# Plasmonic Sensing of Aqueous-Divalent Metal Ions by Biogenic Gold Nanoparticles

Authors: Luisa E. Silva-De Hoyos; Victor Sánchez-Mendieta; Alfredo R. Vilchis-Nestor; Miguel A. Camacho-López; Jésica Trujillo-Reyes; Miguel Avalos-Borja
Journal: Journal of Nanomaterials
 (2019)

---

## Abstract

The chemical interaction between biogenic gold nanoparticles (AuNPs) and several metal (II) ions can be regarded as a practical, twofold, colorimetric, and plasmon resonance sensing method for the recognition of some divalent metal ions in aqueous solutions. The green synthesized AuNPs, usingCamellia sinensis as a reducing agent, were characterized by a surface plasmon resonance (SPR) using UV-Vis spectroscopy, infrared spectroscopy, and transmission electron microscopy. The AuNP colloidal solutions obtained have a pink-reddish color with SPRs centered between 529 and 536 nm. AuNPs with spherical, triangular, and hexagonal shapes were found by TEM analyses. Despite their divergent morphologies, these AuNPs can be employed as colorimetric and plasmon resonance sensors for detection of Ca2+, Sr2+, Cu2+, and Zn2+, primarily, in aqueous solutions. Sensibility studies based on molar concentrations were also performed for these metal ions. Furthermore, solid biogenic AuNPs/cellulosic biocomposites were prepared with the aim of developing portable, fast, and dependable colorimetric sensors; nevertheless, these biocomposites resulted to be good adsorbent materials of metal ions.


---

## Body

## 1. Introduction

Divalent metal ions are responsible for the accomplishment of a great number of biological reactions in live organisms. However, depending on their concentration, many of these metal ions, and also in complex compounds, represent hazardous conditions for humans and other live species. Therefore, increasing interest to develop facile, trustworthy, and inexpensive methods for the detection of metal ions has been noticed since the past decade. Nanomaterials are materials where one or more external dimensions are in the range of 1–100 nm [1]. Thus, it has been demonstrated that nanomaterials have unique physical and chemical properties, which have promoted the advent of nanotechnology since about two decades ago [2]. It has also been proved that nanomaterials are not only man-made since there are several of these materials that are formed naturally and coexist in the environment [3]. As part of the nanomaterials, nanoparticles (NPs) are considered particles with a range of sizes between 1 and 100 nm; these nanostructures exhibit improved properties as compared to the bulk-size material; one of the enhanced properties is the optical properties; these can be modulated by controlling the size, shape, and chemical environments [4]. NPs of noble metals, such as gold and silver, have a great interest in science and technology as they exhibit strong absorption of electromagnetic waves in the visible region of the spectrum due to the surface plasmon resonance (SPR), chemical inertness (AuNPs), stable dispersions (AuNPs), chemical ambient, and biocompatibility (AuNPs). If there is a slight change in the NPs like the size, shape, surface nature, or distance between particles, it can lead to changes in their optical properties. Among the noble metal nanoparticles, gold and silver nanoparticles are perhaps the most studied, due to the relatively easy synthesis methods and to their inherent chemical and optical properties [5, 6]. Thus, these particles can be used in applications such as fabrication of optical devices, surface-enhanced Raman scattering, bioimaging, colorimetric sensors, localized surface plasmon sensing, and fluorescence-enhanced sensors [7]. Localized surface plasmon resonance (LSPR) is known as collective oscillations of free electrons in confined metal systems that require excitations by external electromagnetic waves [8–12]; this happens on extended metal surfaces. LSPR frequencies depend on the size, shape, composition, dielectric constant, and interparticle distance of the NPs [8–14]. Because of these characteristics, NPs are suitable for sensing applications [15]. Due to these relevant applications, researchers around the world seek environmentally friendly process for the synthesis of NPs such as biological methods using microorganisms, plants, or plant extracts. The use of these methods is an alternative to chemical and physical methods. Using plant extracts, the synthesis is carried out extracellularly, the process is relatively easy, and the cost decreases. It is important to mention that the plant extracts contain polyphenols that exhibit the ability of reducing and capping agents [4]. The polyphenols have hydroxyl as well as carboxylic acid groups that play a key role in biosynthesis. Previous studies reported that molecules with two vicinal hydroxyl groups act as reducing agents [16]; in contrast, the role of the carboxylic moiety is to stabilize the nanoparticle as a capping agent, so this group has influence on the control of the shape and size of the metal nanoparticles. Thus, the Camellia sinensis aqueous extract with 59.8±1.8g of total polyphenols, among them, catechol and epicatechin, was highlighted as a biogenic reaction medium to obtain Ag and Au nanoparticles with good control of the morphology and narrower size distribution in contrast to other biosynthetic methods [17–19]. Colorimetric sensing is due to the aggregation of AuNPs with different sizes; this aggregation induces interparticle surface plasmon coupling, resulting in a visible color change from pink to purple. The color change during the aggregation of the NPs provides a way to target different analytes [20]. In this particular work, the type of sensing used is molecular recognition by aggregation; when two NPs are close to each other, they become optically coupled due to the interaction and exhibit an additional resonance. Depending on the proximity between the NPs, the SPR has different changes that leads to a redshift and a color change [13, 21–23]. The extent of the redshift in the SPR depends on the concentration of the metal ions in the solution. The bathochromic shift (red shift) in the SPR band is attributed to the formation of smaller aggregates in the presence of metal ions. When two NPs are close to each other, their SPR is coupled and the new position of the resonance band depends on the number of NPs and their proximity [8, 13, 21–23]. Thanks to the optical properties of the NPs, already mentioned, the use of these for the detection of metal ions in water is a viable and relatively simple method for this application [7, 24–29]. Furthermore, nanoplasmonic devices are known as nanoparticles with plasmonic properties that impregnate solid substrates that can be used in sensing applications. There are a variety of substrates that can be used to immobilize plasmonic nanoparticles such as glass, quartz, silicon, and flexible subtracts such as paper. Also, a diversity of methods can be used to immobilize the nanoparticles on the substrate; in this work, the “dip-coating” method was used to prepare a NPs/cellulosic material composite. Paper or other cellulosic material has been used as a flexible support material, such as supercapacitors and electronic and sensing devices. Some of the advantages are easy fabrication, relatively cheap material, ecofriendly, and biodegradable [30].In this research, biogenic AuNPs were synthesized using a simple one-pot methodology withCamellia sinensis (green tea) extract, as a reducing and capping agent. These AuNP colloidal solutions were used as colorimetric and plasmon resonance sensors of different divalent metal ions in aqueous solution: Mg2+, Ca2+, Sr2+, Cu2+,Zn2+, Hg2+, and Pb2+. In addition, AuNPs/cellulosic material composites were prepared and tested as potential portable-metal ion sensor.


