### *3.1. Analysis of Fresh Olive Oil Samples*

The subset of 11 olive oil samples characterized by a peroxide index < 20 was tested with the portable sensor system. In Figure 3 the emulsion electrical conductance at 23.5 ◦C (Gm,23.5 ◦C) is plotted vs. the free acidity determined by the reference titration technique. A correlation exists between Gm,23.5 ◦C and the olive oil free acidity. The best-fit curve that correlates the two parameters is defined by the following equation:

**Figure 3.** Scatter plot of the emulsion electrical conductance measured at 23.5 ◦C vs. free acidity for the subset of olive oil samples featuring a peroxide index <20 meq O2/kg oil.

Equation (1) was used to estimate the free acidity for all the tested olive oil samples. The values of the estimated free acidity as well as the free acidity determined with the reference titration technique are reported in Table 1. As can be seen the free acidity estimated with the portable sensor system is very close to the value obtained with the reference titration technique, and the error in the estimated free acidity is never higher than 0.23%.

**Table 1.** Estimated values of the free acidity for the subset of olive oil samples featuring a peroxide index <20 meq O2/kg oil.

#### *3.2. Analysis of the Full Set of Olive Oil Samples*

The full set of 17 olive oil samples (6 EVOOs, 3 VOOs and 8 LOOs) was tested with the portable sensor system. In the case of oxidized samples, characterized by a peroxide index > 20, the presence of non-volatile compounds contributes to the increase in the emulsion electrical conductance. This was verified by performing the following experiment: a sample, with a free acidity of 0.42% and peroxide index of 11.07 meq O2/kg of oil, was subjected to UV radiation for a total time of 3 weeks, and the emulsion electrical conductance at 23.5 ◦C was measured at time intervals of 1 week. The results, presented in Table 2, confirm that even after 1 week there was a substantial increase in the emulsion electrical conductance due to the products of oil oxidation.

**Table 2.** Emulsion electrical conductance at 23.5 ◦C as function of UV stress time.

The results for the full set of samples is presented in Figure 4, where each sample is represented by a circle of different color depending on the quality grade (EVOO, VOO and LOO), while the circle diameter represents the emulsion electrical conductance at 23.5 ◦C. In general, samples of lower-quality grades were characterized by higher values of the circle diameter. The results show that by setting a suitable threshold value for the emulsion electrical conductance at 23.5 ◦C (Gm,23.5◦C,TH = 2.7 μS), EVOOs can be distinguished from lower-quality oils (VOOs and LOOs) with good accuracy. In this case, all 11 samples of lower-quality grades (3 VOOs and 8 LOOs) were correctly classified. In the case of EVOOs, five samples were correctly classified, and the only misclassified sample featured a free acidity value (0.76%) that was close to the threshold between EVOO and VOO (0.8%).

**Figure 4.** Emulsion electrical conductance at 23.5 ◦C as function of free acidity and peroxide index for the full set of olive oil samples.

#### **4. Conclusions**

A portable battery-operated sensor system for the evaluation of olive oil quality grade has been presented. The system is characterized by its small size, light weight and quick measurement response. It can be used for the in situ evaluation of olive oil quality grade in small industrial environments that cannot afford an internal laboratory.

The system working principle is based on the measurement of the electrical conductance of an emulsion between a hydro-alcoholic solution and the olive oil sample. The emulsion electrical conductance is mainly affected by the free acidity as well as the oxidation level of the sample. Tests on 17 olive oil samples demonstrated how EVOO samples can be differentiated from lower quality oils with good accuracy.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/CSAC2021-10614/s1.

**Author Contributions:** Conceptualization, M.G.; methodology, M.G. and A.B.; software, M.G.; validation, M.G. and A.B.; investigation, M.G., E.V. and A.B.; data curation, M.G. and A.B.; writing original draft preparation, M.G.; writing—review and editing, M.G., E.V. and A.B.; supervision, T.G.T. and B.R.; project administration, T.G.T.; funding acquisition, T.G.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was developed in the context of the project OLEUM "Advanced solutions for assuring authenticity and quality of olive oil at global scale" funded by the European Commission within the Horizon 2020 Programme (GA no. 635690).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

