**Hydroponics Monitoring through UV-Vis Spectroscopy and Artificial Intelligence: Quantification of Nitrogen, Phosphorous and Potassium †**

**Aníbal Filipe Silva 1,2 , Klara Löfkvist 3, Mikael Gilbertsson 4, Erik Van Os 5, Geert Franken 5, Jos Balendonck 5, Tatiana M. Pinho 6, José Boaventura-Cunha 6,7 , Luís Coelho <sup>2</sup> , Pedro Jorge 1,2 and Rui Costa Martins 2,\***


**Abstract:** In hydroponic cultivation, monitoring and quantification of nutrients is of paramount importance. Precision agriculture has an urgent need for measuring fertilization and plant nutrient uptake. Reliable, robust and accurate sensors for measuring nitrogen (N), phosphorus (P) and potassium (K) are regarded as critical in this process. It is vital to understand nutrients' interference; thusly, a Hoagland fertilizer solution-based orthogonal experimental design was deployed. Concentration ranges were varied in a target analyte-independent style, as follows: [N] = [103.17–554.85] ppm; [P] = [15.06–515.35] ppm; [K] = [113.78–516.45] ppm, by dilution from individual stock solutions. Quantitative results for N and K, and qualitative results for P were obtained.

**Keywords:** nitrogen; phosphorus; potassium; Hoagland; nutrient; spectrophotometry; interferences

#### **1. Introduction**

Fertilizer usage represents an important part of traditional agriculture and crop yield. In a world of growing food (and other agricultural products) demand—estimates indicate up to 50% increase in the 2012–2050 time frame [1]—fertilizer (ab)use is seldomly a go-to solution for crop yield increase. Additionally, although growth rates for arable land are expected to increase within a sustainable manner, if an arable land loss scenario due to climate changing conditions is taken into account [2], further conflicts and competition might arise between protected lands, agricultural exploitation and human expansion.

Considering these concerns—well reflected by the United Nation's 2030 Agenda for Sustainable Development [3]—and also motivated to provide a solution for sustainable agriculture, our group has undertaken the task to develop a technology that is able to help farmers ensure that their crops' needs are being met, through their fertilization procedures. Knowing what is being fed to the crops and what is being taken up, it is possible to reduce water/fertilizer consumption to an optimal level, reducing the operational costs, whilst allowing crops to develop at their optimal speed, towards a bigger crop turnover.

Spectroscopy is, among others, one of the most well-established techniques for chemical identification and quantification. Several chemical determination methodologies that

**Citation:** Silva, A.F.; Löfkvist, K.; Gilbertsson, M.; Os, E.V.; Franken, G.; Balendonck, J.; Pinho, T.M.; Boaventura-Cunha, J.; Coelho, L.; Jorge, P.; et al. Hydroponics Monitoring through UV-Vis Spectroscopy and Artificial Intelligence: Quantification of Nitrogen, Phosphorous and Potassium. *Chem. Proc.* **2021**, *5*, 88. https://doi.org/10.3390/ CSAC2021-10448

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/).

rely on spectroscopy exist (e.g., ICP-AES, LIBS, FTIR, GC-VUV); nevertheless, the following limitations also exist: the sample must be responsive to electromagnetic radiation (absorption/emission); linearity outside the Beer–Bouguer–Lambert Law [4–6] can sometimes be problematic, or the simple fact that spectroscopy is a molecular-level information tool, which can add entropy to the analysis by providing a wider range of information than the one desired. If pure compounds are analysed, little or no interference exists; when more complex mixtures are targeted, interferences might play a key role on the successful outcome. In such cases, in order to obtain an accurate and reliable measurement, interferences have to be taken into account. Chemometrics presents itself as a putative solution to, by employing varying complexity mathematical calculations, together with statistics and algorithms, allow the extraction of relevant information from the superimposed and sometimes—latent data. Linearity-based models are unable to solve the interference pattern between any constituents present; this is the case for either interferents and non-interferents (target analytes), due to the fact that light has a wave-like nature and, hence, the sample information might suffer from constructive or destructive interferences [7]. Nevertheless, new chemometrics methodologies that encompass interferences already allow critical developments to be achieved, e.g., on health-related point-of-care analysis [8].

