1. Introduction
The development of a surface plasmon resonance (SPR) sensor in various applications such as drug discovery, disease monitoring and effluent detection normally originates in the possible interactions between the analyte of interest and its immobilised ligands on the surface of the sensing layer [
1]. Macromolecular interactions which result in successful binding between the analyte and ligands will vary the refractive index in the vicinity of the sensing surface, thus manipulating the response of the SPR sensor. The binding affinity constant, K, is a parameter that quantifies the effectiveness of these macromolecular interactions [
2].
The binding affinity constant provides quantitative information on the binding interactions between the analyte and sensing layer, which explains the sensing performance of an SPR sensor in terms of sensitivity and selectivity. Evaluating the binding affinity under interference of a multiple-analyte system can provide an information on the selectivity of the sensor [
3]. Binding interaction under various conditions, such as different pH values, enables the determination of the right regeneration mechanism of an SPR sensor [
4]. Analysing the binding affinity at varying temperatures can also extract the thermodynamic nature of the macromolecular interactions [
5].
The derivation of the binding affinity constant is normally based on the kinetic and thermodynamic model [
6]. Nevertheless, the SPR technique provides a practical tool for retrieving such a constant by leveraging the Langmuir isotherm model, which is analogous to the kinetic approach [
7]. However, careful interpretation is needed such that the derived value can be regarded as the “apparent binding affinity constant” instead of at equilibrium. This is to accommodate the kinetic controls on the macromolecular interactions that might still be occurring between the analyte and ligands during measurement. The practicality of this technique in deriving the value of K inspired many researchers to study the binding affinity of various types of analyte, including heavy metal ions on different SPR sensors [
8,
9,
10,
11].
Heavy metals refer to any metallic element with atomic density four to five times greater than water, such as lead (Pb), mercury (Hg), arsenic (As) and cadmium (Cd) [
12]. Assuming that atomic density and toxicity are interrelated, heavy metals are considered detrimental to the environment and organisms [
13]. Therefore, their detection plays an important role in monitoring the emission of effluents in our environment. The most common techniques for detecting heavy metals are mass spectroscopy, atomic absorption spectrometry [
14], inductively coupled plasma mass spectrometry (ICPMS) [
15,
16] and microwave induced plasma atomic emission spectroscopy (MIP–AES) [
17,
18]. Although these detection techniques are known for their high sensitivity, the technologies involve expensive instrumentation that is complex to operate and immobile. Therefore, electrochemical techniques such as fluorimetry [
19], colorimetry [
20] and voltammetry [
21,
22,
23] have been studied to provide a cheaper alternative offering simplicity and mobility.
Anodic stripping voltammetry (ASV) is one of the commonly used electrochemical techniques that can achieve a very low limit of detection (LOD), i.e., in ppt levels. For instance, Lu et al. fabricated an ASV glassy carbon electrode (GCE) coated with graphene, Au nanoparticles and CS that can detect down to 1 ppt of Pb
2+ ions [
24]. Although this technique is appealing for its low LOD and portability through miniaturisation, the interference from several species in the analyte can degrade the sensitivity of the electrode. On the other hand, Zhou et al. leveraged the ion interference to simultaneously detect Pb
2+ and Cd
2+ ions using L-cysteine/graphene–chitosan GCE [
25]. Even though simultaneous detection is possible through multiplexing circuitry, an individual electrode still needs to be fabricated and optimised for each analyte, hence limits to the practicality of this approach.
Other than the electrochemical techniques, an SPR sensor is another attractive method that also offers cost-efficiency and simplicity. Although many SPR sensors for heavy metal detection have been developed, their portability has hardly been examined. Nevertheless, recent work by several researchers has shown that the portability of this sensor is viable through fabrication with optical fibre [
26,
27]. Not only that, some SPR sensors based on smartphone platforms have also been studied for the same purpose [
28,
29]. Progressive interest in the portability of SPR sensing technology shows a promising future for this sensor for in situ heavy metal detection. Consequently, various types of SPR sensor for heavy metal detection have been employed in order to further explore the potential of this device.
Recently, Wang et al. presented a study on the competitive adsorption of albumin for the detection of Cu
2+ ions and other heavy metal ions in tap water [
10]. The study exhibited an explicit analysis of the SPR data using the Langmuir isotherm model to obtain the dissociation constant, K
d. Considering the definition of K
d as the reciprocal of K [
7], the values of binding affinity deduced from this study are relatively small, i.e., 2.4 × 10
2 and 4.3 × 10
2 for both Pb
2+ and Hg
2+, in comparison with the values obtained by other SPR sensors in
Table 1. The small values of K obtained by Wang and his co-workers suggests that albumin might not be a good ligand for heavy metal ions.
