An Improved Multiple Competitive Immuno-SERS Sensing Platform and Its Application in Rapid Field Chemical Toxin Screening

Improving the signal-to-noise ratio (SNR) by amplifying the outputting signal or reducing nonspecific binding (NSB) are the key techniques in multiple immunoassay. Aiming at these issues, this paper presents an improved multiple indirect competitive immune surface-enhanced Raman scattering (ci-SERS) assay for the rapid screening of highly toxic rodenticides in food and biological samples, which ensured remarkable accuracy, ultra-sensitivity and reproducibility. The non-fouling polymer brush grafted magnetic beads (the MB@P-CyM) were prepared as multiple competitive recognition substrates after conjugating triplex haptens (the MB@P-CyM-hap). It was demonstrated that the particular 3D hair-like structures of P-CyM not only facilitate conjugate high-density hapten but reduce the steric hindrance from SERS probes recognition, thus enhancing SNB. On the other hand, Au nanoflowers (AuNFs) of high SERS activity were synthesized using a simple one-pot hydrazine reduction. For simultaneously detecting three highly toxic rodenticides, i.e., diphacinone (DPN), bromadiolone (BRD) and tetramine (TET), the obtained AuNFs were fabricated as a SERS-encoded nanoprobe cocktail after successively labeling mono-antibodies/Raman probes. By integrating the MB@P-CyM-hap with the SERS-encoded cocktail, a highly sensitive multiple SERS assay was achieved in less than 2 h with a limit of detection of 0.62 ng mL−1 for BRD, 0.42 ng mL−1 for TET and 1.37 ng mL−1 for DPN, respectively. The recoveries of these rodenticides in spiked food and biological samples were determined and ranged from 72 to 123%. Above all, the proposed modifications show remarkable improvements for high efficient multiple chemical toxin immunoassay.

Introduction
The simple, accurate and rapid screening multiple analytes of interest plays a critical role in early disease diagnosis, food safety supervision as well as environmental surveillance [1][2][3]. Considering the complexity of the real food and biological samples, the key issue for various immune-sensors is in improving the signal-to-noise ratio (SNR), either by amplifying outputting signal or reducing nonspecific binding (NSB), causing false results [4]. Simultaneously, simplifying testing procedures and improving testing speeds are also highly desired. Currently, various highly sensitive signal reporting modules are putting forth new ideas [5,6]. However, strategies to reduce nonspecific binding in the sensing processes are slightly insufficient, [7,8] due to lacking adequate and deep understanding on adsorption/desorption, energy exchange as well as electron transfer occurred Toxics 2022, 10, 605 2 of 11 on nano-interfaces. Surface-enhanced Raman spectroscopy (SERS) is considered as an ideal tool for multiplex detection owing to its technical simplicity, excellent sensitivity and superior encoding capability [9,10]. By synthesizing highly sensitive SERS encoders with anisotropic plasmonic nanostructures, SERS-based multiplex competitive immunoassays have been proposed [11][12][13][14]. However, the immunoassays are proposed in the native "noisy" environments of lower fouling, and improving reproducibility is still not completely realized [7,15,16].
Chemical toxins-induced food poisoning accounted for more than 80% of the total foodborne deaths in China [17]. Among all of the chemical toxins, rodenticides, represented by diphacinone (DPN) [18], bromadiolone (BRD) [19] and tetramine (TET) [20], were the most common toxics, which have caused thousands of accidental or intentional poisoning events [21]. As beneficial complements for instrument assay [22], field screening these rodenticides in one simple test could not only remarkably improve treating efficiency, but it is also in line with future demands for rapid, cheap and high-throughput field screening toxins.
Benefiting from their adjustable properties and flexible spatial structures, non-fouling polymer brushes offer attractive alternatives to engineer sensing interfaces for enhancing specific molecular recognition, rejecting nonspecific adsorption, amplifying the detecting signal as well as maintaining signal stability in complex working matrices [23,24]. In this work, an improved multiple competitive immuno-SERS assay was proposed for rapid screening DPN, BRD and TET (the chemical structures of these toxins are shown in Scheme 1A) in a single no-wash test. A non-fouling polymer brush grafted with magnetic beads (MBs) which were appended with triplex haptens for DPN, BRD, and TET (the chemical structures of these haptens are shown in Scheme 1B) was constructed as a multiple competitive substrate (the MB@P-CyM-hap). It was demonstrated that the hair-like P-CyM acted as the scaffold for conjugating haptens and could remarkably enhance its specific capture capacity for SERS probes by relieving steric hindrance as well as reducing nonspecific adsorption. For enhancing sensitivity, Au nanoflowers (AuNFs) were prepared using a one-step hydrazine reduction in aqueous solutions. The obtained AuNFs were recognized as an outstanding SERS substrate featuring high activity, easy preparation and good reproducibility. After successively labeling with the mono-antibodies (mAb)/Raman probe pairs, a specific SERS probe cocktail for sensing DPN, BRD and TET was constructed. By combining the MB@P-CyM-hap with the SERS sensing cocktail, a rapid and accurate rodenticide screening in a wide range of real samples was achieved. Under the optimized condition, if there are no toxins in the system, all of the SERS probes would bind with the hapten-modified MB and then remove them from the system. Thus, the supernatant shows no SERS signal. On the contrary, when toxins are present in the system, they would bind with the SERS probes preferentially, thus blocking the SERS probe from binding with the hapten modified-MB, which makes the supernatant show a distinct SERS signal (as shown in Scheme 1F,G). Scheme 1. Chemical structures of (A) three rodenticides and (B) their corresponding haptens; (C) polymerization of the CyM on the MB based on the RAFT strategy; (D) synthesis of the AuNFs and their following label processes for constructing the specific SERS-encoding cocktail; (E) the preparation of the MB@P-CyM-hap; (F,G) the sensing principle was based on the multiple competitive immunoassay format: simultaneously mixing the mAb-labeled SERS encoding cocktail and the MB@P-CyM-hap with the rodenticide-spiked sample solutions. After reaching the recognition equilibrium, a magnet field was used to remove the MB@P-CyM-hap/SERS probes from the solutions, and the supernatant-containing SERS cocktails were collected for quantification.
The extinction spectrum was recorded on a Shimadzu UV 3600 UV-vis-NIR spectrophotometer. The morphologies of various nanostructures were observed on a JEM-2100F transmission electron microscope (JEOL Ltd., Tokyo, Japan). A physical property measurement device (Cryogenic, 12 T Magnet) was used to characterize the magnetic properties of the MBs. Thermogravimetric analysis (TGA) was performed with a TGA Instruments device (Model TGA Q500) from room temperature to 800 °C at a heating rate of 10 °C per min under nitrogen gas (40 mL per min). An attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR, INVENIO Bruker) was used to characterize the MBs. The concentration of the AuNFs and the MBs was measured on an ICP-MS (Agilent 8800) and an ICP-OES (IRIS Advantage, Thermo Scientific, St. Louis, MO, USA), respectively. Scheme 1. Chemical structures of (A) three rodenticides and (B) their corresponding haptens; (C) polymerization of the CyM on the MB based on the RAFT strategy; (D) synthesis of the AuNFs and their following label processes for constructing the specific SERS-encoding cocktail; (E) the preparation of the MB@P-CyM-hap; (F,G) the sensing principle was based on the multiple competitive immunoassay format: simultaneously mixing the mAb-labeled SERS encoding cocktail and the MB@P-CyM-hap with the rodenticide-spiked sample solutions. After reaching the recognition equilibrium, a magnet field was used to remove the MB@P-CyM-hap/SERS probes from the solutions, and the supernatant-containing SERS cocktails were collected for quantification.
The extinction spectrum was recorded on a Shimadzu UV 3600 UV-vis-NIR spectrophotometer. The morphologies of various nanostructures were observed on a JEM-2100F transmission electron microscope (JEOL Ltd., Tokyo, Japan). A physical property measurement device (Cryogenic, 12 T Magnet) was used to characterize the magnetic properties of the MBs. Thermogravimetric analysis (TGA) was performed with a TGA Instruments device (Model TGA Q500) from room temperature to 800 • C at a heating rate of 10 • C per min under nitrogen gas (40 mL per min). An attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR, INVENIO Bruker) was used to characterize the MBs. The concentration of the AuNFs and the MBs was measured on an ICP-MS (Agilent 8800) and an ICP-OES (IRIS Advantage, Thermo Scientific, St. Louis, MO, USA), respectively.

Preparation of the MB@P-CyM-hap as Multiple Competitive Substrate
The synthesis of SiO 2 -capped Fe 3 O 4 magnetic beads (MBs) was conducted referring to the previously reported method [25]. The polymer monomer, named cysteine methacrylate (CyM), was synthesized according to the reported methods [26]. The non-fouling polymer brush-grafted MBs (the MB@P-CyM) were synthesized via a typical RAFT process [27]. For grafting the CyM polymer brushes from the MBs, CTA-MBs (0.1 g), AIBN (20 mg) and the CysMA monomer (0.1 g) were dissolved in 10 mL of degassed water/methanol (1:1) solution. After N 2 bubbling for 30 min, the system was sealed and heated under 80 • C. After reaction for 5 h, the MB@P-CyM were collected and then washed by DMF 3 times (as shown in Scheme 1C).

Chemical Conjugation of Triple Haptens on the MB@P-CyM
The non-fouling MB@P-CyM was chemically conjugated with triple hapten (as shown in Scheme 1E) via the EDC/NHS mediated amino-carboxyl coupling reaction. The triple haptens were simultaneously linked on the MB@P-CyM with the feed molar ratio of 1:1:1. The triple hapten mixture with a total amount of 1 mmol L −1 was firstly activated by NHS (10 mg mL −1 ) and EDC (20 mg mL −1 ) in 1.0 mL methanol. After reacting for 60 min, the MB@P-CyM (0.1 g in 5 mL methanol) were added in the solution for conjugation. The reaction was processed for 10 h under room temperature, and then, the liquid supernatant was removed. The obtained MB@P-CyM-hap was washed with acetone repeatedly and finally dispersed in the PBS buffer for further use.
For achieving the competitive substrate with different hapten density, three groups of the hapten mixtures (10, 5, 1 mmol L −1 in 1 mL methanol) with the mole ratio of 1:1:1 were linked on the MB@P-CyM to fabricate high (the MB@P-CyM-hap1), middle (the MB@P-CyM-hap2), and low (the MB@P-CyM-hap3) densities, respectively, and then, we tested their binding capacity with the specific SERS encoders.
Moreover, the amino-derived MBs was also prepared for modifying triple haptens (the MB@hap), and we examined the specific binding capacity. Both the MB@P-CyM-hap and the MB-hap were prepared to share the same Fe concentrations as determined by ICP-OES.