### *Abstract* **Optical Characterization of Acetone-Sensitive Thin Films of poly(vinyl alcohol)-g-poly(methyl acrylate) †**

**Katerina Lazarova 1,\* , Silvia Bozhilova 2, Sijka Ivanova 2, Darinka Christova 2 and Tsvetanka Babeva 1,\***

Organic solvents are widely used as reaction media and/or for the separation and purification of synthetic products in chemical and pharmaceutical industries. Many of those solvents, among them being acetone, are considered to be harmful to human health. The detection of vapors of such volatile solvents present in the air can be achieved using multiple devices and materials [1], but the method of optical detection has a few important advantages, such as room temperature detection without the need for electrical power supply and easy detection when it is based on color/reflectance change. To achieve this, acetone-sensitive copolymers were designed by grafting poly(methyl acrylate) side chains onto poly(vinyl alcohol) precursors. Copolymer aqueous dispersions were used for thin-film deposition on silicon substrates by applying the spin-coating method. Optical properties of the film-refractive index, n, and extinction coefficient, k, as well as thickness, d, were determined from measured reflectance spectra, R, by using the two-stage nonlinear curve fitting method [2]. To evaluate the sensing properties of the films, they were placed in a quartz cell, and the atmosphere inside was constantly changed from air to argon to acetone using a homemade bubbler system (Figure 1).

**Figure 1.** Scheme of the detection of acetone vapor process.

Reflectance spectra were measured before and during exposure to acetone vapors and were used to calculate optical constants and the thickness of the films in the presence of acetone vapors. When exposed to the vapors, the copolymer side chains swelled due to

**Citation:** Lazarova, K.; Bozhilova, S.; Ivanova, S.; Christova, D.; Babeva, T. Optical Characterization of Acetone-Sensitive Thin Films of poly(vinyl alcohol)-g-poly(methyl acrylate). *Chem. Proc.* **2021**, *5*, 41. https://doi.org/10.3390/ CSAC2021-10416

Published: 30 June 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the absorption of acetone, and as a result, the film thickness increased while its refractive index decreased. This led to a shift of reflectance spectrum toward longer wavelengths and a subsequent change of the color of the film (Figure 1). The calculated sensitivity of polymer thin films was about 1.2 × 10−4% per 1 ppm but could be further increased by two approaches. Firstly, the thickness of the polymer films could be optimized in order for the optical response to be maximized. Secondly, different multilayers structures could be designed using polymer films as building blocks.

According to the selectivity experiments, which are in progress, the initial results are very promising: the optical response of the films exposed to relative humidity of up to 80% RH is more than 10 times less as compared to acetone vapors response.

In conclusion, thin films of poly(vinyl alcohol)-g-poly(methyl acrylate) were successfully deposited using the spin-coating method on silicon substrates. A reaction toward acetone vapors and a very weak humidity response were demonstrated by measuring reflectance changes. Optimization approaches for sensitivity enhancement were discussed.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/CSAC2021-10416/s1.

**Funding:** This research received no external funding.

**Acknowledgments:** S. Bozhilova acknowledges the National Scientific Program for young scientists and postdoctoral fellows, funded by the Bulgarian Ministry of Education and Science (MES) with DCM 577/17.08.2018.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

*Proceeding Paper*


**3. Results**

### *3.1. Variation in Leaf Stoichiometry among C. paliurus Populations*

Significant differences were found in leaf N and P concentrations and N:P ratios among different populations of *C. paliurus* (Table 1). For the 30 populations studied, the mean values of N and P concentrations and N:P ratios were 9.57 mg g−1, 0.91 mg g−1 and 10.51, respectively, with a range of 5.89 (Wencheng, Zhejiang)–15.18 (Qingchuan, Sichuan) mg g−1, 0.50 (Wencheng, Zhejiang)–1.18 (Jingde, Anhui) mg g−1, and 7.04 (Jinggangshan, Jiangxi)–18.39 (Laowangshan, Zhejiang) (Table 1), respectively. Moreover, leaf N concentrations were significantly correlated with leaf P concentrations (*R*2 = 0.1815, *p* = 0.0189) (Figure 2).

