Detecting CdSe Nanomaterials with a Fluorescent Schiff Base Ligand

Citation: Sanmartín-Matalobos, J.; Bermejo-Barrera, P.; Pérez-Juste, I.; Fondo, M.; García-Deibe, A.M.; Alves-Iglesias, Y. Detecting CdSe Nanomaterials with a Fluorescent Schiff Base Ligand. Chemosensors 2022,10, 394. https://doi.org/ 10.3390/chemosensors10100394 Academic Editor: Sofian Kanan Received: 24 August 2022 Accepted: 25 September 2022 Published: 28 September 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). chemosensors Article Detecting CdSe Nanomaterials with a Fluorescent Schiff Base Ligand Jesús Sanmartín-Matalobos 1,* , Pilar Bermejo-Barrera 2, Ignacio Pérez-Juste 3, Matilde Fondo 1, Ana M. García-Deibe 1,* and Yeneva Alves-Iglesias 1,2 1Coordination and Supramolecular Chemistry Group (SupraMetal), Department of Inorganic Chemistry, Faculty of Chemistry, Institute of Materials (iMATUS), Universidade de Santiago de Compostela, Avenida das Ciencias s/n, 15782 Santiago de Compostela, Spain 2 Trace Element, Speciation and Spectroscopy Group (GETEE), Department of Analytical Chemistry, Nutrition and Bromatology, Faculty of Chemistry, Institute of Materials (iMATUS), Universidade de Santiago de Compostela, Avenida das Ciencias s/n, 15782 Santiago de Compostela, Spain 3Departamento de Quıímica Física, Facultad de Química, Edificio de Ciencias Experimentales, Universidade de Vigo, 36310 Vigo, Spain *Correspondence: [email protected] (J.S.-M.); [email protected] (A.M.G.-D.) Abstract: We investigated the easily synthesized ligand H 3 L as a fluorescent chemosensor for the detection of CdSe nanoparticles (CdSe NPs) and L-cysteine-capped CdSe quantum dots (CdSe-Cys QDs) in ethanol–water samples. A drastic quenching of the fluorescence emission of H 3 L at 510 nm occurred, as a result of the addition of CdSe NPs and CdSe-Cys QDs. A solution of H 3 L (1.26 ppb) showed sensitive responses to both CdSe NPs and CdSe-Cys QDs, with limits of detection (LOD) as low as 40 and 62 ppb, respectively. Moreover, using a smartphone color recognizer application, the fluorescence intensity response of H 3 L-modified cellulose paper to CdSe-Cys QDs was recorded on a red channel (R), which allowed us to detect CdSe-Cys QDs with LOD = 15 ppb. Interference of some common metal nanomaterials (NMs), as well as metal ions, in the determination of CdSe NMs in solution was studied. The affinity of H 3 L to CdSe NPs and CdSe-Cys QDs was spectroscopically determined. Scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDX), microX-ray fluorescence ( μ -XRF), 1 H-NMR, attenuated total reflection infrared spectroscopy (ATR-IR), and density functional theory (DFT) were also used to investigate the interaction of H 3 L with CdSe NMs. Keywords: CdSe; nanomaterials; quantum dots; Schiff base ligand; chemosensor; fluorescence 1. Introduction Over the past few years, the unique chemical and biological properties of nanomaterials (NMs) has led to their ubiquitous presence in daily life. Amid the varied types of NMs, the II–VI semiconducting NMs have attracted much attention due to their outstanding electronic and optoelectronic properties [ 1 – 3 ]. In particular, one of the most widely used NMs of this type is cadmium selenide, as it has useful properties for biochemical sensors, biomedical imaging, photovoltaic applications, light-emitting diodes (LEDs), laser, thinfilm transistors, or solar cells [ 3 , 4 ]. Another feature of this chalcogenide is its ability to form different nanostructures from simple nanoparticles to quantum dots (QDs), nanowires, nanobelts, or nanotubes, with dissimilar properties [4]. However, a clear negative consequence of this extensive use is the discharge into the environment of such noxious material. The toxicity of CdSe NMs results from the release of their harmful elements in an acidic environment. While cadmium is a well-known toxic element, which