Constructing the mAb-Labeled SERS Probe Cocktail
The AuNFs were prepared using hydrazine reduction in aqueous phase at room temperature for the first time, which was regarded as a seedless and surfactant-free method (as shown in Scheme 1D). In detail, 2.0 mL of 50.0 mmoL −1 HAuCl 4 was mixed with 0.5 mL of 10.0 mmoL −1 TA ethanol solution in 100 mL of D.I. water. After stirring for 5 min, 0.5 mL of the freshly N 2 H 4 aqueous solution was rapidly injected to the solution. The color of the solution changed to blue immediately, indicating the formation of the AuNFs. After standing for 2 h at room temperature, the AuNFs were purified by ultra-centrifuging (8000 rpm for 5 min) and washed twice with water, finally dispersing in 10 mL D.I. water.

Detection of DPN, BRD and TET in Buffer
For examining the sensing performances, 1.0-10.0 µL of the standard toxins was added to the MB@P-CyM-hap or the MB-hap solutions (50 µL), and then, their corresponding SERS probe cocktails were added. After 30 min, the free SERS probe solutions were collected after magnetic separation for Raman interrogation. The results were analyzed by linear regression and used to verify the accuracy of the immunoassay. The selectivity of the assay was examined by using chemical analogue (0.5 µg mL −1 ), and the subsequent SERS measurements were processed consistently with the above procedures. The detailed sample preparation and measurements are given in the Supplementary Materials.

Screening DPN, BRD and TET in Spiked Biological and Food Matrices
The SERS-encoded cocktail (100 µL) was prepared by mixing the AuNF@PEG/TSA-mAb BRD , the AuNF@PEG/DTNB-mAb TET and the AuNF@PEG/DMPI-mAb DPN with the final molar ratio of 2:1:3, and then, the MB@P-CyM-hap solutions (50 µL) were added in EP tubes separately. The negative human serum and urine samples were supplied by the Beijing Center for Disease Prevention and Control (Beijing, China). The negative food samples including milk and chicken were purchased locally. For method validation, different concentrations of BRD, DPN and TET standards were spiked into the real samples to the final concentrations of 5.0 ng mL −1 and 10 ng mL −1 , respectively. Briefly, 1.0 mL or 1 g of samples was extracted with ethyl acetate (1.0 mL) on the vortex for 5 min, and the mixture was centrifuged for 5 min at 3500× g. After being dried using nitrogen flow, the dried residue was reconstituted with PBS for measurement.

SERS Measurement
A Raman spectrometer (inVia Renishaw, UK) was utilized for SERS measurements. The mAb-labeled SERS probe cocktail was dispersed on quartz cells and then measured under the laser excitation wavelength of 785 nm. The laser output was performed by a 50 L × 0.3 NA air objective lens. For each measurement, the laser power and exposure time were set at 10 mW and 5 s exposure with 10 accumulation, respectively.

Characterizations of the Non-Fouling MB@P-CyM-hap
Surface geometries and chemical compositions of nanostructures have close relations with affinity recognitions [5]. In-depth research on the properties of the sensing interface are vital for constructing nanosensors [6,7]. Different from small molecule probes, SERS nanoprobes often possess tens of nanometers, the effect of steric hindrances on recognition and binding should be considered. The P-CyM is verified as an attractive non-fouling interface which contains abundant active sites for conjugating hapten. In this work, the P-CyM brushes were grafted from the MBs using a typical RAFT strategy. As shown in Figure 1A-C, the MB@P-CyM shows good dispersion and uniform morphologies with the inner core of ≈110 nm in diameter, the middle SiO 2 protecting layer of ≈15 nm and the outer P-CyM brushes of ≈20 nm. The hydrophilic P-CyM made the MB@P-CyM have good stability in aqueous solution. Moreover, the saturation magnetization of the MB@P-CyM is measured as 30.5 emu g −1 . The magnetization curves exhibit symmetry and passed through the origin, which ensures a facile separation and reusability of the MB@P-CyM from sample matrices ( Figure 1D). FT-IR spectra ( Figure 1E) of the MB@P-CyM are collected and compared with their precursors. The strong peaks around 1080 cm −1 assigned to Si-O are observed both in the MBs and the MB@P-CyM. Specifically, the stretching vibration of −CH 2 −, −COOH, −amino and the symmetric stretching vibration of C-O associated with P-CyM ligand appear at 2950 cm −1 , 1720 cm −1 , 1340 cm −1 and 1625 cm −1 , respectively in the MB@P-CyM spectra. According to TGA analysis, a distinct mass loss of ≈14% is observed in the MB@P-CyM, which is higher than that of the NH 2 -MBs of ≈2% weight loss ( Figure 1F). exhibits a similar unspecific fouling to that of the MB@P-CyM-hap3. It is found that the MB@P-CyM-hap2 reaches a saturated adsorption within 30 min, suggesting that the flexible hair-like interface of the MB@P-CyM-hap2 facilitated the synergy affinity by reducing steric hindrance. As shown in Figure 1G, the MB@P-CyM-hap2 shows weak interaction with the nonspecific protein (HSA). Moreover, after storing under RT for 6 months under the freeze-dried state, the MB@P-CyM-hap2 shows no obvious adsorption drop, indicating their long-term chemical stability in aqueous solution. All of these results emphasize the vital role of the P-CyM brushes as the recognition interface on the MB. Comprehensively considering these results, the MB@P-CyM-hap2 is selected for further SERS sensing applications.  The MB@P-CyM comprise abundant primary amino groups which enable convenient conjugation with triplex haptens with adjustable density. Different amounts of the hapten mixtures of the mole ratio of 1:1:1 were covalently linked on the MB@P-CyM with low (the MB@P-CyM-hap1), middle (the MB@P-CyM-hap2) and high (the MB@P-CyM-hap3) densities. As a control, the amino-MBs without grafting P-CyM were also conjugated with triple haptens (the MB@hap). For comparing the binding capacities of different modified MB, their concentrations were set as the same levels (in terms of Fe ions). Their specific bindings with the total mAb mixture as well as the nonspecific protein (HSA) were examined. It is found that the adsorption capacities of hapten-modified MB@ P-CyM for the triplet mAb mixture improved with the increase in the hapten density ( Figure 1G). Under physiological condition, the MB@P-CyM-hap2 and the MB@P-CyM-hap3 show comparable binding contents for the total mAb, demonstrating the steric hindrance restrict saturating absorption. For all of these, the MB@P-CyM-hap show a better adsorption capacity than the MB@hap. The anti-fouling performance derived from the P-CyM shows degradation due to the hapten conjugation-induced polarity changes, as it is reflected that both the MB@P-CyM-hap1 and the MB@P-CyM-hap2 exhibit strong resistance to biofouling, with the nonspecific protein absorption as low as 1.2 µg g −1 , but the value increases to 5.4 µg g −1 for the MB@P-CyM-hap3 ( Figure 1G). The MB@hap without grafting the P-CyM exhibits a similar unspecific fouling to that of the MB@P-CyM-hap3. It is found that the MB@P-CyM-hap2 reaches a saturated adsorption within 30 min, suggesting that the flexible hair-like interface of the MB@P-CyM-hap2 facilitated the synergy affinity by reducing steric hindrance. As shown in Figure 1G, the MB@P-CyM-hap2 shows weak interaction with the nonspecific protein (HSA). Moreover, after storing under RT for 6 months under the freeze-dried state, the MB@P-CyM-hap2 shows no obvious adsorption drop, indicating their long-term chemical stability in aqueous solution. All of these results emphasize the vital role of the P-CyM brushes as the recognition interface on the MB. Comprehensively considering these results, the MB@P-CyM-hap2 is selected for further SERS sensing applications.

Fabricating the Specific SERS-Encoding Probes and Examining Their Sensing Capacities
Both experimental and theoretical research have indicated that shapes can determine the properties of particular nanostructures such as sizes. In this paper, uniform AuNFs were synthesized by one-step reduction in an aqueous phase under room temperature. The formation mechanism of the AuNFs is depicted in Scheme 1D. The oxidation product of N 2 H 4 , i.e., N 2 , was generated in aqueous solution, interfering with the growth of Au seeds; thus, the flower-like Au nanostructure is obtained. The AuNFs were completely formed within 30 s and stayed the same afterwards. As shown in the TEM images (Figure 2A), the Au NFs are monodispersed with size distribution between 40 and 70 nm ( Figure 2E). EDS results evidence the sole Au element component of these nanoparticles (Figure 2B,D). The HR-TEM image of the AuNFs is also given in Figure 2B, where the two lattice fringes with the d-spacing of 0.236 and 0.204 nm corresponding to the (111) and (200) planar distances of Au are shown. As shown in Figure 2F, the extinction spectrum of the AuNFs exhibits a distinctive surface plasma resonance (SPR) band at around 570 nm and showed blue color. It indicated that the AuNFs can remarkably enhance the Raman signal of probes (TSA in this research) by having a large surface-to-volume ratio, surface roughness as well as a plethora of nanogaps, which is more sensitive than that of the spherical AuNP of similar diameters. Statistically, the SERS enhancement of different batches of the AuNFs shows a relative standard deviation of 6.7%, indicating that the achieved AuNFs have well reproducible SERS performance.
AuNFs exhibits a distinctive surface plasma resonance (SPR) band at around 570 nm and showed blue color. It indicated that the AuNFs can remarkably enhance the Raman signal of probes (TSA in this research) by having a large surface-to-volume ratio, surface roughness as well as a plethora of nanogaps, which is more sensitive than that of the spherical AuNP of similar diameters. Statistically, the SERS enhancement of different batches of the AuNFs shows a relative standard deviation of 6.7%, indicating that the achieved AuNFs have well reproducible SERS performance.
In the SERS barcoding system, the AuNF@PEG was functionalized with different pairs of mAb and Raman reporters, i.e., the mAbBRD/TSA, the mAbTET/DTNB as well as the mAbDPN/DMPI, respectively. Comparing the original AuNFs with the SPR peak at 572 nm, it slightly shifts to 590 nm after successively labeling the Raman reporters and the mAb ( Figure 2F). This result suggests that both encoding and ligand grafting procedures did not detriment the colloidal stability. For simultaneously screening multiple rodenticides, three typical Raman probes (MBA, DTNB, and DMPI) that generated unique Raman signals upon laser excitation were selected to encode SERS probes for BRD, TET and DPN, respectively. The SERS signals of each AuNF encoder and their mixtures are demonstrated in Figure 2G. It is found that three sensitive and non-interfering SERS peaks are clearly observed, which demonstrate that the cocktail of these SERS nanoprobes could serve as an effective tool for multiplex target screening. These specific SERS nanoprobes have similar negative charges around −24.1 ± 0.3 mV due to the presence of TA ligands, and the PEG ligand could effectively maintain the stability of them in the complex biological media, as it demonstrated that no obvious aggregation of the AuNFs is found in PBS or 10 times diluted human serum for over 1 month. In the SERS barcoding system, the AuNF@PEG was functionalized with different pairs of mAb and Raman reporters, i.e., the mAb BRD /TSA, the mAb TET /DTNB as well as the mAb DPN /DMPI, respectively. Comparing the original AuNFs with the SPR peak at 572 nm, it slightly shifts to 590 nm after successively labeling the Raman reporters and the mAb ( Figure 2F). This result suggests that both encoding and ligand grafting procedures did not detriment the colloidal stability. For simultaneously screening multiple rodenticides, three typical Raman probes (MBA, DTNB, and DMPI) that generated unique Raman signals upon laser excitation were selected to encode SERS probes for BRD, TET and DPN, respectively. The SERS signals of each AuNF encoder and their mixtures are demonstrated in Figure 2G. It is found that three sensitive and non-interfering SERS peaks are clearly observed, which demonstrate that the cocktail of these SERS nanoprobes could serve as an effective tool for multiplex target screening. These specific SERS nanoprobes have similar negative charges around −24.1 ± 0.3 mV due to the presence of TA ligands, and the PEG ligand could effectively maintain the stability of them in the complex biological media, as it demonstrated that no obvious aggregation of the AuNFs is found in PBS or 10 times diluted human serum for over 1 month.

