A polyelectrolyte based ratiometric optical sensor for Arginine and Lysine

Sensors & Actuators: B. Chemical 303 (2020) 127182

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A polyelectrolyte based ratiometric optical sensor for Arginine and Lysine 1

Shrishti P. Pandey , Prabhat K. Singh*

T

Radiation & Photochemistry Division, Bhabha Atomic Research Centre, Mumbai 400 085, India

ARTICLE INFO

ABSTRACT

Keywords: Amino acid sensor Pyrene methyl ammonium chloride Fluorescence sensor Ratiometry Arginine and Lysine Monomer and aggregate

The crucial involvement of amino acids in various physiological processes and their ability to function as effective biomarkers for a variety of diseased conditions has inspired a significant activity in conceptualizing sensor system for amino acids, although there are still limited reports which aim to detect basic amino acids with simpler design yet with high sensitivity and selectivity. Herein, we report a ratiometric optical sensor that is based on Arginine and Lysine-induced disintegration of a supramolecular assembly, leading to significant modulation in the monomer-aggregate equilibrium, which yields a ratiometric signal that has been used for quantification of Lysine and Arginine in aqueous solutions. Our sensor design overcomes many disadvantages that are associated with the existing optical sensing schemes, such as, technically demanding and time-consuming synthetic protocols for probe synthesis, limited aqueous solubility, and more importantly, most of the previously reported sensors operate via single wavelength based measurements, which makes their performance error prone to a variety of analyte-independent factors which can be overcome by ratiometric detection as achieved here. Overall, we have devised a label free, rapid, simple, dual-sensing, ratiometric sensor for the detection of basic amino acids. Importantly, our sensing system also shows response in the human serum matrix.

1. Introduction Amino acids are the prime building units of proteins, and are an important class of biomolecules that are crucially involved in a variety of physiological processes [1–3]. They act as crucial intermediates in primary metabolism in all biological cells [2,3]. Due to their crucial involvement in many different physiological processes, they also function as effective biomarkers in a variety of diseased conditions [4]. Therefore, analysis of amino acids and their selective detection is of prime importance in various types of medical diagnostics, and in many other fields such as nutritional analysis, pathogen recognition, as well as in fundamental research [5,6]. In this regard, immense efforts have been dedicated for the detection of various thiol containing amino acids and several sensors for the detection of these amino acids have been reported [7–11]. However, there are still limited reports on the detection of other biologically significant amino acids such as basic amino acids. Arginine (Arg) and Lysine (Lys) are two such basic amino acids, which are crucially involved in many important biological processes. For example, Arginine plays an important role in wound healing, release of hormones, immune function, cell division and removing ammonia from the body. [12] Also, Arginine taken together with

oryohimbine [13] and proanthocyanidins [14], have been employed in the treatment of erectile dysfunction. The important functions associated with Arginine have led to the development of oral supplementation of Arginine which helps in quick recovery of damaged tissue [15]. However, excess of Arginine can cause elevated levels of stomach acid, especially gastrine and can cause anaphylaxis, or an allergic reaction. Further, due to the vasodilator property of Arginine, reduction in blood pressure may be another side effect of arginine supplementation [15]. Since Arginine is also reported to increase the production of sodium, potassium, phosphate, chloride, creatinine and blood urea nitrogen levels in the body, thus, people associated with liver or kidney problems are highly prone to these chemical imbalances caused by Arginine [15]. On the other hand, Lysine, which is an essential basic amino acid, plays important roles in polyamine synthesis and protein synthesis, [16] as well as in adjustment of the somatotropic hormone in human body [2]. The abnormal metabolism of Lysine, leads to a variety of clinical symptoms, such as saccharopinuria and glutaric aciduria [17]. Therefore, detection of Arginine and Lysine is of great significance in the field of Medical science and nutritional analysis [18,19]. Considering their importance, various methods have been developed for the detection of basic amino acids, such as liquid

Corresponding author. E-mail address: [email protected] (P.K. Singh). 1 On M. Sc. research project from Viva College, Virar, Palghar 401303, India. ⁎

https://doi.org/10.1016/j.snb.2019.127182 Received 2 August 2019; Received in revised form 18 September 2019; Accepted 21 September 2019 Available online 07 October 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.