The magnitude of the binding affinity depends on the strength of the interactions between the heavy metal ions and immobilised ligands of the SPR sensor. Chitosan (CS) is known for its high affinity towards heavy metal ions such as Pb
2+ and Hg
2+ due to abundant amino groups (–NH
2) that bind easily with such cations [
33]. Therefore, CS has been widely used as the sensing layer of SPR sensors for heavy metal detection [
30,
34,
35]. McIlwee et al. studied the performance of CS as the SPR sensor for Fe
3+ ions, and proposed 9.49 × 10
5 M
−1 to describe quantitatively the binding affinity between the cation and CS. Abdi et al. added polypyrrole to the CS nanocomposite to improve its sensitivity to Pb
2+ and Hg
2+ ions [
34]. The binding affinity of the polypyrrole–CS was further analysed using Cu
2+, Zn
2+ and Ni
2+ ions, which yielded the binding affinity constants of 1.3 × 10
4, 2.3 × 10
4 and 1.7 × 10
4, respectively [
31,
32].
Other materials that have recently driven many studies in SPR sensing are graphene-based materials such as graphene oxide (GO), which has a large surface area and high π-conjugation structure that significantly improve the sensitivity of an SPR sensor [
36,
37,
38]. In addition, GO supports the surface plasmon in the visible range, and its planar sheet structure provides extra protection to the SPR sensor [
36]. Apart from that, GO also carries a significant volume of hydroxyl (−OH), carbonyl (C=O) and carboxylic (C(=O)OH) functional groups, which are readily available binding sites for the heavy metal ions [
39].
The abundance of functional groups in CS and GO that are readily available for binding with the heavy metal ions has prompted the development of a CS–GO nanocomposite as an SPR sensor. The first CS–GO SPR sensor, which was fabricated by Lokman et al., demonstrated an outstanding sensitivity of 1.1 °ppm
−1 for 5 ppm Pb
2+ ions. However, the conventional CS–GO SPR sensor on a single Au layer has a limited linearity range of only up to 1 ppm [
40]. Therefore, an advanced CS–GO SPR sensor based on an Au/Ag/Au multi-metallic nanostructure was proposed to provide an enhanced evanescent field which can further detect the increasing concentration of Pb
2+ ions without saturation, thus extending the existing linearity range [
41]. However, previous studies on the Au/CS–GO and Au/Ag/Au/CS–GO SPR sensors did not include work to derive the binding affinity constant.
Therefore, the aim of this study is to quantify the binding affinity of Pb
2+ and Hg
2+ to an Au/Ag/Au/CS–GO SPR sensor by deriving their binding affinity constant, K. It is important to highlight that the Au/Ag/Au/CS–GO SPR sensor was used in this study to ensure a strong and stable evanescent field is maintained in order to detect the whole range of ion concentration, i.e., 0.1 ppm, 0.5 ppm, 1 ppm, 3 ppm and 5 ppm, without saturation. The extended linearity range due to the enhanced evanescent field is important for providing sufficient SPR data for the whole range of heavy metal concentrations, thus enabling the binding affinity constants to be derived accurately. Both cations were considered in this study due to their strict maximum contaminant levels (MCL) in drinking water of 0.015 ppm and 0.002 ppm, as per established by the United States Environmental Protection Agency (US EPA). However, since the Au/Ag/Au/CS–GO SPR sensor was proposed for heavy metal detection in groundwater, the range of concentration was based on the toxicity characteristic leaching procedure (TCLP), which is the lab procedure that sets the regulatory level of leachable effluent such as Pb
2+ and Hg
2+ that might leach into soil and groundwater and hence be used as drinking water. According to the US EPA, the threshold levels for Pb
2+ and Hg
2+ which were determined by this procedure were 5 ppm and 0.2 ppm, respectively [
42]. The Langmuir isotherm model was used to derive the binding affinity constants for both heavy metal ions and the results were compared. This study is essential for validating the performance of such a sensor in detecting the emission of these heavy metal ions in our environment.
To the best of our knowledge, this is the first derivation of K for Pb2+ and Hg2+ ions on a CS–GO SPR sensor that quantitatively describes the interaction between them, thereby confirming the superior performance of such a sensor in heavy metal monitoring. The sensing performances were presented in terms of sensitivity, linearity range, repeatability and signal-to-noise ratio (SNR).