**Characterization and Comparison of the Relative Humidity Response of Hydromorphic Polymers in Long-Period Fiber Grating Structures †**

**Bernardo Dias 1,2,\* , João P. Mendes 1,2,3 , José M. M. M. de Almeida 1,4 and Luís C. C. Coelho 1,2,\***


**Abstract:** Relative humidity monitorization is of extreme importance on scientific and industrial applications, and fiber optics-based sensors may provide solutions where other types of sensors have limitations. In this work, fiber optics' sensors were fabricated by combining Long-Period Fiber Gratings with three different humidity-responding polymers, namely Poly(vinyl alcohol), Poly(ethylene glycol) and Hydromed™ D4. The performance of the multiple sensors was experimentally tested and crossed with numerical simulations, which provide a comparison with the expected response given the optical properties of the materials.

**Keywords:** relative humidity sensors; long-period fiber gratings; hydromorphic polymers; optical sensors

#### **1. Introduction**

The real time monitoring of relative humidity on scientific and industrial applications is of extreme importance and many types of sensors have been developed, mostly based on capacitive or resistive structures which may display some flaws, such as not being immune to electromagnetic radiation, and not fit to extreme and harsh environments (such as underwater applications). The usage of hydromorphic polymers in optical fiber structures is a thoroughly explored field of research with many publications associated [1–4], some of which include the usage of Poly(vinyl alcohol) (PVA), Poly(ethylene glycol) (PEG) or the combination of the two [1]. The polymers display a refractive index (RI) that decreases with the absorption of water molecules, also displaying considerable swelling effects. These changes can be tracked by analyzing the spectral characteristics of specific optical structures such as long-period fiber gratings (LPFG) [5,6]. LPFGs consist of a periodic modulation to the RI of the core of the fiber, resulting in coupling of light from the fundamental core mode to the co-propagating cladding modes, which creates rejection bands in the transmission spectrum at specific wavelengths with high sensitivity to the surrounding RI. By coating LPFGs with the aforementioned materials, this refractometer can be used to measure relative humidity.

Preliminary results regarding LPFGs fabricated in single mode optical fibers and coated with three different humidity-responding polymers PVA, PEG and a Hydrogel (Hydromed™ D4) are presented. In the case of Hydrogel, no such work seems to have been published previously. When placed in an environment with varying humidity, the polymers

**Citation:** Dias, B.; Mendes, J.P.; de Almeida, J.M.M.M.; Coelho, L.C.C. Characterization and Comparison of the Relative Humidity Response of Hydromorphic Polymers in Long-Period Fiber Grating Structures. *Chem. Proc.* **2021**, *5*, 42. https:// doi.org/10.3390/CSAC2021-10461

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/).

used as coating of the LPFG will both swell and change their RI, resulting in changes in the coupling conditions of the light to the cladding modes. Besides the experimental work, in order to have a better understanding of the effect of the coatings in the LPFG, simulations based on coupled-mode theory [7] were implemented, which allow understanding of the range of action of the LPFGs and optimization of their performance.

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

In order to test the capabilities of the polymers, several solutions of different concentrations were made, and thin layers were deposited around LPFGs previously fabricated in single mode fibers (Corning SMF28e) with the electric arc technique following the procedure described in [6]. This allowed the calibration of the wavelength shift and optical power shift induced by the coating process, meaning that for a given LPFG it is possible to choose the concentration of the coating solution that gives both visibility of the rejection band and high sensitivity to the RH variation. At the same time, given that the RH response of the sensor is a result of changes in RI and the thickness of the coating layer, testing LPFGs coated with solutions of different concentrations will also allow exploration of different responses caused by this variation of parameters.

#### *2.1. Polymers Used for Humidity Sensing*

Three different polymers were chosen and compared, a hydrogel (HydroMed™ D4), PVA (polyvinyl alchohol) and PEG (polyethylene glycol).

PVA is a water-soluble polymer, which has been studied extensively in the past as a coating for fiber optics-based humidity sensors [1–4]. It is a hydrophilic polymer that absorbs and desorbs water, with variations in the RI between 1.49 to 1.45 (at 1310 nm) according to [2] or between 1.49 to 1.34 (at 1550 nm) according to [4].

PEG is a polymer derived from petroleum containing ether linkages in its main chain, thus being referred to as a polyether. While there are some studies using this polymer for humidity sensing [8], only one application of this polymer to LPFG was found [1], which relied on a combination of PVA and PEG. The RI of PEG is lower than that of PVA, varying from 1.455 to 1.413 (nD) [8].

HydroMed™ D is a series of ether-based hydrophilic urethanes fabricated by Advan-Source Biomaterials [9]. These materials were designed to respond by expanding when in contact with water molecules, with each product having different responses to different water contents. The polymer is distributed in granular form, which can be dissolved in many different solvents, of which ethanol was chosen.

#### *2.2. Fabrication of the Coating Solutions and Coating Process*

The solutions required different procedures in order to be dissolved. Generally, a solvent with low boiling point such as ethanol is preferred because it will lead to a faster evaporation and consequently the coating process will be faster. Nevertheless, given the low solubilities of PVA and PEG in ethanol, deionized water was used instead, which requires a longer time to fully evaporate and thus deposit the coating. In the case of hydrogel, the solvent was ethanol. Solutions with three different concentrations were produced, namely 10, 7.5 and 5% *wt*/*wt*, prepared by dissolving the high purity granules with the solvent, in the corresponding ratio. The PVA solution was obtained by adding PVA to water in a concentration of 10% *wt*/*wt*, and stirring for 3 h at 60 ◦C. The same procedure was applied to obtain the 5 and 7.5% concentration solutions and in the case of the PEG Scheme 50, 75 and 100% *wt*/*wt*.

The fabricated LPFGs were coated with all the polymers by stretching and dipping horizontally with a small angle between the longitudinal axes of the fiber and a V groove filled with the polymer. The fiber was then left at room temperature to ensure solvent evaporation.

This coating process was done for each sensor, thus justifying the need for different concentrations of the solutions, which have different viscosities and thus will create thicker coatings the more concentrated the solution is. In addition, calibration of the appropriate concentration of the solution to a given LPFG, in order to ensure that the wavelength and optical power shifts do not render the LPFG outside certain desirable parameters (such as minimum wavelength in the interval 1500 to 1600 nm), was performed.

#### *2.3. Humidity Measurements*

In order to calibrate the fabricated sensors, an experimental setup was devised in which the environment's humidity could be controlled and measured (Figure 1).

**Figure 1.** Experimental setup created to measure changes in LPFG spectra in varying values of relative humidity. 1—Interrogation unit; 2—Humidity Chamber; 3—Exterior valve; 4—Humidity valve.

This chamber was made from a container with two valves that connected to the exterior (where the RH was around 50%), and a side container with water connected by a valve to the main one. A small fan was also placed inside the container in order to promote faster diffusion of the water molecules in the air. This setup allowed the variation of internal humidity in varying rates, depending on the fan speed.

The humidity chamber allowed for the insertion of two LPFGs at once, one with the humidity sensor and another for thermal compensation. The two fibers were placed in a stand with weights in their extremities, guaranteeing that the sensor was fully stretched at all times. The fiber was connected to an interrogation unit (HBM Fibersensing FS22 Braggmeter) on the outside, which recorded the spectra at all times. Also inside the container was a humidity and temperature sensor (DHT22), which has a typical accuracy of ±2%RH and ±0.5 ◦C and a working range of 0%RH to 100%RH and −40 ◦C to 80 ◦C. This sensor was connected to an Arduino Uno unit which displayed the humidity and temperature values every 12 s, allowing to record every LPFG sensor spectra along with the respective humidity and temperature value.

#### *2.4. Experimental Procedure*

In order to provide a preliminary characterization of the sensors, the LPFG spectra were taken in descending RH values. Firstly, the valve to the side container with water was opened and the fan was turned on for 15 min. This ensured that the chamber attained the maximum possible values of humidity, which was recorded at 99%RH. After this process, the fan was turned off and after a minute of stabilization, the valve to the external environment was opened, and the measurements initiated. Due to the opened valve, the relative humidity decreased by 1% every 2 to 3 min, which provided an ideal stable decrease in humidity for measurements. The spectra were recorded for every decrease in 1%RH. The data were plotted in real time to determine the spectral evolution and the working range of the fabricated sensor.

#### **3. Results**

#### *3.1. Humidity Measurements*

As described in the experimental procedure, for each fabricated sensor its response to RH variations was tested in its working range for both descending and ascending variations. In this section, the results obtained by direct comparison from LPFGs coated with the same polymers at different concentrations are summarized, in order to choose one that has an optimal performance.

#### 3.1.1. PVA Coated LPFGs

Figure 2 shows the results obtained from LPFGs coated with thin films of PVA from solutions with different concentrations.

**Figure 2.** Variation of the spectra of the three PVA coated LPFG sensors and their measured response: (**a**) Optical Power Shift response; (**b**) Wavelength Shift Response.

The sensor coated with a 5% *wt*/*wt* solution displays very low response to RH variations, meaning that the obtained layer is too thin and making the sensor not suitable. On the other hand, the 7.5 and 10% sensors display very different behaviors, with the 10% *wt*/*wt* one displaying the typical LPFG wavelength shift curve due to the external RI being near the cladding index (nclad = 1.44), but the 7.5% sensor displays a transition characteristic from guided to leaky modes, meaning that in this case the external RI transitioned from n<nclad to n > nclad, which disagrees with the RI values reported in [2] (in which the PVA's RI as a value of 1.45 at 100%RH). The discontinuity seen in Figure 2b shows that at 91%RH the layer of PVA matches the cladding index, meaning that the fiber is suddenly thicker, and the mode has a discontinuous transition. This process also renders the 7.5% sensor not suitable. On the other hand, the 10% *wt*/*wt* sensor shows very good response with high sensitivity especially in the region above 90%RH, but overall good performance in the tested range.

Considering the response of the LPFG coated with 10% *wt*/*wt* PVA solution, a comparison with the *3-*layer coupled-mode theory simulations of LPFGs [7] was established, in order to check if the response of the sensor matches the values of [2] or [4]. Good agreement is seen between the simulations considering the RI variations of 1.345 to 1.43 reported in [4] and the experimental data (Figure 3), showing also that in this case the coating layer can be approximated to an infinite medium, because the penetration depth of the evanescent field of the cladding mode is inferior to the PVA coating thickness. The simulations show that the 10% *wt*/*wt* is working on the high sensitivity zone of the LPFG, justifying the excellent performance of the sensor seen in Figure 2. Given the excellent performance of this sensor, the RH cycle was repeated for increasing RH values and a second LPFG was fabricated, in order to test the reproducibility of this structure. The results shown in Figure 3 show that excellent reproducibility was observed.

**Figure 3.** Comparison between the experimental wavelength shift of two 10% *wt*/*wt* PVA LPFG and the simulations, as function the external refractive index and relative humidity.

#### 3.1.2. PEG-Coated LPFGs

Figure 4 shows the optical power and wavelength shifts in varying humidity for the cases of the PEG-coated LPFGs. In this case, the transition associated with the external medium having n > nclad is seen in all cases, and at lower RH values than PVA (80 to 87%RH), as can be seen in Figure 4b. This result was to be expected, given that the RI of PEG is lower than that of PVA, ranging from around 1.44 to 1.413 in the range of 60 to 99%RH (in the Sodium line) [8], meaning that the transition will happen at lower RH values. This renders the sensors not suitable in wavelength shift response, even though their optical power shift response (Figure 3a) could be used, in the 80 to 95%RH in the case of the 75% *wt*/*wt* coated LPFG.

**Figure 4.** Variation of the spectra of the three PEG-coated LPFG sensors and their measured response: (**a**) Optical Power Shift response; (**b**) Wavelength Shift Response.

#### 3.1.3. Hydrogel Coated LPFGs

Figure 5 shows the optical power and wavelength shifts in varying humidity for the cases of the Hydrogel coated LPFGs. The responses in both wavelength and optical power shift seem similar to the case of the PEG coated LPFGs, with the transition ofn>nclad to n<nclad happening in the range of 83% to 92%RH, which makes the wavelength shifts an unsuitable figure of merit for relative humidity monitoring. On the other hand, the optical power shift presents a suitable curve that enables the use of the structures as sensors, even though the sensitivity is considerably less than both the PVA and PEG cases.

**Figure 5.** Variation of the spectra of the three Hydrogel coated LPFG sensors and their measured response: (**a**) Optical Power Shift response; (**b**) Wavelength Shift Response.

#### **4. Conclusions**

The response curves of LPFGs coated with three different humidity responding polymers (PVA, PEG and a Hydrogel) were obtained, with humidities varying from 60%RH (or 70%RH, depending on the polymer) to 100%RH. Of all the fabricated sensors, the 10% *wt*/*wt*. PVA coated LPFG displays the best properties for relative humidity sensing, which were verified when comparing to numerical LPFG simulations.

Due to the variation of the optical properties of the polymers with varying humidity, namely the fact that the polymer's RI becomes larger than the cladding RI at low RH values, a transition from guided to leaky modes is seen, displaying a non-linear wavelength shift response, which renders it as an unsuitable figure of merit for RH sensing for most sensors. This conclusion shows that further work is needed to quantify the response of the three polymers to humidity variations, in order to optimize the sensors' performance and make them commercially viable. By fabricating new materials, which could possibly result from mixing the ones here studied, the desired optical properties (refractive index slightly below the cladding RI for all RH values and linear response) may be attained, creating a highly sensitive, viable solution for industrial and scientific application.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/CSAC2021-10461/s1.

**Author Contributions:** Conceptualization, B.D., J.M.M.M.d.A., L.C.C.C.; methodology, B.D., J.P.M., J.M.M.M.d.A., L.C.C.C.; formal analysis, B.D., J.M.M.M.d.A., L.C.C.C.; investigation, B.D., J.P.M., J.M.M.M.d.A., L.C.C.C.; writing—original draft preparation, B.D.; writing—review and editing, B.D., J.P.M., J.M.M.M.d.A., L.C.C.C.; supervision, J.M.M.M.d.A., L.C.C.C.; project administration, J.M.M.M.d.A., L.C.C.C. All authors have read and agreed to the published version of the manuscript. **Funding:** This work has received funding from the project "SolSensors—Development of Advanced Fiber Optic Sensors for Monitoring the Durability of Concrete Structures", with reference POCI-01- 0145-FEDER-031220, supported by COMPETE 2020 and the Lisbon Regional Operational Program in its FEDER component, and by the budget of FCT Foundation for Science and Technology, I.P. Luis Coelho acknowledges the support from FCT research contract grant CEECIND/00471/2017.

**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* **Identification, Quantification, and Method Validation of Anthocyanins †**

**Paula Garcia-Oliveira 1,2 , Antia G. Pereira 1,2 , Maria Fraga-Corral 1,2 , Catarina Lourenço-Lopes 1,2 , Franklin Chamorro <sup>1</sup> , Aurora Silva 1,3 , Pascual Garcia-Perez <sup>1</sup> , Fatima Barroso <sup>3</sup> , Lillian Barros <sup>2</sup> , Isabel C. F. R. Ferreira <sup>2</sup> , Jesus Simal-Gandara 1,\* and Miguel A. Prieto 1,2,\***


**Abstract:** Nowadays, anthocyanins have gained scientific and industrial attention due to their biological activities and coloring properties. In this regard, anthocyanins have been proposed for use in the development of new nutraceutical foods to replace synthetic additives as well as to be value-added ingredients. The aim of this study was to evaluate current data on identification and quantification techniques and the validation process of such methods. Our results showed that anthocyanins have been identified by different methods, including nuclear magnetic resonance and chromatography-based techniques. Although problems have been described in this validation, most of the reports showed positive results on the validation parameters, suggesting that the current analytical technology offers a satisfactory identification and quantification of anthocyanins.