In hydroponics, most of these interferences can be attributed to the fertilizers. Fertilizers are mixtures of several different nutrients, mostly in their inorganic salt form (e.g., MgSO4, CaCO3, FeCl3) whilst some might be in aqueous solutions (e.g., Mo, Ba, B). In complex mixtures, some signals might superimpose over others, causing a concentration misevaluation, or resulting in a continuous spectrum of overlapping signals.

This study aims to provide insight on the interferences within a complex matrix orthogonal design consisting of 83 independent concentration Hoagland solution samples. The performed assay further complements on our previous findings [9] on the feasibility of information extraction of highly constrained samples, by using an advanced algorithm self-learning artificial intelligence (SLAI)—in order to find the adequate co-variance modes for accurate model prediction.

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

Hoagland solutions were chosen as a matrix due to their widely accepted status among the agronomical community as being a good model for complex nutrient solutions in hydroponics. Stock Hoagland solutions are composed as described by Table 1.


**Table 1.** Half-strength Hoagland solution individual component concentration.


**Table 1.** *Cont.*

The ratio of ionic forms depends on \* the pH and \*\* also on the O2 level within the nutrient solution.

Three stock solutions for N, P and K were freshly and individually prepared in order for it to be possible to vary each target element (N, P or K) independently. Each stock solution comprised of ionic elements already present in the base matrix, as follows: N— NaNO3/NH3; P—H3PO4; K—KCl. The ratio of NaNO3/NH3 of the stock spiking solutions was the same as the ratio on the Hoagland solution (≈93:7).

Final concentrations of all samples (matrix + individual spikes) were corrected taking into account any variations derived from the preparation of fresh stock solutions each day during the execution of the assay.

The tested final concentration ranges, for the target analytes, were as described in Table 2.



The designed orthogonal matrix was composed by 8<sup>3</sup> samples, each one with an independent N, P and K concentration level. At each corresponding level, the N, P and K corresponding spike was added to the matrix and stirred for 10 s in order to attain full homogeneity. Afterwards, the pH value was registered (Crison GLP 21, Crison Instruments, SA, Barcelona, Spain) and the sample pumped into a custom-built flow cell for spectral data acquisition. Each sample had a final total volume of 30 mL (Hoagland + [N+P+ K] spikes). After data acquisition, the sample was discarded and the system flushed with deionised water.

Data acquisition was performed with an in-house LabView-based developed software (National InstrumentsTM Corp., Austin, TX, USA) for pumping control and data acquisition.

Sample irradiation was performed with a D2 light source (FiberLight® D2 HighPower DTM 10/50S, Heraeus Noblelight GmbH, Hanau, Germany) whereas the detection was performed by a miniaturized spectrometer with a 190–650 nm range (STS-UV-L-50-400, Ocean Insight, Inc., Orlando, FL, USA). Individual components were assembled with customlength 600 nm UV-Vis optical fibres (in-house customization), as depicted in Figure 1.

**Figure 1.** Scheme for individual components arrangement, within the developed prototype.

#### **3. Results**

The obtained results from the execution of this matrix were compiled and are depicted on Figure 2.

The collection of spectral data and cross correlation with the concentration information for each solution was performed. Spectroscopy signals were processed accordingly to [7]. Nevertheless, using advanced signal processing it is possible to train the system to recognize and extract the information from the relevant features, incorporating multi-scale interference into the NPK quantification models.

**Figure 2.** Compiled spectra (83 samples) without (**a**) and with scatter correction (**b**).

The correlation of the different levels among the NPK nutrients of the matrix design, can be represented as displayed by Figure 3a whereas Figure 3b shows the corresponding recorded spectra in the UV-Vis region (*circa* 200–650 nm) of the factorial design samples. As expected, most of the systematic spectral variation occurs at ≈250 to 450 nm, and, to a lesser extent, to 500 nm. This figure provides evidence that information about P and K is present, because, even to the naked eye, one can observe that there are more spectral patterns in the region of ≈250 to 450 nm than the expected nitrogen levels of the experimental design; that is a good indication that the interferences between all the constituents are being registered on the spectra.