## 2. Experiments

### 2.1. Materials

Camellia sinensis (green tea) was obtained from a local store. HAuCl4, CaCl2, CuCl2, PbCl2, ZnCl2, SrCl2, HgCl2, and MgCl2 were acquired as analytical-grade reagents from Sigma-Aldrich. All chemicals were used without further purification, and deionized water was employed on the synthesis and on the sensing experiments. Cellulosic material (Whatman filter paper) was employed as support for the preparation of AuNPs/cellulosic material composites.


### 2.2. Preparation ofCamellia sinensis Aqueous Extract

1 g of commercial green tea (Lagg’s) was boiled for 20 min in 90 mL of deionized water; upon cooling, the infusion was vacuum filtered. The resulting extract was stored for a day on a flask at room temperature and used as a reducing and capping agent [31].


### 2.3. Biosynthesis of AuNPs

The biosynthesis of AuNPs was carried out using 5 mL of 10-3 M of HauCl4 solution and 0.4 mL of Camellia sinensis extract. The synthesis performed was an adaptation of a method previously reported by us [31].


### 2.4. Characterization of AuNPs

The characterization of AuNPs was carried out by the UV-Vis spectroscopy, using a Lambda 650 Perkin-Elmer spectrophotometer. For transmission electron microscopy (TEM) analyses, the samples were prepared by placing drops of the reaction mixture over carbon-coated grids and allowing evaporation. TEM observations were performed on a JEOL 2100 microscope operated at accelerating voltage of 200 kV with a LaB6 filament. To know the amount and shape of the NPs in one sample, approximately 400 NPs per sample were taken into account. Particle size diameters were calculated with the equation davg=Pnidi/Pni, where ni is the number of particles of diameter di.


### 2.5. Infrared Spectroscopy (FTIR) Analyses

An aliquot of 40 mLCamellia sinensis extract and of the biogenic AuNps was, separately, left to dry at room temperature for 48 h. The powders obtained were analyzed from 4000 to 500 nm by using a Bruker spectrophotometer Tensor 27 with MIR source, ATR accessory (model ATR Platinum), and diamond crystal.


### 2.6. Preparation of AuNPs/Cellulosic Material Composites

The biocomposites were obtained through the in situ bioreduction of HAuCl4, using the Camellia sinensis extract, in the presence of a piece of the cellulosic material. Rectangles of 5cm×10cm of filter paper were deposited in a vial with 5 mL of 10-3 M HAuCl4 and 0.4 mL of Camellia sinensis extract; the mix was left standing for 4 h. After that time, the piece of paper was taken out from the solution and air-dried at room temperature.


### 2.7. Characterization of AuNPs/Cellulosic Material Composites

The AuNPs/cellulosic material composites were analyzed by UV-Vis diffuse reflectance spectroscopy (DRS UV-Vis) using a Lambda 35 spectrophotometer of Perkin-Elmer. The spectrum of the cellulosic support was subtracted from the spectra of the Au/biocomposites. Environmental scanning electron microscopy (ESEM) was used to evaluate the morphology of the cellulosic fibers modified with Au nanoparticles before and after the sensing studies using a FEI Quanta FEG 250 microscope. Chemical mapping was performed using an EDS spectroscope from EDAX, attached to the FEG 250 equipment.


### 2.8. Sensing Studies

Sensing experiments were performed with AuNPs prepared with 0.4 mL of reducing agent at room temperature. 1 mL of 10-3 M solutions of CaCl2, CuCl2, PbCl2, ZnCl2, SrCl2, HgCl2, and MgCl2 was added, separately, to 1 mL of the AuNP solution at room temperature. To carry out the study of sensibility of the AuNPs towards detection of the metal ions, different concentrations of each one of these ions: 10-3, 10-4, 10-5, and 10-6 M, were added to the AuNP solution. The sensing and selectivity of the metal ions by the AuNP solution were analyzed by UV-Vis using a Lambda 650 Perkin-Elmer spectrophotometer.The solid biocomposites were put in contact with 1 mL of 10-3 M metal ion solutions. The solid biocomposites were analyzed by UV-Vis using a Lambda 35 of Perkin-Elmer with an integrated sphere, with a resolution of 1 nm, from 300 to 700 nm.


## 2.1. Materials

Camellia sinensis (green tea) was obtained from a local store. HAuCl4, CaCl2, CuCl2, PbCl2, ZnCl2, SrCl2, HgCl2, and MgCl2 were acquired as analytical-grade reagents from Sigma-Aldrich. All chemicals were used without further purification, and deionized water was employed on the synthesis and on the sensing experiments. Cellulosic material (Whatman filter paper) was employed as support for the preparation of AuNPs/cellulosic material composites.


## 2.2. Preparation ofCamellia sinensis Aqueous Extract

1 g of commercial green tea (Lagg’s) was boiled for 20 min in 90 mL of deionized water; upon cooling, the infusion was vacuum filtered. The resulting extract was stored for a day on a flask at room temperature and used as a reducing and capping agent [31].