Sensors & Actuators: B. Chemical 303 (2020) 127182

S.P. Pandey and P.K. Singh

chromatography, [20] HILIC column liquid chromatography [21], electrochemistry [22,23], amperometry [24], and spectroscopic methods [25–28]. However, these methods face certain limitations such as, low sensitivity, poor selectivity, operational difficulties, high cost for analysis, and low-test speed. Among the previously existing methods, Fluorescence spectroscopy has received significant attention owing to its various powerful attributes such as high selectivity, high sensitivity, easy operation, and capability of visual detection [9,29]. Various fluorescence-based methods have been developed to detect different amino acids with different functional side chain groups [9,29–31]. However, limited number of fluorescence sensors have been reported for the detection of Lysine and Arginine [1,15,32–34]. Further, out of the reported probes, very few are found to be soluble in either aqueous solutions or are poorly soluble in water. Furthermore, many of these developed probes are synthetically designed, which consumes a lot of time and effort that significantly impacts the prospect of application of these probes in real life applications. Most importantly, majority of the reported probes operate through single wavelength based measurements which are prone to small variation in experimental conditions such as temperature, probe concentration, pH, path length, etc. In this regard, ratiometric fluorescence sensors are considered to be superior for intensity-based sensors since they can exclude most or all the uncertainties by self-calibration of two emission bands at two different peak positions [35]. However, ratiometric fluorescent sensors for Lysine and Arginine have been scantly reported [15,36]. Therefore, there is an urgent need to develop biosensors that overcome these limitations, and will simplify the detection of basic amino acids. One of the prime requirements for developing a ratiometric sensor is the generation of signals of the sensor system at two distinct wavelengths which can be altered in response to the analyte. There is limited number of strategies to achieve dual emission, for example, probe molecules which include the phenomenon of internal charge transfer (ICT) [37,38], fluorescence resonance energy transfer (FRET) [37–40], through-bond energy transfer (TBET) [41], excited-state intramolecular proton transfer (ESIPT) [39,40], and monomer–excimer formation [37,38] are known to display dual-emission bands. In this regard, synthetic polyelectrolytes are reported to cause aggregation of oppositely charged probe molecules [42,43]. Thus, probe molecules which can display new emission band for their aggregated state could be an ideal choice for this scheme. In this connection, pyrene based molecules are known to display distinct emission band in their excimer or aggregated state, when compared to their monomeric emission band [44–48]. Thus, we chose a cationic pyrene based molecule and a negatively charged polyelectrolyte poly (styrene) sulfonate, to see if monomer-aggregate transition can be achieved, and as expected, a nice monomer-aggregate transition has been observed for a cationic pyrene derivative in the presence of a negatively charged synthetic polyelectrolyte, polystyrene sulfonate. In this work, we have developed a ratiometric optical sensor for basic amino acids that is based on Lysine- and Arginine-induced dissociation of a dye-polyelectrolyte supramolecular assembly which is built-up from a negatively charged polyelctrolyte (Scheme 1), Polystyrene sulfonate (PSS) and a commercially available cationic fluorophore, Pyrene Methyl Ammonium Chloride (PMA). The cationic fluorophore (PMA) in aqueous solution displays strong emission and exists in the monomer form. However, upon the addition of the polyelectrolyte (PSS), the probe molecules (PMA), undergoes aggregation, which displays a distinct emission band. Thus a monomer-aggregate transition is achieved which makes an ideal case for the design of a ratiometric sensor. Upon addition of Arginine and Lysine, the comparatively stronger electrostatic interaction of Arginine and Lysine with PSS leads to the weakening of the interaction of the PMA aggregates with PSS which causes a significant modulation in the monomer-aggregate equilibrium. The changes in the ratiometric signals, thus, achieved, in turn, have been used to quantify Lysine and Arginine in aqueous solutions. In addition, the current sensing system is simple,

Scheme 1. Chemical Structure of Pyrene Methyl Ammonium Chloride (A) and Polystyrene sulfonate (B).

