**Developing an Electrochemical Biosensor for the Detection of Hemagglutinin Protein of Influenza A Virus Subtype H1N1 in Artificial Saliva †**

**Carlos Torres-Méndez 1,\*, Jayendra Ellamathy 1, Maria Ines Mascarenhas 2, Yifan Liu 1, Georgia-Vasiliki Gkountana 2,\*, Patrizia Kühne 2, Javier Sebastián 1, Ivana Jovanovic 2, David Bern 1, Sharmilee Nandi 2, Maike Lüftner 2, Viktoria Langwallner 1, Maria Lysandrou 2, Sam Taylor 1, Klara Martinovic 2, Abdul-Raouf Atif 1, Ehsan Manouchehri Doulabi <sup>2</sup> , Masood Kamali-Moghaddam 2,\* and Gemma Mestres 1,\***


**Abstract:** Influenza A virus belongs to the Orthomyxoviridae family and, to date, is one of the most important pathogens causing acute respiratory infections, such as the recent pandemic of 2009. Hemagglutinin (HA) is one of the surface proteins of the virus that allow it to interact with cellular molecules. Due to the fact that it is the most abundant protein in the virus capsule, it is the best target in the detection of the Influenza A H1N1 virus through biosensing devices. Our aim is to develop an electrochemical biosensor to detect H1 by modifying carbon screen-printed electrodes (CSPE) with gold nanoparticles and to add further functionalization with monoclonal antibodies that are specific to this protein. The electrodes were characterized by the means of cyclic voltammetry, differential pulse voltammetry and electrochemical impedance spectroscopy. Our preliminary results suggest that the selected monoclonal antibodies have acceptable affinity and bind effectively to the H1 protein and that the electrodes have a wide potential window in the presence of [Fe(CN)6] <sup>3</sup>−/4−. In the future, we will continue to develop this biosensor in hope that it will be commercialized and be common in medical procedures during flu seasons and future influenza pandemics.

**Keywords:** influenza virus; voltammetry; screen-printed electrodes; hemagglutinin/HA protein; thiol chemistry

#### **1. Introduction**

In 2009, a novel H1N1 influenza A virus caused a pandemic leading to the death of 151,700–575,400 people worldwide according to the estimates of the Centers for Disease Control and Prevention (CDC) of the United States [1,2]. H1N1 influenza is a subtype of influenza A virus that was previously detected in swine, which causes upper and, in some cases, lower respiratory tract infections in its host [1]. Influenza A virus causes one of the most common respiratory diseases globally, seasonal flu, and together with Influenza B, C,

**Citation:** Torres-Méndez, C.; Ellamathy, J.; Mascarenhas, M.I.; Liu, Y.; Gkountana, G.-V.; Kühne, P.; Sebastián, J.; Jovanovic, I.; Bern, D.; Nandi, S.; et al. Developing an Electrochemical Biosensor for the Detection of Hemagglutinin Protein of Influenza A Virus Subtype H1N1 in Artificial Saliva. *Chem. Proc.* **2021**, *5*, 80. https://doi.org/10.3390/ CSAC2021-10477

Academic Editor: Maria Emília de Sousa

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/).

and D, is a part of the Orthomyxoviridae virus family. Moreover, influenza A virus belongs to the single-stranded RNA viruses. It has a segmented genome that encodes several viral proteins that are important for the pathogenesis of the virus [3,4]. Two of these proteins are important for detecting the virus in human specimens; these are hemagglutinin (HA) and neuraminidase (NA), which are the surface proteins of the virus involved in host invasion [5]. HA is the major protein of H1N1 and it is the protein with which the virus binds to the host's cells and invades them, while NA helps in the viral spreading from cell to cell [5].

So far, most of the detection methods for the influenza A virus are characterized by a long detection time, expensive instruments and reagents, and the need for trained technicians, thus creating an inconvenience for both the patients and the healthcare workers [1,6]. The development of sensitive and rapid detection methods, such as biosensors, is now the focus of many research groups and could be a great solution to the aforementioned problem. A lot of different biorecognition elements can be used for the detection of an analyte. However, antibodies seem to be the most widely used type among these elements.

Antibodies are specialized, Y-shaped proteins that identify pathogens by selectively binding to their membranes [7]. Due to their high specificity and sensitivity, antibodies are ideal biorecognition elements for biosensors [8]. Other biorecognition elements commonly used in biosensors include enzymes, nucleic acids, aptamers and molecular-imprinted polymers [9]. The focus of this paper is the development of an electrochemical antibodybased biosensor for the detection of the influenza A surface protein H1.

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

#### *2.1. Reagents and Materials*

HA H1N1 protein, mouse monoclonal antibodies (mAbs) and rabbit polyclonal antibodies (pAb) were purchased from Sinobiological (Frankfurt, Germany). Secondary goat anti-Rabbit IgG antibodies (Alexa Fluor 568) were purchased from Thermo Fisher (Waltham, MA, USA). Chloroauric acid (HAuCl4), Sulphuric acid (H2SO4), 4 aminothiophenol (4- ATP), ethanol, potassium hexacyanoferrate (II) trihydrate and Potassium hexacyanoferrate (III) were purchased from Sigma Aldrich (Darmstadt, Germany). Carbon screen-printed electrodes (CSPE) were provided by Zimmer & Peacock (Horten, Norway).

#### *2.2. Electrochemical Measurements*

The EmStat Pico Module potentiostat controlled using the PSTrace 5.8 computer software was employed for all cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), chronoamperometric electrodeposition and differential pulse voltammetry (DPV) experiments. The CSPE was used as a three-electrode cell system comprising a carbon working electrode (WE), a carbon counter electrode (CE) and an Ag/AgCl reference electrode (RE). EIS measurements were made at 6 mV ac amplitude in the frequency range of 5.0 mHz to 50 kHz and the equivalent circuit models were fitted using PSTrace software.

#### *2.3. Electrodeposition of Gold Nanoparticles on CSPE*

A modified method from the literature was employed [10], an aqueous solution containing 2 mM HAuCl4 and 0.5 M H2SO4 was used to cover the CSPE, a chronoamperometric method using a constant potential of −0.25 V for 60 s was used to deposit gold nanoparticles on top of the CSPE, and the electrode was washed with abundant deionized water, left to dry at room temperature and identified as AuNP-CSPE.

#### *2.4. Modification of AuNP-CSPE Electrodes with 4-ATP*

A previously reported method was adapted [11], in a typical experiment, and the working electrode was covered with 10 μL of 10 mM 4-ATP solution in ethanol at room temperature (22 ◦C) for 15 min. Nonspecifically adsorbed molecules were flushed off by careful rinsing with ethanol and deionized water. The electrode was dried under a stream of nitrogen and identified as NH2-AuNP-CSPE. The amine functionality in the electrode could be used later to form an amide bond [12] and immobilize the mouse monoclonal antibodies against the HA H1N1 protein.

#### *2.5. Testing of mAb Specificity and Sensitivity*

The enzyme-linked immunosorbent assay (ELISA) was used for this purpose. The protocol used for this indirect sandwich ELISA assay was in accordance with the mAb provider [13].

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

#### *3.1. Electrodeposition of Gold Nanoparticles*

The CSPEs offered a reasonable potential window to study the redox reaction of the [Fe(CN)6] <sup>3</sup>−/4<sup>−</sup> system and showed a symmetric shape, and the distance between the oxidation and the reduction peaks was of 727 mV (Figure 1); this value was much higher than the prediction of the Nerst equation for single electron transfer reactions and was attributed to a drop in potential due to the resistance of the carbon material [14]. When the CSPEs were modified with gold nanoparticles, the reversibility of the [Fe(CN)6] 3−/4− redox system increased as the distance between the oxidation and reduction peaks was 280 mV on the voltammogram; this was attributed to the increase in the surface area of the electrode and to the high conductivity of metallic gold nanoparticles.

**Figure 1.** Cyclic voltammogram of CSPE, AuNP-CSPE and NH2-NP-CSPE in the presence of [Fe(CN)6] 3-/4- obtained at a scan rate of 100 mV/s.

#### *3.2. Electrodeposition Length*

Further study into the gold electrodeposition process as a function of time (Figure 2) showed that longer reaction times than one minute do not increase either the current response of the electrode or the reversibility of the system.

**Figure 2.** Cyclic voltammogram of modified AuNP-CSPEs using different electrodeposition times; the experiment was conducted in the presence of [Fe(CN)6] 3-/4- at a scan rate of 100 mV/s.

#### *3.3. Characterization of NH2-AuNP-CSPE*

The cyclic voltammogram of NH2-AuNP-CSPE showed promising results due to the functionalization of the nanoparticles with the 4-ATP linker molecule (Figure 1), as the reversibility of the system increased and the electron transfer process for the reduction and oxidation reactions of [Fe(CN)6] <sup>3</sup>−/4<sup>−</sup> was facilitated on these modified electrodes due to the π delocalized system of the linker molecule. The electrochemical impedance spectroscopy tests indicated a decrease in impedance after the modification of the electrodes with gold nanoparticles and 4-ATP linker (Figure 3). The Nyquist plot of the bare CSPE can be fitted to an equivalent circuit for a simple electron-transfer reaction and NH2- AuNP-CSPE can be fitted to the classical Randles equivalent circuit comprising the ohmic resistance of the electrolyte solution (RΩ) and the charge transfer resistance (RCT), in series with the Warburg impedance element (diffusion controlled impedance) and in parallel with a double layer capacitance (CDL) (Figure 4). The modified NH2-AuNP-CSPE showed a significantly smaller RCT and is, therefore, highly conductive compared to the bare CSPE.

**Figure 3.** Nyquist plot of CSPE (**A**) and NH2-AuNP-CSPE (**B**) using the frequency range of 5.0 mHz to 50 kHz.

**Figure 4.** Fitting of CSPE to equivalent circuit for a simple electron transfer (**A**) and fitting of the NH2-AuNP-CSPE Randles equivalent circuit (**B**).

#### *3.4. Effect of the [Fe(CN)6] <sup>3</sup>*−*/4*<sup>−</sup> *Concentration*

The effect of the [Fe(CN)6] <sup>3</sup>−/4<sup>−</sup> concentration over the current response was evaluated by the means of CV and DPV (Figure 5), with a higher concentration of the electroactive species in the solution implying that more [Fe(CN)6] <sup>3</sup>−/4 molecules could reach the surface of the NH2-AuNP-CSPE to undergo oxidation and reduction, respectively. In the experiment, higher current responses were observed at high concentrations of [Fe(CN)6] <sup>3</sup>−/4. This experiment provided a visual basis for the expected effect on the final design of the biosensor, where the NH2-AuNP-CSPE is going to be coupled to monoclonal antibodies against the H1 protein and a blocking effect over [Fe(CN)6] <sup>3</sup>−/4 (lowering the current response) could take place once the H1 protein is bound to the antibody-modified electrode.

