Surface plasmon coupled emission as a novel analytical platform for the sensitive detection of cysteine

1 Introduction

Detection techniques in the form of diagnostic protocols are especially important in biological and medical sciences because they are used for sensing different types of disease-related biomarkers such as proteins like troponin, a biomarker for myocardial infarction [1], DNA biomarkers such as p53 and K-RAS gene for lung cancer [2], and other chemical moieties like alcohol and aldehyde functionalities. A good detection technique is characterized by high sensitivity, specificity, and rapidity in the detection of the target biomarkers. The rapid sensing of these disease-related markers can help mitigate the suffering of a patient by allowing a therapist to administer a treatment modality well in advance, to either control or treat the symptoms.

One class of biomarkers are amino acids that are simple organic compounds made from amine (-NH2) and carboxylic acid (-COOH) functional groups, along with a side chain specific to each amino acid. Although many amino acids are the constituents of important functional proteins in our body, the elevation or deficiency of some of these amino acids triggers the onset of certain diseases. For example, osteoarthritis can be detected by calculating the ratio of amount of branched chain amino acid to amount of histidine [3], making the detection of such amino acids important.

Some of the techniques used for the detection of amino acids are based on fluorescence, UV-Vis spectroscopy, crystallography, and in recent times, surface plasmon resonance (SPR). Although amino acids can be efficiently detected by the fluorescence and SPR methods, these sensing platforms have certain limitations. In the case of fluorescence-based techniques, the emission arising from an excited fluorophore is isotropic. A detector, however, collects signals only directed in its line of sight as a result of which, only 1% of the total signal is collected, resulting in low collection efficiencies. In SPR, while the signal collection efficiency is >50%, the optics involved is relatively complicated, and it does not provide a visual indication regarding the occurrence of a sensing event as in fluorescence. Combining the advantages of sensitivity and simplicity of both the SPR and fluorescence-based techniques, Lakowicz et al. have reported surface plasmon coupled emission (SPCE) as a novel phenomenon [4, 5]. SPCE occurs when the emission from excited fluorophores couples to the plasmon modes of a thin metallic film that leads to the generation of highly directional and wavelength-resolved emission. Characterized with high, signal-to-noise ratios, SPCE has been successfully employed toward the high sensitive detection of several biochemical analytes [6, 7].

In the present work, we extended the SPCE platform for the selective differentiation of the thiolated amino acid cysteine (Cys), from the non-thiolated amino acid arginine (Arg); exploiting the unique thiol-silver (Ag) covalent interactions [8], which have been reported extensively with special reference to Cys [9–11]. It is noteworthy that although there are other sulfur containing amino acids like methionine (Met), only the thiolated amino acids containing -SH and -SR groups have high affinity and bind covalently to Ag [12]. Furthermore, as Cys is the only thiolated amino acid in the list of 23 proteinogenic amino acids, it can be differentiated very effectively from the rest, facilitating specificity. Here, we monitor the intensity of the SPCE signal arising from the fluorophore rhodamine b (RhB) on exposure to these two amino acid categories, to perceive the sensing event. The detection of Cys is critical because of its role as a biomarker for HIV [13], cancer [14], and obstructive sleep apnea, which is a risk factor for a number of cardiovascular conditions [15]. Elevated vascular Cys levels have also been shown to be associated with obesity [16]. In this study, we have detected Cys and Arg by monitoring the SPCE signal arising from a conventional SPCE substrate (50 nm Ag coated with RhB) on exposure to varying concentrations of amino acids. In addition to achieving the specific detection of Cys to a nanomolar level, we have also investigated the role of the sample coating process, on sensitivity of detection. We have further compared the sensitivity level achieved between the SPCE and the SPR-based UV-Vis platform.

2 Materials and methods

2.4 SPCE studies

The SPCE measurements were carried out in the Reverse Kretschmann (RK) configuration by affixing the SPCE substrate to a hemi cylindrical prism made of BK7 glass. Glycerol was used as the index matching fluid between the substrate and the prism. The substrate-affixed prism was placed on a calibrated rotary stage and a 532-nm laser was used as the excitation source. The recording of both the SPCE and free space emission was performed by using the USB 4000 OceanOptics© spectrometer (Ocean Optics, FL, USA), where the RhB emission at 580 nm, arising from the Cys/Arg-coated substrates was monitored for the different concentrations of the amino acids. A schematic of the experimental setup and the chemical structures of Arg and Cys (depicting the thiol, -SH bond) is shown in Figure 1.