rapid, selective and sensitive and has been assembled from all commercially available components. The remainder of the paper is organized as follows. In the first section, the formation of PMA aggregates on the surface of negatively charged polyelectrolyte, PSS, has been demonstrated and characterized in detail, whereas in the second section, we have shown the response of the PMA-PSS system towards the basic amino acids, Arginine and Lysine, and its analytical performance has been evaluated in detail. 2. Experimental Pyrene Methyl Ammonium Chloride (PMA) was procured from Sigma Aldrich and the concentration of the dye PMA was calculated from its optical density and the molar absorptivity of the dye (ε276 ∼ 33,390 M−1 cm−1) in water. Polystyrene sulfonate (PSS, Mol wt∼70,000), sodium chloride, other salts and all the required amino acids were purchased from Sigma Aldrich and were used as received. All the measurements were done at pH 7 in aqueous solution. The pH of stock solutions of all the amino acids was adjusted to ∼7. The concentration of PMA used in all the experiments was 35 μM unless otherwise mentioned. Ground-state absorption measurements were carried out using a Jasco UV–vis spectrophotometer (model V-650) and steady-state fluorescence measurements were performed using a Jasco spectrofluorimeter (model FP-8500). All measurements were performed at an ambient temperature of 25 ± 1 °C using a quartz cuvette of 1 cm path length unless otherwise stated. Human serum was obtained from a local hospital and optical measurements in 30% human serum samples were performed in quartz cuvette of 2 mm path length as relatively higher concentration of PMA (170 μM) was used. The pH dependent measurements were performed in the aqueous solution by varying the pH of the solution using NaOH and HCl. Time resolved fluorescence decay traces were measured using a IBH instrument based on the principle of Time Correlated Single Photon counting (TCSPC) which is described in details elsewhere [49–52]. A picoseconds diode laser of 339 nm (∼100 ps, 1 MHz) was used for the measurements of fluorescent decay traces. The Instrument Response function of the current setup is ∼150 ps. In order to obtain the best fits for the obtained data, the collected data was analyzed using the data analyzing software (DAS-6) using single and bi-exponential decay model. A good fit is usually characterized when the χ2 value is close to unity and the weighted residuals are distributed randomly along the zero line along the data channels. Therefore, the quality of the fits obtained was evaluated considering the above mentioned criteria. The multi-exponential function used for fitting the fluorescence decay traces is given as follows [53],

I (t ) = I (0)

2

i

exp( t / i )

(1)

Sensors & Actuators: B. Chemical 303 (2020) 127182

S.P. Pandey and P.K. Singh

The average fluorescence lifetime is calculated according to the following equation [53],

< >=

Ai

i

where Ai =

i i/

i i

(2)

where αi represents the amplitude of the individual decay constants. 3. Results and discussion To understand the interaction between the fluorescent probe, PMA, and the polyelectrolyte, PSS, we have measured the steady-state emission spectra of PMA in the presence of various concentrations of PSS, and the results have been presented in Fig. 1. In aqueous solution, PMA shows a typical structured emission spectra in the range of 350–450 nm with peak positions at 376 nm, 394 nm and 416 nm, which is consistent with previous literature for monomeric pyrene derivatives [44]. However, as evident from Fig. 1, upon addition of PSS, the emission intensity for the monomer bands starts gradually decreasing with increasing concentration of PSS. Interestingly, along with the decrease in the emission intensity of monomer band, a new broad emission band starts appearing with a maximum around 480 nm, which reaches saturation at a [PSS] = 0.2 μM. Such broad emission bands have been reported previously for the pyrene dimmers, excimers and/or aggregates [44–48]. Thus, it is very likely that in the present case, PMA aggregates or excimers are formed in the presence of PSS. To know whether PMA ground-state aggregates or excimers are responsible for the broad emission band at 480 nm, in the presence of PSS, we have performed ground–state absorption measurements for PMA at various concentrations of PSS, and the results have been presented in Fig. 2 The PMA ground-state aggregates should be accompanied by changes in the absorption spectrum whereas PMA excimers should be indicated by insignificant changes in the absorption spectrum in presence of PSS. As it is clearly evident from Fig. 2, the addition of PSS causes significant changes in the absorption spectra of PMA indicating strong interactions of the PMA molecules in the ground state itself. In particular, the monomeric absorption bands of PMA at 320 and 340 nm undergo reduction in absorbance along with a concomitant increase in the absorbance at 352 nm. To quantify the changes in the absorption spectra of PMA in the presence of PSS, we have plotted the ratio of absorbance at 340 nm and 352 nm, which shows a gradual decrease with increasing concentration of PSS and reaches a saturation value at [PSS] = 0.2 μM, similar to the one observed in steady-state emission measurements. To further confirm that the 480 nm emission band originates from ground-state aggregates, we have also collected the excitation spectra of PMA in the presence and absence of PSS. The excitation spectra of PMA in water, when monitored at 394 nm, matches

Fig. 2. (A) Ground-state absorption spectra of PMA (35 μM) in presence of various concentrations of PSS (in μM) (1) 0 (2) 0.09 (3) 0.12 (4) 0.14 (5) 0.16 (6) 0.21 (7) 0.30. (B) Variation of ratio of absorbance at 340 to 352 nm with increasing concentration of PSS.