can induce oxidative stress, DNA damage, and apoptosis, excessive selenium can also induce toxicity [ 5 – 9 ], especially for aquatic life [ 10 – 13 ]. Therefore, the increasing concentrations of these NMs in the environment demands for reliable and affordable approaches for their detection. Chemosensors 2022,10, 394. https://doi.org/10.3390/chemosensors10100394 https://www.mdpi.com/journal/chemosensors Chemosensors 2022,10, 394 2of16 Among the most suitable analytical methods useful for detecting NMs in aqueous samples, fluorescence-based sensing stands out. These methods involve simple solution assay processing, low cost, fast response, and high sensitivity, as well as high selectivity through molecular binding design. In this sense, some optical sensors have been developed for the detection of Ag NPs [ 14 – 16 ], but investigation of fluorescent sensors to detect CdSe NMs remains virtually unexplored [17]. Recently, we explored the chelating potential of two N-tosyl imines for CuO NPs sensing at neutral pH [ 18 , 19 ]. Here, we investigate the dansyl-based fluorescent probe H 3 L (Scheme 1), which was easily synthesized by us [ 20 ], with the aim of detecting CdSe NMs in aqueous solution with short response time and sensing at basic pH. Scheme 1. Schematic representation of H3L, with the fluorophore highlighted in green. H 3 L displays an N,N,O donor set suitable to bind the Cd 2+ ions present on the NM surface. In addition, both carboxylate and sulfonamide groups can contribute to the stabilization of the systems by interaction with the superficial ions of the NMs. Herein, we are interested in studying the changes in the fluorescence emission spectra of H 3 L upon increasing addition of CdSe NPs and CdSe-Cys QDs. Interference of some common NMs, as well as of metal ions, in the determination of CdSe NMs in ethanol/water samples was studied. Binding constants were also determined since these constants characterize the affinity of H 3 L for NMs. Furthermore, interactions of H 3 L with CdSe NPs and CdSe-Cys QDs were experimentally and theoretically (DFT calculations) investigated. 2. Materials and Methods All starting materials and reagents, except H 3 L and CdSe-Cys QDs, were commercially available and were used without further purification. The synthesis and characterization of H 3 L were previously reported [ 20 ]. Fluorescence emission studies were performed on a Shimadzu RF-600 Spectro Fluorophotometer. Diffuse reflectance spectra were measured on a PerkinElmer LAMBDA 1050+ UV/Vis/NIR spectrophotometer equipped with an integrating sphere. Scanning electron microscopy (SEM) was applied to investigate the size and morphology, which was carried out under a ZEISS FESEM ULTRA, along with energydispersive X-ray (EDX) microanalysis. X-ray fluorescence was measured under vacuum (19.6 mbar) using a M4 TORNADO system (BRUKER), with an Rh tube, operating at 50 kV and 200 μ A. The fluorescence maps were collected for a total time of 2 ms per pixel, with a pixel size of 20 μ m. 1 H-NMR spectra (400 MHz) were measured in deuterated solvents using a Varian Inova 400 Spectrometer. Jvalues are given in hertz. The infrared spectra were measured in the range 4000 to 400 cm −1 wavenumber, using a Fourier-transform infrared (FTIR) spectrometer PerkinElmer Spectrum Two coupled with platinum diamond ATR, which consists of a diamond disc as an internal reflection element. 