**Figure 3.** Sample distribution of the NPK full factorial design (**a**), whole matrix data (with scatter correction) of the relevant wavelengths of the obtained spectra, where most of the relevant NPKbearing information is contained (**b**).

The principal component analysis (PCA) (please refer to Figure 4a) scores plot of the corresponding experimental design spectra is shown, where the different colours represent the different levels of total nitrogen. The main variance present in the spectra corresponds to the nitrogen absorbance, where the first principal component is highly correlated to the nitrogen content. It is also possible to see that the K-level information is embedded inside each N-concentration level. Analysis of the second component allows to unveil that information of K-level also carries the information of the different P-levels of the sample matrix (please refer to Figure 4b).

**Figure 4.** Principal components analysis (**a**) and the information of K-levels within the N-levels (**b**, left-hand side) whereas (**b**, right-hand side) demonstrates the information of P embedded within K-level groups.

Using the data obtained from the executed matrix, it was possible to train the selflearning AI of the system in order to quantify N and K with 6.7% (0.997) and 3.8% (0.987), respectively, and to obtain qualitative results for P, as shown in Figure 5.

**Figure 5.** Matrix results for total N (**a**), total P (**b**), total K (**c**) and also pH (**d**). Quantitative results for N and K, whereas qualitative results for P are possible to be inferred.

#### **4. Conclusions**

A NPK spectroscopy-based, AI-supported by a robust self-learning artificial intelligence was developed in order to be able to cope with increasing interference complexity of fertilizer solutions in greenhouses. The obtained results allow to be inferred that the current system's performance is adequate for Hoagland solutions, which are used in research and high-end hydroponic systems.

The assembled system aimed to keep a good balance between cost–benefit, without relinquishing reliability, robustness and accuracy; this objective has been successfully attained.

Further analysis of the results—not within the scope of this manuscript—as well as of unpublished data, allows further developments to be implemented to the system/prototype, in order to enhance its robustness and accuracy.

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

**Funding:** This work was funded by the ERA-NET Cofund WaterWorks2015 Call, within the frame of the collaborative international consortium AGRINUPES. This ERA-NET is an integral part of the 2016 Joint Activities developed by the Water Challenges for a Changing World Joint Programme Initiative (Water JPI/002/2015). R.M. acknowledges Fundação para a Ciência e Tecnologia (FCT) research contract grant (CEEIND/017801/2018). A.F.S. gratefully acknowledges the financial support provided by FCT (Portugal's Foundation for Science and Technology) within grant (DFA/BD/9136/2020).

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

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

#### **References**


### *Abstract* **Detection of Indoor Air Pollutants Using Reactive Sputtering/GLAD of Tin Oxide Thin Films †**

**Achraf El Mohajir 1, Jean-Baptiste Sanchez 1,\*, Mohammad Arab Pour Yazdi 1, Olivier Heintz <sup>2</sup> and Nicolas Martin <sup>1</sup>**


**Abstract:** Indoor air quality is a topic of major importance for public health. Among the numerous chemical compounds that can be found in indoor air, BTEX (i.e., benzene, toluene, ethylbenzene, and xylene) is considered one of the most toxic volatile organic compounds (VOCs). The present contribution is focused on the use of an original approach to produce nanostructured materials based on tin oxide with unexplored features, especially for gas sensors. In this work, we combine two physical vapor deposition techniques based, first, on a pulsing injection of the reactive gas during the deposition and second focused on the glancing angle deposition (GLAD) technique, which enables the structuring of various architectures. These active layers are deposited on a micro-hotplate to produce micro-chemical gas sensors for the detection of BTEX. Here, we have demonstrated the utility of using the GLAD deposition technique and the role of sputtering pressure in obtaining porous sensitive thin films. In particular, we established relationships between deposition parameters and gas sensing performances.