very nicely with the absorption bands observed in ground-state absorption spectra of PMA in aqueous solution (Figure S1, ESI). On the contrary, in the presence of PSS, the excitation spectrum is significantly different when monitored and compared at 394 nm and 480 nm. For 394 nm, the excitation spectrum shows peak positions similar to that obtained for PMA in water, whereas for 480 nm, the peaks in the excitation spectrum matched with that of the absorption spectrum of PMA aggregates in PSS, which was significantly different from that of PMA in water (Figure S2 and S3, ESI). This clearly indicates that the emission at 480 nm originates from PMA aggregates which are formed in the presence of PSS. Thus, these measurements clearly suggest that PSS induces the aggregation of PMA molecules which are responsible for a broad emission band at 480 nm. The formation of PMA aggregates in the presence of PSS can be understood in terms of charge neutralization of cationic PMA on the surface of highly negatively charged PSS, where multiple PMA molecules are bound on the surface of PSS, and undergoes intermolecular pipi stacking interaction due to proximity on the surface of PSS. Such anionic polyelectrolyte induced aggregation of cationic probe molecule has been also reported earlier [42,43]. Excited-state lifetime measurements are very sensitive towards the aggregation state of a molecule, as the formation of aggregated state can cause significant changes in the excited-sate relaxation of a molecule. Thus, to support the observation made in the ground-state absorption and steady-state emission measurements, and to get more insights into the excited-state relaxation of PMA in presence of PSS, we have also carried out excited-state lifetime measurements for PMA in the presence and absence of PSS and the results have been presented in

Fig. 1. (A) Steady–state fluorescence spectra of PMA (35 μM, λex = 340 nm) in presence of various concentrations of PSS (in μM) (1) 0 (2) 0.09 (3) 0.14 (4) 0.16 (5) 0.18 (6) 0.21 (7) 0.24 (8) 0.30. Inset: Variation of ratio of emission at 376 to 480 nm with increasing concentration of PSS. 3

Sensors & Actuators: B. Chemical 303 (2020) 127182

S.P. Pandey and P.K. Singh

Fig. 4. Steady–state fluorescence spectra of PMA (35 μM)-PSS (0.3 μM) complex (λex = 340 nm) in presence of various concentrations of NaCl (in mM) (1) 0 (2) 1.2 (3) 2.4 (4) 4.7 (5) 9.4 (6) 20.8 (7) 47.2 (8) 140.1 (9) 408.9. Inset: Variation of ratio of emission at 376 to 480 nm with increasing concentration of NaCl.

Fig. 3. Transient decay of PMA (35 μM, λex =339 nm, λem = 376 nm) in presence of PSS (in μM) (1) 0 (2) 0.28. The black dotted line represents instrument response function (IRF).

Fig. 3. PMA, in water, shows single-exponential excited-state decay kinetics with a lifetime value of 124 ns. Such long excited-state lifetime for pyrene and pyrene derivatives is well reported in literature [54,55]. However, in the presence of PSS, the excited-state decay traces display a bi-exponential behaviour; one which is very fast (∼6.5 ns) and the other which is very slow (∼124 ns) that is similar to the one observed for the monomeric form of PMA in water. Thus, the fast component can be assigned to the PMA aggregates which are formed in the presence of PSS. Such fast components in the excited-state decay trace for pyrene aggregates has been also reported earlier [54,55]. Thus, excited-state lifetime measurements work as a sensitive indicator for the formation of PMA aggregate molecules. These kind of supramolecular aggregates are formed via reversible non-covalent interactions amongst which hydrophobic interaction, dipole-dipole interaction, ion-dipole interaction, electrostatic interaction and hydrogen bonding may play a significant role. When the guest is mainly hydrophobic in nature (aqueous solubility is less) and the predominant interaction force during supramolecular complexation with the host is hydrophobic interaction, the increase in concentration of salt or ionic strength to the solution increases the strength of interaction between guest and the host molecules since increase in salt concentration increases the polarity or dielectric constant of the medium because of which the hydrophobic dye molecules cannot remain in the water solution and rather prefer the less polar or hydrophobic host cavity leading to increased binding. On the other hand, when the interaction between host and the guest molecules are predominantly iondipole/dipole-dipole or electrostatic in nature, the increased salt concentration results in substantial decrease in the interaction between the opposite charges which ultimately reduces the binding strength between guest and host molecules. Thus, to understand the nature of underlying interactions in the present system, the effect of ionic strength was investigated in the present system by performing the steady-state emission measurements for PMA-PSS complex by increasing the concentration of NaCl in the solution and the results have been presented in Fig. 4. As evident from the figure, the addition of NaCl causes a steady decrease in the emission band at 480 nm whereas concomitantly the monomeric emission band at 376 nm and 394 nm gradually increases. Thus, the ratio of emission intensity for monomer to aggregate emission gradually rises with increasing concentration of NaCl as shown in inset of Fig. 4. These results thus indicate that addition of salt causes dissociation of PMA aggregates from the surface of PSS. These ionic strength dependent results thus indicate that the ionic strength of the medium significantly modulates the interaction between PMA and PSS. In particular, it suggests that PMA and PSS undergo