2.1. Sample Preparation Samples for investigations on the interaction of H 3 L with CdSe NMs were obtained by stirring an ethanol solution of H 3 L at pH = 9 and a suspension of NMs in 2:1 molar ratio, at room temperature, for ca. 15 min. The sample was subsequently air-dried. Chemosensors 2022,10, 394 3of16 2.2. Computational Methods Theoretical calculations on ligand–NMs models were performed employing density functional theory (DFT) methods, as implemented in Gaussian 09 [ 21 ]. Thus, M062X/631G* geometrical optimization of different starting structures of H 3 L was performed. For H 3 L, we tested that the M062X/6-31G* level provided results of comparable quality to those obtained with a more extended basis set as 6-311++G** (not included for brevity), but with a lower computational cost. Models of various sizes have been used to evaluate the interaction of the ligand with CdSe NPs. The geometry for the smallest model, comprising one H 3 L unit and a single CdSe unit, was optimized without restrictions. In the remaining cases, the geometries of the ligand over the surface were freely optimized, keeping a frozen structure for the metal selenide obtained from crystallographic data deposited with the Cambridge Structural Database (CSD). Interaction energies, Eint, for these systems were evaluated as Eint =E complex −(EH3L +E CdSe model ), where E complex is the total energy of the ligand–NM interaction; E H3L is the total energy of the monoanionic or dianionic form of the H 3 L ligand, and E CdSe model is the energy of the different CdSe models indicated above. These interaction energies were evaluated by means of the natural energy decomposition analysis (NEDA) method included in the NBO7.0 program [ 22 ], such that the basis set superposition error was corrected by means of the counterpoise method. 2.3. Synthesis and Characterization of CdSe-Cys QDs CdSe-Cys QDs were synthesized and characterized according to a method previously reported [23,24], which was slightly modified by us. Firstly, 0.4 mmol (31.6 mg) of selenium and 1 mmol (38.6 mg) of sodium borohydride were dissolved in 10 mL of deionized water in a three-neck round-bottom flask, under argon atmosphere and constant stirring. The reduction of the selenium was kept in an ice bath for 2 h. In a second reaction flask, 0.4 mmol (123 mg) of cadmium nitrate tetrahydrate and 2 mmol (358 mg) of L-cysteine hydrochloride monohydrate were dissolved in 80 mL of deionized water. The pH of this solution was adjusted to 10 with a sodium hydroxide solution of 0.1 M. After this, the first solution was added to this second reaction flask, and the color of the resulting solution instantaneously changed from colorless to yellow. This solution was stirred and heated up to 100 ◦ C. Monitoring of the QD size was performed every 10 min by both fluorescence emission [ 23 ] and UV/Vis absorption [ 25 ] spectrometry. After 1 h, the solution was red ( λabs = 451 nm and λem = 650 nm, size of about 2.0 nm), and the reaction flask was subsequently kept in an ice bath to stop the reaction. Evaporation of the solution under reduced pressure led to one-fifth of its volume. Then, small portions of acetone were added to the solution, until it became cloudy, resulting in the precipitation of the CdSe-Cys QDs. The resulting supernatant was separated by centrifugation. Small portions of acetone were added again to this supernatant. This procedure was repeated three times. The precipitated QDs were dried at 40 ◦ C, in an oven. Spectroscopy and microscopy characterizations are shown in Figures S1–S4. Yield = 0.07 g. 1 H-NMR (400 MHz, D 2 O, δ in ppm): 3.66 (t, 1H), 3.17 (dd, 1H), 2.96 (dd, 1H). ATR-IR ( ν in cm −1 ): ν (HNH) 3222, νas (COO − ) 1557, νs (COO − ) 1393, ν (CO) 1302. UV/Vis (water, λ in nm): 451. Fluorescence λ /nm: λem = 632 ( λex = 400, bandwidth of 15 nm). 2.4. Immobilization of H3L onto Amine-Modified Cellulose Paper For the immobilization of H 3 L on cellulose paper (Whatman ® qualitative filter paper, grade 2), we followed a previously reported method [ 26 ], which was slightly modified by us. Cellulose filter paper with a diameter of 55 mm was soaked in an ethanolic 3-aminopropyl)trimethoxysilane solution (1 wt.