Sensing Performance of This Assay
For further verifying the advantages of this modified SERS platform, we examined the sensitivity and selectivity of the assay. As expected, by integrating the MB@P-CyM-hap2 with the SERS probes in this assay, gradually, SERS signal restorations as a function of the increasing concentration of targets are observed, as shown in Figure 3A-C. Similarly, SERS signal-enhancing trends are observed for TET, BRD and DPN, suggesting the potentials of quantitative analysis. Multiple calibration curves are plotted between peak intensities at 1034, 1332, and 2174 cm −1 , i.e., the AuNF@PEG/TSA-mAb BRD at 1034 cm −1 for BRD from 1.0 to 80 ng mL −1 (I 1034 = 1896 × lgC BRD + 3407) with a squared correlation coefficient (R 2 ) of 0.962, and the AuNF@PEG/DTNB-mAb TET at 1332 cm −1 for TET from 0.5 to 50 ng mL −1 (I 1332 = 2367 × lgC TET + 2573, R 2 = 0.988) and the AuNF@PEG/DMPI-mAb DPN at 2174 cm −1 for DPN from 2 to 120 ng mL −1 (I 2174 = 2625 × lgC DPN + 1154, R 2 = 0.95) ( Figure 3). In addition, limits of detection (LOD) were calculated as 0.62 ng mL −1 for BRD, 0.42 ng mL −1 for TET and 1.37 ng mL −1 for DPN based on three times the standard deviations of the control signals, which could meet the requirement of fast food poisoning screening. Moreover, TEM images also provide the evidence of forming the nanoassemblies (SERS nanoprobes binding on the surfaces of the MB@P-CyM-hap2) in the presence of different targets, which illustrates their successful binding ( Figure 3G). The inter-and intraday precisions are monitored as less than 16.24%. On the contrary, the sensing performance using the MB@hap under the same detecting conditions is not good as the MB@P-CyM-hap2 in the aspects of RSD, LOD and linear ranges, suggesting the outstanding advantages of using the non-fouling competitive interfaces. This results reveal that attributing to the particular 3D hair-like structures, the P-CyM interface not only facilitates conjugate highdensity hapten but reduces the steric hindrance between the SERS probes, thus enhancing their recognitions and sensing capacity. Moreover, due to the specificity of the mAb and the introduction of the non-fouling sensing interface, the selectivity of the assay is satisfactory ( Figure 3H).

Sensing Performance of this Assay
For further verifying the advantages of this modified SERS platform, we examined the sensitivity and selectivity of the assay. As expected, by integrating the MB@P-CyM-hap2 with the SERS probes in this assay, gradually, SERS signal restorations as a function of the increasing concentration of targets are observed, as shown in Figure 3A-C. Similarly, SERS signal-enhancing trends are observed for TET, BRD and DPN, suggesting the potentials of quantitative analysis. Multiple calibration curves are plotted between peak intensities at 1034, 1332, and 2174 cm −1 , i.e., the AuNF@PEG/TSA-mAbBRD at 1034 cm −1 for BRD from 1.0 to 80 ng mL −1 (I1034 = 1896 × lgCBRD + 3407) with a squared correlation coefficient (R 2 ) of 0.962, and the AuNF@PEG/DTNB-mAbTET at 1332 cm −1 for TET from 0.5 to 50 ng mL −1 (I1332 = 2367 × lgCTET + 2573, R 2 = 0.988) and the AuNF@PEG/DMPI-mAbDPN at 2174 cm −1 for DPN from 2 to 120 ng mL −1 (I2174 = 2625 × lgCDPN + 1154, R 2 = 0.95) (Figure 3). In addition, limits of detection (LOD) were calculated as 0.62 ng mL −1 for BRD, 0.42 ng mL −1 for TET and 1.37 ng mL −1 for DPN based on three times the standard deviations of the control signals, which could meet the requirement of fast food poisoning screening. Moreover, TEM images also provide the evidence of forming the nanoassemblies (SERS nanoprobes binding on the surfaces of the MB@P-CyM-hap2) in the presence of different targets, which illustrates their successful binding ( Figure 3G). The inter-and intra-day precisions are monitored as less than 16.24%. On the contrary, the sensing performance using the MB@hap under the same detecting conditions is not good as the MB@P-CyM-hap2 in the aspects of RSD, LOD and linear ranges, suggesting the outstanding advantages of using the non-fouling competitive interfaces. This results reveal that attributing to the particular 3D hair-like structures, the P-CyM interface not only facilitates conjugate highdensity hapten but reduces the steric hindrance between the SERS probes, thus enhancing their recognitions and sensing capacity. Moreover, due to the specificity of the mAb and the introduction of the non-fouling sensing interface, the selectivity of the assay is satisfactory ( Figure 3H).

Sample Analysis and Method Validation
Finally, SERS detection and quantification for BRD, TET and DPN in spiked biological and food samples were achieved in less than 2 h. The recoveries of the assay, as shown in Table 1, ranged from 72% to 123%, with CV values of 8.86 to 15.46%, indicating the good accuracy and precision of this method. Table 1. Determination of TET, BRD and DPN in different spiked matrices using the proposed detecting strategy (data are mean value of 3 measurements).

Sample
Toxin

Conclusions
In this work, the improved multiple ci-SERS assay was proposed for the simultaneous rapid screening of TET, BRD and DPN in biological and food samples. The chemical improvements are demonstrated for achieving better sensing results: firstly, the non-fouling polymer brush was introduced on the magnetic colloid as the scaffold for conjugation triple hapten, thus achieving enhanced signal-to-noise ratios. Secondly, the uniform AuNFs of high SERS activity was prepared by a simple one-pot reduction, which ensures the well reproducibility and ultra-sensitivity of this assay. These results suggested that the established assay has advantages of sensitivity, rapid response, easy operation and reliability, and thus, it provides an excellent sensing platform in wide fields. Above all, this assay was anticipated as a user-friendly sensor for on-field toxins screening in wide environmental and food matrices.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/toxics10100605/s1, Figure S1: The synthesis route for the CysMA monomer; Figure S2: The synthesis route for the CTA; Figure S3: The synthesis route for the MB@P-CyM-hap; Figure S4: XRD results of different MBs; Figure S5: TEM images of the SERS encoding cocktail.


s2fieldsofstudy: Chemistry Biology

text:
Design of 4-Substituted Sulfonamidobenzoic Acid Derivatives Targeting Coxsackievirus B3

Abstract A series of novel 4-substituted sulfonamidobenzoic acid derivatives was synthesized as the structural evolution of 4-(4-(1,3-dioxoisoindolin-2-yl)phenylsulfonamido)benzoic acid, which is the known inhibitor of the enterovirus life cycle. Antiviral properties of prepared compounds were evaluated in vitro using phenotypic screening and viral yield reduction assay. Their capsid binding properties were verified in thermostability assay. We identified two new hit-compounds (4 and 7a) with high activity against the coxsackievirus B3 (Nancy, CVB3) strain with potencies (IC50 values of 4.29 and 4.22 μM, respectively) which are slightly superior to the reference compound 2a (IC50 5.54 μM). Both hits changed the heat inactivation of CVB3 in vitro to higher temperatures, suggesting that they are capsid binders, as 2a is. The results obtained can serve as a basis for further development of the lead compounds for novel drug design to combat enterovirus infection.

Introduction
Viruses of the genus Enterovirus belonging to the Picornaviridae family are small, non-enveloped viruses with genomes represented by a single-stranded (+) RNA molecule. According to the modern classification of the International Committee on the Taxonomy of Viruses (ICTV), within the genus enterovirus, four types of enteroviruses (A-D) and three types of rhinoviruses (A-C) are distinguished [1]. Enteroviruses are etiological agents of acute and chronic human diseases, from asymptomatic or mild infection to life-threatening myocarditis, acute flaccid myelitis, cardiomyopathy, poliomyelitis, encephalitis, meningitis, bronchiolitis and pneumonia [2]. When entering the human body, they multiple mainly in the gastrointestinal tract and/or upper respiratory tract, but can penetrate into other tissues and target organs from primary infection sites viruses, causing severe complications. Rhinoviruses are the main cause of the common cold, a mild upper respiratory illness, but they can also provide lower respiratory illnesses such as bronchitis, sinusitis, and pneumonia, as well as worsening asthma, cystic fibrosis, and chronic obstructive pulmonary disease [3].
Enteroviruses are characterized by genetic plasticity due to recombination, high resistance to environmental conditions and the ability to cause outbreaks (in particular in organized groups of children) and epidemics. During the last decade, an outbreak of enterovirus D68 in 2014-2016 in the USA led to severe respiratory diseases in children, which in many cases were associated with acute flaccid myelitis [4]. Enterovirus 71 (EV71) infections are of high importance in the Asia-Pacific region [5].
The development of vaccines to prevent human enterovirus and rhinovirus infection is complicated by the multiplicity of existing virus serotypes. To date, there are vaccines for the prevention of poliomyelitis (live and inactivated) and three inactivated EV-A71 vaccines. The later ones are approved by the National Medical Products Administration of China (NMPA) and are commercially available in China [6]. The enormous efforts of the Global Polio Eradication Initiative have been very successful. However, it is now generally accepted that antivirals will be required for successful polio eradication, in particular to control outbreaks of circulating vaccine-derived polioviruses and to stop the excretion of chronically shedding viruses [7]. Despite significant advances in the field of enteroviruses and rhinoviruses biology, there are currently no approved drugs for the treatment and prevention of diseases caused by these viruses [8]. Among the previously studied enterovirus and rhinovirus inhibitor molecules with a known mechanism of action and documented activity in studies on animal models, the following groups can be distinguished: inhibitors that bind to the viral capsid and prevent its penetration into the cell-capsid binders (pleconaril, pirodavir, vapendavir, pokapavir, disoxaril), viral protease inhibitors (rupintrivir and AG7404), virus polymerase inhibitors (ribavirin, gemcitabine, amiloride), and viral ATPase inhibitors (dibucaine, fluoxetine) [9,10]. Despite being active in preclinical studies, none have been approved due to lack of efficacy, poor pharmacokinetic profile and/or adverse effects in clinical studies [11]. It should be noted that the potential use of drugs originally used for other medical conditions (e.g., fluoxetine, an antidepressant, amiloride, a diuretic) has the potential to cause unpredictable emergence of resistant strains in cases where their intended use coincides with asymptomatic EVI in a patient. Thus, the development of effective direct antivirals targeting enterovirus and rhinovirus lifecycle is highly desirable due to unmet clinical needs.
Capsid binders represent the most investigated group of inhibitors targeting viruses of the Enterovirus genus. Their general mechanism of action is well understood: they bind to a virus capsid, causing conformational changes that disturb the binding of the virus to its receptor on the cell surface [12]. These compounds have a pronounced antiviral activity, but, unfortunately, it has been shown that their use in clinical studies is associated with the emergence of resistant strains [13]. Nevertheless, given the importance of the initial step in the virus life-cycle, the search for targets on the surface of the virus for the design of inhibitors is still promising.
For a long time, it was believed that the hydrophobic pocket at the bottom of the canyon formed around each of the 12 vertices of the icosahedral viral capsid is the only target for capsid binder design. However, in 2019 a new promising target on the surface of the capsid was identified through the collaboration of the Neyts, Butcher, and Sinha research groups [14]. This pocket is located between the VP1-VP3 protomers in a conserved region of the surface of the enterovirus capsid. It was shown that the following amino acid residues line the pocket: VP1 (73, 75-78, 155-157, 159-160, 219, and 234) and VP3 (233-236). The residues support quaternary structural changes in the capsid during virus entry when the virion progresses to an expanded altered particle (A-particle). This pocket was found to be suitable as a target for blocking the life cycle of enteroviruses and rhinoviruses. The identification of the hit compound (4-substituted sulfonamidobenzoic acid, Figure 1) carried out in the same study and the published electron microscopy data on the structure of a new pocket on the surface of the enterovirus capsid created the prerequisites for further successful chemical research in the field of capsid binders.
Life 2022, 12, 1832 3 of 14 VP3 (233-236). The residues support quaternary structural changes in the capsid during virus entry when the virion progresses to an expanded altered particle (A-particle). This pocket was found to be suitable as a target for blocking the life cycle of enteroviruses and rhinoviruses. The identification of the hit compound (4-substituted sulfonamidobenzoic acid, Figure 1) carried out in the same study and the published electron microscopy data on the structure of a new pocket on the surface of the enterovirus capsid created the prerequisites for further successful chemical research in the field of capsid binders.  [14] and [15].
In this study, we explored the possibilities for medicinal-chemical modification of the lead-compound 2a [14] to identify new highly active molecules that exhibit the properties of new capsid-binding agents for the prevention and treatment of infections caused by enteroviruses. The results obtained can be used to develop new capsid-binding agents for the prevention and treatment of diseases caused by enteroviruses.