electrostatic interaction during the formation of PMA aggregate on the surface of PSS which is quite logical since PMA is cationic in nature and PSS is anionic in nature. The electrostatic interaction between PMA and PSS is screened by the addition of salt which leads to dissociation of PMA aggregates from the surface of PSS. Similar effect of salt has been employed and observed previously in other systems to approve the presence of electrostatic interaction between the probe molecules and host of polyelectrolyte nature [56–58]. To support the ionic strength dependent steady-state emission measurements, we have also carried out ground-state absorption measurements for PMA-PSS complex in the presence of various concentration of NaCl and the results have been presented in Fig. 5. This figure clearly shows that the addition of NaCl causes fast recovery (increase) of the monomeric absorption band at 340 nm for PMA. This leads to a steady increase in the ratio of absorbance for monomer and aggregate (OD340/OD352), quite similar to the one observed for the ratio of emission intensity. Thus, ground-state absorption measurements clearly support the conclusion drawn from steady-state emission measurements. To support the results obtained from ground-state absorption and steady-state emission measurements, we have also carried out timeresolved emission measurements for the PMA-PSS system in the

Fig. 5. Ground-state absorption spectra of PMA (35 μM)-PSS (0.3 μM) complex in presence of increasing concentration of NaCl in (mM) (1) 0 (2) 2.4 (3) 4.7 (4) 7.1 (5) 9.4 (6) 11.7 (7) 16.3 (8) 20.8 (9) 29.8 (10) 47.2 (11) 80.3 (12) 140.1 (13) 192.5 (14) 408.9. Inset: Variation of ratio of absorbance at 340 to 352 nm with increasing concentration of NaCl. 4

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S.P. Pandey and P.K. Singh

Fig. 6. Transient Decay of PMA-PSS complex (λex =339 nm, λem = 376 nm) in presence of NaCl (in mM) (1) 0 (2) 9.4. The green dotted line represents the transient decay trace of PMA in water and the black dotted line represents instrument response function (IRF). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 8. (A) Ground-state absorption spectra of PMA-PSS complex at different temperatures (1) 200C (2) 450C (3) 600C (4) 700C. (B) Variation in the ratio of OD at 340 to 352 nm with increasing temperature.

temperature (Inset of Fig. 7). Note that the emission from only PMA in aqueous solution does not yield any observable changes in the emission features of PMA as a function of temperature (Figure S4, ESI). These observations clearly suggest that the rise in temperature leads to the weakening of the forces responsible for supramolecular aggregate formation in the PMA-PSS system, finally causing the gradual breakage of the aggregate assembly leading to the release of free PMA in the solution. To support the temperature dependent emission measurement for the PMA-PSS system, we have also performed temperature dependent ground-state absorption measurements for the PMA-PSS system and the results have been summarized in Fig. 8. It can be seen from the inset of the Fig. 8 that the ratio of absorbance representing monomer to aggregate band (OD340/OD352) rises with increase in temperature which suggests that the PMA-PSS supramolecular assembly is weakened by the rise in temperature and shifts the equilibrium towards the monomer form of PMA. Thus, temperature dependent ground-state absorption measurements mirror the findings obtained with steady-state emission measurements. Therefore, these measurements suggest that temperature leads to an impressive modulation of the monomer-aggregate equilibrium of the PMA-PSS system and provides evidence for the involvement of reversible non-covalent weak interactions in the formation of PMA-PSS supramolecular assembly. Thus, so far, we have established that the negatively charged polyelectrolyte, PSS, induces the aggregation of a cationic fluorescent molecule, PMA and the whole supramolecular assembly is responsive to external stimuli like temperature and ionic strength of the medium and brings out significant modulations in the monomer-aggregate equilibrium in the system. Specifically, considering the involvement of electrostatic interactions in the formation of the present supramolecular aggregate assembly, and based on some previous reports that the cationic side chains of the Arginine and Lysine interacts with the anionic sulphate groups of bio-molecules and supramolecular host molecules [36,59–61], we envisioned that the monomer-aggregate equilibrium in the PMA-PSS system may undergo significant modulation in response to Arginine, a basic amino acid, by virtue of strong electrostatic interaction with the positively charged side chains of Arginine. Since the monomers and aggregates in the PMA-PSS system are associated with distinct and well-separated emission bands, thus, the Arginine induced modulation in the monomer-aggregate equilibrium of the PMA-PSS system may provide a very useful ratiometric detection scheme for the detection of Arginine. Therefore, to see the response of PMA-PSS system towards Arginine, we have performed steady-state emission measurements for PMA-PSS complex in the presence of various concentrations