**Keywords:** BTEX gas sensor; GLAD method; sensitivity; porous surface

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/CSAC2021-10548/s1.

**Citation:** El Mohajir, A.; Sanchez, J.-B.; Arab Pour Yazdi, M.; Heintz, O.; Martin, N. Detection of Indoor Air Pollutants Using Reactive Sputtering/GLAD of Tin Oxide Thin Films. *Chem. Proc.* **2021**, *5*, 89. https://doi.org/10.3390/ CSAC2021-10548

Academic Editor: Elisabetta Comini

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/).

### *Proceeding Paper* **Influence of the Type and Amount of Plasticizer on the Sensory Properties of Microspheres Sensitive to Lipophilic Ions †**

**Aleksandra Kalinowska, Patrycja Matusiak, Sandra Skorupska, Ilona Grabowska-Jadach and Patrycja Ciosek-Skibi ´nska \***

> Chair of Medical Biotechnology, Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland; aleksandra.kalinowska3.dokt@pw.edu.pl (A.K.); patrycja.matusiak08@gmail.com (P.M.); sskorupska@ch.pw.edu.pl (S.S.); ilona@ch.pw.edu.pl (I.G.-J.)

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

**Abstract:** Working parameters of chemical sensors, such as selectivity and sensitivity, can be adjusted by optimizing components of chemosensitive layers, including type and amount of plasticizer in the case of PVC membranes in optodes. Plasticizers are also used in the process of creating micro/nanospheres that are incorporated with chemical indicators to form micro/nano-scale optodes. This study investigated the influence of the type of plasticizer (polar o-NPOE and non-polar DOS) on the optical response of microspheres that are sensitive to lipophilic ions. Moreover, the amount of plasticizer was also adjusted in order to obtain satisfactory sensitivity in the widest linear range. The chemosensory response of the developed microspheres was studied with the use of spectrophotometry and spectrofluorimetry, while size of the optodes was estimated by confocal microscopy.

**Keywords:** plasticizer; microspheres; optode; chemosensors

**Citation:** Kalinowska, A.; Matusiak, P.; Skorupska, S.; Grabowska-Jadach, I.; Ciosek-Skibi ´nska, P. Influence of the Type and Amount of Plasticizer on the Sensory Properties of Microspheres Sensitive to Lipophilic Ions. *Chem. Proc.* **2021**, *5*, 90. https:// doi.org/10.3390/CSAC2021-10487

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/).

#### **1. Introduction**

For many years, ion-selective electrodes (ISE) have been considered important analytical chemistry tools. They are widely used both in environmental analysis and in medical diagnostics [1]. Chemosensitive membranes of the ISEs whose signal is potentiometric can be easily modified by the incorporation of chromoionophores, which gives the possibility for optical transduction. On the other hand, polymer scaffolds of such optode membrane can be replaced by surfactants, which leads to the fabrication of suspension of micro- or nano-sized optodes.

Various achievements in the field of micellization of dyes can be applied for the fabrication of micro- and nano-optodes. Micelles are micro- or nanometer-sized particles that exist solution in equilibrium with the particles or ions. They can be incorporated with additional lipophilic components forming nano/microspheres, aggregates of surfactant molecules with a hydrophilic part being in contact with outer water solution, and a hydrophobic part creating inner lipophilic microenvironment [2]. Micelles are widely used in medicine for drug delivery and increase water solubility and pharmaceuticals' bioavailability. In recent years, the huge potential of micellar systems for various applications in analytical chemistry has been noticed [3].

Working parameters of chemical sensors, such as selectivity and sensitivity, can be adjusted by optimizing components of chemosensitive membranes, including type and amount of plasticizer [4]. A plasticizer is a substance allowing for increasing polymer flexibility, as well as susceptibility to its further processing. In membranes of ISEs and optodes, it forms a lipophilic environment in which receptors and additional components responsible for sensing are suspended. A plasticizer in the simplest sense is considered an

**<sup>\*</sup>** Correspondence: pciosek@ch.pw.edu.pl

organic solvent. There are many types of such compounds, such as animal fats, petroleum fractions, and all kinds of plant extracts. Plasticizers can be used in the process of creating micro/nanospheres incorporated with chemical indicators and stabilized by surfactants. As environmental conditions change, such systems may change their optical properties, becoming optodes in the micro/nanoscale [3,5].