**Keywords:** anthocyanins; plant; extraction; validation

#### **1. Introduction**

Anthocyanins are soluble glycosides linked by an O-glucosidic bond between an aglycone and a sugar molecule. It is a group belonging to the flavonoids that are found naturally in various plant sources, including fruits such as berries or grapes and flowers such as hibiscus, forming part of the secondary metabolites of plants [1]. Therefore, the extraction of anthocyanins is usually carried out from plant matrices. In the last few years, these compounds have attracted great interest due to their diverse beneficial properties. Specifically, anthocyanins present a high antioxidant capacity, which is attributed to the presence of phenolic hydroxyl groups in their chemical structure [2] (Figure 1). Furthermore, several studies have reported that a daily intake of this compound has a preventive and protective effect against cardiovascular diseases, diabetes, and heart disease [3–5]. These compounds also have coloring properties, covering ranges from salmon pink to red and from violet to dark blue. Thus, they are considered as an interesting source of natural colorants. To date, more than 20 structures are known, among which are orange pelargonidin, orange red cyanidin and peonidin, bluish-red delphinidin, and bluish-red malvidin and petunidin (Figure 1).

**Citation:** Garcia-Oliveira, P.; Pereira, A.G.; Fraga-Corral, M.; Lourenço-Lopes, C.; Chamorro, F.; Silva, A.; Garcia-Perez, P.; Barroso, F.; Barros, L.; Ferreira, I.C.F.R.; et al. Identification, Quantification, and Method Validation of Anthocyanins. *Chem. Proc.* **2021**, *5*, 43. https:// doi.org/10.3390/CSAC2021-10680

Academic Editor: Manel del Valle

Published: 14 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/).

**Figure 1.** (**Left**) Base group of anthocyanins' structure. (**Right**) Most common anthocyanins in the nature.

Natural dyes have several advantages, such as non-toxicity and eco-friendliness, as their acquisition has a low environmental impact. Therefore, different industries have a great interest in identifying new and economically viable sources of anthocyanins to use them as new functional ingredients and/or colorants in food [6]. As the range of application of these compounds increases, it is necessary to design efficient extraction methods with better yields and to develop suitable analytical methods for the identification and quantification of anthocyanins. Therefore, the main objective of the study was to evaluate the current data on the identification and quantification techniques used and the validation process of these methods.

#### **2. Identification and Quantification Techniques**

The search for new ingredients of natural origin and the study of their bioactivities has been going on for some decades due to the growing demand for natural products with beneficial health properties, including the group of anthocyanins. Various techniques have been used for the identification and characterization of anthocyanins in different matrices, including mass spectrometry (MS), nuclear magnetic resonance (NMR), and high-performance liquid chromatography (HPLC). In Table 1, several examples of studies employing these techniques have been collected.

**Table 1.** Examples of studies employing different methods to identify and quantify anthocyanins.


**Abbreviation**: delphinidin: DEL; cyanidin: CYA; petunidin: PET; malvidin: MAL; peonidin: PEO.

Regarding the application of MS-based technologies, several studies have obtained suitable results. For example, a work employed electron impact (EI)-MS to evaluate the degradation products of cyanidin-3-*O*-glucoside when the compound was subjected to oxidation radicals. Up to six derived products were detected, and two new molecules, 2-(3,4-dihydroxyphenyl)-4,6-dihydroxybenzofuran-3-carboxylic acid) and 2-*O*-(3,4-dihydroxybenzoyl)-2,4 glucose esters, 6-trihydroxyphenylacetic acid glucose ester, were identified. When analyzing samples of black rice stored for long periods, these two new compounds were identified, being useful to distinguish fresh rice from that stored for a long time [7]. Similarly, another EI-MS study investigated the degradation products of anthocyanin glycosides when they were exposed to the microflora of

pig intestine. According to the results, anthocyanin glycosides underwent significant changes, suggesting that the antioxidant or anticancer activities observed for anthocyanin glycosides are due to these degradation products [9]. Fast atom bombardment (FAB)-MS has been also used in the field of anthocyanins to evaluate different structural modifications. For instance, the antioxidant effect of several anthocyanin fractions isolated from bilberry extracts against pyridinium bisretinoid A2E (a prooxidant compound) was determined by this technology. According to the results, the majority of the anthocyanins were delphinidin 3-galactoside, cyanidin 3-galactoside, delphinidin 3-glucoside, and cyanidin 3-glucoside [10]. In orchid flowers, FAB-MS successfully identified eight pigments, of which two were new structures: 3-*O*-[6-*O*-(malonyl)-βglucopyranoside]-7,3 -di-[6-*O*-(trans-synapoyl)-β-glucopyranoside] and its demalonyl derivative [8].

Regarding NMR techniques, they have been used to identify anthocyanins and study their exact structural characteristics, to establish their mechanisms of action, which can lead to a better application of these compounds as functional ingredients [12]. For example, the structure of two major acylated peonidin derivatives obtained from sweet potato were studied using NMR [14]. Another study evaluated the anthocyanins composition of chokeberry showing the presence of the structure of cyanidin galactoside and cyanidin arabinoside along the second stage of the fruit ripening [15].

Finally, high-performance liquid chromatography (HPLC) is the most widely used method for the identification and quantification of anthocyanins. In general, the analytical parameters used in the literature show very uniform conditions for the identification of anthocyanins. The most used column is C18, while mobile phase composition mainly corresponds to water, acetonitrile, and methanol with acid modifiers, such as formic acid. The acid presence in mobile phases ensures that anthocyanin compounds are going to be mobilized in their cationic flavylium form, which has been described as possessing its highest absorbance, around 520 nm [23]. During the HPLC analysis of anthocyanins, as well as other compounds, the retention times and peak areas can be strongly influenced by the column temperature, mobile phase composition, or the complexity of the matrix in which they are embedded. The detection of anthocyanins is often performed by diode array detectors (DAD), mass-spectrometry detectors (both MS or MS/MS) which are, most of the time, coupled to an electrospray ionization source (ESI) [6,16]. These methodologies have been shown to provide satisfactory results for the identification and quantification of anthocyanins. Nevertheless, the use of ultra-high pressure liquid chromatography (UPLC) provided better resolution, shorter elution times, and lower consumption of mobile phases than conventional HPLC methodologies. UPLC also presents a high performance in the efficiency of the peaks of identification [23]. The anthocyanin profile of diverse vegetal samples has been evaluated by HPLC-DAD. For example, in grape, glucoside derivates of delphinidin, cyanidin, pelargonidin, peonidin, petunidin, and malvidin were identified [20]. Similarly, in grape skin samples, petunidin-3-*O*-glucoside and malvidin-3-*O*-glucoside were the major compounds [21]. HPLC-DAD can be also coupled to MS, which provides a more accurate identification, since mass information is considered in the analysis and data processing. HPLC-DAD-MS has been employed with different matrixes, such as strawberries, where cyanidin-3-*O*-glucoside, pelargonidin 3-glucoside, pelargonidin-3-*O*-rutinoside (tentative), pelargonidin-3-*O*-succinyl-glucoside, or pelargonidin-3-*O*-arabinoside were identified [22]. Regarding HPLC coupled to MS, this approach has been employed to analyze the anthocyanin composition of different samples. For example, cyanidin-3-*O*glucoside, cyanidin-3-*O*-rutinoside, and perlagonidin-3-*O*-glucoside have been identified in *Euterpe edulis* extracts [24]. In strawberry, glucoside derivates of cyanidin, delphinidin, pelargonidin, and malvidin were identified. In muscadine grapes, 3,5-di-*O*-glucoside of cyanidin, delphinidin, and petunidin were identified as the major anthocyanins [25].

#### **3. Validation of Methods**

There are different approaches to develop a validation plan, depending on the type of technique used, the field of application of the method, and the type of samples analyzed. The scientific literature shows many examples of the development and optimization of methods for anthocyanins detection. However, when these techniques are validated, just few of them indicated the guideline used to perform this complex process.

#### *3.1. Selectivity*

To achieve a selective method, analytes are first isolated from another family of analytes or matrix interferences. Pre-treatment of anthocyanin samples includes the use of different techniques such as ultrasound or microwave-assisted extraction (UAE or MAE) or the use of solid phase extraction (SPE) cartridges. When methods for detecting anthocyanins were validated, no selectivity/specificity issues were found; the most selective instrument was ultra-high-performance liquid chromatography (UPLC) coupled to a photodiode array detector (PAD) or to mass spectrometry (UPLC-MS) against spectroscopic ones, according to the literature [6,26].

When analyzing anthocyanins together with non-anthocyanin compounds under similar conditions, resolution issues have been reported. In general, the most common option when using HPLC techniques is the selection of C18 columns and the modification of the mobile phase's acidity, by increasing the percentage of acid or by changing the type of acid [27]. However, other authors have also increased the resolution peak between anthocyanins and non-anthocyanin compounds by performing two different injections using C18 columns with different conditions [28]. The last option described is the use of a fluorinated C18 column, which has been demonstrated to provide better results in terms of peak separation, symmetry, and short analysis time [26].

#### *3.2. Linearity, Limit of Detection (LOD), and Quantification (LOQ)*

For anthocyanins, it has been scientifically demonstrated that they can be detected with calibration curves with ranges from 0.01 to 800 μg/mL, using different techniques. Validation studies in which calibration curves have been carried out with concentrations within these ranges have shown high linearity with an R2 ≥ 0.99. For example, Grace et al. (2019) validated an LC-MS method. All calibration curves showed good linearity in the range of 0.04–40 <sup>μ</sup>g/mL, with a regression coefficient (*r*2) ≥ 0.99 [6]. Fibigr et al. (2017) developed an UHPL-UV method, also achieving similar results [26]. There are few exceptions with low anthocyanin concentrations [6,26,29] or in some specific cases. For example, the quantification of malvidin-3-*O*-glucoside by a spectrophotodensitometry method required a polynomial adjustment instead of a linear one [30].

As mentioned before, anthocyanins have been extensively analyzed using different techniques such as HPLC-DAD, UPLC-DAD, UPLC-UV, HPLC-MS, or capillary zone electrophoresis (CZE), among others, but most of the validation studies have been performed using chromatographic techniques. According to the literature, the ranges of the limit of detection and limit of quantification that have been reported were 0.01–3.7 and 0.03–8.5 μg/mL (ppm), respectively, when using chromatographic-based methods, coupled to diverse detectors [6,26,31]. In general, chromatographic methods achieved good performance results as observed in the studies of Grace et al. (2019) or Fibigr et al. (2017). In the first one, the limits of detection and quantification were 0.06–0.40 μg/mL and 0.12–1.20 μg/mL, respectively [6]. On the second example, quite similar results were reported, with limits of detection and quantification being 0.11–0.14 μg/mL and 0.36–0.47 μg/mL, respectively [26].

#### *3.3. Accuracy and Precision*

In most of the validated methods carried out for anthocyanins, both accuracy and precision are determined by adding known amounts of standard solutions to the samples or by using commercial standards. In general, the results of validation studies for accuracy were very good and the relative standard deviation for repeatability, and intermediate precision ranged from <1% to <10%, meaning that the methods are acceptable for possible routine use. Most of the validation studies employ chromatographic methods [6,26,29]. To cite an example, an LC-MS method has been recently validated. The results showed a good precision for all analytes tested; the relative standard deviation in intra and inter-day was less than 10% in both cases, while the reproducibility of all analytes was under 5% [6]. Thus, this method allowed characterizing simultaneously and selectively different anthocyanins and non-anthocyanidins, being a promising method for the analysis of these compounds.

#### *3.4. Stability and Robustness*

Regarding stability, several factors affect the stability of anthocyanins, such as their chemical structure, pH, light, or storage temperature and time, among others [23,32]. Minimal variations have been shown in studies of anthocyanin stability when these compounds are stored at low temperatures for long periods of time. However, rapid degradation processes were observed when matrixes or purified anthocyanins were stored at room temperature, as reported in the study of Gras et al., who employed UHPLC-PDA to evaluate anthocyanins from black carrot. In the study, significant losses of 8 to 14% were observed when standards (9 μg/L) were stored at room temperature for 24 h, showing that standard solution should be evaluated as soon as possible to avoid inaccurate results [23].

Regarding robustness, to our knowledge, few validation studies have evaluated these parameters. Nevertheless, studies in which it has been carried out displayed no significant differences in the total amount of anthocyanins extracted when the method was exposed to experimental variations such as changes in the pH, temperature, source, age and concentration of samples, variable standards, or solvents. This consistency in the results indicated that validated methods are robust and may be applied for the routine detection of anthocyanins [32,33]. For example, in the study of Canuto et al. (2016), the robustness of the developed reverse phase LC method was estimated introducing small variations in the mobile phase pH, column temperature, and mobile phase flow rate. According to their results, no significant results were detected in the determination of Cyn-3-glu and Plg-3-glu, demonstrating the robustness of the method [32].

#### **4. Conclusions**

There is a wide variety of identification and detection methods that have been demonstrated to be efficient for the analysis of anthocyanins. However, there is a lack of papers in which different methodologies are compared by using the same kind of samples. Therefore, it makes it difficult to conclude which method is more advantageous over others. On the one hand, the literature reviewed suggest that LC-MS represents a quick and efficient technique. However, it may present difficulties regarding the complexity of the sample matrix, which can cause ion suppression. Another issue is the determination of the best anthocyanin source. The variability of the experimental conditions, in terms of extraction protocols, analyzing parameters, and quantification, hinders the selection of the most appropriate matrix for obtaining the most efficient recovery of anthocyanins. Therefore, the standardization of extraction and analytical protocols may be critical to permit the real comparison of these experimental results.

**Author Contributions:** Conceptualization, F.B., L.B., I.C.F.R.F., J.S.-G. and M.A.P.; investigation, P.G.-O., A.G.P., C.L.-L., F.C. and A.S.; writing—original draft preparation, P.G.-O., A.G.P., C.L.-L., F.C. and A.S.; writing—review and editing, M.F.-C. and P.G.-P.; supervision, F.B., L.B., I.C.F.R.F., J.S.-G. and M.A.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** The JU receives support from the European Union's Horizon 2020 research and innovation program and the Bio-Based Industries Consortium. The project SYSTEMIC Knowledge hub on Nutrition and Food Security has received funding from national research funding parties in Belgium (FWO), France (INRA), Germany (BLE), Italy (MIPAAF), Latvia (IZM), Norway (RCN), Portugal (FCT), and Spain (AEI) in a joint action of JPI HDHL, JPI-OCEANS and FACCE-JPI launched in 2019 under the ERA-NET ERA-HDHL (n◦ 696295).

**Acknowledgments:** The research leading to these results was supported by MICINN supporting the Ramón y Cajal grant for M.A. Prieto (RYC-2017-22891); by Xunta de Galicia for supporting the program EXCELENCIA-ED431F 2020/12, the post-doctoral grant of M. Fraga-Corral (ED481B-2019/096), the pre-doctoral grants of P. Garcia-Oliveira (ED481A-2019/295) and A. González Pereira (ED481A-2019/0228), the program BENEFICIOS DO CONSUMO DAS ESPECIES TINTORERA- (CO-0019-2021) that supports the work of F. Chamorro. The authors are grateful to the Ibero-American Program on Science and Technology (CYTED—AQUA-CIBUS, P317RT0003), to the Bio-Based Industries Joint Undertaking (JU) under grant agreement No 888003 UP4HEALTH Project (H2020-BBI-JTI-2019) that supports the work of P. Garcia-Perez and C. Lourenço-Lopes. The authors would like to thank the EU and FCT for funding through the project PTDC/OCE-ETA/30240/2017— SilverBrain—From sea to brain: Green neuroprotective extracts for nanoencapsulation and functional food production (POCI-01-0145-FEDER-030240). The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to CIMO (UIDB/00690/2020); and to the national funding by FCT, P.I., through the institutional scientific employment program-contract for L. Barros contract.