presence and absence of salt and the results have been presented in Fig. 6. It is evident from the figure that the fast component in the PMAPSS system, which represents the PMA aggregates, completely disappears, upon addition of salt to the PMA-PSS system, and recovers to a situation existing for free PMA. Thus, these salt dependent time-resolved emission measurements is in full agreement with the groundstate absorption and steady-state emission measurements, suggesting that the presence of salt dissociates the PMA aggregates from the surface of PSS which can be ascribed to the dominant presence of electrostatic interaction between PMA and PSS. As stated earlier, this type of supramolecular assemblies are formed via various types of reversible non-covalent interactions which makes them responsive towards several external stimuli. One such external stimulus is temperature, which is often employed to test the reversible non-covalent nature of the underlying interactions existing in this kind of assemblies [56–58]. Thus, in the present system, we have also performed temperature dependent steady-state emission measurements for the PMA-PSS complex and the results have been displayed in Fig. 7. As clearly evident from the figure, the rise in temperature leads to a decrease in the emission intensity at the aggregate band (480 nm) along with a simultaneous increase in the emission intensity at the monomer emission band (376 nm and 394 nm). Thus, the ratio of monomerto–aggregate emission steadily increases with gradual increase in

Fig. 7. Steady–state fluorescence spectra of PMA-PSS complex (λexc = 340 nm) at various temperatures (1) 200C (2) 250C (3) 300C (4) 350C (5) 550C (6) 700C. Inset: Variation of ratio of emission at 376 to 480 nm with increasing temperature. 5

Sensors & Actuators: B. Chemical 303 (2020) 127182

S.P. Pandey and P.K. Singh

Fig. 10. (A) Ground-state absorption spectra of PMA-PSS complex in presence of various concentration of Arginine (in mM) (1) 0 (2) 0.12 (3) 0.47 (4) 0.93 (5) 2.04 (6) 0.3.31 (7) 4.89 (8) 6.38 (9) 9.70 (B) Variation of ratio of absorbance at 340 to 352 nm with increasing concentration of Arginine. The red triangles represent the data points, whereas the solid blue line represents the linear fit to the data points. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

obtained due to addition of Arginine (Fig. 9). This is because the extent of modulation of monomer-aggregate equilibrium may not be same in both the cases and, in addition, temperature, apart from converting the PMA aggregates to PMA monomers, also influences the emission yield of individual species such as PMA monomers or PMA aggregates. In general, increase in temperature reduces the emission yield of the molecules. To confirm this, we have investigated the effect of temperature on the emission spectra of PMA monomers, and indeed the increase in temperature reduces the emission yield of PMA monomers (Figure S5, ESI). To support the steady-state emission measurements on the PMAPSS-Arg system, we have also performed ground-state absorption measurements for PMA-PSS system in the presence of varying concentration of Arginine and the results have been presented in Fig. 10. As evident from the figure, the addition of Arginine leads to an increase in the absorbance at 340 nm, which represents the absorption maximum for the monomeric PMA. This observation is in support of the inference drawn from steady-state emission measurements, which suggests that the addition of Arginine leads to breakage of the PMA aggregates from the surface of PSS that leads to an increase in the concentration of free PMA in the solution leading to increased absorbance at the monomeric absorption band. When the ratio of the absorbance at 340 nm (monomer band) and 352 nm (aggregate band) is plotted as a function of Arginine concentration, the ratio shows a reasonable linear variation in the Arginine concentration range of 0–1.6 mM with a linear regression equation of OD340/OD352 = 0.13[Arg/mM]+1.17; R2 = 0.97. The LOD, as calculated above, is found to be 35 μM. As mentioned earlier, the extent of modulation of the monomer-aggregate equilibrium caused by increase in temperature and addition of arginine may not be same. Further, the additional effect of temperature on the spectrum of individual species (PMA monomers and PMA aggregates) apart from modulating the monomer-aggregate equilibrium in the PMA-PSS system, leads to a visually different change in the absorption spectra (Fig. 8) with increase in temperature when compared to the effect of addition of Arginine in the absorption spectra of PMA-PSS system (Fig. 10) To further validate the mechanism of Arginine induced changes in photophysical features of PMA-PSS supramolecular aggregate assembly, we have also performed excited-state lifetime measurements for the PMA-PSS system in the presence of Arginine and the transient decay traces for the PMA-PSS system in the presence of various concentration of Arginine have been presented in Fig. 11. As discussed earlier, PMA in