This study aimed to develop two microsphere types sensitive towards lipophilic ions: anion-sensitive (AS) and cation-sensitive (CS). Each of these systems includes a chromoionophore, an ion exchanger, a surfactant, and a plasticizer. In this work we were focused on selecting an appropriate plasticizer, as well as adjusting its quantity. Changes in chemosensory properties were observed in absorbance and fluorescence mode, while confocal microscopy observations were applied to verify the microspheres' size.

#### **2. Experimental**

#### *2.1. Chemicals*

Sodium phosphate monohydrate, disodium phosphate dodecahydrate, Tris-HCl, Pluronic F127 were supplied by Sigma-Merck (Pozna ´n, Poland). Milli-Q water was used for preparation of all aqueous solutions, including phosphate buffer pH 7.4 and Tris-HCl buffer pH 9.0. Plasticizers (2-Nitrophenyl octyl ether, o-NPOE, Bis(2-ethylhexyl) sebacate, DOS), lipophilic salts (Tridodecylmethylammonium chloride, TDMAC, Potassium tetrakis [3,5-bis(trifluoromethyl)phenyl]borate, KTFPB), chromoionophores I and XI were obtained from Fluka (Selectophore). Freshly distilled tetrahydrofuran, THF (Fluka) was used as a solvent for the microspheres' components. All chemicals were used as received.

#### *2.2. Preparation of Microspheres Suspensions*

Two types of optode microspheres: with generic anion-sensitivity (AS) and with generic cation-sensitivity (CS), were prepared (Figure 1). The composition of the membranes and proposed mechanism of optical signal generation are based on the literature data on optodes [6–10]. AS microspheres contained Chromoionophore XI, TDMAC, o-NPOE or DOS, and Pluronic (F-127), whereas CS microspheres contained Chromoionophore I, KTFPB, o-NPOE or DOS, and Pluronic (F-127). Each of the components included in the given microsphere was weighed and dissolved in 1.5 mL of THF. To ensure that all ingredients dissolve well, the vial was placed in an ultrasonic bath for 5 min. 0.5 mL portions of THF solutions were pipetted to 4.5 mL of deionized water on a vortex. The last step was to remove the solvent by passing compressed air through the solution (the process was carried out for 1 h). Clear particle suspensions were obtained that were applied for further measurements using microtiter plates. For this purpose, 100 μL of the prepared microsphere suspension were pipetted to each well and 100 μL of appropriate analyte solution was added. We examined the chemosensory response in the presence of 0.1 M NaOH, 0.1 M HCl, and calibration solutions appropriate for each type of microspheres (1 μM-0.1 M NaClO4 and 1 μM-0.1 M NH4NO3 for AS and CS optodes, respectively). When protonation degree in AS was determined, microspheres suspensions were two times diluted (50 μL of optode cocktail + 50 μL of deionized water) to avoid recording signals out of range by microtiter plate reader.

**Figure 1.** Schematic representation of the prepared microspheres, their composition, and the mechanisms of target analyte recognition with optical signal generation. The mechanisms by which the spheres operate are based on the extraction of the analyte from the aqueous medium (aq subscript) into the lipophilic interior of the microsphere (m subscript). Each type of microsphere solution is based on a plasticizer emulsion stabilized with a Pluronic non-ionic surfactant having hydrophobic and hydrophilic domains (PPO and PEO, respectively). The plasticizer droplets contain all components needed to extract target analytes (lipophilic salt) and generate a signal (IND) [6–10].