## 2.3. Biosynthesis of AuNPs

The biosynthesis of AuNPs was carried out using 5 mL of 10-3 M of HauCl4 solution and 0.4 mL of Camellia sinensis extract. The synthesis performed was an adaptation of a method previously reported by us [31].


## 2.4. Characterization of AuNPs

The characterization of AuNPs was carried out by the UV-Vis spectroscopy, using a Lambda 650 Perkin-Elmer spectrophotometer. For transmission electron microscopy (TEM) analyses, the samples were prepared by placing drops of the reaction mixture over carbon-coated grids and allowing evaporation. TEM observations were performed on a JEOL 2100 microscope operated at accelerating voltage of 200 kV with a LaB6 filament. To know the amount and shape of the NPs in one sample, approximately 400 NPs per sample were taken into account. Particle size diameters were calculated with the equation davg=Pnidi/Pni, where ni is the number of particles of diameter di.


## 2.5. Infrared Spectroscopy (FTIR) Analyses

An aliquot of 40 mLCamellia sinensis extract and of the biogenic AuNps was, separately, left to dry at room temperature for 48 h. The powders obtained were analyzed from 4000 to 500 nm by using a Bruker spectrophotometer Tensor 27 with MIR source, ATR accessory (model ATR Platinum), and diamond crystal.


## 2.6. Preparation of AuNPs/Cellulosic Material Composites

The biocomposites were obtained through the in situ bioreduction of HAuCl4, using the Camellia sinensis extract, in the presence of a piece of the cellulosic material. Rectangles of 5cm×10cm of filter paper were deposited in a vial with 5 mL of 10-3 M HAuCl4 and 0.4 mL of Camellia sinensis extract; the mix was left standing for 4 h. After that time, the piece of paper was taken out from the solution and air-dried at room temperature.


## 2.7. Characterization of AuNPs/Cellulosic Material Composites

The AuNPs/cellulosic material composites were analyzed by UV-Vis diffuse reflectance spectroscopy (DRS UV-Vis) using a Lambda 35 spectrophotometer of Perkin-Elmer. The spectrum of the cellulosic support was subtracted from the spectra of the Au/biocomposites. Environmental scanning electron microscopy (ESEM) was used to evaluate the morphology of the cellulosic fibers modified with Au nanoparticles before and after the sensing studies using a FEI Quanta FEG 250 microscope. Chemical mapping was performed using an EDS spectroscope from EDAX, attached to the FEG 250 equipment.


## 2.8. Sensing Studies

Sensing experiments were performed with AuNPs prepared with 0.4 mL of reducing agent at room temperature. 1 mL of 10-3 M solutions of CaCl2, CuCl2, PbCl2, ZnCl2, SrCl2, HgCl2, and MgCl2 was added, separately, to 1 mL of the AuNP solution at room temperature. To carry out the study of sensibility of the AuNPs towards detection of the metal ions, different concentrations of each one of these ions: 10-3, 10-4, 10-5, and 10-6 M, were added to the AuNP solution. The sensing and selectivity of the metal ions by the AuNP solution were analyzed by UV-Vis using a Lambda 650 Perkin-Elmer spectrophotometer.The solid biocomposites were put in contact with 1 mL of 10-3 M metal ion solutions. The solid biocomposites were analyzed by UV-Vis using a Lambda 35 of Perkin-Elmer with an integrated sphere, with a resolution of 1 nm, from 300 to 700 nm.


## 3. Results and Discussion

### 3.1. Synthesis and Characterization of AuNPs

Camellia sinensis extract contains various polyphenols: flavonoids (catechol, epicatechin, and caffeine) and phenolic acids (3,4-dihydroxybenzoic acid, vanillic acid, 4-hydroxy-3,5-dimethoxybenzoic acid, and benzoic acid). These components contribute to the antioxidant activities of the extract. The aqueous extract contains 59.8±1.8 (mg of CAE/g dw) total phenols with an antioxidant capacity of 70.1±2.4 (%) [17–19, 32].The addition ofCamellia sinensis extract (0.4 mL) to a 10-3 M HAuCl4 solution led to the appearance of a dark pink color in the solution after 0.5 h of reaction, indicating the formation of AuNPs and the excitation of SP vibration in the NPs (Figure 1(b)). The colloidal solution resulting from the previous experiment was examined by the UV-Vis spectroscopy. Figure 1 shows the characteristic AuNP SPR bands centered between 529 and 536 nm (Figure 1(a)).Figure 1
(a) UV-Vis spectra of biogenic AuNPs measured at different times during the reaction of 0.4 mL ofCamellia sinensis extract with 5 mL of a 10-3 M of HAuCl4. Curve profiles correspond to 0.5 h (black), 1 h (red), 1.5 h (blue), 2 h (pink), 3 h (green), 4 h (purple), and 24 h (violet). (b) Picture of vials containing AuNPs obtained at different reaction times (0 h to 24 h).
(a)
(b)The kinetics of the formation of AuNPs was followed by the UV-Vis spectroscopy (Figure1(a)); it is observed that with the progress of the reaction, the intensity of the signal at about 536 nm increases with the time of reaction, while a new SPR band centered at around 529 nm appears at about 24 h of reaction (Figure 1(a) violet curve). With a longer time of reaction, the main absorption wavelength undergoes a blue shift before stabilizing at 24 h (Figure 1(a) curve from 0.5 to 24 h). These time-dependent features on the spectra are characteristics of size and shape changes on the AuNPs.Figures2(a) and 2(b) show representative transmission electron microscopy (TEM) images of the AuNPs synthesized with 0.4 mL of Camellia sinensis extract at room temperature. Different shapes of the biogenic AuNPs: spherical (82%), triangular (14%), and hexagonal (1%), can be seen. TEM analyses reveal the formation of AuNPs with triangular shapes (Figure 2(b)), with an average lateral size of 50 nm, and also particles with spherical shapes (Figure 2(a)), with an average size of 18 nm.Figure 2
(a, b) TEM micrographs of AuNPs obtained with 0.4 mL ofCamellia sinensis extract. Inset in (a): HRTEM micrograph of biogenic AuNPs obtained with 0.4 mL of Camellia sinensis extract. Inset in (b): SAED pattern of biogenic AuNPs collected after 24 h of reaction using 0.4 mL of Camellia sinensis extract.
(a)
(b)AuNPs obtained with 0.4 mL ofCamellia sinensis extract show a single crystalline nature of metallic gold as revealed in the selected area electron diffraction rings (SAED) (Figure 2(b), inset). These rings could be indexed as a face-centered cubic (FCC) crystalline phase, assigning the family of planes as {111}, {200}, {220}, and {311}, respectively [33]. With this analysis, we know that the triangles are highly [111] oriented (Figure 2(a) inset).TheCamellia sinensis extract and the biogenic AuNPs were characterized by FITR spectroscopy (Figures 3(a) and 3(b)). With these studies, we can confirm the presence of polyphenolic biomolecules on the extract and their interaction with the NP surface. The major constituents of Camellia sinensis extract are caffeine, epicatechin, and benzoic acid among others (59.8±1.8 (mg of CAE/g dw) of total polyphenols). These components are associated with the antioxidant activity (70.1±2.4 (%)).Figure 3
(a) FTIR spectrum of AuNPs obtained with 0.4 mL ofCamellia sinensis extract and 5 mL of 10-3 M of HAuCl4. (b) FTIR spectrum of Camellia sinensis extract.IR bands centered at 3395, 1654, 1639, and 1323 cm-1 on Camellia sinensis extract are characteristics of the -OH, C=O, and C-O groups, respectively, stretching modes of the carboxylic acid present on the biomolecules. Bands centered at 1654 and 1639 cm-1 can be attributable to C=O stretch of the carboxylic acid moiety, except for the presence of the bands centered at 2924 and 2856 cm-1, which may correspond to hydrocarbon skeletons of the biomolecules in green tea, while all the rest of the bands remain the same in both spectra (Figures 3(a) and 3(b)). The band due to C-O stretching at 1323 cm-1 is weaker on the AuNP spectrum, and the one due to torsion out of the plane of the carboxyl dimer at 960–875 cm-1 on the AuNP spectra exhibits significant changes [17–19]. By these observations, we can assume that the polyphenolic biomolecules present in the Camellia sinensis extract are responsible for the reduction of Au ions, and also, they can act as capping or passivating agents to generate the nanostructures.