Fig. 9. (A) Steady–state fluorescence spectra of PMA (35 μM)-PSS (0.3 μM) complex (λexc = 340 nm) in presence of various concentrations of Arginine (in mM) (1) 0 (2) 0.12 (3) 0.23 (4) 0.35 (5) 0.58 (6) 0.93 (7) 1.59 (8) 4.89. (B) Variation of ratio of emission at 376 to 480 nm with increasing concentration of Arginine. The blue triangles represent the data points, whereas the solid red line represents the linear fit to the data points. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

of Arginine and the results have been presented in Fig. 9. As evident from the figure, the gradual addition of the Arginine causes a steady decrease in the emission intensity at 480 nm which corresponds to aggregate emission and simultaneously an increase in the monomer emission intensity at 376 and 394 nm is observed. This observation clearly suggests that Arginine causes the disintegration of PMA aggregates from the surface of polyelectrolyte, PSS, which subsequently leads to the release of the free PMA molecules in the bulk water. This creates an ideal situation for ratiometric output of the present system towards Arginine. To quantify the Arginine concentration dependent changes in the fluorescence response of the PMAPSS, the ratio of emission at 376 (representing monomer emission) and 480 nm (representing aggregate emission) was plotted as a function of Arginine concentration, and the data has been presented in Fig. 9B. The ratiometric response varies linearly with Arginine concentration in a large dynamic range of 0–2 mM. The linear correlation equation for the fitted data was analysed to be I376/I480 = 2.01[Arg/mM]+0.35 with a linear regression coefficient (R2) of 0.994. The detection limit calculated on the basis of 3.3 σ/S [62] is found to be 15 μM where σ represents the standard deviation for 10 blank measurements and S stands for the slope of the calibration curve. Please note that although both increase in temperature as well as the addition of Arginine leads to the modulation of the monomer-aggregate equilibrium in the PMA-PSS system, the extent of changes in the fluorescence intensity (Fig. 7), owing to the modulation in the monomer-aggregate equilibrium, due to increase in temperature, is comparatively less as compared to the one 6

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Fig. 11. Transient decay of PMA-PSS complex (λex =339 nm, λem = 376 nm) in presence of various concentrations of Arginine (in mM) (1) 0 (2) 0.12 (3) 0.23 (4) 4.89. The dark red dotted line represents the Transient decay trace of PMA in water and the black dotted line represents Instrument response function (IRF). Inset: The variation of average excited state amplitude (τavg) with increasing concentration of Arginine. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

water displays a long single-exponential decay kinetics whereas in the presence of PSS, it shows an additional faster decay component which represents the aggregated state of PMA molecules on the surface of PSS. Now, upon addition of Arginine to the PMA-PSS assembly, the contribution of faster component gradually starts diminishing with steady increase in the concentration of Arginine in the solution and finally leads to the situation prevailing for PMA only in bulk water. This is clearly reflected in the calculation of average excited-state lifetime which shows a rise in its value with increase in the concentration of Arginine and finally reaches a pleatue at a higher concentration of Arginine (Inset of Fig. 11). These results can be ascribed to the release of PMA molecules in the aqueous phase which can be attributed to the stronger electrostatic interaction of positively charged guanidinium groups with the negatively charged sulfonate groups of PSS, in comparison to that between PMA and PSS, which causes the release of PMA molecules from the surface of PSS. Thus, excited-state lifetime measurements are in complete agreement with the steady-state emission and ground-state absorption measurements for the PMA-PSSArg system. Thus all these above results clearly indicated that addition of Arginine causes the dissociation of PMA aggregates from the surface of PSS molecules, presumably due to stronger electrostatic interaction between the PSS and Arginine molecules as compared to that between PSS and PMA molecules. These molecular events cause a modulation in the monomer-aggregate equilibrium of the system and leads to a technically advantageous ratiometric response for the Arginine in solution (Scheme 2). After understanding the response of PMA-PSS assembly towards Arginine, we employed another basic amino acid, Lysine (pI = 9.47),