#### *2.3. Examination of the Optical Properties of Microspheres*

Both spectrophotometric and spectrofluorimetric measurements were applied to study the chemosensory properties of the obtained microspheres. They were tested using Synergy 2 Multi-Mode Reader (BioTek Instruments, Inc., Winooski, VT, USA). In order to examine the sizes of the obtained microspheres, a confocal microscope Fluoview FV10i (Olympus, Japanese) was used. The following parameters were used to observe the samples: λex = 473 nm, λem = 490–590 nm for AS microspheres and λex = 635 nm, λem = 660–760 nm for CS microspheres. Measurements of microspheres were made using the FV10i-SW software. Samples were observed using CellviewTM Cell Culture Dish (Greiner Bio One, Germany) with a glass bottom and four-compartments. Three. Hundred microliters of suspension of microspheres were placed in each compartment.

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

#### *3.1. Spectrophotometric Measurements of Chemosensory Properties*

The microspheres prepared for this work, both AS and CS, contain an ion exchanger in their composition, whose task is to exchange ions between the aqueous and organic phases according to equations given in Figure 1 (exchange of lipophilic cations to protons in the case of CS microspheres, coextraction of protons with lipophilic anions in the case of AS microspheres). This processes ensure the change in the protonation degree of the chromoionophores and thus cause a change in the optical properties of the produced system. The sensory response of four independent replicates of microspheres suspensions was tested by recording UV-Vis spectra in the presence of model lipophilic ions—perchlorate ions in the case of AS system, and ammonium ions in the case of CS system (Figures 2 and 3, respectively). The tested solutions were buffered to make the change of the chromoionophore spectrum independent of pH, in order to ensure, that the spectral change was influenced only by the change of the target ion concentration. The spectra were gradually changing following change in ion concentration and thus protonation degree of chromoionophore (represented by arrows in Figures 2a and 3a). Calibration curves were determined for the obtained UV-Vis spectra Figures 2b and 3b). First, signal based on baseline correction was calculated (ratio of absorbances obtained for 454 nm and 380 nm, K = A454/A380, and then only change related to this signal in smallest concentration was presented (K − KC=1μM) for more precise comparison of the two systems using various plasticizers (Figure 2b). The obtained results clearly show that in the AS optode system, the non-polar plasticizer DOS allowed for obtaining responses with a greater degree of sensitivity and enables the determination of anions in almost the entire concentration range.

**Figure 2.** Sensory properties of AS microspheres: (**a**) Absorbance spectrum of microspheres with various plasticizers obtained in the presence of 0.1 M NaOH (deprotonated form of chromoionophore), in 0.1 M HCl (protonated form of chromoionophore), in 0.01 M PBS pH = 7.4 (control sample) and when NaClO4 was added (calibration solutions in concentration range 1 μM–0.1 M); (**b**) calibration curves with signal based on change of baseline correction for K = A454/A380; (**c**) signals stated as change in the protonation degree of chromoionophore for microspheres with various amount of DOS. All calibration solutions were buffered with 0.01 M phosphate buffer pH 7.4. Points of calibration curves were determined as mean ± SD; *n* = 4.

**Figure 3.** Spectrophotometric sensory properties of CS microspheres: (**a**) Absorbance spectrum of microspheres with various plasticizers obtained in the presence of 0.1 M NaOH (deprotonated form of chromoionophore), in 0.1 M HCl (protonated form of chromoionophore), in 0.01M Tris-HCl pH = 9 (control sample) and when NH4NO3 was added (calibration solutions in concentration range 1 μM–0.1 M); (**b**) Calibration curves with signal based on change of baseline correction for K = A610/A570; (**c**) signals stated as change in the protonation degree of chromoionophore for microspheres with various amount of DOS. All calibration solutions were buffered with 0.01 M Tris-HCl buffer pH 9.0. Points of calibration curves were determined as mean ± SD; *n* = 4.

In the case of CS microspheres, baseline correction was calculated using absorbances at 610 nm and 570 nm, and resulting signal was the change of K = A454/A380 value (Figure 3b). For CS microspheres based on DOS and o-NPOE, there was no significant difference in calibration curves—they were comparable. However, in the case of DOS-based optodes, a slightly more comprehensive linear range was observed.