General
The preparation and characterization of starting sulfonyl chloride 1 as well as amine 5 were described previously [15][16][17]. Other reactants and solvents were obtained from commercial sources and used without purification. All reactions were performed in an open flask without any protection from CO2 and H2O. Reactions were monitored using analytical thin layer chromatography (TLC) Macherey-Nagel, TLC plates Polygram ® Sil G/UV254. Visualization of the developed chromatograms was performed by fluorescence quenching at 254 nm. 1 H and 13 C NMR spectra were measured on Bruker AVANCE DPX 400 (400 MHz for 1 H and 101 MHz for 13 C, respectively). All chemical shifts (δ) are given in parts per million (ppm) with reference to solvent residues in DMSO-d6 (2.50 for proton and 39.52 for carbon), and coupling constants (J) are reported in hertz (Hz). Multiplicities are abbreviated as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad. Melting points were determined using an Electrothermal IA 9300 series Digital Melting Point Apparatus. Mass spectra were recorded on microTOF spectrometers (ESI ionization).
In this study, we explored the possibilities for medicinal-chemical modification of the lead-compound 2a [14] to identify new highly active molecules that exhibit the properties of new capsid-binding agents for the prevention and treatment of infections caused by enteroviruses. The results obtained can be used to develop new capsid-binding agents for the prevention and treatment of diseases caused by enteroviruses.

General
The preparation and characterization of starting sulfonyl chloride 1 as well as amine 5 were described previously [15][16][17]. Other reactants and solvents were obtained from commercial sources and used without purification. All reactions were performed in an open flask without any protection from CO 2 and H 2 O. Reactions were monitored using analytical thin layer chromatography (TLC) Macherey-Nagel, TLC plates Polygram ® Sil G/UV254. Visualization of the developed chromatograms was performed by fluorescence quenching at 254 nm. 1 H and 13 C NMR spectra were measured on Bruker AVANCE DPX 400 (400 MHz for 1 H and 101 MHz for 13 C, respectively). All chemical shifts (δ) are given in parts per million (ppm) with reference to solvent residues in DMSO-d 6 (2.50 for proton and 39.52 for carbon), and coupling constants (J) are reported in hertz (Hz). Multiplicities are abbreviated as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad. Melting points were determined using an Electrothermal IA 9300 series Digital Melting Point Apparatus. Mass spectra were recorded on microTOF spectrometers (ESI ionization).

Synthesis and Characterization of Compounds 7a and 7b
The corresponding anhydride (1 mmol) was added to a solution of 4-((4-aminophenyl) sulfonamido)benzoic acid 6 (1 mmol) in glacial acetic acid (2 mL). The reaction mixture was stirred for 24 h (TLC monitoring). It was diluted with water (1 mL), filtered off, and washed with water. The resulting crude product was dried at 50 • C. A mixture of 4-(4-(3-carboxypropanamido)phenylsulfonamido)benzoic acid 6 (0.2 g, 0.5 mmol), TMSCl (0.07 g), and NMP (0.6 mL) was then heated for 2 h in a bath at 160-165 • C, cooled to room temperature, and diluted with water. The precipitate was separated by filtration, washed with water, and dried in air to obtain the target compound. These compounds were purified by flash column chromatography using CH 2 Cl 2 :MeOH (97:3) mixture as an eluent.

Synthesis and Characterization of Compound 7e
Anhydrous zinc acetate (1 mmol) and compound 5 (1 mmol) were added under nitrogen to a mixture of naphthalene-1,8-dicarboxylic anhydride (1 mmol) and 10 mL of anhydrous N-methylpyrrolidone. Next, the mixture was heated to 160 • C and stirred at this temperature for 15 h. After cooling to room temperature, the product was precipitated by adding 5 mL of 5% hydrochloric acid, filtered off, washed first with 5% hydrochloric acid and then with hot water, and dried. The product was obtained as a brown solid, which corresponds to a yield of 94%. The resulting crude product was purified by short-path chromatography on silica gel using ethyl acetate as an eluent.

Viruses and Cells
Coxsackievirus B3 (CVB3, strain Nancy) was obtained from the collection of viruses of the Pasteur Institute (St. Petersburg, Russia). The permissive cell line Vero (ATCC #CCL-81) was used in the study. Infectious titers (in a 50%-tissue culture infection dose, TCID50) were determined in Vero cells for CVB3 by endpoint dilution assay using the following procedure. Permissive cells were seeded into 96-wells plates in Eagles minimal essential medium (MEM) supplemented with 5% fetal bovine serum (FBS). After 24 h, the medium was aspirated, the wells were washed with saline, fresh MEM without FBS was added to the wells and the cells were infected with serial tenfold dilutions of virus stocks (100 µL per well, 4 wells for each dilution). The plates were incubated at +37 • C in 5% CO 2 and observed daily for cytopathic effect (CPE). After 72 h the viral titer was calculated in TCID50 using the method of Spearmen-Carber.

Cytoprotection Assay
Vero cells were seeded in 96-well plates in 24 h before the assay. On the assay day, the cells were washed with saline and tested compounds in appropriate concentrations (300-3 µg/mL) in MEM without FBS were added to cells. No compounds were added to Life 2022, 12, 1832 7 of 14 virus control wells. The plates were incubated for 1 h at 37 • C at 5% CO 2 . The cells were then infected with CVB3 Nancy (m.o.i range of 1-0.001) and incubated for 72 h at 37 • C at 5% CO 2 . No virus was added to the cytotoxicity control wells. Cell viability was then assessed using an MTT test with the following procedure. The cells were washed with saline, and a solution of 3-(4,5-dimethylthiazolyl-2) 2,5-diphenyltetrazolium bromide (ICN Biochemicals Inc., Aurora, OH, USA) (0.5 µg/mL) in MEM was added to the wells (100 µL per well). After 2 h of incubation at 37 • C in 5% CO 2 , the supernatant from wells was discarded, and the formazan residue was dissolved in DMSO (100 µL per well). The optical density of cells was then measured on a Multiskan multifunctional reader (Thermo Fisher Scientific, Shanghai, China) at a wavelength of 540 nm.
The cytoprotective activity of compounds was considered to be their ability to increase the values of OD compared to control wells (using only a virus, without drugs). Based on the results obtained, the values of IC 50 , i.e., the concentration of compounds that result in 50% cells protection were calculated for m.o.i. 0.001 using GraphPad Prism 6.01 software.

Cytotoxicity Assay
The microtetrazolium test (MTT) was used to study the cytotoxicity of the compounds. The permissive cell line Vero was seeded in 96-well plates in Eagles minimal essential medium (MEM) supplemented with 10% FBS. After 24 h, the media were removed, and the wells were washed with saline. Compounds were dissolved in DMSO, and a series of two-fold dilutions of each compound in MEM without FBS were prepared and added to the cells in triplicates. Cells were incubated for 24 h at 37 • C in 5% CO 2 . The MTT was then performed as described above. Next, the optical density of cells was measured on a Multiskan multifunctional reader (Thermo Fisher Scientific, Shanghai, China) at a wavelength of 540 nm and plotted against the concentration of the compounds to generate the dose-response curve. The 50% cytotoxic dose (CC 50 ) of each compound (i.e., the compound concentration that causes the death of 50% of cells in a culture, or decreases the optical density twice, as compared to the control wells) was calculated using a fourparameter logistic nonlinear regression model (GraphPad Prism 6.01).