%) for 1 h, at room temperature. The amine-modified cellulose paper was washed with copious ethanol, and dried under vacuum at 40 ◦ C. N-Hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N’- Chemosensors 2022,10, 394 4of16 ethylcarbodiimide hydrochloride (EDCHCl) were added in equimolar ratio (0.1 mmol) to a dimethylformamide solution (20 mL) of H 3 L (0.1 mmol) to activate it. After stirring at room temperature for 3 h, triethylamine (0.3 mmol) was added to the reaction mixture, and the amine-modified cellulose paper was soaked in it overnight. Then, the immobilized H 3 L onto amine-modified cellulose paper was washed with water and copious ethanol, and then dried under vacuum at 40 ◦C. 3. Results and Discussion H 3 L, in powder form, emits a yellow fluorescence under a UV light source at 365 nm (Figure 1, left bottom). An ethanol solution of H 3 L at neutral pH emitted a maximal green fluorescence at about 510 nm, when it was exposed to visible light with a wavelength of 400 nm (Figure 1, at the bottom right). The use of polar solvents affords better emission and justified the use of ethanol to yield high green emission intensities, with Stokes shifts of about 110 nm. The study of the influence of pH on the fluorescence spectrum of H 3 L (Figure 1, top) showed an increase in fluorescence intensity with increasing pH, which is consistent with the deprotonation of H 3 L. Monodeprotonation of H 3 L is expected at pH values below 7, since the pKa value of the carboxylic group is around 4. Deprotonation of the phenol and sulfonamide groups requires higher pH values, since the pKa of these groups is around 9. Moreover, pH values of about 6 (and lower ones) result in the protonation of the dimethylamino group, which is accompanied by a redshifted of λem to a wavelength of 520 nm (bathochromic shift). This can be due to the increase in the electron-withdrawing power of the sulfonyl group upon protonation of the amine nitrogen atom. Figure 1. Top : Influence of pH on the fluorescence spectrum of H 3 L (4.44 × 10 −4 M) measured in ethanol (pH 2.1–12.2) at room temperature. Bottom : The effect of UV light (365 nm) on an ethanol solution (right) and a powdery sample (left)ofH 3L. 3.1. Fluorescence Emission Studies on H3L Solution upon Addition of CdSe NMs The fluorescence intensity of an ethanol/water (80:20) solution of H 3 L varied linearly with the concentrations of nonfluorescent CdSe NPs, as well as with weakly fluorescent CdSe-Cys QDs (λem = 632 nm), as shown in Figure S2 of the ESI. In order to check the usefulness of H 3 L as a chemosensor for the detection of CdSe nanomaterials at concentration ranges of ppm and ppb, we selected concentrations of H 3 L Chemosensors 2022,10, 394 5of16 of 126 ppb and 1.26 ppb. With [H 3 L] = 126 ppb, fluorescent emission varied linearly in the range 0–11.5 ppm of CdSe NPs and 0–10.5 ppm of CdSe-Cys QDs. The decrease by over 80% and 55% in the fluorescence emission of ethanol/water solutions (in 80:20 v/v) of H 3 L( λem = 540 nm) upon addition of CdSe NPs ( λem = 536 nm) and CdSe-Cys QDs (λem = 536 nm), respectively, are shown in Figure 2. Figure 2. Calibration curve of fluorescence intensity of H 3 L in 80:20 ethanol/water solution at pH 11 with gradual addition of NMs (until the fluorescence intensity stops decreasing). Left :[H 3 L] = 126 ppb. Right :[H 3 L] = 1.26 ppb. NaOH 0.1 M was used to deprotonate H 3 L. Final volume of the solution = 4 mL. Each solution was sonicated for 1 min before being measured. λexc = 400 nm. The limit of detection (LOD) and limit of quantification (LOQ) of H 3 L are expressed as LOD = 3SD/M and LOQ = 10SD/M, where SD is the standard deviation of the response, and M is the slope of the calibration curve [ 27 ]. LODs of CdSe NPs and CdSe-Cys QDs with [H 3 L] = 126 ppb were 460 and 630 ppb, respectively. LOQs of CdSe NPs and CdSe-Cys with [H 3 L] = 126 ppb were 1.52 and 2.11 ppm, respectively. LODs of CdSe NPs and CdSe-Cys QDs with [H 3 L] = 1.26 ppb were 40 and 62 ppb, respectively. LOQs of CdSe NPs and CdSe-Cys with [H 3 L] = 1.26 ppb were 134 and 207 ppb, respectively. The working range of H 3 L was obtained using the LOQ as the minimum value that can be measured, with the highest value of [NMs] at which linearity is still maintained as the maximum value. Therefore, the working ranges for detection of CdSe NPs and CdSe-Cys with [H 3 L] = 126 ppb were 1.5–11.5 and 2.1–10.5 ppm, respectively. 