Thus, a non-polar plasticizer DOS was chosen for further experiments, as the better for both microsphere types. The next phase of the research involved the study of the influence of plasticizer amount on chemosensory properties. Three formulations for AS optodes were prepared that was based on various plasticizer content. Again, UV-Vis spectra were recorded as previously, and calibration curves were determined (Figure 2c). Signals were presented as change in protonation degree, 1-α, of chromoionophore (change of 1-α related to this value obtained for the smallest concentration of the analyte, for clarity of presentation). The observation of these calibration curves revealed that the different amount of plasticizer did not significantly affect the sensory response of the system. The microspheres without plasticizers and with its five-fold amount exhibited slightly better sensitivity compared to the system with the standard amount of plasticizer. Additionally, without the addition of DOS, slightly better quantification range was noticed (10 μM–0.1 M). The narrowest linear range was observed for optode system with the highest amount of DOS. The same procedure was repeated for CS microsphere optodes. Respective calibration curves for three studied systems with various amounts of plasticizer are presented in Figure 3c. The best results in terms of sensitivity and quantification range were obtained for the standard amount of the plasticizer.

#### *3.2. Spectrofluorimetric Measurements of Chemosensory Properties*

In addition to measuring the absorbance, chemosensory properties were also tested in the fluorescence mode. Similarly, as in the case of absorbance, a gradual change in the spectrum of AS and CS microspheres, influenced by the growing concentration of target ions was observed (respective emission spectra and calibration curves presented in Figure 4). In the case of both AS and CS microspheres, DOS was recognized as the better plasticizer, which enables the determination of analytes in a wider concentration range

with high sensitivity. Observation of protonation degree (data not shown) revealed that the widest linear range was obtained when the standard amount of DOS was applied, while its five-fold amount caused increase in the sensitivity.

**Figure 4.** Spectrofluorimetric sensory properties of AS (a, b) and CS (**c**,**d**) microspheres with various plasticizers. Calibration curves (**b**,**d**) obtained on the basis of fluorescence emission spectra (**a**,**c**). All calibration solutions were buffered (0.01 M phosphate buffer pH 7.4 for AS microspheres, 0.01 M Tris-HCl buffer pH 9.0 for CS microspheres). Points of calibration curves were determined as means; *n* = 4.

#### *3.3. Confocal Microscope*

Figures 5 and 6 show pictures of microspheres taken with the use of a confocal microscope. The analyzes confirmed the formation of spherical microspheres in all experimental samples regardless of the amount of plasticizer added. A slight spread of the size of the microspheres was observed. Their diameters oscillated between 2–10 μm in the case of both types of microspheres.

**Figure 5.** Confocal microscope pictures of AS microspheres: (**a**) without plasticizer; (**b**) with a standard amount of plasticizer; (**c**) with its five-fold amount.

Based on the observation of the fluorescence intensity of the population of microspheres, it was found that they are homogeneous in terms of fluorescence intensity, which proves the incorporation of the chromoionophore. Moreover, the chromoionophores are evenly distributed in both AS and CS microspheres.

**Figure 6.** Confocal microscope pictures of CS microspheres: (**a**) without plasticizer; (**b**) with a standard amount of plasticizer; (**c**) with its five-fold amount.

#### **4. Conclusions**

The presented results show that in the case of both AS and CS microspheres used in this work, the type and amount of plasticizer change chemosensory properties of the micro-optodes. For both types of microspheres, non-polar plasticizer DOS allowed for obtaining better results compared to the polar plasticizer in the composition. Thanks to DOS, it was possible to determine perchlorate and ammonium ions in a wide concentration range and the obtained sensitivity was higher compared to microspheres with o-NPOE content. The plasticizer amount should also be carefully adjusted to obtain satisfactory chemosensory characteristics. Generally, in the case of microspheres studied in this work, the standard amount led to the best results. All these observations were confirmed both in spectrophotometric and spectrofluorimetric measurements. The obtained results from the confocal microscope confirm the formation of spherical microspheres in size range of 2–10 μm. No influence of the amount of added plasticizer on the shape and size of the microspheres was observed. In summary, the use of an appropriate amount of a non-polar plasticizer allows for obtaining chemosensory microspheres sensitive to lipophilic ions in a broad quantification range from 10 μM to 0.1 M in both absorbance and fluorescence intensity mode.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/CSAC2021-10487/s1.