Antiviral Activity Determination
The antiviral activity of the compounds was evaluated using viral yield reduction assay. The respective permissive cell line Vero was seeded in MEM supplemented with 5% FBS in 24-well plates. When the cell confluence reached 100%, the compounds tested were dissolved in DMSO, and a series of three-fold dilutions of each compound in MEM without FBS was added to the cells, and incubated at 37 • C in 5% CO 2 . After 1 h, the media were discarded; equal volumes of fresh serial dilutions of each compound and viral suspension in MEM without FBS at MOI 0.0 were added. In control wells, only MEM without FBS was added. No compounds were added in the virus control wells. The plates were incubated at 4 • C for 1 h. The unbound virus was then washed away and, again, three-fold dilutions of each compound (the final concentration 50-0.6 µg/mL) in MEM without FBS were added to the wells. After 24 h of incubation at 37 • C in 5% CO 2 , the infectious titer of viral progeny (in TCID50) for each compound concentration, cell control, and virus control wells were determined in permissive cell lines by endpoint dilution assay as described above.
The titer of virus progeny was plotted against log concentration of the compounds tested to generate the dose-response curve. The 50% inhibition concentration (IC 50 ) of each compound tested (i.e., the compound concentration that decreases the infectious viral progeny titer twice, as compared to the control wells) was calculated using a four-parameter logistic nonlinear regression model (GraphPad Prism 6.01).
Selectivity index (SI) was calculated for each compound tested as a ratio of CC 50 to IC 50 values. It was shown that compound 2b in a base medium in the temperature range from 0 to 50 °C undergoes imide ring opening with the remaining ester function. It has been established that the hydrolysis of ester moiety requires prolonged reflux in ethanol with at least a threefold excess of sodium hydroxide (Scheme 2). Both compounds (3 and 4) were isolated and fully characterized using NMR spectroscopy and HR-mass spectrometry. The presence of two new proton signals (at 10.75 and 13.03 ppm) in 1 H NMR spectrum of 3 in comparison with ester 2b is evidence to the phthalimide ring opening. Characteristics of aromatic protons and carbons also changed. Particularly, the number of signals increased for both carbons and protons (see Sections 2.2 and 2.3, as well as Supplementary Materials).
In addition, in order to change the lipophilic properties and the spatial structure of the ligand periphery, we replaced the phthalimide group with fragments of naphthalic anhydride 7e and anhydrides of cycloalkanedicarboxylic acids 7a-d (Scheme 3). It was shown that compound 2b in a base medium in the temperature range from 0 to 50 • C undergoes imide ring opening with the remaining ester function. It has been established that the hydrolysis of ester moiety requires prolonged reflux in ethanol with at least a threefold excess of sodium hydroxide (Scheme 2). Both compounds (3 and 4) were isolated and fully characterized using NMR spectroscopy and HR-mass spectrometry. The presence of two new proton signals (at 10.75 and 13.03 ppm) in 1 H NMR spectrum of 3 in comparison with ester 2b is evidence to the phthalimide ring opening. Characteristics of aromatic protons and carbons also changed. Particularly, the number of signals increased for both carbons and protons (see Sections 2.2 and 2.3, as well as Supplementary Materials).
In addition, in order to change the lipophilic properties and the spatial structure of the ligand periphery, we replaced the phthalimide group with fragments of naphthalic anhydride 7e and anhydrides of cycloalkanedicarboxylic acids 7a-d (Scheme 3). To perform this modification, the starting compound 5 was used, which was obtained with a total yield of 44% according to the known method [15]. The method was based on commercially available 4-N-acetaminophenylsulfonyl chloride, which was reacted with aminobenzoic acid in the presence of pyridine, followed by hydrolysis of the amide group with sodium hydroxide. Amino derivative 5 had low nucleophilic properties and entered into cyclization reactions to form compounds 7 with difficulty. Attempts to obtain imides 7 directly from amine 5 by prolonged refluxing of the reagents in acetic acid gave a positive result only in the case of compounds 7с and 7d. Compound 7e was successfully synthesized by heating it in N-methylpyrollidone in the presence of zinc acetate, according to the method [18]. Moderate yields of products 7a and 7b were obtained from compounds 6 by the action of trimethylchlorosilane in dimethylformamide, ac- To perform this modification, the starting compound 5 was used, which was obtained with a total yield of 44% according to the known method [15]. The method was based on commercially available 4-N-acetaminophenylsulfonyl chloride, which was reacted with aminobenzoic acid in the presence of pyridine, followed by hydrolysis of the amide group with sodium hydroxide. Amino derivative 5 had low nucleophilic properties and entered into cyclization reactions to form compounds 7 with difficulty. Attempts to obtain imides 7 directly from amine 5 by prolonged refluxing of the reagents in acetic acid gave a positive result only in the case of compounds 7c and 7d. Compound 7e was successfully synthesized by heating it in N-methylpyrollidone in the presence of zinc acetate, according to the method [18]. Moderate yields of products 7a and 7b were obtained from compounds 6 by the action of trimethylchlorosilane in dimethylformamide, according to the method [19].
As a result, the 15 new 4-substituted sulfonamidobenzoic acids and its surrogates were synthesized using the routes described above.

Results of In Vitro Biological Studies
At the beginning of our study, cytoprotection properties of compounds against the pleconaril-resistant strain Coxsackievirus B3 Nancy [20] were determined using a wellknown cytoprotection assay. Cytoprotection assay allows the evaluation of cell survival depending on the concentration of tested compounds after virus infection. Cell survival is assessed by MTT assay based on the measurement of cell respiration. Compound 2a was used for the comparison. The results of the cytoprotection assay are presented in Table 1. As shown in Table 1, 7 compounds out of 15 (46% of total tested) demonstrated substantial cytoprotective activity with an SI that was much more than 10. These compounds were selected for further analysis. Compounds 2f and 3 were the most toxic compounds, while for compounds 7c, 2h, and 4 value of CC 50 exceeded the maximum concentration used in the assay (the same as for the reference compound 2a).
To confirm virus-inhibition activity for selected compounds we then performed a viral yield reduction test. As described above, this test was applied for measuring the titer of viral progeny in the supernatant of the infected culture depending on the compound concentration. Therefore, it is possible to evaluate the direct antiviral activity of the compounds. The results of the viral yield reduction assay are summarized in the Table 2 below. Compound 2a was used for comparison. According to the results of the viral yield assay, two compounds-7a and 4-demonstrated potent inhibiting properties with IC 50 lower than that of the reference compound, 2a. They all had comparable cytotoxicity at the 24 h time point, but the SI's of 7a and 4 were higher than of those of the reference compound (2a).
Next, we checked whether the most active candidates (4 and 7a) belonged to the capsid binder group in the thermostability assay. Capsid thermostability assay is one of the approaches to determine the ability of the compound to bind viral particles. It is known that capsid binders (for example, well-known first in-class capsid binder pleconaril and its derivatives) directly interact with the capsid of non-enveloped viruses and stabilize its structure, preventing the virus from entering the host cell and increasing their stability to heat inactivation.
CVB3 Nancy was pre-incubated with 4, 7a or 2a at concentrations greatly exceeding their IC 50 values to allow for strong binding [14,21]. After that, the viral suspension was gradually heated and rapidly cooled down. The infectious titer of CVB3 corresponding to each treatment condition is presented in Figure 2 below. Next, we checked whether the most active candidates (4 and 7a) belonged to the capsid binder group in the thermostability assay. Capsid thermostability assay is one of the approaches to determine the ability of the compound to bind viral particles. It is known that capsid binders (for example, well-known first in-class capsid binder pleconaril and its derivatives) directly interact with the capsid of non-enveloped viruses and stabilize its structure, preventing the virus from entering the host cell and increasing their stability to heat inactivation.
CVB3 Nancy was pre-incubated with 4, 7a or 2a at concentrations greatly exceeding their IC50 values to allow for strong binding [14,21]. After that, the viral suspension was gradually heated and rapidly cooled down. The infectious titer of CVB3 corresponding to each treatment condition is presented in Figure 2 below. It can be seen that in no treatment virus control CVB3 Nancy virus titer gradually decreased with increasing temperature. No infectious particles were determined at 49.9 °C and above. In contrast to the virus control sample, upon addition of 4, 7a and 2a, the It can be seen that in no treatment virus control CVB3 Nancy virus titer gradually decreased with increasing temperature. No infectious particles were determined at 49.9 • C and above. In contrast to the virus control sample, upon addition of 4, 7a and 2a, the heat inactivation of CVB3 changed to higher temperatures, suggesting that all the compounds tested stabilize the viral capsid and protect the virus from heat degradation. Both 7a and 4 were able to preserve the infectivity of CVB3 to the same extent (to 52.4 • C), which slightly exceeded the activity of the reference compound (2a). Thus, we confirm that these new derivatives of N-sulfonamidobenzoic acid also belong to the capsid binder class of enterovirus inhibitors.

Structure-Activity-Relationship
It was shown that the hydroxyl fragment of the carboxyl groups of aminobenzoic acid is primarily responsible for ligand-receptor binding. Attempts to change the lipophilicity of this fragment by esterification of the carboxyl group (2b) or by the formation of amide (2c) and nitrile (2d), sulfonamide (2e) and acetamide (2e) derivatives negatively affected the antiviral activity of the molecules. In some cases, antiviral activity completely disappeared.
Attempts to replace the terminal aminobenzoic acid while retaining the carboxyl function (examples 2g, 2h, 2i) with linear and cycloaliphatic fragments that are more conformationally mobile do not increase the activity of the target ligands. Taking into account the propensity of the imide fragment to hydrolysis reactions, including in physiological media, we synthesized non-imide analogs of compound 2a and gave compound 4 as a potent ligand (first hit). In addition, the phthalimide moiety was replaced by several cyclic aliphatic imides. Although in most cases the inhibitory activity was lower in comparison with 2a, succinimide 7a demonstrated good potency and was recognized as the second hit compound.
In this regard, it should be concluded that in the course of the work carried out on the medicinal-chemistry modification of the lead-compound, two new, highly active compounds were identified that are superior in activity to compound 2 against the Coxsackievirus B3 Nancy. The risks of degradation of the lead compound upon contact with hydrolytic media with the formation of metabolites (4), which also have an antiviral effect, are shown. In this regard, changing the method of delivery of this innovative substance may be recommended.

Discussion
Enteroviral infection is a serious threat to public health as it affects millions of people of different age groups. Despite the wide distribution of enteroviruses and the economic damage due to cases of temporary disability caused by enterovirus infection, no approved antivirals are available to treat non-polio enteroviral infections, and the vaccination option is available only for polioviruses and EV71. Recently, a new class of capsid binders was described (derivatives of 4-substituted sulfonamidobenzoic acid), which inhibit a variety of enteroviruses by occupying a positively charged surface depression at a conserved VP1-VP3 interprotomer interface in the viral capsid [14].
In this study, we prepared 15 novel compounds bearing 4-substitutedsulfonyl-)4aminobenzoic acid and their surrogates, which are characterized in antiviral assays in vitro.
Structural features of the pharmacophore fragment of the molecule responsible for the binding of the ligand to the viral capsid were identified. It was shown that the hydroxyl fragment of the carboxyl group of aminobenzoic acid is primarily responsible for ligandreceptor binding. Esterification, amidation, or replacement of the carboxyl function with a Life 2022, 12, 1832 13 of 14 nitrile, sulfonamide, or acetamide moiety, or replacement of aminobenzoic acid with amino acids of a similar structure-all led to the disappearance of antiviral activity.
We proved that selected lead-compounds belong to the capsid binder group of inhibitors, as their prototype molecule does. It is widely accepted that viral capsid is undeniably an attractive target for the development of direct-acting inhibitors that can interfere with viral entry. Unfortunately, most capsid binders have a major disadvantage-an obvious low barrier to resistance emergence due to mutations in amino acid residues either directly involved in the formation of the hydrophobic binding site or outside the binding pocket [22,23]. Combinational therapy is believed to be an option to overcome this problem. Cocktails of antivirals are used to target multiple viruses and mitigate the development of antiviral drug resistance or delay its emergence [24][25][26]. The application of synergistic drug combinations allows the concentration of antivirals to be reduced, which in turn could decrease the side effects of individual drugs. Therefore, new drugs and their combinations need to be identified to inhibit different enteroviruses. The results of the present study, where we identified novel capsid-binding small molecules with improved activity and low toxicity in vitro, can be used to develop such therapies.