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

#### **References**


### *Proceeding Paper* **Portable Electrochemical Detection of Illicit Drugs in Smuggled Samples: Towards More Secure Borders †**

**Marc Parrilla 1,2 , Amorn Slosse 3, Robin Van Echelpoel 1,2 , Noelia Felipe Montiel 1,2 , Filip Van Durme <sup>3</sup> and Karolien De Wael 1,2,\***


**Abstract:** Illicit drug consumption is posing critical concerns in our society causing health issues, crime-related activities and the disruption of the border trade. The smuggling of illicit drugs urges the development of new tools for rapid on-site identification in cargos. Current methods used by law enforcement offices rely on presumptive color tests and portable spectroscopic techniques. However, these methods sometimes exhibit inaccurate results due to commonly used cutting agents or because the drugs are smuggled (hidden or mixed) in colored samples. Interestingly, electrochemical sensors can deal with these specific problems. Herein, it is presented an electrochemical device that uses low-cost screen-printed electrodes for the electrochemical detection of illicit drugs by square-wave voltammetry (SWV) profiling. A library of electrochemical profiles is built upon pure and mixtures of illicit drugs with common cutting agents. This library allows the design of a tailor-made script that shows the identification of each drug through a user-friendly interface. Finally, the results obtained from the analysis of different samples from confiscated cargos at an end-user laboratory present a promising alternative to current methods offering low-cost and rapid testing in the field.

**Keywords:** electrochemical sensors; square-wave voltammetry; screen-printed electrodes; illicit drugs; forensics; portable device

#### **1. Introduction**

The consumption of drugs of abuse is causing critical issues in our society due to health issues, crime-related activities and the disruption of the border trade [1]. These illicit drugs can enter the illegal market through external borders (e.g., natural drugs) or by internal production (e.g., synthetic drugs). The smuggling of illicit drugs such as cocaine and heroin in Europe urges the development of new tools for rapid on-site identification in cargos. Besides, the production of synthetic drugs increases internal trafficking, thus demanding simple and straightforward devices to detect illicit drugs in the field. Current methods used by law enforcement offices rely on presumptive color tests [2] and portable spectroscopic techniques (e.g., near-infrared [3] and Raman spectroscopy [4]). However, these methods sometimes exhibit inaccurate results due to commonly used cutting agents or because the drugs are colored. Besides, drug traffickers are generating innovative ways to overcome traditional detection methods such as mixing with conventional goods (e.g., tcharcoal, food) or adding colorants or other substances to avoid the on-site determination by current methods. Therefore, new devices that can overcome the current problems are necessary to cope with the determination of smuggled illicit drugs in common goods.

**Citation:** Parrilla, M.; Slosse, A.; Van Echelpoel, R.; Montiel, N.F.; Van Durme, F.; De Wael, K. Portable Electrochemical Detection of Illicit Drugs in Smuggled Samples: Towards More Secure Borders. *Chem. Proc.* **2021**, *5*, 44. https://doi.org/ 10.3390/CSAC2021-10612

Academic Editor: Nicole Jaffrezic-Renault

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/).

The devices for on-site analysis must be portable, low-cost and user-friendly in order to be implemented and used by law enforcement officers [5]. Electrochemical sensors can provide the aforementioned features, and importantly, they can deal with current challenges, providing more reliable results in comparison to commercially available devices [6,7]. In this direction, portable and wearable electrochemical sensors have been designed for the detection of illicit drugs in different configurations [8], including glove-based sensors [9]. The electrochemical approach is based on the characteristic electrochemical profile (EP) of each compound that reveals the electroactive moieties of the target compound. Following this strategy, cocaine [10], ketamine [11], heroin [12] and synthetic cathinones [13] have been detected by using low-cost screen-printed electrodes (SPEs). Amphetamine is a special case as it is not electroactive in the potential window of commercial carbon SPEs. Therefore, an in situ derivatization by employing 1,2-Naphthoquinone-4-sulphonic acid sodium salt allows for its electrochemical detection [14]. Overall, the most used illicit drugs can be determined by electrochemical methods at certain conditions.

Herein, we present an electrochemical device that uses low-cost SPEs for the electrochemical detection of illicit drugs by square-wave voltammetry (SWV) profiling. A pH strategy based on the profiling of each illicit drug using specific buffers allows detection of the most-encountered illicit drugs (i.e., cocaine, MDMA, heroin and amphetamine). Hence, the electrochemical interrogation of the illicit drugs exhibits the oxidation of the electroactive moieties in each drug at a certain potential, with the exception of amphetamine that uses an in situ derivatization to unravel its oxidation peak. A library of electrochemical profiles is built upon pure and mixtures of illicit drugs with common cutting agents. This library allows the design of a tailor-made script that shows the identification of each drug through a user-friendly interface. Finally, the results obtained from the analysis of different samples from confiscated cargos at different end-users sites present a promising alternative to current methods. Overall, the fast analysis of samples with a portable electrochemical device exhibits a straightforward on-site detection aiming to facilitate the tasks of law enforcement agents in the field, thus providing a more secure border management and a safer society.

#### **2. Methods**

#### *2.1. Materials*

Standards of D,L-amphetamine · HCl, methamphetamine · HCl, 3,4-methylenedioxym ethamphetamine · HCl (MDMA), cocaine · HCl and heroin · HCl, were purchased from Chiron AS, Trondheim, Norway. Standards of paracetamol, caffeine and creatine were provided by National Institute for Criminalistics and Criminology (NICC, Brussels, Belgium). Confiscated samples of amphetamine, MDMA, cocaine and heroin were also provided by the NICC. Analytical grade salts of potassium chloride, potassium phosphate, sodium borate, sodium bicarbonate, sodium acetate and potassium hydroxide were purchased from Sigma-Aldrich (Overijse, Belgium). 1,2-Naphthoquinone-4-sulphonic acid sodium salt (NQS) (>98 %) was purchased from Tokyo Chemical Industry Co., LTD., Tokyo, Japan.

#### *2.2. Methods*

Square wave voltammograms and cyclic voltammograms were recorded using a MultiPalmSens4 or EmStat Pico potentiostats (PalmSens, Houten, The Netherlands) with PSTrace/MultiTrace. Disposable ItalSens SPEs (PalmSens, Houten, The Netherlands), containing a graphite working electrode (Ø = 3 mm), a carbon counter electrode and a (pseudo) silver reference electrode were used for all measurements. The SWV parameters that were used: potential range of 0.0–1.4 V, frequency 10 Hz, 25 mV amplitude and 5 mV step potential. All the square wave voltammograms are background corrected using the PSTrace software.

Electrochemical tests were performed in 20 mM buffer solutions with 100 mM KCl by applying 60 μL of the solution onto the SPE. Phosphate buffer, acetate buffer and hydrogen carbonate buffer were used for the detection of cocaine and heroin, MDMA and amphetamine, respectively. Preanodized SPEs for heroin detection were performed by applying 1.5 V for 60 s in PBS solution at pH 7 by drop casting 60 μL on the SPE [12].

The composition of the confiscated samples was previously analyzed in the forensic laboratory at NICC with gas chromatography-mass spectrometry (GC-MS) to subsequently validate the electrochemical approach. Besides, the confiscated samples were also analyzed by a handheld Raman spectrometer (Bruker Bravo, Ettingen, Germany).

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

#### *3.1. Electrochemical Profiling of Illicit Drugs*

The electrochemical profiling is based on the interrogation by SWV of the target molecules which exhibit an oxidation process at a certain potential. Hence, cocaine oxidation peak might differ from heroin oxidation peak, showing the possibility to identify the target by the specific peak potential [15]. Fortunately, illicit drugs contain moieties which are electroactive at the potential window of carbon SPEs. These moieties are usually secondary or tertiary amines that allow its oxidation, thus showing a peak signal during the electrochemical scan by SWV. However, some illicit drugs share some moieties which can exhibit some overlap in the oxidation potential. In previous works, our research group has optimized the detection of illicit drugs by exploring certain conditions during the SWVs. For example, the anodic pretreatment to unravel the phenolic group oxidation of the 6-monoacetylmorphine (6-MAM) (a byproduct of heroin degradation at pH 12) [12], the cathodic pretreatment to avoid the suppressing effect of some adulterants [13] or the use of different pH (as some moieties are not oxidizable in certain pH at SPE) [11]. Besides, some illicit drugs such as amphetamine need a derivatization step to allow its electrochemical profiling employing low-cost carbon SPEs. In this case, a simple mixing step with NQS launches a chemical reaction to a product that is electroactive at the SPE [14]. An oxidation peak was observed at 1.15 V due to NQS oxidation at the carbon SPE. Since this occurs outside the potential window for illicit drugs (from 0.6 to 1.03 V) in pH 10 it will not affect their identification.

#### *3.2. Generating the Library of Electrochemical Profiles*

Figure 1 shows the SWVs of pure illicit drugs (i.e., cocaine, heroin, MDMA and amphetamine) at pH 5, pH 10, pH 12 and pH 12 using anodic pretreated SPEs to record the specific electrochemical profiles of the molecules at certain conditions. Figure 1A,B shows the differences between anodic pretreated SPEs. Although similar profiles were obtained, a clear peak separation occurred in the MDMA signal. The pH dependence on the oxidation of illicit drugs is dramatically shown in the analysis at pH 5, where only heroin and MDMA exhibited electroactivity (Figure 1C). This fact assists in the proper identification of the unknown sample by a simple dual pH test. Finally, the pH assessment clearly exhibits that amphetamine is not electroactive, and only after the derivatization step (Figure 1D), an oxidation peak appears.

A similar electrochemical approach was performed employing the most encountered cutting agents (i.e., paracetamol, levamisole, lidocaine, caffeine, phenacetin, benzocaine, procaine and lactose [12,14,16]) (Figure 2A–D). Particularly interesting is the effect of the anodic pretreatment on the paracetamol signal exhibiting a sharper oxidation peak (Figure 2B). This permits the proper identification of the 6-MAM peak, thus avoiding peak overlap [12]. Besides, the effect of pH on the electrochemical signal showed a pH dependence as the oxidation peak shifts towards higher potentials at acidic pHs (Figure 2C). As pH 10 with NQS is targeted for the detection of amphetamine, only common cutting agents encountered in amphetamine real samples are explored (Figure 2D). Considering the profiling of the cutting agents, most of the peak potentials do not fall in the same position as the illicit drugs, thus allowing for a suitable identification in real samples.

**Figure 1.** Electrochemical profiles of illicit drugs (0.5 mM) obtained by square-wave voltammetry (SWV) using SPE at different pH: (**A**) pH 12; (**B**) pH 12 using preanodized SPE; (**C**) pH 5; and (**D**) pH 10 including the derivatizing agent NQS.

**Figure 2.** Electrochemical profiles of common cutting agents (0.5 mM) obtained by square-wave voltammetry (SWV) using SPE at different pH: (**A**) pH 12; (**B**) pH 12 using preanodized SPE; (**C**) pH 5; and (**D**) pH 10 including the derivatizing agent NQS.

After building the library of electrochemical profiles with several conditions, a custommade script (Matlab R2018b, MathWorks, Natick, MA, USA) is integrated. This script enhances the peak separation and facilitates the identification of the compounds in the sample. In brief, the script removes the background signal and applies a top-hat filter that provides an enhanced separation of overlapped peaks which permits a successful identification of the substances based on the peak potential of each drug. Therefore, the peak potential of each drug and cutting agent is introduced in the script to properly identify the drug and display it through a user-friendly interface.

#### *3.3. Testing the Portable Electrochemical Device with Confiscated Samples*

The electrochemical device consists of a miniaturized potentiostat with Bluetooth connectivity, a disposable SPE, a sampling container, a disposable spatula and a disposable pipette (Figure 3A). The sampling procedure consists of collecting the powder (either powder, liquid, crystal or impregnated material) with the disposable spatula into a tube containing 15 mL of the suitable buffer (Figure 2B). After shaking thoroughly, a drop of the solution is deposited on the SPE with the disposable pipette (Figure 2C). Subsequently, the operation is started on the user-friendly interface launching the electrochemical method, subsequent data treatment and results display (Figure 2D). For the analysis of confiscated samples, the strategies employing pH 12, preanodized SPE in pH 12, pH 5 and pH 10 with NQS were employed for cocaine, heroin, MDMA and amphetamine, respectively.

**Figure 3.** On-site detection of illicit drugs with the portable electrochemical device. (**A**) Elements of the electrochemical device (1—potentiostat, 2—buffer container, 3—SPE, 4—disposable spatula, 5—disposable pipette, 6—confiscated sample); (**B**) Sampling procedure; (**C**) deposition of the solution on the setup ready for the electrochemical interrogation and (**D**) user-friendly interface showing the results of the analysis.

> The reliability of the electrochemical device was evaluated in 40 confiscated samples provided by NICC (Table 1). After the analysis, the 40 samples were all positive for the corresponding illicit drug using the described sampling method in comparison to the standard methods (GC-MS). Besides, a portable Raman spectrometer was also used as a commonly used method in border settings. The electrochemical reader and portable Raman spectrometer exhibited an accuracy of 100% and 50%, respectively, calculated employing (observed detection by the method/actual detection by the GC-MS) × 100. Therefore, the electrochemical device outperformed the Raman device, particularly in heroin and amphetamine detection. It is worth mentioning that the low performance of the Raman device could be attributed to the colored nature of the samples, thus exhibiting one of the flaws of current methods. Overall, the electrochemical device is positioned as a reliable alternative for its use in the field due to its affordability, reliability and user-friendliness.


**Table 1.** Results of the analysis by the analytical methods and composition of the confiscated samples.


**Table 1.** *Cont.*

#### **4. Conclusions**

In this work, the analysis of confiscated samples from illicit drugs is presented by the use of a portable electrochemical device. First, the construction of a library from several electrochemical profiles of standards of illicit drugs and common cutting agents at different conditions by SPE is performed. After the selection of the suitable conditions and the integration of the peak potentials of each target into a tailor-made script, the electrochemical device is ready for on-site analysis. The examination of 40 confiscated samples with the electrochemical device and a portable Raman spectrometer showed an outstanding performance of the electrochemical device in front of the Raman device according to the GC-MS identification. Overall, the electrochemical device based on SPE is presented as a promising alternative to current rapid and on-site methods for the detection of illicit drugs at border and coast controls.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/CSAC2021-10612/s1.

**Author Contributions:** Conceptualization, M.P.; methodology, M.P., A.S. and N.F.M.; software, R.V.E.; validation, M.P., A.S. and F.V.D.; M.P.; investigation, M.P.; resources, K.D.W. and F.V.D.; data curation, M.P. and A.S.; writing—original draft preparation, M.P.; writing—review and editing, M.P.; visualization, M.P.; supervision, K.D.W.; project administration, K.D.W.; funding acquisition, K.D.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by European Union's Horizon 2020 research and innovation program, grant number 833787, Bordersens.

**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**


### *Abstract* **The Inhibition Study of Cytochrome bd Oxidase Using the Enzyme-Based Electrochemical Sensor †**

**Iryna Makarchuk 1,\*, Anton Nikolaev 1, Alexander Thesseling 2, Lisa Dejon 3, Daniel Lamberty 3, Laura Stief 3, Thorsten Friedrich 2, Petra Hellwig <sup>1</sup> , Hamid R. Nasiri <sup>4</sup> and Frederic Melin <sup>1</sup>**


**Abstract:** Membrane proteins that participate in multiple vital functions of every living organism such as transport, signaling and respiration, provide 80 to 90% of the relevant targets for the pharmaceutical industries. The family of cytochrome bd oxidase enzymes is of great interest for the development of future antibiotics as they are found only in the respiratory chain of the prokaryotes and they are believed to be involved in bacterial adaptability mechanisms. They catalyze the reduction of molecular oxygen in water and oxidation of quinols and contribute to the proton motive force required for ATP synthesis. Due to their hydrophobic nature, membrane proteins are more difficult to handle than soluble proteins. Protein film voltammetry is a very convenient technique, because it allows for working at a very low concentration and for optimizing the electrode surface to the nature of the enzyme. Here, we have developed a biosensor for the study of terminal oxidases based on their immobilization on gold nanoparticles modified with a self-assembled monolayer of thiols. The stability of the protein films can be optimized by varying the nature of thiols and amount of lipids. This enzyme-based electrochemical sensor was successfully used for the inhibition screening of a target-focused library of 34 compounds which belong to the families of quinones, naphthoquinones, phenols, quinolones, coumarins and flavonoids against cytochrome bd oxidase. Moreover, the developed device was applied for the study of the catalytic reaction of the enzyme with small gaseous signaling molecules.