**Figure 2.** The relationship between leaf N and P concentrations for the 30 *C. paliurus* populations.

### *3.2. Soil Properties and Their Relationships with Leaf Stoichiometry*

Soil organic C, total N and total P concentrations varied significantly among the natural populations, ranging from 24.04 (Jianhe, Hunan) to 75.82 (Leigongshan, Guizhou) mg g−1, 1.94 (Yongshun, Hunan) to 5.84 (Leigongshan, Guizhou) mg g−1, and 0.76 (Baise, Guangxi) to 3.52

(Shangcheng, Henan) mg g−1 (Table 2), respectively. Table 3 shows that there were no significant correlations between leaf stoichiometry and the soil properties studied (including soil C, N and P concentrations).

**Table 2.** Soil organic C, total N and total P concentrations for the 30 *C. paliurus* populations.

Note: QLF sample missing (sm). Sample ID was defined from the abbreviation of the location name. Different letters indicate significant differences (*p* <0.05 by Tukey's test) between populations.

### *3.3. Leaf Stoichiometry in Relation to Geographic and Climate Variables*

Leaf stoichiometry of *C. paliurus populations* was significantly related to both geographic and climate variables of their origins. Leaf N concentration was positively correlated with latitude (*R*2 = 0.2747 and *p* = 0.0030 for the linear fit) (Figure 3A), but negatively correlated with mean annual temperature (MAT) (*R*2 = 0.2130 and *p* = 0.0103 for the linear fit) (Figure 4A), mean temperature in January (MTmin) (*R*2 = 0.3077 and *p* = 0.0015 for the linear fit) (Figure 4C), and mean annual frost-free period (MAF) (*R*2 = 0.2319 and *p* = 0.0071 for the linear fit) (Figure 5B). Leaf P concentration was negatively related to longitude (*R*2 = 0.1660 and *p* = 0.0255 for the linear fit) (Figure 3E). Leaf N:P ratios were positively correlated with latitude (*R*2 = 0.2921 and *p* = 0.0020 for linear fit) (Figure 3G), mean temperature in January (MTmin) (*R*2 = 0.1741 and *p* = 0.0218 for the linear fit) (Figure 4I), and mean annual frost-free period (MAF) (*R*2 = 0.1655 and *p* = 0.0257 for the linear fit) (Figure 5F).

**Figure 3.** Relationships between leaf N (**A**–**C**), P (**D**–**F**), N:P ratio (**G**–**I**) and geographic origin (latitude, longitude and altitude) of *C. paliurus* populations.

**Figure 4.** Relationships between leaf N (**A**–**C**), P (**D**–**F**), N:P ratio (**G**–**I**) and temperature (MAT: mean annual temperature, MTmax: mean temperature in July, and MTmin: mean temperature in January) for *C. paliurus* populations.

**Figure 5.** Relationships between leaf N (**A**,**B**), P (**C**,**D**), N:P ratio (**E**,**F**) and mean annual precipitation (MAP), and mean annual frost-free period (MAF) of *C. paliurus* populations.


*4.1. Rheumatoid Arthritis*

An increase in circulating MPs secretion is closely linked to an increase in cytokine production and appears to be a significant factor which affects inflammation development within the synovial membrane in rheumatoid arthritis [46]. In one of the first studies assessing the relationship between MPs and rheumatoid arthritis in a group of 19 patients, Knifjj-Dutmer et al. observed an increased number of circulating PMPs compared to a group of healthy individuals, and a significant relationship between the number of circulating microparticles of platelet origin and the disease activity assessed by the DAS28 score [49]. Moreover, researchers suggested a possible effect of PMPs on the development of cardiovascular diseases in patients with RA, leading to increased mortality linked to vascular complications, compared to the general population [49]. Viñuela-Berni et al. observed an increased number of MPs with CD3, CD14, CD19, CD41, and CD63E antigens in plasma of RA patients with high disease activity (DAS 28 > 5.1) [52]. The link between the intensity of inflammation in RA and the number and activity of released microparticles of endothelial origin was confirmed by Barbati et al. The total pool of circulating MPs and endothelial MPs (EMPs) initially increased, then decreased after four months of anti-TNFα therapy [58]. The microparticles with CD3, CD14, CD19, CD41, and CD63E antigens also stimulated secretion of TNFα and IL-1, IL-17 by monocytes in vitro [52]. The potential role of microparticles in the pathogenesis of rheumatoid arthritis appears to be very complex (Table 3).