Conclusions
Currently, dozens of antiviral therapies are at different stages of development, but there are no approved drugs available for the effective management of severe enteroviral infections. Here, new 4-substituted sulfonamidobenzoic acids have shown high in vitro antiviral activity against enteroviruses group B, realizing their antiviral potential by binding to the virus capsid and preventing infection of the permissive cell culture. The hydroxyl fragment of the carboxyl group of aminobenzoic acid is primarily responsible for ligandreceptor binding. Changing the carboxyl function or replacing the aminobenzoic acid with amino acids of a similar structure is detrimental to antiviral activity. In the absence of registered drugs on the market for the prevention and treatment of non-polio enterovirus infections, the results obtained serve as the basis for the further development of this group of compounds.


SnS2 Nanoparticles and Thin Film for Application as an Adsorbent and Photovoltaic Buffer

Energy consumption and environmental pollution are major issues faced by the world. The present study introduces a single solution using SnS2 for these two major global problems. SnS2 nanoparticles and thin films were explored as an adsorbent to remove organic toxic materials (Rhodamine B (RhB)) from water and an alternative to the toxic cadmium sulfide (CdS) buffer for thin-film solar cells, respectively. Primary characterization tools such as X-ray photoelectron spectroscopy (XPS), Raman, X-ray diffraction (XRD), and UV-Vis-NIR spectroscopy were used to analyze the SnS2 nanoparticles and thin films. At a reaction time of 180 min, 0.4 g/L of SnS2 nanoparticles showed the highest adsorption capacity of 85% for RhB (10 ppm), indicating that SnS2 is an appropriate adsorbent. The fabricated Cu(In,Ga)Se2 (CIGS) device with SnS2 as a buffer showed a conversion efficiency (~5.1%) close to that (~7.5%) of a device fabricated with the conventional CdS buffer, suggesting that SnS2 has potential as an alternative buffer.

Introduction
Global energy consumption is anticipated to increase dramatically over the next few decades. This is primarily due to the expected global population expansion as well as the economic and industrial growth of developing nations, among other factors. According to a new report from the International Energy Agency (IEA), worldwide energy consumption has declined by approximately 1% in 2020 because of the COVID-19 pandemic and is expected to increase by approximately 5% in the coming years [1]. Sustainable energy sources are growing rapidly, but not fast enough to meet the significant increase in global energy demand, resulting in a dramatic increase in coal consumption that threatens to increase carbon dioxide emissions from the energy industry to historic levels. Electricity generation from PV technology is more affordable than that from non-renewable fossil fuel sources. As a result, it is necessary to advance the field of photovoltaic (PV) research by utilizing sustainable materials.
Notably, solar technologies based on Cu(In, Ga)Se 2 (CIGS) thin-film semiconducting materials are available at a price comparable to or lower than that of standard silicon modules [2]. The typical structure of CIGS thin film technology comprises a bottom contact layer made of molybdenum (Mo), a p-type CIGS absorber layer (1-3 µm), a thin n-type cadmium sulfide (CdS) buffer layer, a zinc oxide (ZnO)-based transparent window layer [3], and top metal contacts. Generally, conventional CdS buffer is produced by chemical bath deposition (CBD), which shields the absorber layer from direct current (DC) sputtering damage and alters the surface of the CIGS absorber [4]. In addition, the chemical bath that contains dyes. Indeed, most dyes are chemically stable for their intended purposes and are challenging to break down biologically [17]. As a result, photocatalysis [18,19], chemical oxidation [20], and adsorption [21] are the primary methods for removing dyes from aqueous solutions. Adsorption is viable for removing hazardous dyes from aqueous solutions as it is simple to conduct, inexpensive, environmentally benign, efficient, and requires little energy [22].
To address the two distinct types of challenges faced by the world, the authors suggested a single material as the solution. Tin disulfide (SnS 2 ) is a benign, affordable, and simple binary IV-VI group metal chalcogenide with a band gap of~2.9 eV and an n-type conductivity. It consists of earth abundant elements with easily controllable chemical stoichiometry and high chemical and environmental stability. It showed a peculiar CdI 2 -type layered structure, with tin atoms sandwiched between two layers of hexagonally arranged close-packed sulfur atoms [23]. It forms an Ohmic contact with metals without current losses. In addition, excellent structural flexibility, broader spectral response, and better thermal stability of SnS 2 make it a competitive non-toxic substitute for various applications, such as solar cells, field effect transistors, thin film diodes, high-speed photodetectors, lithium-sodium ion batteries, supercapacitors, catalysts, and gas sensors [24][25][26][27][28]. Because of its appropriate band gap, it transmits most of the solar radiation to the absorber and minimizes the parasitic absorption loss. It has carrier density of the order of 10 19 cm −3 and electron affinity of 4.1 eV [29] to achieve sufficient type-inversion within the absorber surface and to make a favorable conduction band offset (CBO, 0-0.4 eV) with the absorber, respectively [30]. Further, it is chemically stable in both acidic and neutral aqueous solutions [21]. Therefore, SnS 2 has the potential to be a promising non-toxic buffer in solar cells [29,31] and an effective adsorbent for the degradation of organic pollutants [32].
In this paper, we present the synthesis of SnS 2 nanoparticles using a facile chemical precipitation approach. Subsequently, SnS 2 thin films were formed by spin coating the SnS 2 nanoparticles. Further, the SnS 2 nanoparticles were investigated as a possible adsorbent for removing a toxic dye, Rhodamine B (RhB), from an aqueous solution. In addition, SnS 2 thin films were explored as a buffer layer for CIGS solar cells.

Synthesis of SnS 2 Nanoparticles for Adsorbent Application
The procedure for the synthesis of SnS 2 nanoparticles is shown in Figure 1. A simple chemical precipitation method was used to synthesize SnS 2 nanoparticles. For a typical synthesis, 0.1 M of stannous chloride, 0.8 M of thioacetamide, and 1 M of trisodium citrate were aggressively stirred on the magnetic hot plate. The solution was allowed to react for 50 min at a constant temperature of 60 • C with a solution pH of 2. The SnS 2 nanoparticles were collected and cleaned using deionized water before drying in a vacuum oven at 70 • C for 4 h. Finally, the as-synthesized SnS 2 nanoparticles exhibited a golden yellow hue. The SnS 2 nanoparticles/powder was collected in a vial and desiccated for further analyses.
SnS2 nanoparticles. Further, the SnS2 nanoparticles were investigated as a possible adsorbent for removing a toxic dye, Rhodamine B (RhB), from an aqueous solution. In addition, SnS2 thin films were explored as a buffer layer for CIGS solar cells.

Synthesis of SnS2 Nanoparticles for Adsorbent Application
The procedure for the synthesis of SnS2 nanoparticles is shown in Figure 1. A simple chemical precipitation method was used to synthesize SnS2 nanoparticles. For a typical synthesis, 0.1 M of stannous chloride, 0.8 M of thioacetamide, and 1 M of trisodium citrate were aggressively stirred on the magnetic hot plate. The solution was allowed to react for 50 min at a constant temperature of 60 °C with a solution pH of 2. The SnS2 nanoparticles were collected and cleaned using deionized water before drying in a vacuum oven at 70 °C for 4 h. Finally, the as-synthesized SnS2 nanoparticles exhibited a golden yellow hue. The SnS2 nanoparticles/powder was collected in a vial and desiccated for further analyses.

Preparation of SnS2 Thin Film for Photovoltaic Application
The as-synthesized SnS2 nanoparticles were used for the preparation of SnS2 thin films by spin coating. SnS2 thin film deposition was accomplished by sonicating the SnS2 nanoparticles dispersed in 2 mL ethanol. Then, the resultant ink was spread out over a pre-cleaned glass substrate and a Mo/CIGS absorber. The preparation process of the SnS2 thin-film is shown in Figure 2. The SnS2 film deposited on a glass substrate was used to analyze the structural and optical properties, and the film on the Mo/CIGS absorber was utilized as a buffer during the construction of a CIGS solar cell for device analysis. The experiments for the synthesis of SnS2 nanoparticles and thin films were repeated, and both of the experimental results showed good reproducibility.

Preparation of SnS 2 Thin Film for Photovoltaic Application
The as-synthesized SnS 2 nanoparticles were used for the preparation of SnS 2 thin films by spin coating. SnS 2 thin film deposition was accomplished by sonicating the SnS 2 nanoparticles dispersed in 2 mL ethanol. Then, the resultant ink was spread out over a pre-cleaned glass substrate and a Mo/CIGS absorber. The preparation process of the SnS 2 thin-film is shown in Figure 2. The SnS 2 film deposited on a glass substrate was used to analyze the structural and optical properties, and the film on the Mo/CIGS absorber was utilized as a buffer during the construction of a CIGS solar cell for device analysis. The experiments for the synthesis of SnS 2 nanoparticles and thin films were repeated, and both of the experimental results showed good reproducibility.

Characterization Details
Characterization of SnS2 nanoparticles/thin films was done using the following techniques. The phase and elemental purity of the SnS2 nanoparticles were analyzed using a Raman spectrometer (Jobin-Yvon Lab Ram HR 800, Horiba, Kyoto, Japan) and X-ray photoelectron spectroscopy (XPS; K-Alpha, Thermo Fisher Scientific, Altrincham, UK), respectively. The structural, morphological and optical properties of the SnS2 thin films were studied using a Seifert 3003TT X-ray diffractometer (Almelo, The Netherlands) with CuKα radiation (λ = 1.5405 Å), scanning electron microscope (SEM; Hitachi S-4800, Tokyo, Japan) and UV-Vis-NIR spectrophotometer (Cary 5000, Agilent, Santa Clara, CA, USA), respectively. The changes in the absorbance spectra of RhB in aqueous solution in the presence of SnS2 nanoparticles were recorded on a UV-Vis absorption spectrophotometer (Hitachi U-3010, Tokyo, Japan). The photovoltaic performance of the CIGS device with SnS2 thin film as a buffer layer was studied using illumination current density, and the voltage (J-V) characteristics were evaluated using a Solar Simulator (McScience K201 LAB50, Suwon, Korea) under AM 1.5 and 100 mW/cm 2 illumination, where the light intensity was calibrated using an Si standard reference cell.