**Figure 5.** Cyclic voltammogram (**A**) and differential pulse voltammogram (**B**) for NH2-AuNP-CSPE as a function of the [Fe(CN)6] 3-/4- concentration.

#### *3.5. mAb Characterization*

To characterize the specificity of the monoclonal antibody targeting the H1 protein, an indirect sandwich ELISA was conducted. It was shown that the antibody can detect the H1 protein specifically (Figure 6). The approach was tested for different H1 concentrations and the lowest detectable concentration was 10 ng/mL.

**Figure 6.** Indirect sandwich ELISA results using 1:500 dilution of mAb, 1:1000 dilution of pAb and different H1 concentrations as shown in the *x* axis; the *y* axis shows the emission measured after the addition of the secondary antibody. Experiments were conducted twice with replicates of 2–6.

#### **4. Conclusions**

Cost-effective CSPE were modified with gold nanoparticles and 4-ATP, and the electrodes were characterized by means of electrochemical methods in the presence of [Fe(CN)6] <sup>3</sup>−/4<sup>−</sup> complexes. The redox system [Fe(CN)6] <sup>3</sup>−/4<sup>−</sup> was found to be more reversible in the modified electrodes AuNP-CSPE and NH2-AuNP-CSPE than in the bare CSPE. The modified NH2-AuNP-CSPE showed a decrease in impedance compared to CSPE, indicating that the electron transfer process is more favorable in the modified electrode. With the indirect sandwich ELISA, it was shown that the monoclonal antibody specifically targets the HA H1N1 protein and can be further used in the biosensor setup. The amine functionality on the modified electrodes can be exploited to couple mouse monoclonal antibodies against the HA H1N1 protein in future work within this project. In addition to this, aims for future work include the detection of the HA H1N1 protein in artificial saliva using DPV, the establishment of a protocol using bovine serum albumin (BSA) to avoid non-specific binding, and the determination of the sensibility and detection limits of the biosensor.

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

**Funding:** This work was supported by the Disciplinary Domain of Medical and Pharmacy and Disciplinary Domain of Science and Technology at Uppsala University, Swedish Research Council under grant 2020-02258 and Swedish Institute's scholarship to C. Torres-Méndez.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data are included herein and no data deposited in external repositories.

**Acknowledgments:** We are grateful to SensUs organization for all the support. We are also grateful to Quentin Palomar-Marchand for the technical advice provided for the electrochemical measurements and Zimmer & Peacock for providing the carbon screen-printed electrodes used in this study.

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

#### **References**


### *Abstract* **Determination of Chemical Oxygen Demand (COD) Using Nanoparticle-Modified Voltammetric Sensors and Electronic Tongue Principles †**

**Qing Wang and Manel del Valle \***

Group of Sensors and Biosensors, Department of Chemistry, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain; arieswq@163.com

**\*** Correspondence: manel.delvalle@uab.cat

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

**Abstract:** Chemical Oxygen Demand (COD) is a widely used parameter in analyzing and controlling the degree of pollution in water. COD is defined as the amount of molecular oxygen (in milligrams of O2) required to decompose all the organic compounds in 1 L of aqueous solution to carbon dioxide and water. There are many methods reported for COD determination, such as the conventional dichromate titration method. Electro-oxidizing the organic contaminants to completely transform them into CO2 and H2O using sensors is considered the best method for COD estimation. Increasing attention has been paid to electrochemical methods because they are highly sensitive, time-saving, low-cost, and easy to operate. In this sense, copper electrodes have been reported based on the fact that copper in alkaline media acts as a powerful electrocatalyst for the oxidation of aminoacids and carbohydrates, which are believed to be the major culprits for organic pollution. Cyclic voltammetry was the technique used to obtain the voltammetric responses. It is common for different organic compounds to show different cyclic voltammogram shapes and current intensities in different concentrations. In this work, four kinds of electrodes modified with copper (Cu)/copper oxide (CuO)/nickel copper alloy (Ni Cu alloy) nanoparticles were studied for COD analysis; this was done by employing the cyclic voltammetry technique, which involved a Nafion film-covered electrodeposited CuO/Cu nanoparticle electrode (**E1**), a Cu nanoparticle–graphite composite electrode (**E2**), a CuO nanoparticle– graphite composite electrode (**E3**), and a Ni Cu alloy nanoparticle–graphite composite electrode (**E4**). The COD values were determined via the plotted calibration of COD values vs. the current intensity. Glucose, glycine, potassium hydrogen phthalate (KHP), and ethylene glycol—which show different reducibilities—were chosen as the standard substances to play the role of organic contaminants with different degradation difficulties. From the obtained cyclic voltammograms, we can see that glucose is very easily oxidized by those four electrodes, with electrode **E1** displaying the best performance, with a linear range of 19.2~1120.8 mg/L and limit of detection of 27.5 mg/L (calculated based on the formula 3σ/k). In contrast, it is very difficult for the compound KHP to be oxidized by these four electrodes. Nevertheless, the obtained voltammetric profiles presented different shapes with the tested organic compounds, suggesting these four electrodes can compose an electronic tongue array for multivariate analysis. As a result, the main component of river samples—whose degradation could be easy or difficult—can be evaluated via the PCA technique. This evaluation is very helpful for the accuracy of COD detection. The resulting sensor-based method demonstrates great potential not only for estimating the precise value of COD, but for predicting the difficulty of its degradation; this represents a simple, fast, and clean methodology, which is perfectly suited to the present demands of green techniques.

**Keywords:** Chemical Oxygen Demand; copper (oxide) nanoparticle electrodes; nickel copper alloy nanoparticle electrode; cyclic voltammetry; electronic tongue

**Citation:** Wang, Q.; del Valle, M. Determination of Chemical Oxygen Demand (COD) Using Nanoparticle-Modified Voltammetric Sensors and Electronic Tongue Principles. *Chem. Proc.* **2021**, *5*, 81. https://doi.org/ 10.3390/CSAC2021-10442

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/).

**Supplementary Materials:** The poster presentation is available online at https://www.mdpi.com/ article/10.3390/CSAC2021-10442/s1.

**Author Contributions:** Conceptualization, Q.W. and M.d.V.; methodology, M.d.V.; writing—original draft preparation, Q.W.; writing—review and editing, M.d.V.; supervision, M.d.V.; Funding acquisition, M.d.V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Spanish Ministry of Science and Innovation, MCINN (Madrid) through project PID2019-107102RB-C21C. Q.W. acknowledges the concession of a PhD grant of the Chinese Scholarship Council (China). M.d.V. thanks the support from Generalitat de Catalunya through the program ICREA Academia.

**Institutional Review Board Statement:** No applicable.

**Informed Consent Statement:** No applicable.

**Data Availability Statement:** The data presented in this study are available in this article and supporting Information.

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

### *Proceeding Paper* **New Half Metal Perovskite NbScO3 for Spintronic Sensing Applications †**

**Amall Ahmed Ramanathan**

Department of Physics, The University of Jordan, Amman 11942, Jordan; amallahmad@gmail.com

† Presented at the 1st International Electronic Conference on Chemical Sensors and Analytical Chemistry,

1–15 July 2021; Available online: https://csac2021.sciforum.net/.

**Abstract:** Half-metallic ferromagnetic (HMF) materials demonstrate 100% spin polarization at the Fermi level, making them promising candidates for spintronic sensing applications. In this work, the full potential linearized augmented plane wave (FP-LAPW) density functional theory (DFT) method is used to calculate the electro-magnetic properties of the transition metal perovskite NbScO3 using the generalized gradient approximation (GGA) and the modified Becke-Johnson (mBJ) approximation for the exchange correlations. The electronic band structures for the two spin orientations using GGA, predict NbScO3 to be an HMF with an integer magnetic moment of 2.0 μB and hence a promising candidate for spintronics. The new half metal perovskite shows metallic behavior in the majority spin and semiconducting in the minority spin channel with a direct Γ−Γ band gap of 1.870 eV. The integer magnetic moment of 2.0 μB is also preserved with mBJ exchange potential. The band structure, however, shows indirect gaps R−Γ and X−Γ of 2.023 eV and 0.780 eV in the minority and majority channels, respectively indicating NbScO3 to be a magnetic semiconductor. The results indicate the suitability of NbScO3 for spintronics as the necessary conditions are satisfied.

**Keywords:** half metal; band structure; spintronics; sensors; information technology; perovskite

#### **1. Introduction**

The rapid technological advancements in the last decade call for smart and sustainable lifestyle management, with sensors playing a vital role [1–4]. Electron spin is fast becoming a very useful tool in sensing devices that are based on spintronics. Spintronics is a science in which the electron spin instead of the charge is used as the information carrier, providing the advantages of low energy consumption, high speed data processing and circuit integration density [5–7]. Among today's various proposed information transfer methodologies like molecular/nano electronics and quantum technologies, spintronics stands out due to the fact that it is compatible with conventional electronics making it easy to extend the existing well known electronic techniques to spintronic circuits. HMFs, due to their exceptional electronic structure, satisfy the needs for spintronic applications. The electrons of one spin direction behave as metals and those of the other spin direction act as semiconductors. Recently, quite a few new perovskites have been predicted to be half-metals [8–10].

Transition metals (TM) are of special interest, and a variety of interesting magnetic properties have been identified, as seen from recent research results. Depending upon the local environment non-magnetic materials have become magnetic due to their presence [11–13]. TM perovskites have piqued the interest of the scientific community due the intriguing nature of the TM ion interplay with the oxide or halide ion [14,15] with the great possibilities of different electronic and magnetic properties.

Unlike the majority of previous research, wherein the TM occupies the B site, in this work we switched the sites, and the TM Niobium occupies the A site with some very interesting magneto-electronic results. The purpose of the paper is to give the essential and accurate theoretical characterization using DFT\_FP-LAPW of the perovskite NbScO3 which is being investigated for the first time for potential use in spintronics and sensing.

**Citation:** Ramanathan, A.A. New Half Metal Perovskite NbScO3 for Spintronic Sensing Applications. *Chem. Proc.* **2021**, *5*, 82. https:// doi.org/10.3390/CSAC2021-10628

Academic Editor: Ye Zhou

Published: 7 July 2021

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

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

#### **2. Method and Materials**

The full-potential linearized-augmented plane wave (FP-LAPW) method, as implemented in the WIEN2k [16] package, is used to calculate the spin polarized ground states of the cubic perovskite NbScO3 within the DFT [17,18] formalism. The Perdew, Burke and Ernzerhof (PBE) [19] generalized gradient approximation (GGA) is used to calculate the optimized structures for a 10 × 10 × 10 k-point grid. The optimized lattice constant value is then used to evaluate the electronic and magnetic properties with the more accurate mBJ exchange correlation of Trans Blaha [20] at a denser k-point grid of 15 × 15 × 15. Kmax, which provides the magnitude of the largest K vector in the plane-wave expansion is set to 8. The muffin-tin radii were set to 1.60 a.u for Sc and O atoms and 2.7 a.u for Nb. The tetrahedron method [21] with 120 k points in the irreducible Brillouin zone is employed for integrations within the self-consistency cycle (SCF). The convergence tolerance thresholds for SCF is less than 10−<sup>4</sup> Ry for energy and 10−<sup>4</sup> for electron charges.