**Keywords:** membrane proteins; bioelectrochemistry; inhibition

**Supplementary Materials:** The poster presentation is available online at https://www.mdpi.com/ article/10.3390/CSAC2021-10555.

**Author Contributions:** F.M., H.R.N. and P.H. designed research and analyzed data. I.M. and A.N. performed the electrochemical experiments and analyzed data. I.M. wrote the abstract and prepared the poster. T.F. and P.H. raised funding. A.T. and T.F. provided the protein samples. L.D., D.L., L.S., A.S. and H.R.N. provided the inhibitors. All authors reviewed and edited the abstract and poster.

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

**Informed Consent Statement:** Not applicable.

**Citation:** Makarchuk, I.; Nikolaev, A.; Thesseling, A.; Dejon, L.; Lamberty, D.; Stief, L.; Friedrich, T.; Hellwig, P.; Nasiri, H.R.; Melin, F. The Inhibition Study of Cytochrome bd Oxidase Using the Enzyme-Based Electrochemical Sensor. *Chem. Proc.* **2021**, *5*, 45. https://doi.org/ 10.3390/CSAC2021-10555

Academic Editor: Nicole Jaffrezic-Renault

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/).

**Data Availability Statement:** The data presented in this study are available in [I. Makarchuk, A. Nikolaev, A. Thesseling, et al, Identification and optimization of quinolone-based inhibitors against cytochrome bd oxidase using an electrochemical assay, Electrochimica Acta 381 (2021) 138293, https://doi.org/10.1016/j.electacta.2021.138293] and in the Supplementary Materials here.

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

### *Proceeding Paper* **Electrochemical Measurement System for Chlorides in Drinking and Wastewater †**

**Diana A. Toriz-Gutiérrez 1, Humberto Ramírez-Gasca 2, Luis E. Cárdenas-Galindo <sup>1</sup> and Eloisa Gallegos-Arellano 2,\***


**Abstract:** This paper presents a system for the measurement of chlorides in drinking and wastewater, based on an electrochemical process using a selective electrode as a transducer, which was developed by this group. The measurement for the concentration is carried out by introducing the implemented electrode (considered as reference) in the sample that will be analyzed; then a current is passed producing a potential difference in the system. Different aqueous solutions of sodium chloride (NaCl) were used, ranging between 35 and 3546 μg of chloride ions (Cl−). As a data acquisition and monitoring system for the analysis, an ATmega 328P microcontroller was used as the main capture element for subsequent interpretation through graphics. The experimental results show that it was possible to detect a potential difference in the electrochemical measurement system that corresponded to 35 μg of chloride ions (Cl−), making clear the detection process and the selectivity of chloride ions. It is important to mention that with this measurement system and the applied methodology, results are obtained in real time using a small sample volume and without generate ng extra liquid waste, compared to the application of the traditional analytical titrimetric method. Finally, this chloride measurement system is inexpensive and can be used in drinking and wastewater measurements.

**Keywords:** measurement system; data acquisition; electrodes; electrochemical process; ion selectivity; chloride ions

#### **1. Introduction**

Measurement by electrometric methods continues to be useful in the analytical identification processes of ion-selective electrodes (ISEs) of potentiometric sensors [1,2] because only small dimensions are involved and measurements can be made requiring little sample volume with respect to the activity of the ions in solution [3,4]. With the increasing demand for environmentally friendly technologies and in situ measurements, these designs facilitate quick readings and detection of low concentrations [1,2], as observed when obtaining measurements of concentrations of 35 μg/L in NaCl solutions [5].

Due to the diverse range of applications and reduced operating times, there is a need for measurement electrodes that are selective and able to detect a specific ion of a species that interacts with it using potential difference [4,6]. Several electrodes have been developed for selective ion determination which involve use of an ionophore to generate exchange in a solution enabling determination of the influence of the exchange ion percentage [7–10].

In this study, a chloride measurement system for drinking and wastewater is proposed based on an electrochemical process using a selective electrode as a transducer which was

**Citation:** Toriz-Gutiérrez, D.A.; Ramírez-Gasca, H.; Cárdenas-Galindo, L.E.; Gallegos-Arellano, E. Electrochemical Measurement System for Chlorides in Drinking and Wastewater. *Chem. Proc.* **2021**, *5*, 46. https://doi.org/ 10.3390/chemproc2021005046

Academic Editor: Nicole Jaffrezic-Renault

Published: 17 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/).

developed by this group. The ionophore that is proposed is an AgNO3 solution with an extra modification that relates to the use of a metal that interacts with a natural organic membrane, modifying the attraction of the ion, which enhances the selectivity to the desired ion (Cl−) [3,11], in addition to the copper reference electrode [9].

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

The experimental arrangement of the electrochemical measurement system, shown in Figure 1, is made up of two essential blocks: the electrochemical half-cell and the data acquisition system.

**Figure 1.** Experimental arrangement of the electrochemical measurement system. (**a**) Electrochemical half-cell; (**b**) Data acquisition system; (**c**) Temperature and potential difference data obtained directly from the measuring device.

For the half-cell (Figure 1a), a 3D design was produced and printed to ensure the electrode separation was constant and to reduce movements in the experimental arrangement. The electrochemical half-cell was constructed with an active noble metal electrode (X) and a copper reference electrode, which are submerged in different solutions, such as AgNO3 and NaCl, respectively, according to Creus (2011) [12] and previous research by Toriz, Loredo and Marcial [13].

The measurement of chloride ions (Cl−) was achieved through reaction in the half-cell taking as analytes NaCl solutions at different concentrations, in which an ionic migration was generated through an organic (natural) membrane. The organic membrane and the inert metallic material (X) allow the potential difference to remain active during the measurement.

The coefficients of total ionic activity are related to the individual coefficients of ionic activity, detecting free energy in the system that connects the thermodynamics of the reaction with the values of the electromotive force (*emf*) [10] and that can only be applied in reversible reactions. This can be represented by Equation (1).

$$\Gamma^{\circ} = \frac{-\Delta F^{\circ}}{nF} \tag{1}$$

where E◦ is the potential difference, Δ*F*◦ is the total free energy of the system, *n* is the number of moles used in the reaction, and ᆼ = Farad (free energy value = 96,500 Joules: approximate value = 23,052 cal).

*ε*

The electromotive force (*emf*) depends on the concentration of the cation according to the Nernst equation [14]. According to the literature, the relationship between the signs Δ*F*◦ and E◦ correspond directly to a spontaneous reaction (Maron and Prutton) [9].

Referring to a standard *emf* when the activity is a unit, the fundamental equation can be related to the thermodynamic equation and the *emf* values. The values of E◦ can be tabulated for each reaction of a half-cell and added algebraically to obtain the value of E◦.

To calculate the total free energy of the system, the relationship with thermodynamics is considered, calculating the *emf* from enthalpy and entropy tables for the reference half-cell [9,10], where T = 298 K, applying the relationship described in Equation (2):

$$
\Delta F^{\odot} = \Delta H - T\Delta S \tag{2}
$$

The calculation of the total Δ*F*◦ corresponds to the sum of the cathodes of the system; the reaction of each cell is stated as in Equation (3):

$$
\Delta F^{\odot} = \mu\_{A\text{g}^{\circ}} + \mu\_{\text{Cu}^{\circ}} + \mu\_{\text{Cl}^{-}} - \mu\_{A\text{g}\text{Cl}} - \mu\_{\text{Ni}} \tag{3}
$$

Substituting the values for the half-cell reaction of the system (total Δ*F*◦), Equation (4) is obtained:

$$
\Delta F^{\odot} = 1000(0 + 23.6 + (-40.02) - (-30.36) + 7.2) - 298(17.67 - 23.6 + 13.17 - 22.9 - 7.2) \tag{4}
$$

Solving Equation (4), the results presented in Equation (5) are obtained:

$$
\Delta F^{\circ} = 21140 + 6812.28 = 27952.28 \tag{5}
$$

Considering *n* = 1 and substituting the values in the equation, the standard potential of the system will be defined by Equation (6):

$$
\epsilon^\circ = \frac{27952.28}{23052} = 1.2126 \, volt \tag{6}
$$

The value obtained from Equation (6) represents the *emf* of the ionic activity with respect to the constant concentration of the ionic activity of the cell. It provides the voltage difference with respect to the element. This theoretical value was obtained experimentally using the measurements presented in Figure 1c. The relationship of this value and the concentration was used to establish the scale.

The data acquisition system is mainly comprised of an ATMEGA328P microcontroller that multiplexes two analog channels to store the temperature data and the potential difference of the half-cell in the microSD memory module for subsequent graphic analysis. The data from the potential difference of the electrodes are acquired through an instrumentation amplifier with unity gain to reduce the effects of electrical noise that may exist in the system (Figure 1b). The system also has an LCD module that prints the values of the analog channels sampled in the microcontroller (temperature and potential difference of the half-cell) every 5 s to verify changes in real time. When acquiring measurements with this system, the data id was saved in an Excel file where the first column shows the temperature and the second column the potential difference (Figure 1c).

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

The data stored in the microSD memory shown in Figure 1b were treated by a moving average filter with an N length window, which allows smoothing of the signal obtained from the potential difference measured in the half-cell.

The most significant data captured at different concentrations are shown in Figure 2, in which it is observed that the signals reached a stable state in the potential difference of each, which can be used for their subsequent characterization. The stabilization of the voltage occurs approximately in the same period, after the ionic migration of the oxidation-reduction reaction (transient response).

**Figure 2.** Graph of *emf* measurement at different concentrations of NaCl.

The data show the behavior of the concentration and the *emf* obtained for each concentration [5,15]. A scale was established to observe the change in electric potential as a function of the ionic activity in the solution [16].

Figure 3 shows that the values of the half-cell (E◦) are proportional to the logarithm of the ion concentration in the solution, in addition to presenting a Nernstian slope, as shown in Equation 7, which presents an r2 coefficient of 0.9953 and a correlation of 0.99.

**Figure 3.** Graph of half-cell values (E◦) as a function of the logarithm of the concentration.

It is important to mention that the value of the half-cell (E◦ = 1.2126 V) corresponds to the standard electrode data and confirms a reversible reaction and the ion selectivity; while the value of the half-cell l (E◦ = −40.47) represents the value of the ion that will be detected in the analyte.

$$
\varepsilon^{\circ} = -37.54 \ln(\text{C}) - 40.47 \tag{7}
$$

It is of note that it was possible to optimize the resolution of the analog-digital converter of the ATMEGA 328P microcontroller so that it can detect potential differences of 1.05 mV, in addition to increasing the number of samples per second that are processed through this internal microcontroller module.

#### **4. Conclusions**

In this study, it was possible to implement a novel chloride ion measurement system using a selective membrane (natural) and a support structure to assemble a half-cell based on a design made in a 3D printer. The half-cell obtained E◦ = 1.2126 V; this data corresponds to the value reported for a standard electrode, ion-selective and for a reversible reaction. The indicated value of the ion-selective in the half cell (E◦ = −40.47), represents chloride ions (Cl−).

The behavior of ionic activity at different concentrations shows a logarithmic trend and a typical Nernstian response and is verified with the coefficient (r2 of 0.9953) of the fitted curve.

Based on the measurements made, the results obtained and the functionality of the assembled device, it was determined that the electrode can be used as a potential chloride ion meter for wastewater and drinking water.

**Author Contributions:** Conceptualization, D.A.T.-G., H.R.-G., L.E.C.-G. and E.G.-A.; methodology, D.A.T.-G., H.R.-G., L.E.C.-G. and E.G.-A.; software, D.A.T.-G., H.R.-G., L.E.C.-G. and E.G.-A.; validation, D.A.T.-G., H.R.-G., L.E.C.-G. and E.G.-A.; formal analysis, D.A.T.-G., H.R.-G., L.E.C.-G. and E.G.-A.; investigation, D.A.T.-G., H.R.-G., L.E.C.-G. and E.G.-A.; resources, D.A.T.-G., H.R.-G., L.E.C.-G. and E.G.-A.; data curation, D.A.T.-G., H.R.-G., L.E.C.-G. and E.G.-A.; writing—original draft preparation, D.A.T.-G., H.R.-G., L.E.C.-G. and E.G.-A.; writing—review and editing, D.A.T.-G., H.R.-G., L.E.C.-G. and E.G.-A.; visualization, D.A.T.-G., H.R.-G., L.E.C.-G. and E.G.-A.; supervision, D.A.T.-G., H.R.-G., L.E.C.-G. and E.G.-A.; project administration, D.A.T.-G., H.R.-G., L.E.C.-G. and E.G.-A.; funding acquisition, D.A.T.-G., H.R.-G., L.E.C.-G. and E.G.-A. 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.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We want to thank Nicolás Flores Sámano and for help in the design and 3D printing, Roxana Robles García and María Guadalupe Martínez Muñoz for their help in the laboratory work and experimental tests, and finally Diana Priscila Vicencio Toriz for the English revision.

**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* **Poly(bromocresol purple)-Based Voltammetric Sensor for the Simultaneous Quantification of Ferulic Acid and Vanillin †**

**Anastasiya Zhupanova \* and Guzel Ziyatdinova**

Analytical Chemistry Department, Kazan Federal University, Kremleyevskaya, 18, 420008 Kazan, Russia; Ziyatdinovag@mail.ru

**\*** Correspondence: Zhupanova.Nastya@mail.ru

† Presented at the 1st International Electronic Conference on Chemical Sensors and Analytical Chemistry, 1–15 July 2021; Available online: https://csac2021.sciforum.net/.

**Abstract:** Natural phenolic antioxidants are extensively studied compounds due to their positive health effect and wide distribution in human diets. The simultaneous occurrence in samples requires selective methods for their determination. Electrochemical sensor based on the polyaminobenzene sulfonic acid functionalized single-walled carbon nanotubes (f-SWCNT) and electropolymerized bromocresol purple has been developed for the simultaneous quantification of ferulic acid and vanillin. The electrode has been characterized by scanning electron microscopy (SEM) and electrochemical methods, and the effectivity of the developed modifier has been confirmed. Thus, the novel sensitive voltammetric sensor is simple to fabricate, reliable, cost-effective, and can be applied for foodstuff screening.

**Keywords:** electrochemical sensors; carbon nanomaterials; electropolymerization; dyes; natural phenolics; antioxidants

Natural phenolic antioxidants are extensively studied compounds in modern electroanalysis due to their positive health effect and wide distribution in the human diet [1]. Simultaneous occurrence in samples requires selective methods for their determination. Among a wide range of natural phenolics, vanillin and its biological precursor ferulic acid [2] are of practical interest. High-performance liquid [3,4] and thin-layer chromatography [5] are usually applied for this purpose. Both phenolics under consideration are electrochemically active, which makes it possible to use electrochemical methods for their quantification. Although various types of electrochemical sensors have been developed for the simultaneous quantification of natural phenolics of different classes [6–8], ferulic acid and vanillin are not considered as analytes.

Thus, the current work is focused on the development of an electrochemical sensor based on a poly(bromocresol purple)-modified electrode for the simultaneous quantification of ferulic acid and vanillin.

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

Bromocresol purple (90% purity), 99% vanillin from Sigma-Aldrich (Steinheim, Germany), and 99% ferulic acid from Aldrich (Steinheim, Germany) were used. Their standard 10 mM solutions were prepared in ethanol (rectificate). The exact dilution was used for the preparation of less concentrated solutions.