Growth of SnS2 Nanoparticles
The following growth process is hypothesized for the synthesis of SnS2 nanoparticles. The chelating agent trisodium citrate complexes the Sn 4+ ions from stannous chloride in the first stage. The following stage involves the release of S 2− ions into the bath as a result of thioacetamide hydrolysis. The precipitation interaction between Sn 4+ and S 2− leads to the production of SnS2 nanoparticles in the final stage. The following equations describe the growth stages of the entire process: H2S + H2O ⇌ H3O + + HS − The phase purity of the prepared nanoparticles was confirmed using Raman

Characterization Details
Characterization of SnS 2 nanoparticles/thin films was done using the following techniques. The phase and elemental purity of the SnS 2 nanoparticles were analyzed using a Raman spectrometer (Jobin-Yvon Lab Ram HR 800, Horiba, Kyoto, Japan) and X-ray photoelectron spectroscopy (XPS; K-Alpha, Thermo Fisher Scientific, Altrincham, UK), respectively. The structural, morphological and optical properties of the SnS 2 thin films were studied using a Seifert 3003TT X-ray diffractometer (Almelo, The Netherlands) with CuKα radiation (λ = 1.5405 Å), scanning electron microscope (SEM; Hitachi S-4800, Tokyo, Japan) and UV-Vis-NIR spectrophotometer (Cary 5000, Agilent, Santa Clara, CA, USA), respectively. The changes in the absorbance spectra of RhB in aqueous solution in the presence of SnS 2 nanoparticles were recorded on a UV-Vis absorption spectrophotometer (Hitachi U-3010, Tokyo, Japan). The photovoltaic performance of the CIGS device with SnS 2 thin film as a buffer layer was studied using illumination current density, and the voltage (J-V) characteristics were evaluated using a Solar Simulator (McScience K201 LAB50, Suwon, Korea) under AM 1.5 and 100 mW/cm 2 illumination, where the light intensity was calibrated using an Si standard reference cell.

Results and Discussion
3.1. SnS 2 Nanoparticles for Adsorbent Application 3.1.1. Growth of SnS 2 Nanoparticles The following growth process is hypothesized for the synthesis of SnS 2 nanoparticles. The chelating agent trisodium citrate complexes the Sn 4+ ions from stannous chloride in the first stage. The following stage involves the release of S 2− ions into the bath as a result of thioacetamide hydrolysis. The precipitation interaction between Sn 4+ and S 2− leads to the production of SnS 2 nanoparticles in the final stage. The following equations describe the growth stages of the entire process: The phase purity of the prepared nanoparticles was confirmed using Raman spectroscopy. The Raman spectrum of the as-synthesized nanoparticles in the region of 100-400 cm −1 is shown in Figure 3. The spectrum reveals that the as-prepared SnS 2 nanoparticles exhibited a broad peak at 310 cm −1 , which corresponds to the Raman mode associated with the hexagonal structure of SnS 2 . The phonon mode at 310 cm −1 is attributed to the vertical plane vibration (A 1g ) of the Sn-S bonds [31]. The detected Raman phonon mode was consistent with that of the previous reports [29]. 100-400 cm −1 is shown in Figure 3. The spectrum reveals that the as-prepared SnS2 nanoparticles exhibited a broad peak at 310 cm −1 , which corresponds to the Raman mode associated with the hexagonal structure of SnS2. The phonon mode at 310 cm −1 is attributed to the vertical plane vibration (A1g) of the Sn-S bonds [31]. The detected Raman phonon mode was consistent with that of the previous reports [29].

Elemental Purity Confirmation of SnS2 Nanoparticles
XPS was used to characterize the composition of the prepared nanoparticles. No significant peaks representing the contaminants were found in the spectra, suggesting that the impurity level was less than the XPS resolution limit (1 at %). Figure 4a shows a typical high-resolution spectrum of the as-synthesized SnS2 nanoparticles with binding energy ranging from 0 to 1000 eV. The wide spectrum revealed numerous peaks, such as S 2s, double S 2p, Sn 3s, doublet Sn 3p, doublet Sn 3d, and Sn 4d, indicating that the synthesized nanoparticles contain Sn and S elements. The peaks linked to C and O were possibly caused by ambient pollution. No other peaks corresponding to other elemental impurities were found in the spectrum. Figure 4b,c show the narrow XPS scans of the Sn 3d and S 2p peaks, respectively. A core-level scan of the Sn 3d doublet revealed two peaks with binding energies of 486.3 eV and 494.6 eV, corresponding to the Sn 3d5/2 and Sn 3d3/2 energy levels of Sn atoms in the Sn 4+ valence state, respectively. The peak splitting energy of Sn 3d5/2 and Sn 3d3/2 levels was approximately 8.3 eV. The binding energies of Sn 3d doublets and their spin energy separations were similar to the values reported [33]. A complete spectrum of the S 2p core level revealed two peaks with binding energies of 162.5 eV and 161.2 eV, which correspond to the S 2p5/2 and S 2p3/2 energy levels of S atoms in the Sn 2− valence state, respectively, which is consistent with the energies of the S 2− -Sn 4+ bond, confirming the presence of a single-phase SnS2 in the synthesized nanoparticles [34].

Elemental Purity Confirmation of SnS 2 Nanoparticles
XPS was used to characterize the composition of the prepared nanoparticles. No significant peaks representing the contaminants were found in the spectra, suggesting that the impurity level was less than the XPS resolution limit (1 at %). Figure 4a shows a typical high-resolution spectrum of the as-synthesized SnS 2 nanoparticles with binding energy ranging from 0 to 1000 eV. The wide spectrum revealed numerous peaks, such as S 2s, double S 2p, Sn 3s, doublet Sn 3p, doublet Sn 3d, and Sn 4d, indicating that the synthesized nanoparticles contain Sn and S elements. The peaks linked to C and O were possibly caused by ambient pollution. No other peaks corresponding to other elemental impurities were found in the spectrum. Figure 4b,c show the narrow XPS scans of the Sn 3d and S 2p peaks, respectively. A core-level scan of the Sn 3d doublet revealed two peaks with binding energies of 486.3 eV and 494.6 eV, corresponding to the Sn 3d 5/2 and Sn 3d 3/2 energy levels of Sn atoms in the Sn 4+ valence state, respectively. The peak splitting energy of Sn 3d 5/2 and Sn 3d 3/2 levels was approximately 8.3 eV. The binding energies of Sn 3d doublets and their spin energy separations were similar to the values reported [33]. A complete spectrum of the S 2p core level revealed two peaks with binding energies of 162.5 eV and 161.2 eV, which correspond to the S 2p 5/2 and S 2p 3/2 energy levels of S atoms in the Sn 2− valence state, respectively, which is consistent with the energies of the S 2− -Sn 4+ bond, confirming the presence of a single-phase SnS 2 in the synthesized nanoparticles [34]. Nanomaterials 2022, 12, x FOR PEER REVIEW 6 of 14 The adsorption of RhB dye from an aqueous solution was carried out at room temperature (approximately 25 °C). Typically, 0.4 g of SnS2 nanoparticles was added to a 1 L aqueous solution containing 10 ppm of RhB. The absorbance of RhB was monitored using UV-Vis spectroscopy for 30-180 min. The absorption spectrum of the RhB solution containing SnS2 nanoparticles is shown in Figure 5a. The absorbance spectra indicated here were taken for few drops of RhB solution after being centrifuged at a high speed (10,000 RPM for 15 min). So, the SnS2 nanoparticles in the sample are resting at the bottom of the sample, meaning there is no peak (at ~427 nm) related to SnS2. The intensity of the absorption band at 552 nm decreased linearly with the increase in time, showing that the RhB solution gradually degraded over the SnS2 adsorbent. The absorption intensity of the peak decreased by approximately 35% in the presence of SnS2 nanoparticles at 30 min. After 60 min, the degree of decolorization was 78%. After 90 min, the degree of

SnS 2 Nanoparticles as Adsorbent for Organic Pollutants (RhB Dye)
The adsorption of RhB dye from an aqueous solution was carried out at room temperature (approximately 25 • C). Typically, 0.4 g of SnS 2 nanoparticles was added to a 1 L aqueous solution containing 10 ppm of RhB. The absorbance of RhB was monitored using UV-Vis spectroscopy for 30-180 min. The absorption spectrum of the RhB solution containing SnS 2 nanoparticles is shown in Figure 5a. The absorbance spectra indicated here were taken for few drops of RhB solution after being centrifuged at a high speed (10,000 RPM for 15 min). So, the SnS 2 nanoparticles in the sample are resting at the bottom of the sample, meaning there is no peak (at~427 nm) related to SnS 2 . The intensity of the absorption band at 552 nm decreased linearly with the increase in time, showing that the RhB solution gradually degraded over the SnS 2 adsorbent. The absorption intensity of the peak decreased by approximately 35% in the presence of SnS 2 nanoparticles at 30 min. After 60 min, the degree of decolorization was 78%. After 90 min, the degree of decolorization increased to 90%, and then at 180 min, it reached 96%, suggesting that the majority of the RhB had been degraded. Discoloration of the solution might occur as a result of the dye chromogen being destroyed. Similar degradation of RhB with the increasing Zn doping concentration in SnS 2 nanoparticles was observed in a previous report [35].  The degradation of the RhB solution agrees with the pseudo-first-order kinetics [36,37], ln(C/C0) = −kt, where C0 and C are the initial and actual concentrations of RhB, k is the rate constant, and t is the degradation time. The normalized concentration of the solution equals the normalized maximum absorbance; therefore, the remaining ratio C/C0 is replaced with A/A0. The C/C0 with respect to reaction time obtained by monitoring the RhB absorption peak at 552 nm is shown in Figure 5b. It is clear that, during the initial 30 min, the concentration of RhB decreased by 65%. Further, the concentration of RhB decreased by 80% and 85% at 120 min and 180 min, respectively. Therefore, within 180 min of reaction time, SnS2 nanoparticles showed the highest adsorption capacity (21.25 mg/g) for RhB, indicating that it is an appropriate adsorbent for the removal of RhB.
The adsorption capacity of SnS2 nanoparticles was compared to that of other reported nanoparticles and nanocomposites in Table 1, which demonstrated that the adsorption performance of the SnS2 nanoparticles synthesized in the present work was similar to that of other nanocomposites and superior to that of other NP adsorbents.  The degradation of the RhB solution agrees with the pseudo-first-order kinetics [36,37], ln(C/C 0 ) = −kt, where C 0 and C are the initial and actual concentrations of RhB, k is the rate constant, and t is the degradation time. The normalized concentration of the solution equals the normalized maximum absorbance; therefore, the remaining ratio C/C 0 is replaced with A/A 0 . The C/C 0 with respect to reaction time obtained by monitoring the RhB absorption peak at 552 nm is shown in Figure 5b. It is clear that, during the initial 30 min, the concentration of RhB decreased by 65%. Further, the concentration of RhB decreased by 80% and 85% at 120 min and 180 min, respectively. Therefore, within 180 min of reaction time, SnS 2 nanoparticles showed the highest adsorption capacity (21.25 mg/g) for RhB, indicating that it is an appropriate adsorbent for the removal of RhB.
The adsorption capacity of SnS 2 nanoparticles was compared to that of other reported nanoparticles and nanocomposites in Table 1, which demonstrated that the adsorption performance of the SnS 2 nanoparticles synthesized in the present work was similar to that of other nanocomposites and superior to that of other NP adsorbents.