**Table 3.** Potential role of microparticles in the development of rheumatoid arthritis.

C—complement component; CCL—C-C motif chemokine ligand; CXC—CXC chemokines; FLS—fibroblast-like synoviocyte; IL—interleukin; MCP—monocyte chemoattractant protein; MMP—metalloproteinase; MPs—microparticles; RA—rheumatoid factor; TNF—tumor necrosis factor; VEGF—vascular endothelium growth factor.

Apart from serum, microparticles have been detected in other body fluids of the patients. Viñuela-Berni et al. observed an increased number of MPs with CD3, CD14, and CD19 antigens in the urine of RA patients with high disease activity [52]. Boilard et al. [8] analyzed samples of synovial fluid

in RA patients and found it to contain large numbers of PMPs (slightly less than 2 × 105 CD41+ MPs/μL). Synovial fluid of RA patients contained (much less than PMPs) MPs with surface antigens of neutrophils, monocytes, and T-cells. It was an interesting observation to determine a group of neutrophils present in rheumatoid synovial fluid, with simultaneously present leukocyte CD45 antigen and a platelet CD41 antigen. The immunofluorescence signal was a result of attaching to neutrophils of the bodies corresponding to PMPs rather than whole platelets [8]. Interestingly, the number of PMPs determined in rheumatoid fluid in this study was much higher than in serum of RA patients in the study by Knijff et al. [49], where it was 600 per μL. Michael et al. [54] determined the number of MPs in synovial fluid in RA patients and found a considerably larger number of MPs derived from granulocytes, monocytes, and T-cells than in patients with degenerative joint disease and in the control group. Moreover, the number of granulocyte-derived MPs in RA patients was significantly larger in the patients with aCCP antibodies.

It is not completely clear how platelets infiltrate synovial fluid, although undamaged platelets, platelet aggregates, and platelets attached to leukocytes have been found in studies with RA patients for many years [66–68]. It is probable that collagen, fibrinogen, proteolytic enzymes, inflammatory cytokines, and shear forces in a joint could stimulate the production of platelet-derived MPs. It is also possible that, owing to their size, microparticles can penetrate synovial fluid and synovial membranes because the number of platelet microparticles in synovial fluid in RA patients is considerably larger than the number of PMPs in peripheral blood, which may suggest locally increased microparticle release from activated platelets in vessels near joints. Platelets can be activated locally by stimulation through collagen, a specific platelet receptor containing glycoprotein VI (GPVI) [69]. In a study by Boilard et al., platelet activation mediated by the GPVI, conducted in vivo on an animal model with transgenic K/BxN mice, induced release of microparticles containing both forms of interleukin 1 (IL-1): IL-1α and IL-β, stimulating production and secretion of IL-6 and IL-8 by fibroblast-like synoviocytes (FLS). The findings of the study suggest the pro-inflammatory potential of PMPs and their active participation in pathogenesis of rheumatoid arthritis [8]. Activation of platelet receptor, GPVI, leads to activation of spleen tyrosine kinase (SYK) in platelets and B-cells, further activating Bruton's tyrosine kinase (BTK), which plays a key role in the activation of B-cells, which is essential in their proper function and development. Bruton's kinase inhibition with the BTK selective inhibitor was examined by Hsu et al. and found to reduce collagen-induced production of PMPs [70]. In another study, BTK blockade in activated platelet culture also resulted in a decrease in production of microparticles and inhibition of production and release of IL-6 and IL-8 [71,72].

Apart from platelet-derived microparticles, synovial fluid in RA patients was found to contain MPs from monocytes, granulocytes, T- and B-cells, and erythrocytes [50,73,74]. Monocyte-derived MPs, as determined by Headland et al., were present in a much larger amount in synovial fluid than in plasma of RA patients. Researchers obtained some interesting findings on an animal model, where they observed a protective effect of monocyte-derived microparticles on articular cartilage, which is associated with decreasing secretion of IL-8 and prostaglandin E2 [53].