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

#### *3.1. Structural and Electronic*

The cubic NbScO3 perovskite has the space group Pm-3m (#221) symmetry, and the atoms occupy the positions 1a (0, 0,0), 1b ( <sup>1</sup> 2 , 1 2 , 1 <sup>2</sup> ) and 3c (0, <sup>1</sup> 2 , 1 <sup>2</sup> ) sites of Wyckoff coordinates for Nb, Sc and O atoms, respectively, as depicted in Figure 1 in an inset image.

**Figure 1.** The energy-volume optimization and Murnaghan fit for the perovskite NbScO3.

The lattice constants are optimized using the Murnaghan equation of state [22] with only volume optimization as the structure is cubic is required. The energy vs. volume optimization gives the minimum equilibrium energy state lattice parameter and is presented in Figure 1. The optimized values of the lattice constant and bulk modulus obtained are 3.985 Å and 160.679 GPa, respectively. This lattice constant value of NbScO3 is used alongside the GGA-PBE and mBJ exchange correlations at denser grids to calculate the electronic band structures along the high symmetry points. The GGA band structure is shown in Figure 2 for both the spin Dn (minority) and spin Up (majority) orientations.

**Figure 2.** The NbScO3 electronic band structures for (**a**) the minority (Spin Dn) and (**b**) the majority (Spin Up) channels with PBE-GGA.

We see from the figure that NbScO3 shows typical semi-conducting behavior in the minority spin with a direct Γ−Γ gap of 1.87 eV, and that it is metallic in the majority spin resulting in a half metal ferromagnetic behavior (HMF). HMFs have 100% spin polarization and can intrinsically provide single-spin channel electrons, which are very useful in spintronics.

The band structure of NbScO3 with the mBJ exchange potential on the other hand shows it to be a magnetic semiconductor as seen from the Figure 3 plots for the spin Dn and spin Up states.

**Figure 3.** The NbScO3 electronic band structures for (**a**) the minority (Spin Dn) and (**b**) the majority (Spin Up) channels with mBJ.

The minority spin and majority spin have indirect R−Γ and X−Γ gaps of 2.02 and 0.78 eV, respectively. Magnetic semiconductors combine the advantages of both magnets and semiconductors, and form the basis for spintronics. Magnetic semiconductors can be used for spin generation, injection, and spin manipulation and detection. Since the mBJ exchange potential provides very reliable and accurate band structures in comparison to that of GGA or hybrid functionals, the correct behavior of NbScO3 would be a magnetic semiconductor.

The half metal gap or spin flip energy EHM is defined here as the minimum energy required to flip a minority-spin electron from the valance band maximum to the majority spin Fermi level. The predicted band gaps and EHM for the GGA and mBJ exchange potential are listed in Table 1.

**Table 1.** The electronic band gaps in the minority and majority spin channels for the NbScO3 perovskite.


#### *3.2. Magnetic*

The band-structure plots in the previous section have clearly shown the magnetic nature of NbScO3, and to fully understand the origin and hybridization of the atomic orbitals, the total and partial density of states (TDOS/PDOS) are calculated using the mBJ exchange potential. These are depicted in Figure 4.

**Figure 4.** The NbScO3 electronic DOS in both minority (bottom part) and majority (top part) spin channels (**a**) the TDOS for the compound and the atom constituents (**b**–**d**) show the PDOS in the different orbitals for the Nb atom, the Sc atom and the oxygen atom, respectively.

We can clearly observe the semiconducting and magnetic nature of NbScO3from the plots. The minority channel shows the wide band gap, with no states at EF (Fermi-energy), whereas the majority channel has the valence band edge at the Fermi energy and one can say that NbScO3 is an intrinsic magnetic semiconductor. The role of Sc in the magnetism is negligible as indicated by the spin polarized PDOS plot for Sc. The main contribution to the magnetism comes from the 'd' orbital of Nb and the 'Px' and 'Py' orbitals of O2. Additionally, the significant difference in the majority and minority TDOS can be clearly seen; resulting in the total integer magnetic moment of 2 μB typical of HMF as given in Table 2. The table also lists the atom-wise and interstitial magnetic moments of NbScO3 with the GGA and mBJ exchange correlations.


**Table 2.** The total and atom projected magnetic moments of the NbScO3 perovskite in units of μB.

#### **4. Conclusions**

In conclusion, the FP-LAWP investigation of the perovskite NbScO3 has predicted the system to be a HMF and an intrinsic magnetic semiconductor using the GGA and mBJ exchange potentials, respectively. Moreover, the considerable size of the bandgap and magnetic moment obtained confirms the feasibility of NbScO3 for spintronic applications. In addition, the large value of EHM supports the robustness of this system for spintronics and sensing applications, whereby a significant role is played by the spin of the electrons in the sensor design.

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

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data can be provided upon reasonable request.

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

#### **References**


### *Proceeding Paper* **Group 14 Metallafluorenes for Lipid Structure Detection and Cellular Imaging †**

**Helena J. Spikes, Shelby J. Jarrett-Noland, Stephan M. Germann, Wendy Olivas, Janet Braddock-Wilking and Cynthia M. Dupureur \***

> Department of Chemistry & Biochemistry, University of Missouri St. Louis, St. Louis, MO 63121, USA; hjs7c5@mail.umsl.edu (H.J.S.); sjdzd@umsystem.edu (S.J.J.-N.); smg8v5@mail.umsl.edu (S.M.G.); olivasw@umsl.edu (W.O.); wilkingj@umsl.edu (J.B.-W.)

**\*** Correspondence: cdup@umsl.edu; Tel.: +1-314-516-4392

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

**Abstract:** Fluorescent compounds have been shown to be useful in probing lipid dynamics, and there is ongoing interest in nontoxic, photostable, and sensitive dyes. Recently, we evaluated a number of 2,7-disubstituted-alkynyl(aryl)-3,6-dimethoxy-9,9-diphenyl sila- and germafluorenes for their potential as cellular fluorescent probes. These compounds exhibit remarkable quantum yields in hydrophobic environments and dramatic increases in emission intensity in the presence of surfactants. Here, we show that they exhibit significant emission enhancements in the presence of small unilamellar vesicles and are nontoxic to *E. coli*, *S. aureus*, and *S. cerevisiae.* Furthermore, they luminesce in *S. cerevisiae* cells with strong photostability and colocalize with the lipid droplet stain Nile Red, demonstrating their promise as lipid probes.

**Keywords:** fluorescence; lipid; metallafluorene

**Citation:** Spikes, H.J.; Jarrett-Noland, S.J.; Germann, S.M.; Olivas, W.; Braddock-Wilking, J.; Dupureur, C.M. Group 14 Metallafluorenes for Lipid Structure Detection and Cellular Imaging. *Chem. Proc.* **2021**, *5*, 83. https://doi.org/10.3390/ CSAC2021-10455

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/).

#### **1. Introduction**

The introductory understanding of the role of biological membranes is that they are barriers and are used to regulate transport and for energy processes. Only fairly recently has there been a developing understanding that membranes are dynamic in their lipid composition and properties, and that these local differences participate in cellular processes in a profound way and have been linked to disease states [1,2], including cellular stress [3].

Fluorescence spectroscopy is an accessible and powerful tool for the sensing of molecules and their behaviors, including binding interactions, conformational changes, and catalytic activities, in both *in vitro* and *in vivo* via cellular imaging [4]. Due to their ability to respond to changes in molecular environment, molecules that exhibit intramolecular charge transfer (ICT) or excited-state intramolecular proton transfer (ESIPT) are particularly attractive as probes of lipids, their interactions, and dynamics [5,6].

Probes commonly used for such purposes include Nile Red, dansyl, NBD [7], and F2N12S [6]. These vary with respect to relevant properties such as the excitation and extinction wavelength, extinction coefficient, working concentration (sensitivity), photostability, and quantum yield, all of which can impact their utility. When this is coupled with the rapidly expanding research area, it is no surprise that a call for more probes to meet expanding needs is prominently articulated [5].

Recently, we evaluated a small library of sila- and germafluorenes (metallafluorenes or MFs) containing alkynyl(aryl) substituents at the 2,7-position ([8,9]; Figure 1) for their potential as fluorescent probes of surfactants. These compounds are soluble and luminescent in aqueous solution and exhibit high quantum yields and dramatic emission enhancements in the presence of various surfactants (5–25-fold) [10]. These results suggest that MFs could have biological applications. Here, we examine the sensitivity, toxicity, and

photostability of MFs toward lipids both *in vitro* and *in vivo* and demonstrate the potential of these compounds as lipid probes. Indeed, they are sensitive to DOPC small unilamellar vesicles (SUVs) with significant fluorescence enhancements. These dyes show no toxicity to Gram-positive bacteria, Gram-negative bacteria, and yeast cells and demonstrate high photostability. When compared to the commercially available lipid droplet dye Nile Red, these MFs show strong colocalization with more punctate staining, demonstrating their potential as lipid probes.

**Figure 1.** Structures of 2,7-disubstituted sila- and germafluorenes used in this study. **1** silicon based 4-ethynyl-1,1 -biphenyl substitutent; **2** germanium based 2-ethynyl-6-methoxynaphthalene substitutent; **3** silicon based 4-ethynyltoluene substitutent; **4** silicon based 1-ethynyl-3-fluorobenzene substitutent.

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

#### *2.1. Materials*

Phospholipids were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Twohundred-proof ethanol was purchased from Decon Labs (King of Prussia, PA, USA). DMSO and Nile Red were obtained from Millipore Sigma (Milkwaukee, WI, USA). p-Xylene was obtained from ThermoFisher (Waltham, MA, USA). All chemicals were of reagent grade and were used as received without further purification. Compounds **1**–**4** were synthesized as previously described using an appropriate alkynyl(aryl) precursor in a palladium-catalyzed Sonagashira cross-coupling reaction [8,9] and dispensed from stocks in DMSO as previously described [10].

#### *2.2. Preparation of Small Unilamellar Vesicles (SUVs)*

At 25 ◦C, a stock concentration of 4.2 mM DOPC was prepared by drying under inert gas and then resuspended in 10 mM Tris buffer. After 30 min, DOPC was sonicated for 27 min at 25 ◦C until cloudy. The DOPC-SUV solution was then passed through an Avanti Mini Extruder eleven times to make uniformly sized 0.1 μm DOPC-SUVs at 25 ◦C. DOPC-SUVs were then diluted to 0.1 mM in a quartz cuvette for fluorescence measurements [11,12].