SnS 2 Thin Films for Photovoltaic Application
The results obtained in Section 3.1 concluded that the synthesized SnS 2 nanoparticles have a high degree of composition and phase purity. Therefore, the as-synthesized SnS 2 nanoparticles were spin coated to obtain the SnS 2 thin film. The as-synthesized SnS 2 nanoparticles showed a granular morphology with the particle size of~52 nm and the asprepared SnS 2 thin film on CIGS showed a tiny grain texture with uniformity, as displayed in Figure 6. The structural and optical properties of the SnS 2 films are described in the following sections.

SnS2 Thin Films for Photovoltaic Application
The results obtained in Section 3.1 concluded that the synthesized SnS2 nanoparticles have a high degree of composition and phase purity. Therefore, the as-synthesized SnS2 nanoparticles were spin coated to obtain the SnS2 thin film. The as-synthesized SnS2 nanoparticles showed a granular morphology with the particle size of ~52 nm and the asprepared SnS2 thin film on CIGS showed a tiny grain texture with uniformity, as displayed in Figure 6. The structural and optical properties of the SnS2 films are described in the following sections.

Crystal Structure of SnS2 Thin Film
X-ray diffraction was used to determine the crystal structure of the as-grown SnS2 film, and the matching profile is shown in Figure 7. The diffraction pattern indicated that the SnS2 film was polycrystalline, with significant peaks at 15.02°, 28.18°, 32.54°, and 49.69° corresponding to the (0 0 1), (1 0 0), (1 0 1), and (1 1 0) planes, respectively, and exhibited a hexagonal crystal structure [20]. The SnS2 peaks observed in the XRD profile are consistent with those found in the standard JCPDS card No. 23-0677. The film lacked any peaks associated with other secondary phases such as SnS and Sn2S3, suggesting that the SnS2 film generated in this work is free of contaminants [45].

Crystal Structure of SnS 2 Thin Film
X-ray diffraction was used to determine the crystal structure of the as-grown SnS 2 film, and the matching profile is shown in Figure 7. The diffraction pattern indicated that the SnS 2 film was polycrystalline, with significant peaks at 15.02 • , 28.18 • , 32.54 • , and 49.69 • corresponding to the (0 0 1), (1 0 0), (1 0 1), and (1 1 0) planes, respectively, and exhibited a hexagonal crystal structure [20]. The SnS 2 peaks observed in the XRD profile are consistent with those found in the standard JCPDS card No. 23-0677. The film lacked any peaks associated with other secondary phases such as SnS and Sn 2 S 3 , suggesting that the SnS 2 film generated in this work is free of contaminants [45].  Figure 8a shows the optical transmittance spectrum of the as-grown SnS2 film recorded in the wavelength range of 300-1000 nm. A sharp decrease in the transmittance spectrum revealed that the as-grown SnS2 film showed an average optical transmittance  Figure 8a shows the optical transmittance spectrum of the as-grown SnS 2 film recorded in the wavelength range of 300-1000 nm. A sharp decrease in the transmittance spectrum revealed that the as-grown SnS 2 film showed an average optical transmittance of approximately 90% above the fundamental absorption edge. The average transmittance difference between CdS and SnS 2 films in the visible light region is around 11%, which is a small discrepancy that could be attributed to scattering losses. The steep absorption edge in the spectrum indicates that the film has a direct optical transition between the parabolic bands. To evaluate the energy band gap (E g ) of the thin film, (αhυ) p was plotted against the photon energy (hυ), where p is an integer, whose value indicates the type of optical transition occurring in the thin film. In the present study, the nature of the transition was found to be direct with p = 1/2, and the energy band gap was calculated using plots of (αhυ) 2 versus hυ (Figure 8b), where the extrapolation of the (αhυ) 2 plot onto the hυ axis provides the band gap. The evaluated optical energy band gap of the as-prepared SnS 2 film was approximately 2.8 eV, which is consistent with previously reported findings [23]. Figure 8a,b show the transmittance spectra and corresponding band gap graph of the conventional CdS buffer. Although the transmittance of the standard CdS buffer was similar to that of the SnS 2 film, the absorption edge of the CdS film was at a longer wavelength than that of the SnS 2 film. The band gap determined from the (αhυ) 2 versus hυ plot of CdS was approximately 2.2 eV [46], which is marginally less than the band gap of the SnS 2 film. In other words, SnS 2 has a wider band gap than the conventional CdS buffer, which enables the transmission of a more significant fraction of the solar spectrum to the absorber.

SnS2 Thin Film as a Buffer in CIGS Solar Cell
In this study, two CIGS thin-film solar cells were fabricated with structures as glass/Mo/CIGS/CdS (conventional buffer)/i-ZnO/AZO/Ag/Ni and glass/Mo/CIGS/SnS2 (buffer from the present work)/i-ZnO/AZO/Ag-Ni using conventional CdS buffer and SnS2 buffer, respectively. Our earlier article [47] detailed the procedure for fabricating a CIGS absorber. Initially, a two-stage technique was used to construct a CIGS light absorber on an Mo substrate. Subsequently, the chemical bath deposition (CBD) method produced a 70 nm thick CdS buffer. The complete deposition conditions for CBD-grown CdS are described in a previous report [48]. A 50 nm thick layer of SnS2 buffer was spin-coated onto the CIGS absorber following the procedure reported in Section 2.2. The devices were then finished by radio frequency (RF)/DC sputtering of i-ZnO/ZnO/Al transparent conducting layers and e-beam evaporation of an Ni/Al grid.
The J-V characteristics of the CIGS devices made with conventional CdS buffer (CIGSCdS) and SnS2 buffer (CIGSSnS2) are shown in Figure 9. The CIGSCdS exhibited an efficiency of 7.5% with an open-circuit voltage (VOC) of 0.51 V, a short circuit current

SnS 2 Thin Film as a Buffer in CIGS Solar Cell
In this study, two CIGS thin-film solar cells were fabricated with structures as glass/Mo /CIGS/CdS (conventional buffer)/i-ZnO/AZO/Ag/Ni and glass/Mo/CIGS/SnS 2 (buffer from the present work)/i-ZnO/AZO/Ag-Ni using conventional CdS buffer and SnS 2 buffer, respectively. Our earlier article [47] detailed the procedure for fabricating a CIGS absorber. Initially, a two-stage technique was used to construct a CIGS light absorber on an Mo substrate. Subsequently, the chemical bath deposition (CBD) method produced a 70 nm thick CdS buffer. The complete deposition conditions for CBD-grown CdS are described in a previous report [48]. A 50 nm thick layer of SnS 2 buffer was spin-coated onto the CIGS absorber following the procedure reported in Section 2.2. The devices were then finished by radio frequency (RF)/DC sputtering of i-ZnO/ZnO/Al transparent conducting layers and e-beam evaporation of an Ni/Al grid.
The J-V characteristics of the CIGS devices made with conventional CdS buffer (CIGS CdS ) and SnS 2 buffer (CIGS SnS2 ) are shown in Figure 9. The CIGS CdS exhibited an efficiency of 7.5% with an open-circuit voltage (V OC ) of 0.51 V, a short circuit current density (J SC ) of 27 mAcm −2 , and a fill factor (FF) of 54%, while the CIGS SnS2 showed an efficiency of 5.1% with a V OC of 0.41 V, J SC of 26 mA cm −2 , and FF of 49%. The performance parameters of CIGS SnS2 and CIGS CdS are summarized in Table 2. The results indicated that the CIGS SnS2 device with the SnS 2 buffer prepared in this study exhibited comparable results to that of the CIGS CdS . However, the CIGS SnS2 device exhibited a considerable drop in shunt resistance (R sh ), suggesting the direct contact between the window layer and absorber possibly raised from plasma damage during window layer preparation and shunt paths caused by pinholes in SnS 2 buffer [49]. As FF is very sensitive to R sh among the device parameters [50], it is reduced by approximately 5% in the case of the CIGS SnS2 device. On the other hand, the effect of R sh is less considerable on the J SC , which is mainly governed by optical and recombination losses. The optical losses are caused by the reflection from the device surface and absorption by the AZO window and SnS 2 buffer layers. Electron-hole pairs are generated when photons hit the CIGS absorber, and some of the produced charge carriers are not collected, but are lost as a result of recombination. The recombination loss is indicative of the quality of the SnS 2 /CIGS junction (measure of V OC ) and is most sensitive to buffer thickness [49]. The plasma damage of SnS 2 and pinholes may create the AZO/CIGS interface as strong recombination centers. This kind of recombination loss may decrease with the increasing SnS 2 thickness. According to a previous report [33], the increase in the thickness of the CdS buffer layer resulted in increased light absorption loss by CdS, but decreased reflection loss. Therefore, SnS 2 thickness optimization is necessary to minimize the photocurrent loss by recombination to ensure the quality of the CIGS/SnS 2 junction and to improve the performance characteristics of the CIGS SnS2 device. increased light absorption loss by CdS, but decreased reflection loss. Therefore, SnS2 thickness optimization is necessary to minimize the photocurrent loss by recombination to ensure the quality of the CIGS/SnS2 junction and to improve the performance characteristics of the CIGSSnS2 device.

Conclusions
Tin disulfide (SnS 2 ), a simple binary metal chalcogenide, was proposed as a viable adsorbent for removing toxic dyes from water and as a buffer for Cd-free thin-film solar cells owing to its abundance, low-cost, non-toxicity, and chemical stability. The current study explored the synthesis of SnS 2 nanoparticles and the deposition of SnS 2 thin films using a chemical precipitation method and spin-coating technique, respectively. The XPS and Raman studies indicated the existence of Sn and S in the synthesized nanoparticles together with a pure SnS 2 phase (characteristic Raman mode at 310 cm −1 ). The XRD and optical studies showed that the as-synthesized SnS 2 thin films had a hexagonal crystal structure with (001) as the preferred orientation and an optical band gap of 2.8 eV. At 180 min of reaction time, 0.4 g/L of SnS 2 nanoparticles demonstrated 96% decolorization and 85% adsorption capacity for RhB (10 ppm), revealing that the majority of the RhB was degraded; therefore, SnS 2 is a suitable adsorbent. The fabricated CIGS device with SnS 2 (50 nm) as buffer exhibited an open circuit voltage (V OC ) of 0.41 V, a short circuit current density (J SC ) of 25.67 mA cm −2 , a fill factor (FF) of 49%, and a conversion efficiency (η) of 5.1%, demonstrating that SnS 2 is an alternative buffer. The authors intend to dope the nanoparticles and optimize the thickness of the SnS 2 buffer layer in future studies to (i) increase the adsorption capacity of SnS 2 nanoparticles and (ii) improve the performance of the CIGS SnS2 device.