Polyaminobenzene sulfonic acid functionalized single-walled carbon nanotubes (f-SWCNT) (*d* × *l* is 1.1 nm × 0.5–1.0 μm) were purchased from Sigma-Aldrich (Steinheim, Germany). A homogeneous 1.0 mg mL−<sup>1</sup> suspension of f-SWCNT was obtained by ultrasonic dispersion for 30 min in dimethylformamide.

**Citation:** Zhupanova, A.; Ziyatdinova, G. Poly(bromocresol purple)-Based Voltammetric Sensor for the Simultaneous Quantification of Ferulic Acid and Vanillin. *Chem. Proc.* **2021**, *5*, 47. https://doi.org/ 10.3390/CSAC2021-10441

Academic Editor: Ye Zhou

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/).

All reagents had chemical-grade purity. Double distilled water was used for the measurements. The experiments were carried out at laboratory temperature (25 ± 2 ◦C).

Electrochemical measurements were carried out on the potentiostat/galvanostat Autolab PGSTAT 302N with FRA 32M module (Eco Chemie B.V., Utrecht, The Netherlands) and NOVA 1.10.1.9 software. The 10 mL glassy electrochemical cell with a working glassy carbon electrode (GCE) with a 7.07 mm<sup>2</sup> geometric surface area (CH Instruments, Inc., Bee Cave, TX, USA) or modified electrode, a silver-silver chloride saturated KCl reference electrode, and a platinum wire as the counter electrode was used.

An "Expert-001" pH meter (Econix-Expert Ltd., Moscow, Russian Federation) equipped with the glassy electrode was applied for pH measurements.

Scanning electron microscopy (SEM) was carried out on the high-resolution field emission scanning electron microscope MerlinTM (Carl Zeiss, Oberkochen, Germany) at the accelerating voltage of 5 kV and emission current of 300 pA.

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

#### *3.1. Characterization of the Electrodes*

Bromocresol purple forms a nonconducting film which is confirmed by the disappearance of the oxidation peak with an increase of the cycles number and which is typical for the electropolymerization of phenolics [9].

The conditions of bromocresol purple potentiodynamic electropolymerization (monomer concentration, number of scans, supporting electrolyte pH, electrolysis parameters) have been optimized in order to find the best voltammetric response of the co-existed ferulic acid and vanillin. The peak potential separation of 170 mV on the polymer-based electrode is not affected by electropolymerization conditions, while oxidation currents change significantly. The best response has been obtained for the poly(bromocresol purple) obtained by 10-fold potential cycling from 0.1 to 1.2 V with a scan rate of 100 mV s−<sup>1</sup> from the 25 μM monomer solution in 0.1 M phosphate buffer pH 7.0.

The electrodes have been characterized by SEM (Figure 1). The data obtained confirm the successful immobilization of the nanomaterial on the electrode surface.

(**a**) (**b**) (**c**) **Figure 1.** SEM images for (**a**) CGE; (**b**) CGE/f-SWCNT; and (**c**) CGE/f-SWCNT/Poly(bromocresol purple).

> The effective surface area of the electrodes has been evaluated using 1.0 mM [Fe(CN)6] 4− as a redox probe under conditions of cyclic voltammetry and chronoamperometry (for GCE). The Cottrell equation for GCE and Randles–Sevcik equation for the cyclic voltammetry data have been applied. A 5.1-fold increase of the effective surface area for the polymer-modified electrode vs. bare GCE has been obtained, which leads to the ferulic acid and vanillin oxidation current increasing. Electrochemical impedance spectroscopy (EIS) has been performed in the presence of [Fe(CN)6] <sup>4</sup>−/3<sup>−</sup> as a redox probe at 0.23 V. EIS data show a 7.2-fold lower charge transfer resistance for the poly(bromocresol purple)-based sensor in comparison to GCE, which means an increase of the electron transfer rate.

#### *3.2. Simultaneous Quantification of Natural Phenolic Antioxidants*

The well-resolved oxidation peaks of the ferulic acid and vanillin at 0.732 and 0.903 V, respectively, with a potential separation of 170 mV have been obtained on the created sensor (Figure 2).

**Figure 2.** Cyclic voltammogram of 10 μM mixture of ferulic acid and vanillin on the poly(bromocresol purple)–based electrode in Britton−Robinson buffer pH 2.0.

The analytes' electrooxidation parameters have been studied. Both phenolics are oxidized under a diffusion control and irreversible with the participation of two electrons and protons (Figure 3).

**Figure 3.** Electrooxidation scheme of (**a**) vanillin and (**b**) ferulic acid.

The developed sensor has been operated under conditions of differential pulse voltammetry. The pulse parameters have been optimized, and it has been found that a modulation amplitude of 75 mV and modulation time of 25 ms provide the best response of the target analytes. The sensor allows a direct simultaneous quantification of ferulic acid and vanillin in the ranges of 0.1–5.0 and 5.0–25 μM for both analytes (Figure 4) with detection limits of 72 and 64 nM, respectively. The accuracy of determination has been tested on the model

mixtures of ferulic acid and vanillin. The relative standard deviation and recovery values obtained confirm the absence of a random error and the precision of the developed sensor.

**Figure 4.** Calibration plots for (**a**) ferulic acid and (**b**) vanillin based on the differential pulse voltammetry data on the poly(bromocresol purple)-based electrode in Britton—Robinson buffer pH 2.0.

Thus, the novel sensitive voltammetric sensor is simple to fabricate, reliable, costeffective, and can be applied for the foodstuff screening.

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

**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.

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

#### **References**


### *Proceeding Paper* **Label-Free Anti-Human IgG Biosensor Based on Chemical Modification of a Long Period Fiber Grating Surface †**

**João P. Mendes 1,2,3,\* , Luís C. C. Coelho 2,4 , Viviana P. Pereira <sup>1</sup> , Manuel A. Azenha 1,3 , Pedro A. S. Jorge 2,4 and Carlos M. Pereira 1,3**


**Abstract:** This work introduces a method specially developed to produce a biorecognition element based on modified Stöber silica nanoparticles by the covalent immobilization of the human IgG. The sensing structure is based on long period fiber gratings (LPFG), specially developed to allow the interaction of the electromagnetic wave with the target analytes through its evanescent field. The surface was modified by the immobilization of the IgG-modified nanoparticles serving has recognition elements for specific target molecules. The resulting configuration was tested in the presence of anti-human IgG, recording the refractometric response of the modified LPFG in contact with different amounts of analyte. The selectivity of the sensor was also assessed.

**Keywords:** optical fiber; long period gratings; evanescent field; chemical immobilization; biosensor

#### **1. Introduction**

Biosensors are powerful allies for food safety, drug discovery, environmental monitoring and clinical diagnosis [1–3]. The sensing methodology comprises a bioreceptor, a recognition element and a transducer whose properties changes upon analyte binding [4]. Typically, the bioreceptor is immobilized on the surface of the transducer and the binding event can be, e.g., mechanically, electrically or optically transduced [5,6] producing an increase in mass, a change in electrical resistivity or changes in the refractive index at the surface of the used material allowing to be measured. In recent years, optical biosensors are an active field of research worldwide, presenting rapid progress [7–9]. In this perspective, optical biosensors based on refractometric sensing schemes have been developed with great successes in the last few decades [7]. Moreover, optical fibers (OF) based on evanescent wave sensing are an excellent platform to develop high-stability and high-sensitive optical biosensors [10]. The quantitative and/or qualitative measurements result from the interaction of the biorecognition element with the evanescent field of light at the fiber surface. Its good biocompatibility makes them appropriate for biochemical functionalization, creating very sensitive structures targeting viruses, drugs and proteins [11]. In recent years, several authors have reported transduction scheme's using optical fibers for optical biosensing. Lobry et al. demonstrated a plasmon-assisted tilted fiber Bragg gratings (TFBGs) based biosensor for non-enzymatic D-glucose using polydopamine-immobilized concanavalin A [12]. More recently, Liyanage et al. developed a label-free sensitive tapered optical fiber plasmonic biosensor targeting microRNAs. The sensing platform comprises different types of gold nanoparticles immobilized on the surface of the fiber to enhance the evanescent

**Citation:** Mendes, J.P.; Coelho, L.C.C.; Pereira, V.P.; Azenha, M.A.; Jorge, P.A.S.; Pereira, C.M. Label-Free Anti-Human IgG Biosensor Based on Chemical Modification of a Long Period Fiber Grating Surface. *Chem. Proc.* **2021**, *5*, 48. https://doi.org/ 10.3390/CSAC2021-10454

Academic Editor: Huangxian Ju

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/).

mode, followed by self-assembled ssDNA probes [13]. Using a long-period fiber grating (LPFG) platform, Liu et al. demonstrated the use of a LPFG coated with graphene oxide (GO) nanosheets using the changes in resonant intensity to measure different concentrations of hemoglobin adsorbed by the GO layer [14]; Chiavaioli et al. reported a D-shaped single mode optical fiber (SMF) nanocoated with different metals for IgG/anti-IgG assays, reaching limit of detections (LOD) around to the femtomolar values [15]; and Dey et al. presented a sensitivity-enhanced LPFG near the turning point by etching the fiber with hydrofluoric acid to detect anti-mouse IgG [16]. In a similar approach to this work, Liu et al. demonstrated an LPFG-coated with silica nanoparticles modified with gold nanoparticles (AuNPs). The gold surface was modified with anti-IgM receptors and the sensing platform was tested to understand its suitability for the detection of human IgM antibodies [17].

In this work, a method based on silica nanoparticles (prepared based on Stöber [18] method), immobilized in the surface of commercial SMF28 OF, serving as recognition elements for specific target molecules is presented. The nanoparticles surface was functionalized by the introduction of an aminosilane (APTMS) followed by the covalent immobilization of the immunoglobulin G from human serum (human-IgG). The antibody was activated by the EDC/NHS protocol to allow the interaction of the amine exposed groups, located on the surface of the silica nanoparticles, with the activated carboxyl acid groups of the human-IgG molecules. The resulting template was immobilized onto the surface of an OF by electrostatic interactions between the negative charges of the fiber surface and the positively charged amine groups located in the IgG molecules. The sensing structure is based on LPFGs, specially developed to allow the interaction of the electromagnetic wave with the target analytes through its evanescent field. The refractometric system comprises a Braggmetter unit (HBK, FiberSensing, Darmstadt, Germany) working in a wavelength range from 1500 to 1600 nm and a reference LPFG to correct possible false interactions. The resulting configuration was tested in the presence of anti-human IgG, recording the refractometric response of the modified LPFG in contact with different amounts of analyte.

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

#### *2.1. Chemical Reagents*

Silica nanoparticles were prepared following the Stöber method [19], using tetraethyl orthosilicate (TEOS; Sigma-Aldrich, St. Louis, MO, USA; ≥98%) and ammonium hydroxide solution (NH4OH; Sigma-Aldrich, 28% m/m) as reagents. The functionalization of the nanoparticles surface was attained using the following reagents: (3-aminopropyl) trimethoxysilane solution (Sigma-Aldrich, St. Louis, MO, USA; 97%), anhydrous toluene (Sigma-Aldrich, St. Louis, MO, USA; 99.8%), phosphate buffered saline (PBS; pH 7.4, tablets, Sigma-Aldrich, St. Louis, MO, USA), 2-(*N*-morpholino)ethanesulfonic acid (MES; Sigma-Aldrich, St. Louis, MO, USA), *N*-(3-dimethylaminopropyl)-*N* -ethylcarbodiimide hydrochloride (EDC; Sigma-Aldrich, St. Louis, MO, USA; ≥99%), *N*-hydroxysuccinimide (NHS; Sigma-Aldrich, St. Louis, MI, USA; ≥98%) and immunoglobulin G from human serum (human-IgG; Sigma-Aldrich, St. Louis, MO, USA; ≥95%). For the LPFG surface cleaning were used sodium hydroxide anhydrous (NaOH; Sigma-Aldrich, St. Louis, MO, USA; ≥98%) and hydrochloric acid (HCl; Sigma-Aldrich, St. Louis, MO, USA; 37%). For surface activation were used sulfuric acid (H2SO4; Sigma-Aldrich, St. Louis, MO, USA; 95%–98%) and hydrogen peroxide solution (H2O2; Sigma-Aldrich, St. Louis, MO, USA; 30%) to perform piranha solution. For the affinity and selectivity assays were used the human IgG, the human serum albumin (HAS; Sigma-Aldrich, St. Louis, MO, USA; ≥98%) and the anti-human IgG (Fab specific; antibody produced in goat, Sigma-Aldrich). Ultrapure water (type II-analytical grade, <1 <sup>μ</sup>S·cm−1) and ethanol (Labchem, Zelienople, PA, USA; 96%) were also used.

#### *2.2. Synthesis of the SiO2 Nanoparticles*

The SiO2 nanoparticles were synthesized according with the Stöber method. Briefly, in a proper container the ethanol, the ultra-pure water and the TEOS, were mixed in that order. The mixture was sonicated for 20 min and was added, under stir, the ammonia hydroxide and the final mixture was stirred for 24 h at room temperature. The resulted solution was centrifuged at 6000 rpm for 10 min and the beads were redispersed/centrifuged (five times) in deionized water and acetone. The resulted beads were dried at 40 ◦C for 12 h in the oven. The average size of the nanoparticles was determined by W130i Dynamic Light Scattering (DLS, AvidNano, Wycombe, UK), showing an average diameter ranging from 300–400 nm and were evaluated by attenuated total reflectance (FTIR-ATR, Bruker, Billerica, MA, USA).

#### *2.3. Immobilization of the Biorecognition Molecule onto the SiO2 Surface*

The dry beads were incubated in freshly prepared APTMS solution 2% (*v*/*v*) in anhydrous Toluene for 24 h at room temperature in a closed container, using 10 mg/mL of beads concentration. After incubation, the nanoparticles were centrifuged at 6000 rpm for 10 min and redispersed/centrifuged for five times in acetone and ethanol. From this step resulted amino-functionalized silica nanoparticles that were verified by ATR-IR. The beads were redispersed in the PBS solution in a concentration of 5 mg/mL. A solution of the biorecognition element in MES buffer (pH 5.5) and the activation of the template was prepared by adding EDC (10× molar excess) to NHS (10× molar excess) and incubating for 30 min at room temperature. The previous prepared beads solution was added to the template solution and the pH was adjusted to 7.4–8.0. The incubation carried out for 4 h at room temperature without stirring (just swirled the mixture every half-hour). The resulted modified nanoparticles were centrifuged at 6000 rpm for 10 min and redispersed/centrifuged in deionized water. The nanoparticles were assessed by FTIR-ATR.

#### *2.4. Working Principle of the Evanescent Wave Based Sensors, Long-Period Fiber Grating Fabrication and Surface Modification*

In this work, a long-period fiber grating was microfabricated on the optical fiber surface. This grating works as a wavelength selective filter, displaying a spectrum with several resonances resulting from the combination of the mode of the core and the different cladding modes [20]. Figure 1 shows a description of a LPFG on an optical fiber and the resultant transduced optical signal from the interaction between the recognition molecule and the target.

Moreover, the LPFGs were fabricated by the induced electric-arc technique following the protocol published by Rego (2016) [20] by creating a modulation in the propagating mode refractive index which, in this case, is achieved point-by-point through electric arc discharges with a current of 9 mA and a duration of 1 s along 30 to 50 mm with a period of 415 μm. Afterwards, the LPFG was chemically modified by the immobilization of the biorecognition molecule on the fiber surface. The sensitive section of the optical fiber was cleaned witha2M NaOH solution for 10 min followed by immersion in a 0.5 M HCl solution for 2 h. After washing with deionized water, the sensitive surface was activated with piranha solution (3:1 *v*/*v*) for 1 h at 60 ◦C. Finally, the LPFG was washed

with deionized water and kept in the oven for 10 min to completely dry and was cooled with pure nitrogen. The process is schematically presented in Figure 2.

**Figure 2.** Schematic figure of the LPFG surface chemical modification: (**a**) Bare LPFG; (**b**) activated surface with negative electrical charges; and (**c**) modified surface by the template immobilization.