Figure 1:

A schematic depicting the reverse Kretschmann (RK) configuration to monitor the SPCE signal arising at the surface plasmon angle (θ_SP) on exposure to the different amino acids, namely, non-thiolated Arg and thiolated (-SH group) Cys that forms high-affinity Ag-S covalent bonds, facilitating its sensitive detection.

3 Results and discussion

The intensity of the SPCE signal arising from RhB at 580 nm was monitored from both the spin-coated and self-assembled amino acid thin film stacks.

Spin coating: On performing SPCE measurements for the spin-coated substrates, it was observed that as the concentration of the amino acid increased, the intensity of the SPCE signal reduced for both Cys and Arg. For the Ag substrates spin coated with Cys, the “percentage quenching” with respect to the control signal (SPCE substrate with no amino acid) was found to vary from 29% (for 0.25 μm) to 89% (for 0.25 m), represented in Figure 2A. The quenching of control signal intensity was also observed in the case of the Arg-coated substrates, from 23% (for 0.25 μm) to 72% (for 0.25 m) represented in Figure 2B.

Figure 2:

Plot depicting variation of SPCE signal intensity with varying amino acid concentrations. (A) Spin-coated Cys/Ag system. (B) Spin-coated Arg/Ag system. (C) Self-assembled Cys/Ag system. (D) Self-assembled Arg/Ag system.

Self-Assembly: For the substrates coated with self-assembly of amino acids, a greater degree of signal quenching was observed. For Cys, the percentage quenching of the control signal was found to vary from 67% (for 0.25 μm) to 100% (for 0.25 m) as shown in Figure 2C. For Arg, the percentage quenching of the control signal varied from 23% (for 0.25 μm) to 83% (for 0.25 m), represented in Figure 2D. It was clear that the reduction in the signal intensity was far more drastic in the case of Cys than Arg.

The SPCE signal was radically quenched in the self-assembled Cys system compared to any other system. The quenching profile for the Arg system, however, was independent of the coating protocol followed; as shown in Figure 2B and D. A summary of these results is represented in Table 1. All the studies were performed in triplicate and were found to yield similar results.

These results indicate that in comparison to Arg, the detection of Cys was more specific and sensitive especially at the lowest concentration. The following mechanism elucidates the sensitivity of the SPCE platform especially toward the detection of Cys.

When Cys molecules are introduced onto the Ag film, they bond to the Ag surface due to the high affinity thiol-Ag covalent interaction, which renders them quite stable [10–12]. As a result, the mobility of the electrons on the surface of Ag film is reduced drastically through formation of localized Ag-S bonds, which damps the SPR feature in the Ag-Cys system [18]. Consequently, the Cys molecules hinder the efficient transfer of energy of RhB emission to the surface plasmon modes of Ag resulting in the quenching of the SPCE signal. Naturally, as substantiated in our results, with an increase in amino acid concentration, more Cys molecules bind to the surface of the silver film leading to greater quenching of the SPCE signal. Arg molecules, on the contrary, do not show any affinity for the Ag substrate, and hence, in the wash procedures, especially for the lower concentrations, a significant number of Arg molecules are lost from the Ag surface, which allows relatively unhindered transfer of energy from RhB to the Ag plasmon modes. Thus, lower quenching was observed in the Arg system, compared to the Cys system. With respect to the spin coating versus the self-assembly process, spin coating, itself, removes a large number of molecules due to the centrifugal force irrespective of whether it is Cys or Arg; hence, a similar percentage quenching was observed between the two. Self-assembly, on the other hand, allows the formation of uniform chemically bound layers of Cys as opposed to Arg that is reflected in the high percentage quenching of SPCE signal observed in Cys compared to Arg.

It is important to, however, note that the quenching percentage increases with concentration even in the case of Arg. This can be understood upon consideration that the concentrations of millimolar and molar are very high, not encountered in biomedical scenarios. At these concentrations, Arg had lower solubility in water and, hence, was deposited on the surface of Ag via spin coating and especially the self-assembly process. These deposits disabled the efficient transfer of fluorescence energy to the plasmon modes as mentioned before that led to quenching. Importantly, in sensing studies such as ours, the aim is to sensitively detect the lowest concentrations of analytes possible. In lieu of this, our studies clearly show that Cys is sensitively detected at the nanomolar range, not reported before, with greater than twofold sensitivity than Arg at the same concentration levels, where the physical particulate effects are negligible. It is pertinent to reiterate here that the covalent interaction and affinity between the thiolated Cys and Ag is unique and is not observed in other non-thiolated sulfur-containing amino acids [12], thereby facilitating the sensitive and specific detection of Cys as observed in our results.