#### *2.3. Spectroscopy*

Absorbance spectra were recorded on a Shimadzu 1800 (Kyoto, Japan) with slits (bandpass) set to 1 nm. Emission spectra were collected in an acid-washed quartz cuvette on a Fluorolog-3 (SPEX) spectrofluorimeter (Horiba Scientific, Piscataway, NJ, USA). The temperature was maintained at 25 ◦C with a thermostatted cell holder equipped with a magnetic stirrer. Emission spectra were collected with the indicated excitation wavelength and slits (bandpass). MF photostability in xylene was observed at the indicated emission maximum.

#### *2.4. Microbial Toxicity*

Culture tubes containing LB media or YPD media were inoculated with *Escherichia coli* (Gram-negative), *Staphylococcus aureus* (Gram-positive), or *Saccharomyces cerevisiae*, respectively. Compounds **1**–**4** were added such that the final DMSO concentration was 2–10% and the MF at its solubility limit in the media. The tubes were incubated at either 37 ◦C (bacteria) or 30 ◦C (yeast) overnight and visually inspected for growth.

#### *2.5. Confocal Laser Scanning Microscopy*

Samples were prepared by smearing a small amount of cells onto a glass microscope slide and heat-fixed by passing the slide through a flame no more than 5 times. Then, **1**–**4** or NR was applied to heat-fixed cells at 15 μM and incubated at room temperature for 15 min for MFs and 10 min for Nile Red. Slides were then rinsed with 2–3 mL of deionized water, topped with coverslips, and sealed with clear nail polish. Cells were imaged with a Zeiss LSM 900 (Zeiss, Oberkochen, Germany) confocal microscope with an excitation wavelength of 405 nm. For photostability, the sample was illuminated with 1% laser power and images collected periodically.

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

#### *3.1. Spectroscopic Studies*

To assess their sensitivity to a biologically relevant membrane, the emission spectra of **1**–**4** were compared in the absence and presence of DOPC-SUVs. As shown in Figure 2, fold-enhancements range from two- to sevenfold, with **1** and **2** showing the most dramatic changes.

**Figure 2.** Emission spectra of MFs **1**–**4** (as numbered in Figure 1) in the absence (dashed) and presence (solid) of 0.1 mM DOPC-SUVs. Conditions: 1 μM MF, 0.1 mM DOPC, 10 mM Tris pH 8, 25 ◦C. The excitation wavelength was 387 nm and the slits (bandpass) set to 1.0 nm. Three minute incubation.

The photostability of these MFs was initially probed by observing the emission signal as a function of time in xylene, which is used to mimic the interior of membranes [13]. As summarized in Figure 3, these signals are remarkably stable over two hours of continuous excitation. Together, the responsiveness to SUVs and photostability in xylene indicate promise for MFs as probes of lipids *in vivo*.

**Figure 3.** Photostability *in vitro*. Compounds **1**–**4** (as defined in Figure 1) were diluted into p-xylene and excited continuously. Conditions: 1 μM MF, 0.1–0.4% DMSO. **1**: excitation at 387 nm, slits 1 nm; **2**: excitation at 390 nm, slits 0.8 nm; **3**: excitation at 376 nm, slits 0.9 nm; **4**: excitation at 376 nm, slits 0.9 nm.

#### *3.2. Microbial Toxicity Studies*

To assess their potential for use in cellular imaging, MFs were screened for toxicity against microorganisms. For yeast, *E. coli* and *S. aureus*, no inhibition of growth was observed at the MF solubility limit in media (at least 50 μM).

#### *3.3. Imaging of S. cerevisiae with Metallafluorenes*

To determine if these MFs can be used to stain cells, **1**–**4** were introduced to yeast cells and subsequently imaged using confocal microscopy. Figure 4 illustrates that in all cases, the MF emission intensity is visible inside fixed yeast cells.

**Figure 4.** Confocal imaging of MFs **1**–**4** in yeast cells. Conditions: 15 μM MF as indicated, 63×. The excitation wavelength was 405 nm and scan range 400–600 nm. Numbers refer to MFs as defined in Figure 1.

To assess the MF photostability in yeast cells, excitation was applied and fluorescence was observed as a function of time. As summarized in Figure 5 for **1** and **2**, fluorescence persisted for over 2 min, with **2** showing greater photostability. See Supplemental Figure S1 for photostability studies of **3** and **4**.

**Figure 5.** Photostability of **1** and **2** in yeast cells. See Methods for details. *S. cerevisiae* were stained for 15 min with 15 μM **1** or **2** and then imaged periodically during continuous excitation. Magnification is 63×. Numbers at left refer to compounds as defined in Figure 1.

Finally, to determine where these MF localize in yeast, we costained with Nile Red, a well-known lipid droplet stain [14]. As shown in Figure 6, **1** yields more punctate images and colocalizes with this probe, demonstrating clear specificity for *S. cereviseiae* organelles, including the vacuole and possibly lipid granules. See Supplemental Figure S2 for a colocalization study of **2** and **4**.

**Figure 6.** MF **1** colocalizes in yeast with Nile Red. Red, Nile Red; green, **1**; top right, transmitted light; bottom right, yellow indicates colocalization. 15 μM probe at 63× magnification.

#### **4. Conclusions**

We show here that these metallafluorenes have good photostability and are sensitive to lipid structures *in vitro*, demonstrating impressive fold enhancements in the presence of SUVs. Furthermore, they are non-toxic to cells and can enter cells and colocalize with Nile Red, a lipid probe. In addition, the higher extinction coefficients of MFs and competitive quantum yields [10] make them more sensitive. All of these observations bode well for the application of MFs as lipid probes both *in vitro* and *in vivo*. The synthetic scaffolding of these MFs provides convenient tuning of desired properties by changing the 2,7 substituent. This feature facilitates designs that incorporate optimal solubility, emission spectra, dipole moment, and solvatochromism for specific applications.

#### **5. Patents**

WO/2020/210416; PCT International Patent Application No.: PCT/US2020/027355.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/CSAC2021-10455/s1, Figure S1: Photostability of 3 and 4 in Yeast Cells, Figure S2: 2 and 4 Colocalize with Nile Red in Yeast.

**Author Contributions:** Conceptualization, C.M.D., H.J.S. and S.J.J.-N.; investigation, H.J.S. and S.J.J.-N.; resources, C.M.D., J.B.-W., S.M.G. and W.O.; data curation, S.J.J.-N.; writing—original draft preparation, C.M.D. and H.J.S.; writing—review and editing, C.M.D. and S.J.J.-N.; supervision, C.M.D.; project administration, C.M.D.; funding acquisition, J.B.-W. and C.M.D.; H.J.S. and S.J.J.-N. contributed equally to the preparation of this manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Science Foundation (CHE-1362431 to J.B.-W.).

**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* **QSPR Modelling of Potentiometric HCO3** *−***/Cl***−* **Selectivity for Polymeric Membrane Sensors †**

**Nadezhda Vladimirova \* , Julia Ashina and Dmitry Kirsanov**

Institute of Chemistry, Saint Petersburg State University, 198504 Saint Petersburg, Russia; y.ashina@spbu.ru (J.A.); d.kirsanov@gmail.com (D.K.)

**\*** Correspondence: hada96@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:** Since the development process of new sensors is long and tedious, it would be very helpful to develop a model that could predict sensor properties based on active compound structure without the actual synthesis and characterization of the corresponding sensors. In this work, the model for the prediction of logK (HCO3 −/Cl−) was constructed based on 40 ligand structures suggested in the literature for carbonate sensing. Substructural molecular fragments (SMF) were used to describe the structure of compounds, where fragments were considered as sequences of bonds and atoms. The projection on latent structures (PLS) method was used to calculate the regression model.

**Keywords:** membrane sensor; carbonate ionophore; QSPR; PLS

#### **1. Introduction**

Polymeric membrane electrodes offer numerous advantages, and their properties can be tuned in a wide range by the modification of membrane composition. However, the process of the selection of an appropriate ligand to construct the sensor for a particular task is time-consuming and requires ligand synthesis, sensor preparation, and characterization. It would be very helpful for researchers to make a model that allows the prediction of sensor properties of an electrode based on the structure of the employed ionophore.

Using the computational chemistry, different characteristics of chemical compounds can be predicted. Quantitative relations between physical or chemical properties of organic compounds and their chemical structures can be set with the QSPR (quantitative structure property relationship). This methodology is widely applied nowadays, e.g., in pharmaceutical investigations; specifically, a search for new drugs [1]. There are QSPR models for various materials, such as nanomaterials [2], catalysts [3], and ionic liquids [4]. Recently, an application of QSPR for predicting the sensor properties of membrane electrodes was suggested [5]. It was possible to relate the structure of the organic ligand with the selectivity constant of the corresponding membrane sensor for Ca2+/Mg2+.

This study aims to expand this approach to anion-selective sensors, where ligand selection is much more challenging than in the case of metal cations, due to the wide variability of the geometries of inorganic ligands, and their hydrolysis in the case of weak acids.

Among anions, there are ones with important biological and industrial roles. An example of such anions is carbonate. Therefore, it is a prospective object for predicting selectivity to carbonate against chloride anions by means of QSPR.

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

#### *2.1. Dataset*

The dataset of 40 samples was composed with literature sources and experimental data. A summary table with compositions of all samples and literature sources can be found

**Citation:** Vladimirova, N.; Ashina, J.; Kirsanov, D. QSPR Modelling of Potentiometric HCO3 −/Cl− Selectivity for Polymeric Membrane Sensors. *Chem. Proc.* **2021**, *5*, 84. https://doi.org/10.3390/ CSAC2021-10621

Academic Editor: Ye Zhou

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/).

in the supplementary materials. Whereas the number of anionic (especially carbonate) ionophores is small due to the reasons that we have already discussed earlier, there are a few articles about carbonate ionophores. A great part of the data was extracted from IUPAC review "POTENTIOMETRIC SELECTIVITY COEFFICIENTS OF ION-SELECTIVE ELECTRODES", with a summary table of anions existing in 2002 [6]. Considering the significant shortage of carbonate ionophores, we had to add in the table ionophores with Cl−/HCO3 − selectivity that were converted to HCO3 −/Cl− selectivity according to the Nikolsky–Eisenman equation:

$$E = E\_I^0 + \frac{RT}{z\_I F} \ln(a\_I + \sum K\_{IJ} a\_J^{\frac{z\_I}{z\_J}}).\tag{1}$$

All of the structures of ionophores are available in Table S1 in Supplementary Materials. We also made sure that all these data were obtained in the narrow range of pH (7.0–8.6) for understanding which particular ionic form prevailed in an examined solution.

All the membranes described in the literature are based on polyvinylchloride (PVC) as a polymer, and one of three plasticizers: nitrophenyl octyl ether (NPOE), dioctyl adipate (DOA) and dioctyl sebacate (DOS), which is also known as bis(2-ethylhexyl) sebacate (BEHS). Ion-exchanger is tridodecylmethylammonium (TDMA) with Cl− or NO3-counter-ion.