Microparticles exhibit high pro-coagulation activity mediated by the TF/VII factor, thereby stimulating thrombin production. They can also contribute to developing proatherogenic vasculitis and to the formation of "rice bodies" within joints as a manifestation of local coagulation processes [64]. In their study with RA patients, concerning non-differentiated arthritis, Berckmans et al. found incubation of MPs obtained from synovial fluid in the presence of FLS obtained by the biopsy of the synovial membrane to result in an increase in production and release by synoviocytes of IL-6, IL-8, monocyte chemoattractant protein 1 (MPC-1), RANTES (regulation on activation normal T-cells expressed and secreted) chemokine, and vascular endothelium growth factor (VEGF) [63]. A local increase in VEGF secretion can stimulate angiogenesis within articular tissues in RA, especially at its early stages [74]. Stimulation of angiogenesis in joints affected by RA can also be mediated by chemokines. Reich et al. observed a stimulating effect of leukocyte-derived MPs on production and release by rheumatoid synoviocytes of proangiogenic CXC chemokine with an ELR motive—a sequence

of three amino acids: Glutamic acid-leucine-arginine (Glu-Leu-Arg). In their study, the authors observed increasing mRNA expression for ligands of chemokine CXC ELR+: CXCL1, CXCL2, CXCL3, CXCL5, and CXCL6 [65].

Matrix metalloproteinases (MMP) are responsible for processes of extracellular matrix component transformation and degradation. Distler et al. demonstrated that MPs derived from B-cells and monocytes can stimulate rheumatoid FLS additionally to synthesis of metalloproteinases 1, 3, 9, and 13, which participate in the destruction of the extracellular matrix of cartilaginous and bone tissue in RA. In this study, the microparticles under study stimulated the production of IL-6, IL-8, MCP-1, and MCP-2 by fibroblasts [62].

The complement system, comprising approximately 40 proteins, plays an important role in the regulation of innate immune response by stimulating phagocytosis and intensification of an inflammatory reaction. The system is activated in a cascade manner. Removing apoptotic and necrotic cells is one of the functions of the complement system [75]. These cells activate the complement system mainly through a conventional pathway, as a result of a reaction of the C1q component with the CH2 domain of the antibody Fc fragment [76–78]. Microparticles with membranous features of apoptotic and necrotic cells (with exposed phosphatidylserine, phosphatidylethynolamine, oxidated phospholipids) can participate in the development of inflammation in RA by activating the complement cascade. It has been shown that MPs formed from apoptotic Jurkat leukemia cell [79] and stimulated neutrophils [80,81] can bind the C1q and, thereby, activate the complement in vitro through a conventional activation pathway. Biro et al. found C1q, C4, and/or C3 components on microparticles isolated from synovial fluid and—to a lesser extent—on microparticles obtained from serum [60]. It seems that microparticles, especially those derived from platelets, exposing the CD41 antigen, can also take part in the formation of immune complexes (IC), usually described as structures containing antibodies, antigens, and complement components. Cloutier et al. used high resolution flow cytometry and transmission electron microscopy and detected MPs in IC present in synovial fluid in RA patients, forming specific mpIC [61].

Microparticles in rheumatoid synovial fluid can undergo the process of protein citrullination—posttranslational deimination of arginine residues catalyzed by peptidylarginine deiminase (PAD), which results in the production of antibodies against cyclic citrullinated peptides (aCCP). For platelet microparticles described in the study by Cloutier et al., with expression of the FcγRIIa receptor, mpIC were formed not by connecting antibodies to this receptor, but by binding MPs to citrullinated proteins, such as fibrinogen and vimentin. The researchers confirmed that platelet MPs can react with aCCP antibodies in a mechanism which follows citrullination of PMPs surface proteins and by binding microparticles and citrullinated proteins. The paper also describes a stimulating effect of platelet mpIC on the production of leukotrienes by neutrophils. The researchers made an interesting observation by comparing the amount of mpIC in synovial fluid collected from RA and PsA patients. Despite the presence of MPs and immunoglobulins in synovial fluid of PsA patients, the amount of detected mpIC was nearly twenty times smaller than in RA patients (2000 ± 900 mpICs/μL vs 39,400 ± 9400 mpICs/μL) [82]. Unlike the previous study, two other studies found no proof of any relationship between the amount of circulating microparticles and immune complexes containing MPs in RA patients and the indices conventionally used to assess the disease activity [49,82]. This may indicate a highly effective vascular and reticular-endothelial mechanism of IC elimination or local formation of mpIC in joints affected by inflammation.