### 3.2. Colorimetric Sensing Using AuNPs

This method of detection is a simple, sensitive, sometimes selective, rapid, and portable; complies with green chemistry principles; and can be used with a wide range of analytes. Colorimetric assays are based on the colloidal solution color change, which happens when the NPs switch from dispersed to aggregated particles (Figure4(c)). These aggregates are mediated by certain analytes, such as metal ions. The biomolecules present on the surface of the AuNPs, in this case carboxylic groups of the capping agents, form aggregates with the metal ions by means of metal-ligand interactions, better known as complex formation (Figure 4(b)), causing thus a color change in the AuNP colloidal solution.Figure 4
(a) UV-Vis spectra of biogenic AuNPs (synthesized with 5 mL of a 10-3 M HAuCl4 solution and 0.4 mL of Camellia sinensis extract) interacting with Mg2+, Hg2+, Sr2+, Pb2+, Ca2+, Cu2+, and Zn2+ metal ions. (b) TEM images of (1) AuNPs, (2) AuNPs-Zn2+, (3) AuNPs-Sr2+, (7) AuNPs-Ca2+, and (8) AuNPs-Cu2+. (c) Picture of vials having AuNPs (synthesized with 5 mL of a 10-3 M HAuCl4 solution and 0.4 mL of Camellia sinensis extract) and the different metal ions probed, respectively.
(a)
(b)
(c)The addition of the different divalent metal ions (Mg2+, Hg2+, Sr2+, Pb2+, Ca2+, Cu2+, and Zn2+) to the AuNP colloidal solution, obtained with 0.4 mL of Camellia sinensis extract, had a visible color change from pink to purple-grayish (Figure 4(c)) particularly with four different metal ions: Zn2+(2), Sr2+(3), Ca2+ (7), and Cu2+(8). The color change that occurs in the solution is due to the switch from individual state to aggregates, in this case is mediated by specific metal ions (Figure 4(c)) [24, 34]. This is an interesting result, since all the metals here used have similar complexing behavior [35]. Therefore, no matter the similarities in the ionic radii of the metal ions studied: Mg2+ (72 pm), Ca2+ (100 pm), Sr2+ (118 pm), Cu2+ (73 pm), Zn2+ (74 pm), Hg2+ (102 pm), and Pb2+ (119 pm), it seems that Mg2+, Hg2+, and Pb2+ cannot form complexes with the AuNPs as easy as the other metal ions; so, the formation of complexes, and subsequent aggregation of AuNPs, can be attributed to the chemical affinity, through electrostatic interactions, of the moieties in the biomolecules surrounding the AuNPs towards the corresponding metal ions.