Due to the small number of carbonate ionophores examined, we had to compile the resulting database with membranes that differ not just in ionophores, but plasticizers as well. We took different plasticizers into account and added their dielectric constant (also known as relative permittivity) as a descriptor for adjusting our model and making it more comprehensive.

The selectivity coefficients of these ionophores varied from −5.8 to 6.2 on the logarithmic scale. The average selectivity logK (HCO3 <sup>−</sup>/Cl−) was −1.425, and the median value was −2.6.

#### *2.2. Descriptors*

We used substructural molecular fragments (SMF) for encoding molecular structures in a matrix. A molecular structure can be described with this method by dividing a molecule into all possible fragments and writing the number of these fragments into the matrix. These SMFs were obtained by using "ISIDA QSPR" software [7]. There are two approaches for obtaining a molecule's SMF in ISIDA: sequences of atoms and/or bonds (topological path) and selected ("augmented") atom (atom-centered fragments) with its environment that can be atoms, bonds, or both (Figure 1). In this work, atom and bond sequences were applied. A molecule was represented as a graph and its descriptors were, consequently, subgraphs.

Hence, ISIDA SMF descriptors are numbers of fragments (or subgraphs) in a molecule with each element of the descriptor associated with one of the detected possible fragments. Only the shortest paths from one atom to the other were used. It should be noted that the length of sequences is limited. The minimal and the maximal lengths are 2 and 15, respectively.

#### *2.3. Projection on Latent Structures (PLS) Modelling*

In order to relate molecular descriptors of ligands with selectivity coefficients of the corresponding sensors, we employed the PLS regression algorithm. PLS regression searches for a set of components are known as latent vectors that perform a synchronous decomposition of X and Y, with the clause that these components explain, as much as possible, the covariance between X and Y. Data matrix size was 40 × 1855, where 40 is the number of samples (ligands) and 1855 is the number of descriptors.

**Figure 1.** Two approaches to obtaining ISIDA SMF: topological path and atom-centered fragments.

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

The molecular descriptors obtained for the chosen ligands were calculated with ISIDA QSPR software [7]. The 40 ionophore structures and their properties, specifically substructural molecular fragments (SMF) and the permittivity of membranes, were used as descriptors. The PLS model relating the descriptors with selectivity was evaluated according to the following parameters: root mean square error (RMSE) and squared determination coefficient (R2). The results of QSPR modelling are shown in Figure 2. Each point in the graph corresponds to an item in the database, whereas straight lines represent the resulting models. Blue and red colours correspond to training and test samples, respectively.

**Figure 2.** "Measured vs. predicted" plot for the QSPR model for predicting the selectivity of membrane sensors.

It can be seen that the derived model allows for a semi-quantitative estimation of the selectivity coefficients, based on the ligand structure. As far as each compound is an individual molecule, which consists of a variety of molecular fragments, regression coefficients allow evaluating the contribution of each fragment (represented as an independent variable in the matrix) in the selectivity of the sensors. The largest coefficients signify the variables (in this case, SMF, which are the encoded fragments of molecules) with the most important impact. The fragments with the largest contribution in the absolute value of the selectivity logK (HCO3 −/Cl−) of potentiometric membrane sensors are shown in Figure 3.

0ROHFXODUIUDJPHQWV

**Figure 3.** Fragments with the largest contribution in logK (HCO3 −/Cl−) values, where '-' is a single bond and '=' is a double bond.

As follows from the graph, the fragment C=C-C=C-C-C-F makes the largest negative contribution, and it is part of a longer fragment C-C=C-C=C-C-C-F with a smaller contribution. The shortest fragment with negative contribution C-C=C-C=C is included in the remaining fragments with the largest negative contribution. The fragment with positive contribution C=C-C=C-Hg contains mercury in its composition. These observations provide valuable information for the further design of the ligands with required selectivity.

#### **4. Conclusions**

Despite some problems with anions' ionophores that are described more fully in the introduction, we were able to collect the database that allowed making a QSPR model while satisfying RMSE and R<sup>2</sup> for a relatively small amount of data. We found the fragments with the highest impact in selectivity logK (HCO3 −/Cl−) of potentiometric membrane sensors, and we believe that this will help in the future search for the new ionophores. It appears that semi-quantitative prediction of sensor selectivity is possible based on the ligand structure.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/CSAC2021-10621/s1, Table S1: QSPR modelling of potentiometric HCO3 −/Cl− selectivity for polymeric membrane sensors supporting information.

**Author Contributions:** Conceptualization, D.K.; methodology, D.K.; formal analysis, D.K. and N.V.; investigation, N.V.; writing—original draft preparation, N.V.; writing—review and editing, D.K. and J.A.; visualization, N.V.; supervision, D.K.; funding acquisition, J.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by RFBR, grant number 20-33-70272.

**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* **Room Temperature Hydrogen Sensing Based on Tapered Optical Fiber Coated with Polyaniline (PANI) †**

**Mohammed Majeed Alkhabet 1,2 , Saad Hayatu Girei 1,3 , Abdul Hadi Ismail 4, Suriati Paiman <sup>5</sup> , Norhana Arsad <sup>6</sup> , Mohd Adzir Mahdi <sup>1</sup> and Mohd Hanif Yaacob 1,\***


**Abstract:** This work demonstrates a hydrogen (H2) sensor at room temperature made of tapered optical fibers coated with a polyaniline (PANI) nanofiber. A transducing platform was constructed using a multimode optical fiber (MMF) with a 125 μm cladding and a 62.5 μm core diameter. In order to enhance the light evanescent field surrounding the fiber, the fibers were tapered from 125 μm in diameter to 20 μm in diameter with 10 mm waist and coated PANI using the drop casting technique. Various characterization techniques, such as field emission scanning electron microscopy (FESEM), energy dispersive X-ray (EDX), differential X-ray (XRD), and atomic force microscopy, have been used to establish the PANI's properties. When H2 is subtracted, the optical properties of the PANI layer change, resulting in a change in light absorption. The fabricated sensor was tested by exposing it to H2 at different concentration from 0.125% to 1%. In this case, the sensitivity, response, and recovery times were 15.928/vol%, 110 s, and 160 s, respectively. The improved hydrogen sensor holds great promise for environmental and industrial applications due to its ability to operate at room temperature.

**Keywords:** hydrogen (H2); tapered optical fiber; polyaniline (PANI); drop casting technique

**1. Introduction** Hydrogen (H2), due to its high fuel efficiency, abundance, uncontaminated character, and sustainability, is one of the possible solutions to the impending energy crisis [1]. It is also a reliable gas for misdiagnosis in power transformers [2,3]. H2 has also been used in other sectors, such as aerospace engineering, mineral refineries, oil exploration, chemical processing, cryogenic refrigeration, and many more [4]. However, the high diffusion coefficient (0.16 cm2/s in air), low ignition energy (0.018 mJ), wide explosion concentration range (4–75%), and high heat of combustion (285.8 kJ/mol) convert them to gases, which are explosive and potentially dangerous for use, transport, and storage [5].

On the other hand, optical sensors rely on optical fibers, which have unique characteristics such as lightness, small size, electromagnetic interference resistance, instability, and stiffness in harsh environments [6]. Due to their peculiar properties, optical fibers are perfect candidates for detection in harsh environments [7].

**Citation:** Alkhabet, M.M.; Girei, S.H.; Ismail, A.H.; Paiman, S.; Arsad, N.; Mahdi, M.A.; Yaacob, M.H. Room Temperature Hydrogen Sensing Based on Tapered Optical Fiber Coated with Polyaniline (PANI). *Chem. Proc.* **2021**, *5*, 85. https:// doi.org/10.3390/CSAC2021-10415

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/).

Recently, the scientific community has made significant progress on the synthesis and application of conducting polymers [8–10]. One of the promising materials for gas sensing is polyaniline (PANI), which among polymers has peak environmental stability. It is currently known as the only conducting polymer that is stable in air [11]. As a sensor, PANI is particularly helpful because it is sensitive at room temperature [12,13], and it can be applied to detect a range of gases in combination with additional nanomaterials [14].

Several optical hydrogen sensors using PANI as an energy transformer have been reported in recent years. Most of them rely on fiber gratings (FBGs) [15] and plastic optical fiber [16]. To make fiber optic sensors sensitive to their surroundings, most of them must be functionalized [17]. In this research, PANI coated with dissolved, tapered optical fibers is used to detect hydrogen gas.

#### **2. Experiments**

#### *2.1. Tapered Optical Fiber Fabrication*

The H2 gas sensor was developed using a multimodal tapered fiber optic (MMF) with cladding and core dimensions of 125 m and 62.5 m, respectively, as a transducing platform. For reduction, the Vytran glass processing system (Vytran GPX-3400, Thorlabs, Inc., Trenton, NJ, USA) was employed. The machine operates on a heating and pulling principle, with a graphite filament acting as a heating source to create the desired tapered profile geometry. The MMF had a 125 mm cladding diameter that tapered to a 20 mm waist diameter, a 10 mm waist length, and a 5 mm tapering top. The picture of the tapered optical fiber created with the tapered area is shown in Figure 1.

**Figure 1.** Scanning electron microscopy (SEM) micrograph of a tapered multimode optical fiber's transition area (MMF).

#### *2.2. PANI Functionalization on Tapered Optical Fiber*

Aniline was purified by distillation before polymerization. The purified aniline (0.16 M) was instantly dissolved in pre-prepared 0.05 M perchloric acid (HClO4) [18,19] (Merck, 70–72%) to avoid atmospheric oxidation. In another volumetric flask, 0.16 M of ammonium peroxodisulfate was also dissolved in HClO4, and both mixtures were rested overnight. Ammonium peroxodisulfate solution was carefully added to the aniline solution with continuous stirring at room condition. The oxidative polymerization reaction of the mixture was left stirring for 24 h. The obtained PANI (dark green precipitate) was filtered and washed with ethanol (EtOH) until a colorless supernatant liquid can be observed in order to minimize the amount of unreacted monomers and oligomers [20]. It is then dried at 60 ◦C until a constant weight is obtained. A drop casting technique was used to coat the tapered optical fibers. To guarantee full evaporation of the aqueous medium, a drop of mixture (about 20 μL) was placed into the base of the tapered optical fiber by using a micropipette, and the sample was heated in the oven at 80 ◦C for 40 min.

A light source (tungsten halogen, HL-2000, Ocean Optics, Dunedin, FL, USA) with a coverage wavelength of 360 nm to 2500 nm and a spectrophotometer (USB 4000, Ocean Optics) comprised the experimental phase of the gas optical sensing system. It can detect anything between 200 and 1100 times per second. The optical gas detection system's experimental setup includes a customized gas chamber for measuring the optical absorption spectrum. The PANI-coated sensor was placed in a close gas unit and purged with a centrifuge at a gas flow rate of 200 sccm from a computer managed mass flow controller. Figure 2 depicts the H2 sensor's experimental setup.