#### *2.5. Affinity and Selectivity Assays*

The modified LPFGs were tested in the presence of the anti-human IgG in different concentrations ranging from 1.5 × <sup>10</sup>−<sup>2</sup> to 9 <sup>μ</sup>g/mL to attest the affinity of the sensing platform. To verify the selectivity of the modified optical fiber, the sensing scheme was exposed to a 9 μg/mL of human IgG, HSA and anti-human IgG solutions, in the same experimental conditions. In order to obtain the most trustable values, was used a bare LPFG as a reference signal. All data will be presented as the differential between sensing LPFG and reference LPFG.

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

#### *3.1. SiO2 Nanoparticles Bare, SiO2-NH2 and SiO2-NH2-IgG FTIR-ATR Spectra*

To confirm the introduction of the new functional groups after each step of the SiO2 nanoparticles surface (nanoSiO2) modification, were made FTIR-ATR analysis. Figure 3 shows the spectra of the bare SiO2 (nanoSiO2), the aminated SiO2 (nanoSiO2-NH2) and the obtained nanospheres after the incubation of the IgG (nanoSiO2-NH2-IgG). In the main figure, the absorption peaks at 3000–3500 cm−<sup>1</sup> are related to the stretching -OH bands, the absorption peaks at 1000–1150 cm−<sup>1</sup> are assigned to the Si-O-Si asymmetric stretching bands and the peaks at 800–950 cm−<sup>1</sup> are appointed to the asymmetric bending of Si-OH. In the Figure 3a, the asymmetric deformation vibration of the -NH2 at around 1550 cm−<sup>1</sup> is displayed, suggesting that the amino groups were successfully fixed in the silica nanoparticle surface. Figure 3b show a peak at around 2900 cm−<sup>1</sup> that are attributed to the presence of methyl groups of the APTMS structure. Finally, Figure 3c shows the carboxylate peak at 1650 cm−1, assigned to the presence of the IgG molecule. This evaluation is similar to the evaluation made by Feifel and his co-worker when the authors proved the possibility to create electro-active cytochrome C multilayers by using carboxyl-modified SiO2 nanoparticles [21]. Moreover, Hernandez-Leon et al. also showed parallel spectra when the authors modified a core-shell SiO2 nanobeads for capture low molecular weight proteins and peptides [22].

**Figure 3.** Obtained absorbance spectra from FTIR-ATR analysis of bare SiO2 nanoparticles (black line), after aminofunctionalized SiO2 surface (inset graphs **a** and **b**; red line), and after IgG immobilization (inset graph **c**; blue line).

#### *3.2. Affinity and Slectivity Assays*

The SiO2/IgG-modified LPFG probe was tested in the presence of different concentrations of anti-human IgG to attest the affinity of the sensing platform. The sensing LPFG (sensLPFG) was placed in an experimental chamber as well as the reference LPFG (refLPFG). Both gratings were exposed to a freshly prepared standard target solutions ranging from 1.5 × <sup>10</sup>−<sup>2</sup> to 9 <sup>μ</sup>g/mL in PBS. After 10 min of exposure time, the LPFGs were washed three times with fresh PBS and three times with deionized water. All data were obtained measuring the LPFGs in deionized water at 22 ◦C. Figure 4a show the experimental data of the wavelength shift (sensLPFG–refLPFG) versus the anti-human IgG. Data are reported as a mean value with standard deviation (n = 3). Other similar works were described recently, such as the interferometric optical fiber biosensor for IgG/anti-IgG immunosensing presented by Wang et al., reporting a limit of detection around of 50 ng/mL [23]. Another approach was demonstrated by Han et al. that combined a Bragg acoustic reflector with an Au electrode and an aluminum nitride piezoelectric thin film, to develop a biosensor for anti-human IgG detection by immobilization of the human IgG antibody onto the modified Au electrode. The sensing platform was able to detect anti-human IgG concentrations smaller than 0.4 mg/mL [24]. The sensing platform presented in this work is able to detect the referenced target below to 0.1 μg/mL. Additionally, the data resulting from the linear fitting of Figure 4a, displaying a sensibility (S) (i.e., the slope) about |S| = 59 pm/(μg/mL).

To validate the specificity of the sensing platform, the same protocol was followed in the presence of human IgG antibody, the HSA protein and, finally, the anti-human IgG. Figure 4b show the resulting data after 10 min of incubation time for each target in 9 μg/mL (in PBS). The results are reported as a mean value with standard deviation (n = 3). These results showed the specificity of the built sensing platform to the proposed target, revealing a very relevant wavelength shift when exposed to it. By the other side, the shifts showed by the LPFG in the presence of the other targets are not relevant.

**Figure 4.** (**a**) Resonance shift vs. the anti-human IgG concentration (1.5 <sup>×</sup> <sup>10</sup>−<sup>2</sup> to 9 <sup>μ</sup>g/mL). Data is reported as a mean value (n = 3) with standard deviation; (**b**) Resonance shift obtained by 10 min incubation in 9 μg/mL in human IgG, HSA protein and anti-human IgG. Data is reported as a mean value (n = 3) with standard deviation.

#### **4. Conclusions**

In this work, a sensing platform for the detection of the anti-human IgG antigen was developed by chemical modification of long period fiber grating surface. The sensing methodology is based on refractometric changes due to the interactions between the biorecognition molecule and the target. The surface of the optical fiber was changed by immobilization of IgG- modified silica nanoparticles. The FTIR-ATR spectra proved that the biorecognition molecule was successfully attached onto the SiO2 nanoparticles surface and specificity assays demonstrated the selectivity of the method. The use of IgG-modified nanoparticles can bring some advantages, increasing the number of receptors available to interact with the target.

The low-cost and easy-to-use optical sensor reported here can detect anti-human IgG concentrations below 0.1 μg/mL by promoting specific antibody/antigen interactions. In the next step, we aim to imprint molecularly the analogue synthetic molecule of this template. The goal is to produce highly sensitive and selective molecularly imprinted polymers using the template of this work, combining them with highly sensitive optical platforms.

**Supplementary Materials:** The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/CSAC2021-10454/s1.

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

**Funding:** This work was financed by National Funds through the Portuguese funding agency, FCT—Fundação para a Ciência e a Tecnologia, within projects "UIDB/50014/2020" and "UIDB/00081/ 2020 (CIQUP)"and ANI through project "FAMEST"—POCI-01-0247-FEDER-024529 under program P2020|COMPETE.

**Acknowledgments:** João Mendes would like to thank FCT for the PhD research grant SFRH/BD/ 130674/2017 and Luís Coelho acknowledges the support from FCT research contract grant CEECIND/ 00471/2017.

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

#### **References**


### *Proceeding Paper* **Precipitation of Iron Oxide in Hydrogel with Superparamagnetic and Stimuli-Responsive Properties †**

**Alice Mieting 1,\*, Sitao Wang 1, Mia Schliephake 2, Daniela Franke <sup>1</sup> , Margarita Guenther <sup>1</sup> , Stefan Odenbach <sup>2</sup> and Gerald Gerlach <sup>1</sup>**


**Abstract:** In this work, we present a template-based preparation of iron oxide-containing hydrogels (ferrogels) with ionic sensitive and superparamagnetic properties. The influence of the cross-linked template polyacrylamide and the concentration of the iron salts and sodium hydroxide on the precipitation of the iron oxide particles is investigated with respect to the stability of the ferrogels. Scanning electron microscope images show cubic particles, which can be semiquantitatively classified in three groups of particle size with respect to the dilution level. Magnetic hysteresis curves reveal a sigmoidal shape without remanence and coercivity for all samples. The higher cross-linked ferrogels, in comparison with the lower cross-linked ferrogels, possess a steady-state degree of swelling in ultrapure water and a stimuli-sensitive deswelling over a wide range of varying ionic strengths. Thus, they are suitable candidates for applications in sensing and microfluidics.

**Keywords:** stimuli-responsive hydrogel; superparamagnetic; iron oxide; coprecipitation; ferrogel

#### **1. Introduction**

Hydrogels are cross-linked, usually hydrophilic polymers, which are suitable candidates for applications in sensor [1] and actuator technology [2] due to their stimuli-sensitive swelling and viscoelastic properties. The incorporation of iron oxide particles into hydrogels results in novel composite materials with enhanced chemical and physical properties. Various approaches have demonstrated the sensitive and adsorptive properties of iron oxide particles with respect to heavy metal ions [3–5], pH [6], and biomolecules [7,8], as well as photocatalytic activity [9,10].

The aim of this work is the investigation of how the mechanical stability of the template structure and the concentration of the synthesis solutions influence the properties of in situ precipitated iron oxide particles. Understanding the structure–property relations of such novel composite materials relates to ongoing topics of scientific applications and research in engineering [11] and biomedicine [12] as well as in the treatment of contaminated water [13].

In this study, the wet chemical precipitation of iron oxides from iron salts with sodium hydroxide in the stoichiometric ratio of magnetite (FeII(FeIII)2O4) is investigated in two various cross-linked hydrogels: a higher cross-linked hydrogel, which has already been used in piezoresistive sensors [14], named sensor hydrogel/ferrogel, and a lower crosslinked hydrogel, which is used in actuator setups [15], named actuator hydrogel/ferrogel

**Citation:** Mieting, A.; Wang, S.; Schliephake, M.; Franke, D.; Guenther, M.; Odenbach, S.; Gerlach, G. Precipitation of Iron Oxide in Hydrogel with Superparamagnetic and Stimuli-Responsive Properties. *Chem. Proc.* **2021**, *5*, 49. https:// doi.org/10.3390/chemproc2021005049

Academic Editor: Nicole Jaffrezic-Renault

Published: 17 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/).

(Figure 1A). It is expected that in template-based precipitation the differently crosslinked hydrogels will affect the particle shape and size and in turn the swelling properties as well as the magnetic properties of the resulting ferrogels. On the other hand, different dilution levels of iron salts and sodium hydroxide will be used to investigate a suitable synthesis concentration with respect to the sensitivity and stability of the ferrogels (Figure 1B).

**Figure 1.** Scheme of the precipitation of iron oxide in hydrogel using iron chloride and sodium hydroxide solutions (**A**). Overview of the transformation of a sensor hydrogel to a ferrogel by soaking the samples in iron salt solution with orange-yellow coloring and forming black-brown-colored ferrogels in sodium hydroxide at different dilution levels (**B**).

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

#### *2.1. Synthesis of Iron Oxide in Hydrogels*

The monomer acrylamide (AAm), crosslinker *N*,*N* -methylene-bis-acrylamide (BIS), ammonium peroxodisulfate (APS), ferric chloride hexahydrate (FeCl3·6H2O), ferrous chloride tetrahydrate (FeCl2·4H2O), and sodium chloride (NaCl) were purchased from Sigma-Aldrich, Saint Louis, MO, USA. *N*,*N*,*N* ,*N* -tetramethylethylenediamine (TEMED) was purchased from Carl Roth, Karlsruher, Germany.

#### 2.1.1. Sensor Hydrogels

For the synthesis of sensor hydrogel, 1.6 M (8 mmol, 0.5686 g) AAm and 1.5 mol% (0.12 mmol, 0.0185 g) BIS were dissolved in 4.156 mL ultrapure water and placed in an ice bath to cool. Polymerization was initiated by adding 300 μL of 0.072 M APS solution (0.022 mmol APS) and 2.1 mol% (0.168 mmol, 25.4 μL) TEMED. The cooled solution was placed in glass tubes with a diameter of about 6 mm, sealed, and left overnight at room temperature for polymerization. The polymerized polyacrylamide (PAAm) hydrogels were removed from the glass tubes and washed in ultrapure water for 5 days. Discs of about 2 mm thickness were cut from each of the cylindrical samples for in situ precipitation of the iron oxides in hydrogel.

#### 2.1.2. Actuator Hydrogels

The actuator hydrogel was synthesized and handled in the same procedure as the sensor hydrogel, but with the following composition: 2.8 M (14 mmol, 1.0 g) AAm and 0.03 mol% (4.2 μmol, 0.7 mg) BIS were dissolved in 3.8 mL ultrapure water. Polymerization was initiated by adding 300 μL of 0.15 M APS solution (0.045 mmol APS) and 0.48 mol% (0.1 mmol, 10.2 μL) TEMED.

#### 2.1.3. Coprecipitation of Iron Oxide in Hydrogels

In order to prepare for precipitation, the sliced native hydrogel discs were rinsed with nitrogen in ultrapure water free of oxygen.

The first step was to disperse the iron chloride solution into the hydrogel. Thus, 12 mL of a 3 M mixture of iron (III) chloride hexahydrate (24 mmol, 6.4870 g) and iron (II) chloride tetrahydrate (12 mmol, 2.3860 g) was prepared in a 2:1 molar ratio. After that, a 1:10 dilution and a 1:100 dilution, each in 10 mL, were made from the 3 M iron salt solution. Each sensor and actuator hydrogel was placed in 5 mL of the appropriate iron chloride concentration for 24 h.

For the precipitation of iron oxide, 12 mL of an 8 M sodium hydroxide solution (96 mmol, 3.8397 g) was prepared, and a 1:10 dilution and a 1:100 dilution, each in 10 mL, were made from it. The iron salt-soaked hydrogels were transferred to 5 mL of the appropriate concentration of sodium hydroxide and left overnight. Finally, the ferrogels were washed until the pH of the water was neutral.

In general, the precipitation was performed under nitrogen atmosphere, and all solutions and the ultrapure water were used in a degassed condition. However, the following experiments and investigations were carried out to characterize the hydrogels under ambient conditions so that oxidation of magnetite (Fe3O4) to maghemite (γ-Fe2O3) is suggested.

#### *2.2. Characterization Methods*

#### 2.2.1. Scanning Electron Microscopy (SEM)

A piece of each sample was air-dried and sputtered with a 5 nm thick gold layer for subsequent secondary electron imaging of the embedded iron oxide particles with a SEM (Zeiss Supra 40VP; Schottky emitter) at a fixed stage width and 7 keV.

#### 2.2.2. Vibrating Sample Magnetometer (VSM)

The magnetization of the air-dried samples was measured on a Lake Shore VSM 7407 in the magnetic field range of ±17.5 kOe at room temperature.

#### 2.2.3. Swelling Experiments

In order to measure the impact of the changed environmental conditions on the hydrogels, the respective masses of the samples were weighed before and after addition of the stimulus. Without stimulus, the sample was in ultrapure water and had a mass *m*0. The mass *mi* of the sample with stimulus was determined after 24 h or after the corresponding long-term point. The degree of swelling was calculated as follows:

$$Q = \frac{m\_i - m\_0}{m\_0} \times 100\%.\tag{1}$$

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

#### *3.1. Morphological Properties*

The scanning electron microscope images in Figure 2 of the sensor and actuator ferrogels of each dilution level show cubic particles between 50 and 300 nm and, in some cases, 1 μm in size. Sensor and actuator ferrogels of undiluted concentrations (Figure 2a,b) and the actuator ferrogel of dilution level 1:10 (Figure 2e) additionally exhibit particles smaller than 20 nm distributed in the sample in a lawnlike manner and cannot be resolved with the currently used equipment. Due to the nonplanar arrangement of the particles, quantitative evaluation was not performed.

(**a**) (**b**) (**c**) (**d**) (**e**) (**f**)

**Figure 2.** Representative SEM images of sensor ferrogels (**a**–**c**) and actuator ferrogels (**d**–**f**) of the different dilution levels. Scale bar: 1 μm.

#### *3.2. Magnetic Properties*

Magnetization curves with an S-shaped profile without hysteresis gaining magnetic saturation characterize superparamagnetic behavior [16,17]. For all prepared ferrogels, the magnetization curves show a sigmoidal curve shape without coercivity and without remanence (Figure 3).

**Figure 3.** Magnetic hysteresis loop of the sensor ferrogels (**a**) and the actuator ferrogels (**b**) of the different dilution levels. Inset in the diagrams shows the curves at a low magnetic field.