### 3.3. Plasmon Resonance Sensing

The polyphenolic compounds present on the plant extract, such as caffeine, epicatechin, and benzoic acid, are known to form complexes with heavy metal ions in addition to transition metal cations. Some metal cations preferentially bind with the phenolic groups of the biomolecules [16]. Thus, the addition of the different metal ions employed: Mg2+, Hg2+, Sr2+, Pb2+, Ca2+, Cu2+, and Zn2+, to the AuNP solution resulted in a bathochromic shift in the plasmon resonance, from 529 to 539 nm with Zn2+, from 529 to 540 nm with Sr2+, from 529 to 548 nm with Pb2+, from 529 to 544 nm with Ca2+, and from 529 to 558 nm with Cu2+ (Figure 4(a)). The extent of the shift depends on the concentration of the ions in the AuNP solution [16]. The shift in the plasmon resonance absorption band and the visual color change of the solution (Figures 4(a) and 4(b)) can be attributed to the formation of aggregates of the NPs in the presence of metal cations. To confirm the presence of aggregates, TEM studies were carried out with the AuNPs only and with AuNP metal ions (Sr2+, Pb2+, Ca2+, Cu2+, and Zn2+) (Figure 4(c)). AuNPs are dispersed in the absence of metal ions (Figure 4(b), image 1), whereas, in the presence of metal ions, formation of the aggregates can be observed (Figure 4(b), images 2, 3, 7, and 8). So, the complexation of the metal ions with the phenolic hydroxyl groups present in the surface of the NPs can bring the Au particles close together. This proximity induces the coupling of the plasmon resonance resulting thus in a bathochromic shift.In our previous article on this topic [36], AuNPs were synthesized by using Citrus paradisi (grapefruit) extract as a bioreducing agent. These nanoparticles possessed a unique ability to detect by plasmon resonance sensing: Hg2+, Ca2+, and Zn2+. However, in this actual work, the AuNPs, synthesized with Camelia sinensis (green tea), could detect, in addition, two very important divalent metal ions: Cu2+ and Pb2+ ions by LSPR. Hence, this comparison of sensing results evidences that the chemical nature of the biomolecules surrounding the biogenic AuNPs is a key factor in the plasmonic sensing and selectivity of divalent-aqueous metal ions.


#### 3.3.1. Sensibility Studies

To know the sensibility effect of the biogenic AuNPs obtained with 0.4 mL ofCamellia sinensis extract towards Ca2+, Cu2+, Sr2+, and Zn2+ metal ions, solutions of these metals, with concentrations from 10-3 to 10-6 M, were added to the AuNP solutions [16, 26, 31]. The degree of the bathochromic shift of the LSPR depends on the concentration of the metal ions in the NP solution [16]. When 10-3 M metal ion solutions were added to the colloidal AuNPs, there were bathochromic shifts of the plasmon resonance: 12 nm, from 534 to 546, for Ca+2 (Figure 5(a)); 15 nm, from 534 to 549 nm, for Cu2+(Figure 5(b)); 8 nm, from 534 to 542 nm, for Sr2+ (Figure 5(c)); and 10 nm, from 534 to 544 nm, for Zn2+ (Figure 5(d)).Figure 5
Absorption spectra changes of AuNPs upon addition of increasing concentrations, from 10-3 to 10-6 M of (a) Ca2+, (b) Cu2+, (c) Sr2+, and (d) Zn2+.
(a)
(b)
(c)
(d)For all cases, as the metal concentration decreases below 10-3 M, no significant shifts were observed. Consequently, it seems that when these metal ion concentration is in the order of 10-3 M, aggregates of AuNPs are formed more readily, throughout complexation with these metal ions, resulting in a smaller interparticle gap and therefore greater bathochromic shifts in the plasmon resonance.


### 3.4. Synthesis and Characterization of Biocomposites (AuNPs/Cellulose)

Au nanoparticles were synthesized by the addition of 0.4 mL ofCamellia sinensis extract to a HAuCl4 solution 10-3 M, as described in Experiments. After immersion of the cellulosic material into the Au colloidal solution, its white color turns into purple (Figure 6(b)), which is associated with the plasmon absorption response of the Au nanoparticles supported on the cellulosic substrate, as confirmed by the DRS UV-Vis spectrum in Figure 6(a).Figure 6
(a) DRS UV-Vis spectrum of the cellulosic support decorated with Au nanoparticles. (b) Picture of Au/cellulosic material composites obtained using 0.4 mL ofCamellia sinensis aqueous extract.
(a)
(b)Direct assembly of metal nanoparticles on cellulose substrate is promoted via electrostatic interaction between the nanoparticles and substrate. The electrostatic deposition of metal nanoparticles has been proved successfully to anchor metal nanoparticles onto natural cellulose fibers [37–40] In this case, the formation of the Au/biocomposite can be explained by the self-assembling of Au nanoparticles (positively charged) and the negatively charged functional groups of the cellulose (-OH group).The presence of the Au nanoparticles supported on cellulosic fibers is observed in the FESEM micrographs in Figure7. EDS spectrum (Figure 7, inset) shows the existence of Au nanoparticles on the surface of the cellulosic fibers, which are uniformly distributed, as confirmed by the chemical mapping in Figure 7, indicating thus the effectiveness of the assembly of the Au nanoparticles on the cellulosic support. The deposition of Au nanoparticles on the surface of the natural cellulose material was demonstrated via electrostatic assembly. Au-decorated fibers were immersed into 1 mL of 10-3 M solutions of Mg2+, Hg2+, Sr2+, Pb2+, Ca2+, Cu2+, and Zn2+ ions, to evaluate the sensibility performance. The specimens were imaged using FESEM, as can be observed in Figure 8. Well-dispersed Au-nanoparticles were found to pack on the fiber surface, which do not present any morphological change after the immersion into the ion solutions. However, any visible color change was released after the contact with metallic ions. Energy-dispersive X-ray spectroscopy studies (spectra and chemical mapping) [41], presented in Figure 8, were collected for each specimen. The presence of the metal ion signal, with a homogeneous distribution around all the Au/biocomposite, is evident, indicating the retention of the metal ions. This suggests that, even though the Au/biocomposite cannot be used as a naked-eye detector for metal ions, it certainly can be considered a functional material to be employed to remove metal ions from aqueous solutions.Figure 7
FESEM images at different magnifications (a, b) of the surface of cellulosic fibers coated with Au biogenic nanoparticles. The inset shows the EDS spectrum collected in (b). (c) Elemental mapping of the Au/biocomposite showing the distribution of Au nanoparticles.Figure 8
FESEM micrographs, electron dispersive X-ray spectra, and chemical mapping of the Au/biocomposite after the contact with metal ions: Zn2+ (a, b), Hg2+ (c, d), Sr2+ (e, f), Cu2+ (g, h), Mg2+ (i, j), Pb2+ (k, l), and Ca2+ (m, n), respectively. (o) Picture of the cellulosic material coated with Au nanoparticles after interaction with the different metal ion solutions.