**Figure 2.** H2 sensor experimental setup.

#### *2.3. Materials Characterization*

FESEM (JSM-7600F, Musashino, Akishima, Tokyo, 8558, Japan) was used to examine the films' morphology, while EDX analysis was used to assess their original composition. XRD examination indicated material identification, crystallization, and the PANI phase transition (APD 2000). FESEM images of PANI nanoparticles are shown in Figure 3. The PANI is mostly made up of uneven grains and chips with sharp edges, as can be observed. Furthermore, the structure seems to be completely porous, creating very small polyaniline particles that can expand the liquid–solid interface [21,22].

Figure 4a shows the XRD pattern of the PANI, which shows an amorphous nature in a partly crystalline condition with a diffraction peak at 22.34◦. (200). Due to the repeating of benzenoid and quinoid rings in the PANI chains, this pattern displays poor conductive polymer crystallinity.

Figure 4b shows the UV–vis spectra of PANI structures. Spectroscopic scanning was performed at a wavelength range from 300 to 1000 nm. The UV–vis spectrum is useful for measuring the amount of conjugation, and for this reason, conductive polyaniline samples exhibit a broad absorbance, referred to as a free carrier tail, at wave lengths greater than about 800 nm. As the conjugation length becomes longer, the peak shifts to broader wavelengths and becomes extremely large [23].

Figure 4c shows the EDX pattern of PANI, which revealed that the key elements in PANI film are C, N, O, and Si, as observed by their peaks. A silica fiber was employed as the substrate shared the silicon (Si) peak.

The atomic force microscope (AFM) can verify the average surface roughness and thicknesses of PANI. A 10 × 10 μm section of the boundary area was scanned for AFM analysis. The average surface roughness values of the PANI were ≈23.4 nm, as shown in Figure 5a,b. As part of this study, the thicknesses of the PANI coatings were measured. As shown in Figure 5c, measurements were taken by surrounding parts of fiber with aluminum tape and then assessing the thickness differences between coated and uncoated fiber. The average thickness of the PANI coatings was 690 nm.

**Figure 3.** FESEM micrograph of Polyaniline (PANI).

**Figure 4.** (**a**) XRD pattern of PANI; (**b**) shows the UV–vis spectra of PANI structures; and (**c**) shows the EDX measurement of PANI.

**Figure 5.** Two-dimensional topography AFM images (**a**) PANI; (**b**) 3D topography AFM images of PANI; and (**c**) 3D topography AFM images of the boundary area between the uncoated and coated fiber for PANI sensing layer.

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

The absorption spectra of the developed sensor coated with PANI relative to synthetic air and 1.00% H2 at room temperature were observed. As shown in Figure 6a, the PANIcoated sensor shows notable changes in absorption, particularly in the wavelength range of 550 to 850 nm. The sensor performance of the PANI-coated sensor was monitored in terms of cumulative absorbance, which is the product of a combination of response curves over a particular wavelength range. Figure 6b shows the dynamic response of PANI-coated based sensors to H2 concentrations in air ranging from 0.125% to 1.00% at room temperature. The response and recovery times of the developed PANI-coated based sensor were 110 s and 160 s, respectively. The absorbance change was approximately 4% at 0.125% H2 and 19% higher with 1.00% H2. Compared with the results of previous studies [24,25], the PANIcoated based sensor showed greater H2 absorbance and recovery, as well as advanced compromise differences.

The repeatability of the PANI-coated based sensor was tested by exposing it to three cycles of 1.00% H2. Overall, the PANI-coated based sensor showed a strong and stable absorbance response as well as high repeatability towards H2.

**Figure 6.** (**a**) Absorbance versus optical wavelength, (**b**) dynamic absorbance curves, and (**c**) repeatability of PANI-coated based sensor towards H2.

Figure 7a shows the absorption versus H2 concentration for PANI-coated based sensors. The PANI-coated based sensors had a sensitivity of 15.928/vol% and a linearity slope of 98%. When measuring gas-sensing properties, selectivity is an important key to consider. As shown in Figure 7b, the sensor's absorption properties toward ammonia (NH3) and methane gas (CH4) at a concentration of 1.00% were investigated. The PANI-coated based sensor had a very high NH3 absorption response but a substantially lower response for the other gases. Furthermore, the adsorption of PANI based materials was highly selective for polar molecules such as NH3, whereas sensitivity was low for non-polar molecules such as H2 and CH4 [26].

**Figure 7.** (**a**) Absorbance changes at different H2 concentration for PANI-coated based sensor and (**b**) the selectivity of PANI-coated based sensor.

#### **4. H2 Mechanism of PANI Coated on Tarped Optical Fiber**

The H2 sensor with PANI mechanism consists of two parts, as illustrated in Figure 8. The first is the physical absorption of gas molecules in the PANI, which causes changes in the refractive index of the optical fiber's surface, which in turn will result in changes in the amount of light transmitted in the fiber. A higher RI would allow more light to escape, lowering the intensity of light detected by the spectrophotometer. The charge transfer between the adsorbent and the PANI molecules is the second step. The charge transfer from the electron donating H2 gas to PANI changes the surface chemistry of the sensor layer as it is absorbed into the walls and sides of PANI. This involves changes in the sensor layer's optical properties, as the light that traveled through the fiber is absorbed by environmental changes, causing spectrum shifts.

**Figure 8.** Hydrogen PANI-sensing mechanism.

#### **5. Conclusions**

By using a drop casting technique, this study proved that optical fiber sensors could be fabricated from Polyaniline (PANI). The response of the established sensor to various concentrations of H2 gas at room temperature was used to measure its efficiency. When exposed to 1.00 % H2 in synthetic air, the PANI-coated based sensor enhanced its ab-sorption response by 19%, according to these findings. The selectivity investigation indicated that the PANI-based optical sensor responded strongly towards ammonia, methane, and hydtogen chemicals. The findings suggest that an affordable and easy methodology may be utilized to enhance an effective, accurate, and repeatable H2 sensor in real-world atmospheric conditions.

**Supplementary Materials:** The poster presentation is available online at https://www.mdpi.com/ article/10.3390/CSAC2021-10415/s1.

**Author Contributions:** Conceptualization, M.M.A., and M.H.Y.; methodology, M.M.A., S.H.G., A.H.I., and M.H.Y.; writing—original draft preparation, M.M.A.; review and editing, M.H.Y., M.A.M., S.P., and N.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** Universiti Putra Malaysia funded this research, grant number GP-IPS/2019/9674900.

**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**


### *Proceeding Paper* **Aqueous Medium Fluoride Anion Sensing by Fluorophore Encapsulated UiO-66 Type Zirconium Metal–Organic Framework †**

**Rana Dalapati and Ling Zang \***

Department of Materials Science and Engineering, Nano Institute of Utah, University of Utah, Salt Lake City, UT 84112, USA; rana.dalapati@utah.edu

**\*** Correspondence: lzang@eng.utah.edu

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

**Abstract:** A well-known fluorophore molecule, pyrene was encapsulated into a stable metal organic framework by in situ encapsulation method. The existing metal-organic framework (MOF) called UiO-66 (UiO = University of Oslo) served as host material for pyrene fluorophore. The fluorescence of pyrene was quenched after encapsulation inside the porous host. Recovery of quenched fluorescence was accomplished by anion induced host dissolution, followed by the release of the fluorophore molecule. Using this anion induced dissolution, a selective sensing of fluoride anion in pure aqueous was achieved.

**Keywords:** metal-organic framework; pyrene; fluoride anion sensing

#### **1. Introduction**

For the past few decades, a tremendous effort has been dedicated by the scientific community towards the development of a modest strategy for the selective and precise sensing of anions [1], as they perform a key role in biological systems, health, and environment [2]. Among the anions present in biological systems, the smallest fluoride anion has drawn significant attention, due to its biological and environmental impact [3]. Currently, the presence of fluoride in drinking water and commercial household products is the emerging concern for public health [4]. Although fluoride is considered as a micronutrient [5], the excess uptake of fluoride can cause fluorosis [6], and even chronic renal failure [7]. As such, there is an urgent necessity for selective and precise determination of fluoride anions in fluoride-contaminated water.

Metal-organic framework (MOF), a new class of porous materials, have received tremendous attention for their potential applications in gas storage [8], chemical separation [9], catalysis [10], and drug delivery [11]. The UiO-66 framework (UiO = University of Oslo) is one typical Zr-MOF, constructed with Zr6O4(OH)4 clusters and 1,4 benzenedicarboxylate, BDC linkers [12]. With a higher surface area, thermal resistivity, and an exceptional structural stability in water, this becomes an ideal molecular host material [13]. The triggered release of a guest molecule by host dissolution is one of the efficient strategies for molecular recognition [14]. Recently Bein et al. reported fluoride sensing by using the hybrid composite of the metal-organic framework NH2-MIL-101(Al) and fluorescein [15].

Herein, we report a selective and precise sensing of fluoride ions in a pure aqueous medium by a fluoride triggered release of pyrene fluorophore from Zirconium based MOF, UiO-66 [16]. First, in one step we have synthesized pyrene encapsulated UiO-66. Where the Zr-O or μ3-oxo bond of UiO-66 framework acts as a reactive probe and the pyrene molecule acts as a signal transductor. Upon encapsulating, the inside of the pore of the framework fluorescence of pyrene was found to be completely quenched. The addition of the fluoride

**Citation:** Dalapati, R.; Zang, L. Aqueous Medium Fluoride Anion Sensing by Fluorophore Encapsulated UiO-66 Type Zirconium Metal–Organic Framework. *Chem. Proc.* **2021**, *5*, 86. https://doi.org/10.3390/ CSAC2021-10551

Academic Editor: Ye Zhou

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/).

ion provoked the decomposition of the host UiO-66 and the released pyrene provides a turn-on fluorescence.

#### **2. Methods**

All the starting materials were of reagent grade and used as they were received from the commercial suppliers.

#### *2.1. Synthesis*

Syntheses of the pyrene containing UiO-66 framework were performed as reported by Biswas et al. with modification [17]. In brief, a mixture of ZrCl4 (72.24 mg, 0.31 mmol), Benzene-1,4-dicarboxylic acid (H2BDC) (0.31 mmol), pyrene (10 mg, 0.05 mmol), and formic acid (1.2 mL, 3.18 mmol) in Dimethylacetamide (DMA) (3 mL) was placed in a Pyrex tube. The tube was sealed and heated in a preheated heating block to 150 ◦C for 24 h. The reaction mixture was then cooled to room temperature. Finally, the precipitate was collected by filtration, washed with acetone, and dried in an air oven (60 ◦C).

#### *2.2. Fluorescence Titration Measurement*

For fluorescence titration measurements, a stock solution of pyrene@UiO-66 (1 mg/mL) was diluted in water (final concentration of 99 μg/mL) in a quartz glass cuvette at room temperature. A 4 mm solution of different anions was used. All the titration fluorescence emission was monitored using an excitation wavelength of 337 nm.