A very important role in pathogenesis of RA is played by activated B-cells. Data from the study by Messer et al. suggest the role of microparticles in synovial fluid in inducing the release of B-cell activating factor (BAFF), thymic stromal lymphopoietin (TSLP), and antileukoprotease (SLPI) by fibroblast-like synoviocytes. MPs present in synovial fluid stimulated the secretion of BAFF to the same extent as IFN-γ used as a control. The effect was observed both among the RA patients—study participants—and in patients with degenerative joint disease, which indicated the activity of MPs in stimulating B-cells, regardless of the disease type. The main difference in this study was quantitative. The number of MPs in synovial fluid in RA patients was considerably larger than in individuals with no joint inflammation. The study assessed the ability of MPs from THP-1 monocytic–macrophagic cell line and from the CEM lymphocyte line to synthesize and release BAFF, TLSP, and SLPI by activated FLS. Microparticles of monocytic–macrophagic origin had high inflammatory activity, which indicates the important participation of monocytes in initiation of an inflammatory response. MPs from activated T-cells which, in turn, stimulate the secretion of IL-6 and IL-8, did not stimulate the release of BAFF, but of TLSP and SLPI by synoviocytes. Those same MPs, did not affect the release of BAFF, TLSP, or SLPI after being treated with actinomycin D, which suggested no effect of MPs from apoptotic T-cells on activation of B-cells [59].


**Abstract**

Corrosion inhibitors are widely used in the production process due to their significant effect. In this chapter, the formulation of typical corrosion inhibitors in acid solution, near-neutral solution, alkaline solution, and oil and gas systems will be discussed, respectively. Firstly, the importance of pickling corrosion inhibitors for thermal equipment in industrial production in different situations is discussed in the "Inhibitors for acid solution" section, and the types of pickling inhibitors are mainly applied in different kinds of acid media, such as sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, hydrofluoric acid, citric acid, and sulfamic acid. Secondly, in the "Inhibitors for near-neutral solution" section, the cooling water inhibitors principally include chromate, stabilized phosphate, alkaline zinc/organic, molybdate, all organic, soft and lean water, and environmental and closed-loop programs. The hot-water inhibitor is also mentioned here. Then in the "Inhibitors for alkaline solution" section, boiler water inhibitor, oxygen scavenger, and corrosion inhibitors for condensate line are talked over. Finally, in the "Inhibition for oil and gas systems" section, drilling fluid, fracturing acidizing, oil and gas well, and oil field which produced water treatment inhibitors are introduced here. The corrosion inhibition mechanism and development trend of inhibitors are also discussed.

**Keywords:** inhibitors for acid solution, inhibitors for near-neutral solution, inhibitors for alkaline solution, inhibition for oil and gas systems

## **1. Introduction**

Pickling is widely used in various production processes, such as for pickling and rust removal of metal pipes, plates, and wires, and products in the metallurgical and mechanical industries, scale cleaning of heat exchange equipment, heat transfer equipment, and cooling equipment in various industrial sectors, especially for pickling of thermal equipment such as boilers in the electric power sector. It is meaningful using pickling, for example, from a socioeconomic point of view, fuel consumption due to fouling can be reduced; from the perspective of environmental protection, fuel exhaust and atmospheric pollution are reduced; from a safety point of view, thermal equipment such as boilers and heat exchangers can be used well. As we know, various types of dirt are gradually formed in the process, and the poor thermal conductivity of the dirt causes the local temperature of the furnace tube to rise, which reduces the strength of the steel and often causes a pipe burst accident and affects the operation of the boiler. Therefore, pickling plays a very important role in the boiler operation of power plants. In addition, the use of industrial circulating cooling water technology to increase the

concentration of running water can save a lot of industrial water. In circulating cooling water, in order to avoid corrosion and scaling of equipment, it is often necessary to add water stabilizer. Also, in the process of crude oil and natural gas collection, the current drilling and oil recovery technology requires fracturing and acidification of limestone oil and gas fields with high concentration of hydrochloric acid solution. As the 15–28% hydrochloric acid solutions are often used in the oil wells, the corrosion problem of tubing and oil production equipment is serious in deep well acidification technology due to the high temperature in the well. Therefore, metal materials and products are subjected to various environments during production, storage, transportation, and use. Metal products are easy to vary from different degrees of corrosion. Corrosion problems in tubing and oil production equipment are serious, sometimes causing fracture accidents, resulting in construction failure.