**Author Contributions:** Conceptualization, P.C.-S. and I.G.-J.; methodology, design of experiments, A.K. and S.S; investigation, P.M., A.K. and S.S.; Original draft preparation, A.K. and S.S.; resources, supervision, project administration, funding acquisition, review and editing of the manuscript were completed by P.C.-S. All authors have read and agreed to the published version of the manuscript.

**Funding:** National Science Centre (Poland): UMO-2018/30/E/ST4/00481.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** This work was financially supported by National Science Centre (Poland) within the framework of the SONATA BIS project No. UMO-2018/30/E/ST4/00481. Aleksandra Kalinowska acknowledges financial support from IDUB project (Scholarship Plus program).

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

#### **References**

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### *Abstract* **Intrinsically Coloured Red Aromatic Polyamides †**

**Patricia Peredo-Guzmán \* , Miriam Trigo-López \* , Saúl Vallejos \* , Félix García \* and José Miguel García \***

Chemistry Department, Faculty of Science, Burgos University, Plaza Misael Bañuelos s/n. 09001 Burgos, Spain **\*** Correspondence: pdperedo@ubu.es (P.P.-G.); mtrigo@ubu.es (M.T.-L.); svallejos@ubu.es (S.V.);


**Abstract:** Aromatic polyamides or aramids are materials with exceptional thermal and mechanical properties. For this reason, they are considered high-performance materials with many applications in fields such as civil security (bullet-proof body armour or fire, chemical, and saw protection suits), transport (automotive and aerospace), and civil engineering, among many others. The remarkable properties arise from the high cohesive energy due to their chemical structure, including the rigidity of the main chain due to the wholly aromatic structure conjugated with the amide groups, the high average bond energy, and a strong and highly directional interchain hydrogen bonds between the amide moieties. Although the natural yellowish colour of the fibres is used, generally, most of the applications require coloured fibres. However, aramid fibres have poor dyeing properties for the same reasons that make them thermally and mechanically resistant, and traditional dyeing methods, such as dope dyeing, are inefficient and aggressive, which impairs the fibres' properties. The ideal colour fastness of fibres is achieved by intrinsically, inherently, or self-coloured polymers by introducing a dye motif or chromophore monomer in the chemical structure of the polymer. In addition, the colour hue can be controlled by tuning the chromophore monomer molar content in the final composition. In previous research, we successfully obtained inherently blue-coloured aramids, with blue chromophore motifs unable to migrate and evenly distribute along the polymer chain and maintain their high-performance properties, and our aim now is to obtain red-coloured aramids prepared in the same fashion.

**Keywords:** red aromatic polyamides; chromophore monomer; self-coloured polymers; high-performance polymers

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/CSAC2021-10421/s1, Poster S1: Intrinsically coloured red aromatic polyamides.

**Author Contributions:** Conceptualization, M.T.-L. and F.G.; methodology, S.V.; formal analysis, P.P.-G.; investigation, P.P.-G. and M.T.-L.; resources, J.M.G.; data curation, P.P.-G.; writing—original draft preparation, P.P.-G. and M.T.-L.; writing—review and editing, P.P.-G. and M.T.-L.; visualization P.P.-G.; supervision, F.G. and S.V.; project administration, J.M.G.; funding acquisition, M.T.-L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Spanish Ministerio de Ciencia e Innovación, grant number PID2019-108583RJ-I00/AEI/10.13039/501100011033.

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

**Informed Consent Statement:** Not applicable.

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

**Citation:** Peredo-Guzmán, P.; Trigo-López, M.; Vallejos, S.; García, F.; García, J.M. Intrinsically Coloured Red Aromatic Polyamides. *Chem. Proc.* **2021**, *5*, 91. https://doi.org/ 10.3390/CSAC2021-10421

Academic Editor: Nicole Jaffrezic-Renault

Published: 30 June 2021

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**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/).

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