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

#### *3.1. Material Characterization*

Pyrene encapsulated UiO-66 (pyrene@UiO-66) was synthesized by a single step in situ encapsulation method. Initially, a certain amount of pyrene was added with ZrCl4 and H2BDC during the synthesis process. The pyrene@UiO-66 material was well characterized by various instrumental techniques, such as X-ray powder diffraction (XRPD), Fouriertransform infrared spectroscopy (FT-IR), and N2 sorption analysis.

XRPD experiments showed that UiO-66 and pyrene@UiO-66 possessed very similar XRPD patterns (Figure 1a). The similarities between the patterns simulated and found during the experiments of XRPD confirmed the formation of pure UiO-66.

**Figure 1.** (**a**) XRPD pattern of (i) pyrene@UiO-66; (ii) only UiO-66; (iii) simulated pattern of UiO-66 MOF. (**b**) Nitrogen adsorption (filled symbols) and desorption (empty symbols) isotherms of UiO-66 (circle) and pyrene@UiO-66 (square) collected at −196 ◦C.

The N2 sorption isotherms of pyrene@UiO-66 (Figure 1b) exhibited an insufficient decrease in the surface area, compared to UiO-66, which indicated the successful encapsulation of the pyrene into the pore framework of UiO-66. Pyrene encapsulated UiO-66

showed a surface area of 898 cm3/g, which was lower than the guest-free UiO-66 material (955 cm3/g).

#### *3.2. Anion Sensing Experiment*

The fluorescence emission spectra of pyrene@UiO-66 in water was recorded upon the gradual addition of sodium (Na+) salts of various anions (F−, Cl−, Br−, I−, NO2 −, NO3 −, AcO−, S2O3 <sup>2</sup>−, HSO3 −, SO4 <sup>2</sup>−, HSO4 −, SO3 <sup>2</sup>−, ClO4 −, SCN−, and HCO3 −). Figure 2 shows the "turn-on" response of F− anion towards pyrene@UiO-66 in water. There were almost no changes observed in the fluorescence emission spectra for all other anions. The bar plot in Figure 3a summarized the selectivity of pyrene@UiO-66 towards F− anion over all other anions.

**Figure 2.** Change in fluorescence intensity with the gradual addition of the F- solution to a suspension of pyrene@UiO-66 in aqueous medium.

**Figure 3.** (**a**) Change in the fluorescence intensity of pyrene@UiO-66 upon the incremental addition of different anions. (**b**) Change in the fluorescence intensity of pyrene@UiO-66 upon the addition of F- solution in the absence and presence of different anions. (F<sup>−</sup> (1), Cl<sup>−</sup> (2), Br<sup>−</sup> (3), I<sup>−</sup> (4), NO2 − (5), NO3 − (6), AcO− (7), S2O3 <sup>2</sup><sup>−</sup> (8), HSO3 − (9), SO4 <sup>2</sup><sup>−</sup> (10), HSO4 − (11), SO3 <sup>2</sup><sup>−</sup> (12), ClO4 − (13), SCN− (14), and HCO3 − (15)).

To examine the sensitivity of the pyrene@UiO-66 sensor material towards fluoride ions, even in the presence of other known interfering ions generally present in water, competitive experiments were performed by monitoring the fluorescence emission intensity of pyrene@UiO-66 in the absence and presence of other anions. During these experiments, solutions of interfering anions were added first to a water of pyrene@UiO-66, followed by

the addition of the F− anion. The change of the fluorescence intensity of pyrene@UiO-66 upon the addition of the F− anion, in absence and presence of other interfering anions, are displayed in Figure 3b. In all cases, the interfering anions did not show any interference in their sensing of the F− anion.

#### *3.3. Mechanism for Anion Sensing*

Until now, few mechanisms have been proposed for anion sensing via metal-organic framework. Some include: (1) anion induced coordination to metal-oxygen cluster [18], (2) hydrogen bonding formation with solvated framework [19], and (3) anion induced structural decomposition [15]. To understand the mechanism of fluoride sensing, XRPD and FT-IR measurement was carried out. To check the fluoride induced UiO-66 framework decomposition, MOF material was soaked in the fluoride anion solution. From Figure 4a it was shown that after the fluoride treatment, the characteristic diffraction peak for UiO-66 framework vanished, which confirmed the collapse of the framework in presence of fluoride. However, no change was observed in XRPD pattern after treatment with other anions in aqueous solution (data not shown).

**Figure 4.** (**a**) The XRPD pattern and (**b**) FT-IR spectra of pyrene@UiO-66 before and after fluoride treatment.

The UiO-66 structure consisted of an octahedron of zirconium atom. These octahedrons were capped by μ3-oxo and μ3-hydroxy groups in an alternating fashion. Carboxylate group from benzenedicarboxylate linker (H2BDC) connected these octahedral edges. The peak in FT-IR (between 1100–1000 cm<sup>−</sup>1) ~1020 cm−<sup>1</sup> could be assigned as Zr-OH bending vibration [20], which vanished after fluoride treatment (Figure 4b). Thus, the initial replacement of the hydroxyl group may be responsible for the fluoride sensitivity of the MOF. A peak in FT-IR near 747 cm−<sup>1</sup> and 663 cm−<sup>1</sup> was responsible for a Zr-μ3-oxo bond that almost disappeared, which also suggested fragmentation of the μ3-oxo bond. Observation suggested that fluorescence enhancement occurred via zirconium and fluoride coordination, which led to the release of pyrene from the framework host.

#### **4. Conclusions**

We have demonstrated that fluoride induced UiO-66 framework decomposition can be successfully used as a selective sensing probe for the same. Although this system is not reversible in nature, the simple one-step synthesis protocol, high stability, and low toxicity makes this material a promising candidate for fluoride sensing in an aqueous medium.

**Author Contributions:** Conceptualization, L.Z. and R.D.; methodology, R.D.; formal analysis, R.D.; investigation, R.D.; resources, R.D.; data curation, R.D.; writing—original draft preparation, R.D.; writing—review and editing, R.D.; visualization, R.D.; supervision, L.Z.; project administration, R.D.; funding acquisition, L.Z. All authors have read and agreed to the published version of the manuscript. **Funding:** The authors are highly grateful to University of Utah for instrumental facilities and other support.

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

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** The support from the University of Utah is acknowledged.

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

#### **References**


### *Proceeding Paper* **Customized Screen-Printed Electrodes Based on Ag-Nanoseeds for Enhanced Electroanalytical Response towards Cd(II), Pb(II) and As(V) in Aqueous Samples †**

**Karina Torres-Rivero 1,2,\* , Clara Pérez-Ràfols 3, Julio Bastos-Arrieta <sup>4</sup> , Núria Serrano 3,5 , Vicenç Martí 1,2,6 and Antonio Florido 1,2**

	- **\*** Correspondence: karina.torres.rivero@upc.edu
	- † Presented at the 1st International Electronic Conference on Chemical Sensors and Analytical Chemistry, 1–15 July 2021; Available online: https://csac2021.sciforum.net/.

**Abstract:** Electrochemical analysis based on screen-printed electrodes (SPEs) represents a great alternative to conventional analytical methods such as ICP-MS or LC-MS due to their portability, sensitivity, selectivity, and cost-effectiveness. In addition, the functionalization of SPEs with nanomaterials has been reported to provide an enhanced analytical performance. In this regard, silver nanoparticles (AgNPs) were synthesized and appropriately characterized, showing spherical silver nanoseeds (Ag-NS) with a diameter of 12.20 ± 0.04 nm. Using the drop-casting methodology, the synthesized AgNPs were used to modify screen-printed carbon nanofiber electrodes (SPCNFEs). Ag-NS deposition onto the electrode surface was confirmed by scanning electron microscopy (SEM). Furthermore, the analytical response of the modified electrodes (Ag-NS-SPCNFE) was evaluated for the determination of trace Pb(II), Cd(II), and As(V) using differential pulse anodic stripping voltammetry (DPASV), obtaining detection limits of 3.3, 3.7, and 2.6 μg L−1, for Pb(II), Cd(II) and As(V), respectively. Finally, Ag-NS-SPCNFE was tested towards the determination of As(V) in a spiked tap water sample, showing a good agreement with concentrations determined by ICP-MS.

**Keywords:** screen-printed electrodes; Ag nanoparticles; anodic stripping voltammetry; lead determination; cadmium determination; arsenic determination

#### **1. Introduction**

Water contamination caused by heavy metal ions (HMIs) is a concerning issue due to their high toxicity, non-biodegradability, bioaccumulation, and adverse health effects in humans [1]. In particular, for As, Cd, and Pb, the World Health Organization (WHO) has established the maximum allowed concentration in drinking water as 10 μg L−1, 3 μg L<sup>−</sup>1, and 10 μg L−1, respectively [2]. The determination of these low concentration levels requires very sensitive analytical techniques, such as flameless atomic adsorption spectroscopy (FAAS) [3], inductively coupled plasma mass spectrometry (ICP-MS) [4,5], and hydride generation atomic fluorescence spectrometry (HG-AFS) [6]. However, these

**Citation:** Torres-Rivero, K.; Pérez-Ràfols, C.; Bastos-Arrieta, J.; Serrano, N.; Martí, V.; Florido, A. Customized Screen-Printed Electrodes Based on Ag-Nanoseeds for Enhanced Electroanalytical Response towards Cd(II), Pb(II) and As(V) in Aqueous Samples. *Chem. Proc.* **2021**, *5*, 87. https://doi.org/ 10.3390/CSAC2021-10469

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/).

analytical techniques require expensive equipment and highly trained personnel, extended analysis time, and high operating costs.

In contrast, electrochemical techniques and anodic stripping voltammetry (ASV) allow relatively fast determination of trace HMIs with easy-handling and low-cost equipment [7,8]. In particular, electrochemical sensors represent a versatile tool for monitoring different samples in the environmental field. In addition, the literature has reported how modifying their surface with nanomaterials enhanced the electrochemical reactivity and sensitivity to specific analytes [9], allowing lower detection limits and higher sensitivity for stripping techniques [10–12].

The use of metallic nanoparticles (MNPs) to modify screen-printed electrodes (SPEs) can reduce the electron transfer resistance at the electrode surface, decreasing the electron transfer limited process and consequently catalyzing the electrode's response at low analyte concentrations [13–15]. Nanoparticles exhibit higher reactive surface influenced by the exposed atoms disposition resulting in more electrocatalytically active sites [16].