3.2. Fluorescent Paper Detection of H3L toward CdSe-Cys QDs To facilitate detection of CdSe-Cys QDs, without the need to use a spectrofluorometer, we immobilized H 3 L onto cellulose paper. The immobilization results in a slight color change of the paper, from white to pale yellow, which is visible with the naked eye. The ligand-modified cellulose paper upon soaking in CdSe-Cys QDs water solutions for 15 min exhibited a continuous color change from light green to bluish green under 365 nm ultraviolet light, when the CdSe-Cys QDs concentration increased from 5 to 100 ppb (Figure 3). Chemosensors 2022,10, 394 6of16 (a) (b) (c) (d) (e) Figure 3. ( a ) Commercial cellulose paper under visible light. ( b ) Schematic representation of the ligand-modified cellulose paper. ( c ) Ligand-modified cellulose paper under visible light. ( d ) Variation of red coordinate in the RGB space of fluorescent images (excitation at 365 nm) of the ligand-modified cellulose paper upon soaking in CdSe-Cys QDs solution (5–100 ppb) for 15 min. Images (under UV light) of the fluorescent paper are included to show the color change. ( e ) Decrease in the absorbance of the band at 270 mm, with the increase in CdSe-Cys QDs concentration from 100 ppb to 2 ppm. Since slight color changes cannot be easily observed with the naked eye, we digitized the color information (RGB value) of photographs corresponding to the paper under UV exciting radiation for analysis through the color recognizer application of a smartphone [ 28 , 29 ]. A series of photos were taken for three replicates of each assay with a smartphone under 365 nm ultraviolet light. The red–green–blue (RGB) values corresponding to these photos were obtained through the color recognizer application (RGB Color Detector) for further statistical analysis [ 30 , 31 ]. The RGB coordinates of five points on distinct parts of the paper for three replicas were obtained. Since the R coordinate shows sensitive response to CdSe-Cys QDs, the fluorescence intensity response, as an average of values for the measurements made, was recorded on the red channel. As Figure 3shows, there was a good linear relationship between the red coordinate and the CdSe-Cys QDs concentration (R2= 0.9857). The LOD and LOQ calculation results were 15 and 50 ppb, respectively. We also explored the use of UV/Vis/NIR spectrophotometry to discern the color change of the ligand-modified cellulose paper with the increase in the concentration of CdSe-Cys QDs. The ligand-modified cellulose paper upon soaking in CdSe-Cys QDs water solutions for 2 h exhibited a continuous decrease in the absorbance of each band of the diffuse reflectance spectrum, which showed three dominant signals located around 420, 330, and 270 nm. As the decrease in the absorbance was clearest in the band at 270 nm, we used this wavelength to study the response of the ligand-modified cellulose paper to the CdSe-Cys QDs concentration. Figure 3(right) shows the linear decrease in the absorbance Chemosensors 2022,10, 394 7of16 of the band at 270 mm, when the CdSe-Cys QDs concentration increased from 100 ppb to 2 ppm. The LOD and LOQ calculation results were 245 and 815 ppb, respectively. 