## 3.1. Synthesis and Characterization of AuNPs

Camellia sinensis extract contains various polyphenols: flavonoids (catechol, epicatechin, and caffeine) and phenolic acids (3,4-dihydroxybenzoic acid, vanillic acid, 4-hydroxy-3,5-dimethoxybenzoic acid, and benzoic acid). These components contribute to the antioxidant activities of the extract. The aqueous extract contains 59.8±1.8 (mg of CAE/g dw) total phenols with an antioxidant capacity of 70.1±2.4 (%) [17–19, 32].The addition ofCamellia sinensis extract (0.4 mL) to a 10-3 M HAuCl4 solution led to the appearance of a dark pink color in the solution after 0.5 h of reaction, indicating the formation of AuNPs and the excitation of SP vibration in the NPs (Figure 1(b)). The colloidal solution resulting from the previous experiment was examined by the UV-Vis spectroscopy. Figure 1 shows the characteristic AuNP SPR bands centered between 529 and 536 nm (Figure 1(a)).Figure 1
(a) UV-Vis spectra of biogenic AuNPs measured at different times during the reaction of 0.4 mL ofCamellia sinensis extract with 5 mL of a 10-3 M of HAuCl4. Curve profiles correspond to 0.5 h (black), 1 h (red), 1.5 h (blue), 2 h (pink), 3 h (green), 4 h (purple), and 24 h (violet). (b) Picture of vials containing AuNPs obtained at different reaction times (0 h to 24 h).
(a)
(b)The kinetics of the formation of AuNPs was followed by the UV-Vis spectroscopy (Figure1(a)); it is observed that with the progress of the reaction, the intensity of the signal at about 536 nm increases with the time of reaction, while a new SPR band centered at around 529 nm appears at about 24 h of reaction (Figure 1(a) violet curve). With a longer time of reaction, the main absorption wavelength undergoes a blue shift before stabilizing at 24 h (Figure 1(a) curve from 0.5 to 24 h). These time-dependent features on the spectra are characteristics of size and shape changes on the AuNPs.Figures2(a) and 2(b) show representative transmission electron microscopy (TEM) images of the AuNPs synthesized with 0.4 mL of Camellia sinensis extract at room temperature. Different shapes of the biogenic AuNPs: spherical (82%), triangular (14%), and hexagonal (1%), can be seen. TEM analyses reveal the formation of AuNPs with triangular shapes (Figure 2(b)), with an average lateral size of 50 nm, and also particles with spherical shapes (Figure 2(a)), with an average size of 18 nm.Figure 2
(a, b) TEM micrographs of AuNPs obtained with 0.4 mL ofCamellia sinensis extract. Inset in (a): HRTEM micrograph of biogenic AuNPs obtained with 0.4 mL of Camellia sinensis extract. Inset in (b): SAED pattern of biogenic AuNPs collected after 24 h of reaction using 0.4 mL of Camellia sinensis extract.
(a)
(b)AuNPs obtained with 0.4 mL ofCamellia sinensis extract show a single crystalline nature of metallic gold as revealed in the selected area electron diffraction rings (SAED) (Figure 2(b), inset). These rings could be indexed as a face-centered cubic (FCC) crystalline phase, assigning the family of planes as {111}, {200}, {220}, and {311}, respectively [33]. With this analysis, we know that the triangles are highly [111] oriented (Figure 2(a) inset).TheCamellia sinensis extract and the biogenic AuNPs were characterized by FITR spectroscopy (Figures 3(a) and 3(b)). With these studies, we can confirm the presence of polyphenolic biomolecules on the extract and their interaction with the NP surface. The major constituents of Camellia sinensis extract are caffeine, epicatechin, and benzoic acid among others (59.8±1.8 (mg of CAE/g dw) of total polyphenols). These components are associated with the antioxidant activity (70.1±2.4 (%)).Figure 3
(a) FTIR spectrum of AuNPs obtained with 0.4 mL ofCamellia sinensis extract and 5 mL of 10-3 M of HAuCl4. (b) FTIR spectrum of Camellia sinensis extract.IR bands centered at 3395, 1654, 1639, and 1323 cm-1 on Camellia sinensis extract are characteristics of the -OH, C=O, and C-O groups, respectively, stretching modes of the carboxylic acid present on the biomolecules. Bands centered at 1654 and 1639 cm-1 can be attributable to C=O stretch of the carboxylic acid moiety, except for the presence of the bands centered at 2924 and 2856 cm-1, which may correspond to hydrocarbon skeletons of the biomolecules in green tea, while all the rest of the bands remain the same in both spectra (Figures 3(a) and 3(b)). The band due to C-O stretching at 1323 cm-1 is weaker on the AuNP spectrum, and the one due to torsion out of the plane of the carboxyl dimer at 960–875 cm-1 on the AuNP spectra exhibits significant changes [17–19]. By these observations, we can assume that the polyphenolic biomolecules present in the Camellia sinensis extract are responsible for the reduction of Au ions, and also, they can act as capping or passivating agents to generate the nanostructures.