MNPs-modified sensors have been reported to allow the detection of arsenic [17,18], lead [19,20], and cadmium [21,22] at the level of a few μg L<sup>−</sup>1, fulfilling the WHO guidelines for drinking-water quality [2]. These sensors were based on screen-printing technology, offering significant advantages over conventional voltammetric sensors such as low-cost, disposable character, portability, and commercial availability [1,9]. Thus, in this work, the voltammetric determination of HMIs, based on the use of carbon-nanofiber-based screenprinted electrodes (SPCNFEs) modified with silver nanoparticles (Ag-NPs), is proposed. Ag-NPs were synthesized in the shape of silver nanoseeds (Ag-NS), and the resulting modified electrodes were microscopically and analytically characterized for the determination of As(V), Pb(II), and Cd(II) by means of differential pulse anodic stripping voltammetry (DPASV). In addition, the applicability to real sample analysis was demonstrated through the direct determination of As(V) in spiked tap water samples.

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

#### *2.1. Apparatus*

DPASV measurements were carried out with either a Multi Autolab/M204 Modular Multi Potentiostat/Galvanostat or an Autolab PGSTAT204, attached to a Metrohm 663 VA Stand, as well as a personal computer with NOVA 2.1 software package to control the potentiostat and perform the required data treatment. All the electrochemical instrumentations and software were acquired from Metrohm (Herisau, Switzerland).

A Crison Basic 20 pH meter (Hach Lange Spain, L'Hospitalet de Llobregat, Spain) was used for pH measurements.

Ag-NS and SPCNFE modified with Ag-NS electrodes were characterized using a JEM-2010 transmission electron microscope (TEM) from JEOL (Tokyo, Japan) and a Gemini scanning electron microscope (SEM) from ZEISS® (Jena, Germany), respectively. Size distribution histograms were calculated using the ImageJ version 1.51 m software by the National Institute of Health (NIH, Bethesda, MD, USA).

ICP-MS measurements were carried out by means of inductively coupled plasma mass spectrometer model 7800 by Agilent Technologies (Santa Clara, CA, USA).

Commercial SPCNFEs, including working (4 mm disk), counter, and reference electrodes, were purchased from Dropsens (Llanera, Spain).

#### *2.2. Preparation of Working Electrodes*

The working electrode (WE) was a SPCNFE modified with silver nanoseeds (Ag-NS-SPCNFE). Ag-NS were first synthesized following a seed-mediated methodology, combining aqueous trisodium citrate (5 mL, 2.5 mmol L−1), aqueous poly sodium styrenesulfonate (PSSS) (0.25 mL, 500 mg L−1), aqueous sodium borohydride (NaBH4) (0.3 mL, 10 mmol L<sup>−</sup>1) freshly prepared, followed by the addition of aqueous silver nitrate (AgNO3) (5 mL, 0.5 mmol L−1) using a syringe pump at a rate of 2 mL min−<sup>1</sup> under continuous

stirring [12,23]. Then, SPCNFEs were modified by drop-casting, dropping 40 μL of Ag-NS onto the working electrode, and evaporating the solvent at 50 ◦C for 30 min.

#### *2.3. Electrochemical Measurements*

DPASV measurements of Pb(II) and Cd(II) were carried out at a deposition potential (Ed) of −1.4 V, applied under stirring conditions during a deposition time (td) of 180 s in 0.1 mol L−<sup>1</sup> acetate buffer (pH 4.5) and scanning the potential from −1.4 to 0.0 V. For As(V) determination, the experimental conditions used were Ed of −1.3 V and td of 120 s in 0.01 mol L−<sup>1</sup> HCl pH 2 with a potential scan from −1.3 to −0.65 V.

A step potential of 5 mV, a pulse time of 50 ms, and a pulse amplitude of 50 mV were employed in all cases. All experiments were performed at room temperature (22 ± 1 ◦C) and without oxygen removal.

For real sample analysis, tap water samples were collected from the local water distribution network in Barcelona (Spain) and spiked with 20 μg L−<sup>1</sup> of As(V). Prior to electrochemical analysis, water samples were diluted and acidified with 0.01 mol L−<sup>1</sup> of HCl (pH 2.0), resulting in a final concentration of 10 μg L−<sup>1</sup> of As(V). Sample analysis was carried out by means of the standard addition method, performing four successive As(V) additions from a standard solution of 1 mg L−1. DPASV measurements were recorded under the above-mentioned electrochemical conditions.

#### **3. Results**

#### *3.1. Microscopic Characterization*

Ag-NS synthesis was microscopically confirmed by both SEM (Figure 1a) and TEM (Figure 1b). As it can be deduced from the TEM image, most Ag-NS presented a spherical shape. On the other hand, SEM images were used to calculate the corresponding size distribution histogram (Figure 1c), which was computed from 400 Ag-NSs. The obtained results show that the synthesized Ag-NS presented an average diameter of 12.2 ± 0.4 nm. These structures are in good agreement with the reported shapes of Ag-NPs [24,25].

**Figure 1.** Microscopic characterization of Ag-Nanoseeds (**a**) SEM micrograph. (**b**) TEM micrograph and (**c**) corresponding size distribution histogram [15].

SEM micrographs were also obtained for a bare SPCNFE (Figure 2a) and an Ag-NS-SPCNFE (Figure 2b) to assess the modification of SPCNFEs by drop-casting. Compared to the non-modified carbon nanofiber surface of the bare electrode, Ag-NS can be spotted as white dots deposited onto the carbon nanofibers in the modified electrode (Ag-NS-SPCNFE), thus confirming the successful modification of the working electrode.

**Figure 2.** SEM micrographs for (**a**) Bare SPCNFE, and (**b**) Ag-NS-SPCNFE modified using the drop-casting methodology [12,15].

#### *3.2. Electrochemical Characterization*

DPASV measurements were carried out in solutions containing either Pb(II), Cd(II), or As(V). Well-defined voltammetric peaks were obtained in all cases, with peak potentials of ca. −0.65 V, −0.75 V, and −1.0 V for Pb(II), Cd(II), and As(V), respectively (see Figure 3a).

**Figure 3.** (**a**) DPASV voltammograms of Pb(II), Cd(II), and As(V) at 25 μg L−<sup>1</sup> and (**b**) their calibration plots at the previously mentioned conditions using Ag-NS-SPCNFE.

Individual calibration curves of Pb(II), Cd(II), and As(V) were obtained by DPASV by increasing metal ion concentration in the ranges 1.9 to 150.0 μg L<sup>−</sup>1, 0.6 to 120.6 μg L<sup>−</sup>1, and 1.0 to 50.1 μg L<sup>−</sup>1, respectively. The obtained data were used to calculate the corresponding analytical parameters (i.e., sensitivity, limit of detection (LOD), limit of quantification (LOQ), and linear range), which are displayed in Table 1.

**Table 1.** Calibration data for the individual determination of Pb(II), Cd(II), and As(V) using Ag-NS-SPCNFE and the corresponding buffer and DPASV parameters (see the experimental section for more details).


<sup>a</sup> The lowest value of the linear range corresponds to the LOQ. <sup>b</sup> The standard deviations are expressed in parentheses.

From this data, LODs and LOQs were calculated by using the Miller and Miller procedure [26,27].

As shown in Table 1, good linear response between the peak heights and the concentration of the different analytes was achieved using the Ag-NS-SPCNFE. LODs were at μg L−<sup>1</sup> levels in all cases, and similar or even lower to other LODs reported in the literature. For example, LODs of 3.30 and 4.43 μg L−<sup>1</sup> for Pb(II) and Cd(II), respectively, were reported using a graphene/polyaniline/polystyrene (G/PANI/PS) nanoporous fibermodified screen-printed carbon electrode [28]. Additionally, the obtained LOD for As(V) is considerably lower than that reported using boron-doped diamond electrodes and ASV (12 μg L−1) [29]. However, it is important to mention that Ag-NS-SPCNFE for the determination of As(V) presented a more restricted linear range in which the highest value is limited to a lower concentration value (until 40.0 μg L<sup>−</sup>1), compared to the one reached by Nagaoka et al. (until 100 μg L<sup>−</sup>1) [29].

In terms of sensitivities (nA μg−<sup>1</sup> L), which were calculated as the slope of the calibration curves, Ag-NS-SPCNFE exhibited higher sensitivity toward As(V) (260 nA μg−<sup>1</sup> L). In the case of Pb(II) and Cd(II), the sensitivities were significantly lower (103 and 22 nA μg−<sup>1</sup> L, respectively).

#### *3.3. Application to the Analysis of Spiked Tap Water*

The applicability of Ag-NS-SPCNFE for real sample determination was evaluated through the determination of As(V) in a spiked tap water sample. The determination of As(V) was performed in triplicate by the standard addition calibration method. Representative voltammograms are shown in Figure 4. As it can be observed, a well-shaped As(V) peak and a good correlation between peak area and concentration were acquired. Sample concentration calculated by extrapolation was 10.04 μg L−<sup>1</sup> (SD:0.37 μg L−1), which is in good agreement with values obtained by ICP-MS (10.7 μg L−1, SD:0.20 μg L−1), as an analytical reference technique. These results confirm the suitability of Ag-NS-SPCNFE for the analysis of real samples.

**Figure 4.** DPASV measurements of As(V) in a spiked tap water sample on Ag-NS-SPCNFE at pH 2.0 applying an Ed of −1.30 V and a td of 120 s. Inset: As(V) standard addition plot [12].

#### **4. Conclusions**

In this work, a DPASV method for the determination of trace Pb(II), Cd(II), and As(V) based on the modification of SPCNFE with Ag-NS has been proposed. The Ag-NSs were synthesized, microscopically characterized, and used for the modification of SPCNFEs.

The analytical performance of the modified electrode was evaluated for the three studied analytes. It was demonstrated that Ag-NS-SPCNFE is suitable for determining Pb(II), Cd(II), and As(V) at low μg L−<sup>1</sup> levels, showing wider linear ranges for Pb(II) and Cd(II) but lower sensitivities as compared to As(V). Regarding previous studies of Pb(II), Cd(II), and As(V) determination, the LODs achieved in this investigation are equal or lower than other LODs previously reported.

The suitability of Ag-NS-SPCNFE for real sample analysis was demonstrated for the determination of As(V) in spiked water samples, achieving comparable results to those obtained by ICP-MS measurements with good reproducibility.

**Author Contributions:** K.T.-R., J.B.-A. and A.F. carried out the synthesis and microscopic characterization of silver nanoparticles. K.T.-R., C.P.-R. and N.S. carried out the modification of screen-printed electrodes with nanoparticles, the voltammetry measurements, and the data treatment. All authors contributed to the writing, revision, and critical discussion of the results presented in the final version of the manuscript. A.F., V.M. and N.S. were responsible for the student supervision. A.F. and V.M. dealt with the project administration and funding acquisition. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research has been funded by Ministerio de Ciencia, Innovación y Universidades, and European Union Funds for Regional Development (FEDER), projects CTM2015-68859-C2-2-R and CGL2017-87216-C4-3-R, as well as by the Generalitat de Catalunya (Project 2017SGR312 and 2017SGR-311).

**Data Availability Statement:** Not applicable.

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

#### **References**


#### *Proceeding Paper*