The magnetization curve for the sensor ferrogel of dilution level 1:100 is not plotted in Figure 3a because the mass of this sample could not be determined due to sample instability in the dried state, making a quantitative comparison of the curves impossible. Except for the actuator ferrogel of dilution level 1:1, the saturation magnetization in both ferrogel types decreases with the dilution level, reflecting the lower content of magnetic particles in the ferrogel.

#### *3.3. Reversibility of Ferrogel Swelling*

Figure 4 depicts the swelling degrees of the ferrogels and native hydrogels alternating in ultrapure water and 1 M NaCl. Both ferrogels show a deswelling of around 80% in solution with increased ionic strength. It can be concluded that in aqueous solution, the ferrogels are in the swollen state due to electrostatic repulsions of charged surface groups of the iron oxides. An increased ionic strength in the solution leads to electrostatic neutralization of the ionized groups of the iron oxide with the dissolved ions.

**Figure 4.** Swelling cycles showing the repeatability of the deswelling in 1 M NaCl solution and swelling in ultrapure water (0 M NaCl) of the sensor ferrogels (**a**) and actuator ferrogels (**b**).

Deswelling of the ferrogel occurs due to a reduction in electrostatic repulsions within the composite material comparable to the swelling behavior of pH or ion-sensitive hydrogels with fixed ionic groups in the polymer network [18]. In contrast, the native hydrogels show minor swelling under increased ionic strength due to osmotically induced swelling mechanisms.

The sensor ferrogels obtain their initial steady-state degree of swelling in water after three water/NaCl cycles (Figure 4a).

Due to the increased volume in water after synthesis, it was not possible to remove the actuator ferrogels from the sample containers so that detecting the mass could just start in the deswollen state. Furthermore, the actuator ferrogels show an increase in swelling degree during the second cycle in water and reach a swelling degree up to 50–100% in the third water cycle (Figure 4b).

#### *3.4. Sensitivity of the Sensor Ferrogel to Ionic Strength*

Figure 5 depicts the swelling levels of the sensor ferrogels over a wide range of varying ionic strengths from nM to 5 M.

**Figure 5.** Swelling sensitivity of sensor ferrogels in NaCl solutions versus ionic strength.

A decay of the swelling curves between 1 mM and 1 M NaCl shows the concentration range of the deswelling that applies to all three dilution levels. Interesting for sensory applications is the flattened curve starting at 1 nM to 1 mM of the 1:100 sample. For this ferrogel, there seems to be an optimal balance between the charge density of ionized iron oxide particles under changing ionic strength and the mechanical stability of the hydrogel, so it can be used as a stimuli-responsive ferrogel in sensory applications.

#### **4. Conclusions**

The use of different cross-linked hydrogels as templates for the wet chemical precipitation of iron oxide producing fairly homogeneous cubic-shaped particles with superparamagnetic characteristic curves independent of the applied concentration of iron salts and base was presented. The ion-sensitive swelling properties of the sensor ferrogels and the reversibility of their swelling make them suitable candidates for applications in piezoresistive sensors. Their magnetic properties allow applications under magnetic field control in microfluidics and medicine. Due to their strong swelling in water, the lower cross-linked actuator ferrogels could be used as adsorption materials for the remediation of contaminated water.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/chemproc2021005049/s1.

**Author Contributions:** Conceptualization, A.M., D.F. and M.G.; methodology, A.M., S.W. and M.S.; writing—original draft preparation, A.M.; writing—review and editing, D.F., M.S., S.O. and G.G.; supervision, D.F. and M.G.; project administration, G.G.; funding acquisition, G.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the German Research Foundation (DFG) in the course of the Research Training Group "Hydrogel-Based Microsystems" (RTG 1865).

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We thank the Institute of Electronic Packaging Technology (German abbreviation IAVT) at TU Dresden for the access to their scanning electron microscope (SEM).

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

#### **References**


### *Proceeding Paper* **Critical Variables Influencing the Ultrasound-Assisted Extraction of Bioactive Compounds—A Review †**

**Anxo Carreira-Casais 1, Maria Carpena <sup>1</sup> , Antia G. Pereira 1,2 , Franklin Chamorro <sup>1</sup> , Anton Soria-Lopez 1, Pascual Garcia Perez <sup>1</sup> , Paz Otero 1, Hui Cao 1, Jianbo Xiao 1, Jesus Simal-Gandara 1,\* and Miguel A. Prieto 1,2,\***


**Abstract:** Ultrasound-assisted extraction (UAE) is a novel methodology, belonging to the so-called "Green Chemistry", which has gained interest in recent years due to the potential to recover bioactive compounds, especially those from plant matrices. It is widely recognized that the extraction of molecules by UAE gives rise to higher or similar yields than those obtained by traditional extraction methods. UAE has certain advantages inherent to Green Chemistry extraction methods, such as short extraction time and low solvent consumption. The aim of this review is to critically present the different variables and parameters that can be modified in UAE, such as ultrasound power, time, temperature, solvent, and solid to solvent ratio that influence yield and extraction performance.

**Keywords:** ultrasound-assisted extraction; critical variables; power; temperature; time; solvent

#### **1. Introduction**

Ultrasound-assisted extraction (UAE) is a technique that belongs to the group of novel extraction methods, together with microwave assisted extraction (MAE), enzyme assisted extraction (EAE) or high-pressure assisted extraction (HPAE) [1,2]. UAE promotes the extraction of compounds of interest, lowering the consumption of resources, such as solvent and energy, whereas achieving remarkably higher extraction yields [3,4]. In addition, UAE is a multipurpose method that lends itself to be combined with other extraction methods, both conventional and novel [5]. UAE has been applied to obtain extracts rich in bioactive compounds, such as phenolic compounds, pigments, polysaccharides, and amino acids, among others from plant matrices [1,3,6,7].

This methodology is based on the principle of cavitation, which leads to cell collapse of the matrix and allows the release of their inner substances. Several variables are relevant for the performance of UAE, including the solid–liquid ratio, the type of solvents used, the extraction time and the ultrasound power applied. Besides, ultrasound power and extraction time are closely linked to a fifth important factor, which is temperature. In practical terms, a correct optimization of these variables is essential to obtain a correct performance, resulting in a maximal extraction yield. In addition, temperature can affect the integrity of the bioactive compounds, since most of them are thermolabile. Considering that high ultrasound power linked to long extraction periods may lead to sample damage, temperature control is essential for a correct design of the cooling reactor and the optimization of UAE extraction protocols. Keeping all this in mind, this critical review is focused on

**Citation:** Carreira-Casais, A.; Carpena, M.; Pereira, A.G.; Chamorro, F.; Soria-Lopez, A.; Perez, P.G.; Otero, P.; Cao, H.; Xiao, J.; Simal-Gandara, J.; et al. Critical Variables Influencing the Ultrasound-Assisted Extraction of Bioactive Compounds—A Review. *Chem. Proc.* **2021**, *5*, 50. https:// doi.org/10.3390/CSAC2021-10562

Academic Editor: Huangxian Ju

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/).

the influence of all the variables that affect UAE, to analyze the critical factors involved in the optimization of this technique. In addition, response surface methodology (RSM) can be a representative tool to generate meta-models. RSM allows to analyze and optimize several variables at the same time and minimize the loss of matrices [8,9].

#### **2. Variables Influencing Ultrasound-Assisted Extraction**

To obtain good extraction yields, an optimization of the variables that influence the process is required. Among the variables that affect UAE, there are three types of parameters, as follows: physical, medium-dependent, and matrix-dependent parameters. Regarding the first, physical parameters are related to the ultrasonic waves applied during UAE and the equipment used. In this sense, those attributed to ultrasonic waves are ultrasound power, frequency, and ultrasound intensity (UI), whereas those related with ultrasound equipment are extraction time, and shape and size of the ultrasonic reactor. Medium-dependent parameters are related with the space in which ultrasound waves are transmitted from the emitting source to the matrix. Solvent properties, temperature and the presence of gases are examples of medium-dependent parameters. Finally, matrixdependent parameters are those that have a significant influence in the extraction of target compounds and considerably affect the effectiveness of the extraction. Type of matrix, structure, pre-treatment, particle size, or solid–liquid ratio are examples of those parameters [3,10]. Therefore, a correct design of the process, optimization of the variables, and appropriate equipment is needed to obtain extraction yields comparable to those obtained by the so-called traditional methods [11].

Regarding the ultrasound power, the use of high values usually improves extraction yields due to the generation of strong shear forces, so it is considered as one of the critical parameters to be optimized. Furthermore, higher ultrasound power reduces the time of extraction. For example, a study showed good results of the extraction of β-d glucans at a high extraction power (590 W), in only 58 min [12]. However, the use of high ultrasound power without control can overheat the reactor producing degradation of labile compounds and solvent evaporation [13]. In addition, the higher ultrasound power, the higher UI, so that when UI reaches the maximum value can produce liquid agitation and the consequent loss of ultrasound wave and the reduction of cavitation efficiency [14].

Regarding the extraction time, UAE allows to obtain good extraction yields with relatively short processing times (maximum 60 min) since longer times may cause undesirable changes in the extracted compounds. In this sense, optimized time commonly ranges between 20–60 min, minimizing the energy consumption and reducing the compounds' exposure to the process [15]. For example, one study shows that 7.25 min are enough to extract pigments from annatto seeds [16], while 37 min are needed to extract betacyanin and betaxanthin in bougainvillea flowers [17]. In the case of amino acid extraction, shorter extraction time was needed (6 min) [18,19], whereas other authors were able to extract polysaccharides from purple glutinous rice bran (*Oryza sativa*) with an extraction time of 20 min at 70 ◦C [20] (Table 1).

The type or polarity of the solvent used is closely linked to the nature of the compounds to be extracted. In addition, due to current concern for the environment, eco-friendly solvents are preferred. Predominantly, an aqueous medium is generally chosen for the extraction of polar compounds used in food matrices, while in the case of other organic compounds, ethanol, and methanol are usually employed. However, despite the use of methanol tends to obtain better extraction yields, ethanol is preferably chosen because of its lower toxicity [10]. For example, distilled water and ethanol are usually used for pigments extraction, while water is the most common solvent for the extraction of polysaccharides and amino acids [12,20,21] (Table 1). Furthermore, European Directive 2010/59/EU lists the solvents that can be used for the extraction of compounds from foodstuffs, as well as their uses and limitations [22]. In addition to the suitable solubility of the compounds of interest, it is also important to consider the vapor pressure, the surface tension, and the viscosity of the solvent, since those may affect cavitation and the extraction yield [3]. The solid-to-solvent ratio used for each compound does not follow a certain pattern. It depends especially on the type of solvent and the matrix used. Table 1 shows that for the extraction of phenolic compounds, the most used solvent is ethanol and the solid-to-solvent ratio varies between 0.025 g/mL [23] and 0.1 g/mL [24], whereas ratios vary between from 0.058 g/mL for the extraction of betacyanin and betaxanthin in bougainvillea flowers [17] to 0.14 g/mL for the extraction of a natural pigment from annatto seeds [16].

Finally, high extraction temperatures not only affect the extraction yield but could also have negative effects due to the possible degradation of thermolabile compounds [14]. For this reason, the cooling system must allow the extraction of compounds avoiding the overheating of the medium by controlling the temperature of the system. For example, it is possible to extract phenolic compounds with temperatures up to 75 ◦C, β-d glucans at 81 ◦C or amino acids at 70 ◦C with optimal yields [12,18,25]. The increase in the temperature caused by the ultrasound probe itself is fundamentally produced when high ultrasound power is applied. The temperature increase produces a decrease in both viscosity and surface tension and induces an increase in the vapor pressure. Thus, too high temperatures can be harmful for the propagation of ultrasounds through the medium [13]. For these reasons, the optimization of the extraction temperature must be focused on both protecting the structure and function of the target components and improving the extractive properties of the solvent. Generally, the temperature does not exceed 80 ◦C, and it commonly works around 50 ◦C (Table 1).



Abbreviations: TFC: total flavonoid content. UPA: ultrasonic power amplitude. T: temperature; ET: extraction time; PSMP: perilla seed meal polysaccharides; NI: not included; Dw: dry weight.

#### **3. Conclusions**

UAE is a useful method for obtaining different compounds of interest from plant matrices, since remarkably higher extraction yields are obtained with short extraction times. This extraction method belongs to "Green Chemistry" because it allows to decrease the consumption of resources, such as solvent and energy. However, it is still necessary to optimize the more relevant variables that influence the effectiveness of UAE, such as ultrasound power, extraction time and temperature, type of solvent, and solid-to-solvent ratio. Different studies have shown extraction yields of different bioactive compounds, such as phenolic compounds, polysaccharides, pigments, and amino acids, using UAE with short extraction times (maximum 60 min), medium ultrasound power (between 200–500 W), temperatures around 50 ◦C (maximum 80 ◦C), and environmentally friendly solvents (distilled water and ethanol). Therefore, UAE can be appropriately applied to obtain bioactive compounds through an efficient and eco-friendly process, considering and optimizing the different critical variables that affect the process.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/CSAC2021-10562/s1.

**Author Contributions:** Conceptualization, A.C.-C., A.G.P., M.C., P.O.; F.C., and A.S.-L.; methodology, P.O., and P.G.P.; validation, P.O.; formal analysis, P.O., and P.G.P.; writing—original draft preparation, A.C.-C., P.O., and M.C.; writing—review and editing, A.C.-C., A.S.-L., M.C., P.O., and M.A.P.; visualization, M.C., and A.G.P.; supervision, P.O., H.C., J.X., J.S.-G., and M.A.P.; project administration, M.A.P., and J.S.-G. All authors have read and agreed to the published version of the manuscript.

**Funding:** The JU receives support from the European Union's Horizon 2020 research and innovation program and the Bio Based Industries Consortium. The project SYSTEMIC Knowledge hub on Nutrition and Food Security has received funding from national research funding parties in Belgium (FWO), France (INRA), Germany (BLE), Italy (MIPAAF), Latvia (IZM), Norway (RCN), Portugal (FCT), and Spain (AEI) in a joint action of JPI HDHL, JPI-OCEANS and FACCE-JPI, launched in 2019 under the ERA-NET ERA-HDHL (n◦ 696295).

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data sharing not applicable.

**Acknowledgments:** The research leading to these results was supported (1) by MICINN supporting the Ramón y Cajal grant for M.A. Prieto (RYC-2017-22891) and the FPU grants for A. Carreira-Casais (FPU2016/06135) and A. Soria-Lopez (FPU2020/06140), (2) by Xunta de Galicia for supporting the predoctoral grants of A.G. Pereira (ED481A-2019/0228) and M. Carpena (ED481A 2021/313), and (3) by the program BENEFICIOS DO CONSUMO DAS ESPECIES TINTORERA-(CO-0019-2021) that supports the work of F. Chamorro. The authors are grateful to the Ibero-American Program on Science and Technology (CYTED—AQUA-CIBUS, P317RT0003), and to the Bio Based Industries Joint Undertaking (JU) under grant agreement No 888003 UP4HEALTH Project (H2020-BBI-JTI-2019) that supports the work of P. Otero and P. Garcia-Perez.

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

#### **References**


### *Proceeding Paper* **Plants of the Family Asteraceae: Evaluation of Biological Properties and Identification of Phenolic Compounds †**

**Marta Barral-Martinez <sup>1</sup> , Paula Garcia-Oliveira 1,2 , Bernabe Nuñez-Estevez 1,2, Aurora Silva 1,3 , Tiane C. Finimundy <sup>2</sup> , Ricardo Calhelha 2, Marija Nenadic 4, Marina Sokovic <sup>4</sup> , Fatima Barroso 3, Jesus Simal-Gandara <sup>1</sup> , Isabel C. F. R. Ferreira <sup>2</sup> , Lillian Barros 2,\* and Miguel A. Prieto 1,2,\***


**Abstract:** The present study focused on the biological analysis of five plants: *Achillea millefolium*, *Arnica montana*, *Calendula officinalis*, *Chamaemelum nobile* and *Taraxacum officinale*. The results indicated that *A. montana* extracts showed the highest content of phenolic compounds. Regarding the biological properties, *A. millefolium* had outstanding antioxidant activity, while *C. officinalis* had the highest rate of antimicrobial and antifungal activity. The anti-inflammatory and cytotoxic activities reflected that *C. nobile* showed the highest effect. In enzyme assays, *C. nobile* and *C. officinalis* extracts showed the highest inhibitory effects on acetylcholinesterase and butyrylcholinesterase enzymes. Overall, this study provides scientific evidence for the evaluation of the potential of medicinal plant extracts for the development of new products.