## 3.2. Colorimetric Sensing Using AuNPs

This method of detection is a simple, sensitive, sometimes selective, rapid, and portable; complies with green chemistry principles; and can be used with a wide range of analytes. Colorimetric assays are based on the colloidal solution color change, which happens when the NPs switch from dispersed to aggregated particles (Figure4(c)). These aggregates are mediated by certain analytes, such as metal ions. The biomolecules present on the surface of the AuNPs, in this case carboxylic groups of the capping agents, form aggregates with the metal ions by means of metal-ligand interactions, better known as complex formation (Figure 4(b)), causing thus a color change in the AuNP colloidal solution.Figure 4
(a) UV-Vis spectra of biogenic AuNPs (synthesized with 5 mL of a 10-3 M HAuCl4 solution and 0.4 mL of Camellia sinensis extract) interacting with Mg2+, Hg2+, Sr2+, Pb2+, Ca2+, Cu2+, and Zn2+ metal ions. (b) TEM images of (1) AuNPs, (2) AuNPs-Zn2+, (3) AuNPs-Sr2+, (7) AuNPs-Ca2+, and (8) AuNPs-Cu2+. (c) Picture of vials having AuNPs (synthesized with 5 mL of a 10-3 M HAuCl4 solution and 0.4 mL of Camellia sinensis extract) and the different metal ions probed, respectively.
(a)
(b)
(c)The addition of the different divalent metal ions (Mg2+, Hg2+, Sr2+, Pb2+, Ca2+, Cu2+, and Zn2+) to the AuNP colloidal solution, obtained with 0.4 mL of Camellia sinensis extract, had a visible color change from pink to purple-grayish (Figure 4(c)) particularly with four different metal ions: Zn2+(2), Sr2+(3), Ca2+ (7), and Cu2+(8). The color change that occurs in the solution is due to the switch from individual state to aggregates, in this case is mediated by specific metal ions (Figure 4(c)) [24, 34]. This is an interesting result, since all the metals here used have similar complexing behavior [35]. Therefore, no matter the similarities in the ionic radii of the metal ions studied: Mg2+ (72 pm), Ca2+ (100 pm), Sr2+ (118 pm), Cu2+ (73 pm), Zn2+ (74 pm), Hg2+ (102 pm), and Pb2+ (119 pm), it seems that Mg2+, Hg2+, and Pb2+ cannot form complexes with the AuNPs as easy as the other metal ions; so, the formation of complexes, and subsequent aggregation of AuNPs, can be attributed to the chemical affinity, through electrostatic interactions, of the moieties in the biomolecules surrounding the AuNPs towards the corresponding metal ions.


## 3.3. Plasmon Resonance Sensing

The polyphenolic compounds present on the plant extract, such as caffeine, epicatechin, and benzoic acid, are known to form complexes with heavy metal ions in addition to transition metal cations. Some metal cations preferentially bind with the phenolic groups of the biomolecules [16]. Thus, the addition of the different metal ions employed: Mg2+, Hg2+, Sr2+, Pb2+, Ca2+, Cu2+, and Zn2+, to the AuNP solution resulted in a bathochromic shift in the plasmon resonance, from 529 to 539 nm with Zn2+, from 529 to 540 nm with Sr2+, from 529 to 548 nm with Pb2+, from 529 to 544 nm with Ca2+, and from 529 to 558 nm with Cu2+ (Figure 4(a)). The extent of the shift depends on the concentration of the ions in the AuNP solution [16]. The shift in the plasmon resonance absorption band and the visual color change of the solution (Figures 4(a) and 4(b)) can be attributed to the formation of aggregates of the NPs in the presence of metal cations. To confirm the presence of aggregates, TEM studies were carried out with the AuNPs only and with AuNP metal ions (Sr2+, Pb2+, Ca2+, Cu2+, and Zn2+) (Figure 4(c)). AuNPs are dispersed in the absence of metal ions (Figure 4(b), image 1), whereas, in the presence of metal ions, formation of the aggregates can be observed (Figure 4(b), images 2, 3, 7, and 8). So, the complexation of the metal ions with the phenolic hydroxyl groups present in the surface of the NPs can bring the Au particles close together. This proximity induces the coupling of the plasmon resonance resulting thus in a bathochromic shift.In our previous article on this topic [36], AuNPs were synthesized by using Citrus paradisi (grapefruit) extract as a bioreducing agent. These nanoparticles possessed a unique ability to detect by plasmon resonance sensing: Hg2+, Ca2+, and Zn2+. However, in this actual work, the AuNPs, synthesized with Camelia sinensis (green tea), could detect, in addition, two very important divalent metal ions: Cu2+ and Pb2+ ions by LSPR. Hence, this comparison of sensing results evidences that the chemical nature of the biomolecules surrounding the biogenic AuNPs is a key factor in the plasmonic sensing and selectivity of divalent-aqueous metal ions.


### 3.3.1. Sensibility Studies

To know the sensibility effect of the biogenic AuNPs obtained with 0.4 mL ofCamellia sinensis extract towards Ca2+, Cu2+, Sr2+, and Zn2+ metal ions, solutions of these metals, with concentrations from 10-3 to 10-6 M, were added to the AuNP solutions [16, 26, 31]. The degree of the bathochromic shift of the LSPR depends on the concentration of the metal ions in the NP solution [16]. When 10-3 M metal ion solutions were added to the colloidal AuNPs, there were bathochromic shifts of the plasmon resonance: 12 nm, from 534 to 546, for Ca+2 (Figure 5(a)); 15 nm, from 534 to 549 nm, for Cu2+(Figure 5(b)); 8 nm, from 534 to 542 nm, for Sr2+ (Figure 5(c)); and 10 nm, from 534 to 544 nm, for Zn2+ (Figure 5(d)).Figure 5
Absorption spectra changes of AuNPs upon addition of increasing concentrations, from 10-3 to 10-6 M of (a) Ca2+, (b) Cu2+, (c) Sr2+, and (d) Zn2+.
(a)
(b)
(c)
(d)For all cases, as the metal concentration decreases below 10-3 M, no significant shifts were observed. Consequently, it seems that when these metal ion concentration is in the order of 10-3 M, aggregates of AuNPs are formed more readily, throughout complexation with these metal ions, resulting in a smaller interparticle gap and therefore greater bathochromic shifts in the plasmon resonance.