3.3. Affinity of H3L to CdSe NMs Since the reaction of H 3 L with both CdSe NPs and CdSe-Cys QDs resulted in a decrease in its fluorescence emission, we investigated the quenching mechanism using Stern–Volmer plots (F 0 /F=1+K SV [NMs]). Figure 4shows that the value of the quenching constant (K SV = slope) decreased with increasing temperature, which is a sign of static quenching [ 32 ]. It must be noted that, for static quenching ( τ0 / τ = 1), the K SV value matched that of the binding constant (K B ) for the interaction under study. Thus, we found that the affinity of H 3 L to the NMs studied at room temperature was as follows: CdSe NPs (K B = 4.42 × 10 3 L · mg −1 ) > CdSe-Cys QDs (K B = 2.30 × 10 3 L · mg −1 ). Values of Gibbs free energy (Table 1), which were determined from K B , indicate that CdSe-H 3 L NPs ( − 20.46 kJ · mg −1 ) were more stable than CdSe-Cys-H3L QDs (−18.87 kJ·mg−1). Figure 4. Plots of the intensities of the fluorescence spectra of H 3 L (10 −4 M) vs. the concentration of the quenchers CdSe NPs ( top ) and CdSe-Cys QDs ( bottom ) in an 80:20 ethanol/water solution. Slopes of the curves at 293, 303, and 313 K are Stern–Volmer constants K SV (K SV =k qτ , where τ is the lifetime of the fluorophore) at the cited temperatures. Chemosensors 2022,10, 394 8of16 Table 1. Values of Gibbs free energy and quenching constant for the reaction of H 3 L with CdSe NPs and CdSe-Cys QDs. Species ΔGKsv 293 K kJ·mol−1 (kJ·mg−1) Ksv 293 K M−1 (L·mg−1) Ksv 303 K M−1 (L·mg−1) Ksv 313 K M−1 (L·mg−1) CdSe NPs −20.08 (−20.46) 3790 (4420) 3150 (3600) 2610 (2750) CdSe-Cys QDs (−18.87) (2300) (1800) (1230) Considering the higher affinity of H 3 L for CdSe NPs compared to CdSe-Cys QDs, we studied the number of binding sites for H 3 L on the surfaces of both CdSe NPs and CdSe-Cys QDs. The number of interaction binding sites (n) was determined using the Scatchard equation (Equation (1)), where F 0 and Fare the relative fluorescence of H 3 L in the absence and presence of NMs, respectively, K B is the binding constant, and [Q]is the quencher concentration [ 33 ]. The Scatchard equation linear graph of H 3 L and NMs is shown in Figure 5. logF0−F F=log KB+nlog[Q](1) Figure 5. The Scatchard plot for binding H 3 L to CdSe NPs ( bottom ) and CdSe-Cys QDs ( top ) at 293, 303, and 313 K and pH 11. Chemosensors 2022,10, 394 9of16 The number of binding sites (n) was around five at 293 K, suggesting five binding sites for H 3 L on the CdSe NP surface. However, the number of binding sites was about two for H 3 L on the CdSe-Cys QD surface. This is not surprising considering that many of the surface cadmium ions interact with cysteine through the sulfur atom. The interaction ratio slightly increased for all the interactions with rising temperature, probably because temperature favors the reactivity of surface metal ions. 3.4. Selectivity In view of the higher affinity of H 3 L to CdSe NPs, we studied the selectivity of H 3 Las a probe for the cited NPs. As a criterion for interference, a ± 10% variation of the average fluorescence intensity at the respective concentration of CdSe NPs was used. The selectivity of H 3 L as a probe for CdSe NPs was tested in the presence of some common NMs such as Cu NPs, CuO NPs, ZnO NPs, and TiO 2 NPs, as well as some common metal ions in water, such as the following hard acids: Na+,K +,Mg 2+,Ca 2+,Fe 3+, and Al3+ (Figure 6). Figure 6. Fluorescence responses of H 3 L (2.5 nM, 1.26 ppb) toward CdSe NPs (8 ppm) in the presence of various NPs ( bottom ) such as Cu NPs (8 ppm), CuO NPs (4 ppm), TiO 2 NPs (4 ppm), and ZnO NPs (4 ppm) and some common metal ions ( top ) such as Na + (8 ppm), K + (8 ppm), Fe 3+ (8 ppm), Mg 2+ (2 ppm), Ca 2+ (2 ppm), and Al 3+ (2 ppm). All experiments were performed in 4 mL of an ethanol–water solution in 80:20 v/v(pH 11) under λexc = 400 nm. Results of three replicas of each assay showed that H 3 L possessed selectivity toward CdSe NPs in the presence of Cu NPs, which could be tolerated in concentration of at least 8 ppm concentration, when the concentration of CdSe NPs was 8 ppm. 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