As mentioned earlier, in order to gauge the sensitivity of SPCE platform, we chose the SPR-based UV-Vis platform for comparison that has shown good sensitivity in detection of analytes conjugated to metal nanoparticles of silver and gold [19]. Previous reports have already shown the application of SPR-based platforms for the detection of Cys [8–10, 12]. In a similar manner to the SPCE measurements, on exposure to the different concentrations of Cys and Arg, the percentage quenching of the control peak intensity of AgNP SPR was monitored; shown in Figure 3A and B. It was determined that the percentage quenching of the SPR signal was 27% for 0.25 μm, 40% for 0.25 mm, and 57% for 0.25 m concentrations of the Cys solution, respectively. Similarly, the percentage quenching of the Ag SPR peak on exposure to Arg was determined to be 13% for 0.25 μm, 30% for 0.25 mm, and 43% for 0.25 m concentrations of the Arg solution. The summary of the results obtained by the comparison of SPR versus SPCE-based techniques is represented in Table 2.

Figure 3:

Variation of SPR peak profile of AgNPs on exposure to different concentrations of (A) Cys and (B) Arg.

It is straightforward to note from these numbers that the SPCE platform exhibits a response that is twofold more sensitive than the SPR platform within the same concentration range. Another important observation is the comparison of the percentage quenching between Cys and Arg in the SPR platform itself. For the least concentration of (0.25 μm), Cys is still found to quench the SPR signal by twofold compared to Arg, which again confirms the greater affinity that thiolated Cys has toward silver, in comparison to Arg. In previous reports, Cys has been detected by the SPR-based colorimetry platform in the range of 5.0×10^-7–1.0×10^-5m [20]. In our studies using the SPCE platform, we are able to detect Cys to the level of 0.25 μm or 250 nm. Thus, the SPCE platform undoubtedly offers a much higher degree of sensitivity than the conventionally used platforms for detection.

The mechanism of interaction of Cys with AgNPs follows the same principle as described previously in the case of Cys-Ag thin films involving covalent Ag-S interactions [9, 12]. A higher Cys concentration meant a larger coverage of the surface area of AgNPs that led to greater quenching of the SPR peak. In addition to the quenching of absorbance; from Figure 3A, we can also clearly observe broadening of the Ag SPR peak that can be attributed to the aggregation of the AgNPs due to covalent bonding with Cys molecules. The aggregation naturally increases with increasing Cys concentration, as was observed in our studies. Figure 3B shows that, on exposure to Arg, only quenching of the Ag SPR peak is seen, and conversely, peak broadening is not observed. This reflects the relative lack of chemical affinity of Arg molecules toward Ag NPs. However, in both these scenarios, the Ag NPs unbound to Cys and Arg molecules still contribute toward the intensity of the Ag SPR peak, which is significant.

Thus, although quenching occurred, the percentage quenching with respect to the control signal was not as significant as observed in SPCE. This superior sensitivity of the SPCE platform, in comparison to the SPR platform, stems from the fact that SPCE is a near-field phenomenon, wherein only the fluorophores that are in the proximity of a metal surface couple to the surface plasmon modes of the metal [21]. The contribution of emission from the distal fluorophores is, hence, rejected [22], facilitating the canceling of the background noise from RhB molecules unbound to the amino acids.

In this manner, the attributes of highly directional plasmon coupled emission and the innate advantage of background noise cancelation characteristic to the SPCE platform, rendered it a more sensitive tool for the detection of Cys.

4 Conclusions

In this work, we employed SPCE as a novel sensing platform for the sensitive and specific detection of the thiolated amino acid Cys exploiting the unique, strong covalent thiol-silver interactions, which is not associated with non-thiolated amino acids. In this study, we were able to detect Cys to a nanomolar concentration, not reported before, thereby, substantiating the high sensitivity associated with the SPCE platform in comparison to the SPR-based UV-Vis platform. As the sensing is highly specific to Cys, especially at very low concentrations, this platform can be extended toward the detection of Cys biomarkers associated with diseases like HIV, cancer, thrombosis, cardiovascular problems and even obesity. Thus, the high sensitive detection of Cys on the simplistic and economical SPCE platform is of medical relevance, promising the early and rapid detection of Cys-associated medical conditions.