**Keywords:** medicinal plants; beneficial effects; biological properties; phenolic compounds

#### **1. Introduction**

Currently, medicinal plants have great relevance due to their reported beneficial health properties. Many studies reflect that their biological properties, such as antioxidant, antitumor and antimicrobial activities, are related to different bioactive compounds, including phenolic compounds. Although some of their mechanisms of action are unknown, in many cases it has been shown that various natural phenolic compounds are related to bioactive properties, and this has aroused the interest of the scientific community [1]. Several medicinal plants are still used for therapeutic purposes, employed in different formulas (decoctions, infusions, ointments, etc.) but, in general, their use has been reduced. However, these plants can be re-valorized for the recovery of bioactive compounds with applications in the food, cosmetic and pharmaceutical industries [2]. In particular, plants from Asteraceae family are promising candidates, due to their beneficial properties and bioactive compounds.

The present study focused on five medicinal plants from the Asteraceae family, namely, *Achillea millefolium* L., *Arnica montana* L., *Calendula officinalis* L., *Chamaemelum nobile* L. and *Taraxacum officinale* (L.) Weber ex F. H. Wigg., all belonging to the Asteraceae family. These plants have been widely used in traditional medicine for the treatment of various disorders, but their use has been reduced. *A. millefolium*, *T. officinale* and *C. officinalis* are the most

**Citation:** Barral-Martinez, M.; Garcia-Oliveira, P.; Nuñez-Estevez, B.; Silva, A.; Finimundy, T.C.; Calhelha, R.; Nenadic, M.; Sokovic, M.; Barroso, F.; Simal-Gandara, J.; et al. Plants of the Family Asteraceae: Evaluation of Biological Properties and Identification of Phenolic Compounds. *Chem. Proc.* **2021**, *5*, 51. https://doi.org/10.3390/ CSAC2021-10486

Academic Editor: Manel del Valle

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/).

studied of these plants, and some of their terpenoids, flavonoids, phenolic acids and carotenoids have been described as bioactive compounds [3,4]. The bioactive compounds of *C. nobile* have not been studied in depth, although this plant is well is known to be especially beneficial for digestive health. Bioactive compounds identified so far include terpenoids, flavonoids, coumarins and other compounds such as esters of angelic and tyglic acids, among others [5]. However, the main bioactive compounds in *A. montana* have been demonstrated to be the so-called sesquiterpene lactones, which are related to its anti-inflammatory effects [6]. On this basis, the study focused on the determination of phenolic compounds and the evaluation of the biological properties of these plants, to deepen knowledge about the bioactive compounds and evaluate their possible use in future bio-based applications.

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

#### *2.1. Sample Extraction*

The samples were acquired in 2020 from Soria Natural and Pinisan and were received at room temperature, dried and crushed to facilitate and improve the efficiency of the extraction processes. Then, the samples were sieved with a sieve (pore size < 2 mm). The samples were extracted by solid–liquid extraction. A 5 g sample of each species was extracted with 100 mL of methanol–water (60:40 *v*/*v*). Extraction was carried out at 45 ◦C for 3 h. Then, the extracts were freeze-dried using Telstar LyoAlfa 15 equipment to obtain dry extracts that were used in the subsequent analyses.

#### *2.2. Determination of Phenolic Compounds*

The identification of phenolic compounds was carried out using a Dionex UltiMate 3000 UPLC system (Thermo Scientific, San Jose, CA, USA) [7]. The determination was performed using a diode array detector (DAD) and mass spectrometry (MS) (LTQ XL mass spectrometer, Thermo Finnigan, San Jose, CA, USA) working in negative mode. Data acquisition was carried out with an Xcalibur® data system (Thermo Finnigan, San Jose, CA, USA). The phenolic compounds were identified according to their chromatographic characteristics, by their retention, absorption spectra and mass characteristics in comparison to the obtained standard compounds and the literature. For quantitative analysis, calibration curves were prepared with appropriate standards. The results were expressed in mg per g of dry extract. Analyses were performed in triplicate.

#### *2.3. Determination of the Main Biological Properties*

#### 2.3.1. Assessment of Antioxidant Activity

To evaluate the antioxidant activity, the lipid peroxidation inhibition in porcine (*Sus scrofa*) brain homogenates was analyzed, evaluating the decrease in thiobarbituric acid reactive substances (TBARS), as previously described in Pineda et al. [8]. Brain tissue was homogenized in Tris-HCl buffer (20 mM, pH 7.4) and then centrifuged at 3000 *g* for 10 min. An aliquot of the supernatant was incubated with the extracts at different concentrations in the presence of FeSO4 (10 mM) and ascorbic acid (0.1 mM) for 1h at 37 ◦C. Trichloroacetic acid (28%) and thiobarbituric acid (2%) were added to stop the reaction at 80 ◦C, and stirred for 20 min. After centrifugation, the color intensity of the malondialdehyde complex in the supernatant was measured via its absorbance at 532 nm. Using the dose–response values of the results obtained, a parameter that summarized the potential antioxidant effect of each sample was obtained, i.e., the concentration necessary to produce 50% of the antioxidant response (EC50) [7].

#### 2.3.2. Assessment of Antimicrobial Activity

The dried extracts were dissolved in distilled water (10 mg/mL) and the procedure described by Sokovi´c et al. [9] was followed. The activity was studied against threeGramnegative bacteria: *Escherichia coli*, *Salmonella typhimurium* and *Enterobacter cloacae* and three Gram-positive bacteria: *Bacillus cereus*, *Listeria monocytogenes* and methicillin-resistant

*Staphylococcus aureus* (MRSA). For antifungal assays, six micromycetes were tested: *Aspergillus fumigatus* (human isolate), *Aspergillus niger* (ATCC 6275), *Aspergillus versicolor* (ATCC11730), *Penicillium funiculosum* (ATCC 36839), *Trichoderma viride* (IAM 5061) and *Penicillium verrucosum var. cyclopium* (food isolate). The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum fungicidal concentration were determined.

#### 2.3.3. Assessment of Anti-Inflammatory Properties

The dried extracts were dissolved in distilled water (8 mg/mL) and serial dilutions (1–8 mg/mL) were prepared and tested using a RAW 264.7 murine macrophage cell line. Lipopolysaccharide was used to stimulate inflammation and the production of nitric oxide was measured as described previously [10]. The results obtained were expressed as EC50 values (μg/mL) and dexamethasone was used as a positive control.

#### 2.3.4. Cytotoxic Properties

Cytotoxicity was assessed using four tumor cell lines: AGS (human gastric adenocarcinoma cell line), CaCo (Caucasian colon adenocarcinoma), MCF-7 (break adenocarcinoma cell line), NCI- H460 (lung cancer). The Vero cell line was used as a control. Cytotoxic activity was measured using the sulforhodamine B assay [11]. The results obtained were expressed as GI50 values, i.e., the concentration of extract that inhibited 50% of net cell growth, and ellipticin was used as a positive control.

#### 2.3.5. Enzymatic Activity

A previously developed colorimetric method was used [12]. This consists of detecting the inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activity via the increase in yellow coloring due to the production of thiocholine. These two enzymes have been reported to be involved in neurological disorders. In addition, inhibition of AChe has been recognized as a possible avenue for the symptomatic treatment of Alzheimer's disease [13]. The assay was carried out using three buffers: A with 50 mM Tris–HCl, pH 8; B with 50 mM Tris–HCl, pH 8, 0.1% BSA and C with 50 mM Tris–HCl, pH 8, 0.1 M NaCl and 0.02 M MgCl2. The inhibitory capacity of the extracts was tested at concentrations of 1 and 2 mg/mL.

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

The phenolic profile of the selected plants showed great variability, both in quantity and in the identified phenolic compounds (Table 1; Figure 1). The plant with the highest content of phenolic compounds was *A. montana*, with a concentration of 119 mg/mL, where the most representative compound was 5-*O*-caffeolyquinic acid. The extracts of *C. nobile* presented a total phenolic content of 100 mg/mL and, in this case, the major compound was the flavonoid luteolin-*O*-pentosylhexoside. *A. millefolium* extracts achieved a total phenolic content of 81 mg/mL, and the most representative compound was 3-*O*-caffeoylquinic acid. *T. officinale* extracts had a phenolic content of 18 mg/mL and were rich in 3-*O*-caffeoylquinic acid. Finally, *C. officinalis* extracts had the lowest phenolic content, at 14.1 mg/mL, with 3-*O*-caffeoylquinic acid as the major compound. These results coincide with those of other studies, e.g., in the case of the plant *A. montana*, where 5-*O*-caffeoylquinic acid has also previously been reported as the main phenolic acid in the ethanolic extract of this plant [14]. However, it should be noted that the content of phenolic acids can be influenced by external factors such as the type of solvent used in the extraction process, and it is also related to the growing conditions of the plants, as mentioned in previous studies on *A. montana* and other species belonging to the Asteraceae family [15].


**Table 1.** Total phenolic content and main phenolic compounds identified (mg/mL).

**Figure 1.** Representative chromatogram of the phenolic compounds identified: (**A**) *A. millefolium*; (**B**) *A. montana*; (**C**) *C. officinalis*; (**D**) *C. nobile*; (**E**) *T. officinale*.

Regarding antioxidant activity, the extracts of *A. millefolium* showed exceptional activity, with an EC50 value of 0.013 mg/mL. The extracts of *A. montana*, *C. nobile* and *C. officinalis* showed similar EC50 values (0.2, 0.2 and 0.25 mg/mL, respectively). Finally, the extracts of *T. officinale* showed the lowest antioxidant activity with an EC50 of 0.035 mg/mL. These results are presented in Figure 2. In previous studies, this assay has been employed to evaluate the antioxidant activity of *A. millefolium*, *C. officinalis* and *C. nobile*, reporting significant results. To the best of our knowledge, no study has used the TBARS assay to evaluate *A. montana* and *T. officinale*, but their antioxidant properties have been corroborated by various studies showing positive results [16–18].

**Figure 2.** Antioxidant activity in traditional plants of the family Asteraceae: *A. millefolium*, *A. montana*, *C. officinalis*, *C. nobile* and *T. officinale*.

Regarding antimicrobial activity, all the plant extracts displayed significant antimicrobial effects, with *C. officinalis* being the most remarkable. This plant presented MIC values ranging from 0.25 to 0.5 mg/mL for all the tested bacteria and fungi. MBC and MFC values ranged between 0.5 and 1 mg/mL. The most susceptible bacteria were the Gram-positive species, while *T. viride* was the most susceptible fungus. *T. officinale* also showed relevant antibacterial potential, while *C. nobile* was also effective against fungi species. The antimicrobial potential of these species has been previously confirmed. Focusing on *C. officinalis*, a study reported that petal extracts of this plant showed comparable antibacterial effects against Gram-positive and Gram-negative bacteria using the disk diffusion method [19]. The results found in the literature are very similar to those obtained experimentally and therefore corroborate the hypothesis that the *C. officinalis* plant could be used as a possible source of antimicrobial compounds.

According to the results (Figure 3), *C. nobile* extracts showed the greatest effects in both assays, with EC50 values of 15.21 μg/mL for anti-inflammatory activity and GI50 values between 54 and 10.3 μg/mL in the case of cytotoxic activity. *A. millefolium* also showed significant results, with an EI50 of 30 μg/mL for anti-inflammatory activity and GI50 values ranging between 42 and 125 μg/mL. Considering the *C. nobile* results, the anti-inflammatory and the cytotoxic properties of this plant have been reported previously, showing positive results [20,21], and therefore this plant could be a promising source of anti-inflammatory and cytotoxic extracts.

**Figure 3.** (**A**) Anti-inflammatory and (**B**) cytotoxic activity of *A. millefolium*, *A. montana*, *C. officinalis*, *C. nobile* and *T. officinale*.

Finally, *C. nobile* showed the highest inhibitory effects on AChE activity for the extract concentrations tested (1 and 2 mg/mL), causing an inhibition of >35% and >60%, respectively. In the case of the BuChE enzyme, *C. officinalis* caused an inhibition of >50% in both concentrations tested. *C. nobile* also showed a remarkable inhibitory effect against this enzyme, with an inhibition of >40% at 2 mg/mL and >20% at 1 mg/mL. To the best of our knowledge, no previous studies have evaluated the enzymatic activity of the selected plants.

#### **4. Conclusions**

All the plants studied had diverse phenolic compositions and biological activities. Regarding phenolic compounds, *A. montana* extracts showed the highest content. Regarding bioactivities, *A. millefolium* showed high antioxidant activity and *C. officinalis* showed the best antimicrobial and antifungal activities. In the case of anti-inflammatory and cytotoxic activities, *C. nobile* extracts achieved the best results. Finally in the enzyme assays, both *C. nobile* and *C. officinalis* extracts showed the highest inhibitory effects. Therefore, this study provides scientific evidence of the potential of medicinal plants as a source of extracts and bioactive compounds that may be considered for the development of new products.

**Supplementary Materials:** The poster presentation is available online at: https://www.mdpi.com/ article/10.3390/CSAC2021-10486/s1.

**Author Contributions:** Conceptualization, M.B.-M., P.G.-O., L.B. and M.A.P.; methodology, M.B.- M., B.N.-E., A.S., F.B., M.N., M.S., T.C.F., R.C. and I.C.F.R.F.; software, M.B.-M., B.N.-E. and A.S.; validation, F.B., I.C.F.R.F., M.A.P. and L.B.; formal analysis, A.S., F.B., M.N., M.S., T.C.F., R.C. and I.C.F.R.F.; investigation, M.B.-M., P.G.-O., A.S. and F.B.; writing—original draft preparation, M.B.-M. and P.G.-O.; writing—review and editing, M.B.-M. and P.G.-O.; visualization, P.G.-O., T.C.F. and L.B.; supervision, M.A.P., L.B. and J.S.-G. All authors have read and agreed to the published version of the manuscript.

**Funding:** The JU receives support from the European Union's Horizon 2020 research and innovation program and the Bio Based Industries Consortium. The project SYSTEMIC Knowledge hub on Nutrition and Food Security has received funding from national research funding parties in Belgium (FWO), France (INRA), Germany (BLE), Italy (MIPAAF), Latvia (IZM), Norway (RCN), Portugal (FCT) and Spain (AEI) in a joint action of JPI HDHL, JPI-OCEANS and FACCE-JPI launched in 2019 under the ERA-NET ERA-HDHL (No. 696295).

**Acknowledgments:** The research leading to these results was supported by MICINN by supporting the Ramón y Cajal grant for M.A. Prieto (RYC-2017-22891); by Xunta de Galicia by supporting the program EXCELENCIA-ED431F 2020/12 and the pre-doctoral grant of P. García-Oliveira (ED481A-43 2019/295); by the EcoChestnut Project (Erasmus+ KA202) that supports the work of B. Nuñez-Estevez; by the program Grupos de Referencia Competitiva (GRUPO AA1-GRC 2018) that supports the work M. Barral-Martínez; by the Bio Based Industries Joint Undertaking (JU) under grant agreement No 888003; by the UP4HEALTH Project (H2020-BBI-JTI-2019) that supports the work of P. Garcia-Perez and by the Ibero-American Program on Science and Technology (CYTED—AQUA-CIBUS, P317RT0003). The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to CIMO (UIDB/00690/2020); and to the national funding by FCT, P.I., through the institutional scientific employment program contract for L. Barros.

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

#### **References**