Corrosion inhibitor is an anticorrosion chemical; it can be added to the corrosive environment to cause physical and chemical interaction with the metal surface, thereby significantly reducing the corrosion of the metal material. At the same time, the use of the corrosion inhibitor does not require special equipment, nor does it need to change the properties of the metal. Compared with other anticorrosion methods, corrosion inhibitors are easy to use, cost-effective, and widely used in industrial production and social life. Therefore, it is widely used in various industrial processes, such as pickling, cooling water systems, acidification of oil and gas wells, water injection in oil fields, storage and transportation of metal products, and so on. With the advancement of society and the enhancement of human environmental awareness, the development and application of corrosion inhibitors have received more and more attention. In recent years, countries around the world have paid great attention to the research and application of corrosion inhibitors, which can be used alone or in combination with other anticorrosion materials.

Herein, in view of the current requirements, the characteristic formulation of corrosion inhibitors in acid solution, near-neutral solution, alkaline solution, and oil and gas systems is discussed. The types of pickling inhibitors in different kinds of acid media, inhibitors for the cooling water, alkaline solution, and oil and gas systems are principally talked about in details, respectively.

#### **2. Inhibitors for acid solution**

In various corrosive media, acidic gases and liquids are of strong corrosive medium. The rate of metal corrosion in acidic medium is much faster than that in other media, especially in sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, hydrofluoric acid, citric acid, and sulfamic acid, which are commonly used in pickling and removing iron oxide, descaling and decontaminating of boiler equipment, and so on. In addition, high level of H2S and CO2 in oil and gas wells is also the corrosive acidic medium. Since acid has a corrosive effect on metal equipment, especially the corrosion effect of inorganic acid is more serious, and the released hydrogen will diffuse into the interior of the metal, causing hydrogen embrittlement of the equipment to be washed. In addition, as a large amount of acid gas will generate, it will deteriorate working conditions inevitably. Therefore, a suitable corrosion inhibitor should be added during pickling to inhibit the corrosion of the metal in the acidic medium, reduce the amount of acid used, improve the pickling effect, and prolong the service life of the thermal equipment. **Table 1** shows the commonly used corrosion inhibitors in corrosive acidic medium.

**155**

**Inhibitor** Advanced pyridine alkali

4-Methylpyridine residual

Nitroaniline 1-Phenyl-3-thioformamide

Reactant of amine and alkynol

Methenamine + CuCl2

Methenamine + SbCl2

Furfural Methenamine + KI

Methenamine + thiourea + Cu2+

1,3-Dibutyl-2-thiourea + OP

**Table 1.**

*Operating conditions and properties of some common corrosion inhibitors.*

**Acid concentration and temperature**

12% HCl + 5% HF, 40°C

10% HCl + 6% HF, 30°C

2–3 mol/L HNO3

20–35% HNO3

5–15% HCl, 93°C

2–25% HCl 10–15% HCl 0.2–6 mol/L HCl

20% H2SO4 10% H2SO4 10–20% H2SO4, 60–80°C

**Inhibitor dosage**

0.2% 0.2% 0.002 mol/L

0.0005% 0.01–0.25% 0.6 + 0.02% 0.8 + 0.001% 5–10/230 mL

0.6% (8:1) 0.14 + 0.097 + 0.003%

0.5 + 0.25%

**Inhibition efficiency**

<0.1 mm/a <0.1 mm/a High efficiency High efficiency

99.4%

99% 99% High efficiency

99% 99% High efficiency

*Formulation of Corrosion Inhibitors*

*DOI: http://dx.doi.org/10.5772/intechopen.88533*

**Metals** Carbon steel Carbon steel

Copper Aluminum Carbon steel Carbon steel

Carbon steel

Copper and alloy

Carbon steel

Carbon steel

Carbon steel