## 3.3.1. Sensibility Studies

To know the sensibility effect of the biogenic AuNPs obtained with 0.4 mL ofCamellia sinensis extract towards Ca2+, Cu2+, Sr2+, and Zn2+ metal ions, solutions of these metals, with concentrations from 10-3 to 10-6 M, were added to the AuNP solutions [16, 26, 31]. The degree of the bathochromic shift of the LSPR depends on the concentration of the metal ions in the NP solution [16]. When 10-3 M metal ion solutions were added to the colloidal AuNPs, there were bathochromic shifts of the plasmon resonance: 12 nm, from 534 to 546, for Ca+2 (Figure 5(a)); 15 nm, from 534 to 549 nm, for Cu2+(Figure 5(b)); 8 nm, from 534 to 542 nm, for Sr2+ (Figure 5(c)); and 10 nm, from 534 to 544 nm, for Zn2+ (Figure 5(d)).Figure 5
Absorption spectra changes of AuNPs upon addition of increasing concentrations, from 10-3 to 10-6 M of (a) Ca2+, (b) Cu2+, (c) Sr2+, and (d) Zn2+.
(a)
(b)
(c)
(d)For all cases, as the metal concentration decreases below 10-3 M, no significant shifts were observed. Consequently, it seems that when these metal ion concentration is in the order of 10-3 M, aggregates of AuNPs are formed more readily, throughout complexation with these metal ions, resulting in a smaller interparticle gap and therefore greater bathochromic shifts in the plasmon resonance.


## 3.4. Synthesis and Characterization of Biocomposites (AuNPs/Cellulose)

Au nanoparticles were synthesized by the addition of 0.4 mL ofCamellia sinensis extract to a HAuCl4 solution 10-3 M, as described in Experiments. After immersion of the cellulosic material into the Au colloidal solution, its white color turns into purple (Figure 6(b)), which is associated with the plasmon absorption response of the Au nanoparticles supported on the cellulosic substrate, as confirmed by the DRS UV-Vis spectrum in Figure 6(a).Figure 6
(a) DRS UV-Vis spectrum of the cellulosic support decorated with Au nanoparticles. (b) Picture of Au/cellulosic material composites obtained using 0.4 mL ofCamellia sinensis aqueous extract.
(a)
(b)Direct assembly of metal nanoparticles on cellulose substrate is promoted via electrostatic interaction between the nanoparticles and substrate. The electrostatic deposition of metal nanoparticles has been proved successfully to anchor metal nanoparticles onto natural cellulose fibers [37–40] In this case, the formation of the Au/biocomposite can be explained by the self-assembling of Au nanoparticles (positively charged) and the negatively charged functional groups of the cellulose (-OH group).The presence of the Au nanoparticles supported on cellulosic fibers is observed in the FESEM micrographs in Figure7. EDS spectrum (Figure 7, inset) shows the existence of Au nanoparticles on the surface of the cellulosic fibers, which are uniformly distributed, as confirmed by the chemical mapping in Figure 7, indicating thus the effectiveness of the assembly of the Au nanoparticles on the cellulosic support. The deposition of Au nanoparticles on the surface of the natural cellulose material was demonstrated via electrostatic assembly. Au-decorated fibers were immersed into 1 mL of 10-3 M solutions of Mg2+, Hg2+, Sr2+, Pb2+, Ca2+, Cu2+, and Zn2+ ions, to evaluate the sensibility performance. The specimens were imaged using FESEM, as can be observed in Figure 8. Well-dispersed Au-nanoparticles were found to pack on the fiber surface, which do not present any morphological change after the immersion into the ion solutions. However, any visible color change was released after the contact with metallic ions. Energy-dispersive X-ray spectroscopy studies (spectra and chemical mapping) [41], presented in Figure 8, were collected for each specimen. The presence of the metal ion signal, with a homogeneous distribution around all the Au/biocomposite, is evident, indicating the retention of the metal ions. This suggests that, even though the Au/biocomposite cannot be used as a naked-eye detector for metal ions, it certainly can be considered a functional material to be employed to remove metal ions from aqueous solutions.Figure 7
FESEM images at different magnifications (a, b) of the surface of cellulosic fibers coated with Au biogenic nanoparticles. The inset shows the EDS spectrum collected in (b). (c) Elemental mapping of the Au/biocomposite showing the distribution of Au nanoparticles.Figure 8
FESEM micrographs, electron dispersive X-ray spectra, and chemical mapping of the Au/biocomposite after the contact with metal ions: Zn2+ (a, b), Hg2+ (c, d), Sr2+ (e, f), Cu2+ (g, h), Mg2+ (i, j), Pb2+ (k, l), and Ca2+ (m, n), respectively. (o) Picture of the cellulosic material coated with Au nanoparticles after interaction with the different metal ion solutions.


## 4. Conclusion

In summary, we have developed a simple, sensitive, and to some extent selective, colorimetric, and plasmonic sensor for divalent metal ions in aqueous solution based on biogenic and unmodified AuNps prepared with green tea as the reducing and passivating agent. This dual AuNP colloidal sensor worked very well for colorimetric detection of Ca2+, Sr2+, Cu2+, and Zn2+ and also as a plasmon resonance sensor for these same metal ions, mainly. The no detection of metal ions such as Mg2+, Hg2+, and Pb2+, which have complex characters and ionic radii similar to the ones detected, seems to be more a situation of chemical affinity of the biomolecules surrounding the AuNPs towards the different metal ions. Sensibility studies demonstrate that a 10-3 M solution of Ca2+, Sr2+, Cu2+, and Zn2+ is optimal for the sensing of these ions. Lower concentrations preclude the formation of complexes between AuNPs and metal ions, switching off thus naked-eye and plasmon resonance sensing. Further studies are underway regarding the limit of minimum concentration detectable. Moreover, solid biogenic AuNPs/cellulosic biocomposites were obtained with a simple in situ dip-coating methodology, expecting to get portable, fast, and reliable colorimetric sensors; nonetheless, these biocomposites resulted to have better functionalities as materials for adsorption of metal ions in an aqueous medium rather than metal ion sensors.



---

*Source: 9846729-2019-02-17.xml*