Fig. 12. (A) Steady–state fluorescence spectra of PMA-PSS complex (λexc = 340 nm) in presence of various concentrations of Lysine (in mM) (1) 0 (2) 0.24 (3) 0.69 (4) 1.15 (5) 2.03(6) 6.38 (7) 11.18 (B) Variation of ratio of emission at 376 to 480 nm with increasing concentration of Lysine. The red triangles represent the data points, whereas the solid blue line represents the linear fit to the data points. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

which is also positively charged at the biological pH, and is expected to interact with PMA-PSS system in a manner similar to that of Arginine. Fig. 12 presents the steady-state emission spectra of PMA-PSS system in the presence of various concentration of Lysine. As evident from the figure, the changes in the emission features of PMA-PSS system, upon addition of Lysine, is akin to that of Arginine, i.e., the emission intensity at aggregate emission band, 480 nm, steadily decreases along with an increase in the emission at monomer emission band (376 nm and 394 nm). This again indicates the disassembly of PMA-PSS aggregate upon addition of Lysine which can be attributed to the stronger electrostatic interaction of positively charged side chains of Lysine with the negatively charged sulfonate groups of PSS. This Lysine induced modulation in the monomer-aggregate equilibrium leads to a steady increase in the ratio of monomer to aggregate emission as a function of Lysine concentration which registers a linear variation in a large concentration range of 0–2 mM (Fig. 12B). The linear regression equation was found to be I376/I480 = 1.51[Lys/mM]+0.38 with a regression coefficient (R2) of 0.985. The detection limit for Lysine was calculated to be 19 μM, which is slightly higher than that obtained for Arginine. Similarly, we also performed ground-state absorption measurements for PMA-PSS system in the presence of Lysine and the results have been summarized in Figure S6 (ESI). Similar to the case of Arginine, it can be seen from the figure S6 (ESI) that the absorbance ratio for the monomer to aggregate band shows a linear variation in the concentration range of 0.2–2 mM with a linear regression equation of OD340/OD352 = 0.07[Lys/mM]+1.17; R2 = 0.95. The LOD was calculated to be

Scheme 2. Schematic representation of Arginine induced dissociation of PMA aggregates from the surface of polystyrene sulfonate (PSS). 7

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S.P. Pandey and P.K. Singh

Fig. 13. Transient decay of PMA-PSS complex (λex =339 nm, λem = 376 nm) in presence of various concentrations of Lysine (in mM) (1) 0 (2) 0.06 (3) 0.47 (4) 9.70. The grey dotted line represents the Transient decay trace of PMA in water and the black dotted line represents Instrument response function (IRF). Inset: The variation of average excited state lifetime (τavg) with increasing concentration of Lysine.

Fig. 14. Variation of emission intensity (at 480 nm) for the PMA-PSS complex as a function of pH.

65 μM which is slightly higher than the one obtained for Arginine Fig. 13 illustrates the decay traces for the PMA-PSS system in the presence of various concentrations of Lysine, and as evident from the figure, the gradual addition of Lysine, leads to an incremental decrease in the contribution of the shorter component, which represents the breakage of PMA aggregate from PSS and subsequent release of PMA in the bulk aqueous solution. At a higher concentration of Lysine, the transient decay traces reaches a situation that was found for PMA in the bulk aqueous solution. This situation in quantitatively described by the trend in the variation of the average excited-state lifetime, which initially registers an increase in its value with increasing concentration of Lysine, and finally saturates to a value at a higher concentration of Lysine, which is similar to that of PMA in bulk water. Overall, these results strongly suggest that Arginine and Lysine causes a significant modulation in the monomer-aggregate equilibrium of the PMA-PSS system by virtue of stronger electrostatic interaction between the Arginine or Lysine with the sulfonate groups of PSS as compared to that between PMA and PSS. One important requirement of a new sensing system is the pH range over which it can operate. An ideal sensing system should be able to operate over a wide pH range including the biological pH. To evaluate this aspect for the current sensing system, we have performed pH dependent emission measurements for the PMA-PSS system and the results have been presented in Fig. 14. As evident from the figure, the emission intensity at the aggregate band (480 nm) largely remains unchanged with variation in pH from 4 to 9. Thus, these results demonstrate the broad useful pH range satisfied by the current sensing platform. Another very important aspect to be assessed, for a sensor system, is the selectivity of the sensing system towards the targeted analyte. Thus, to evaluate the selectivity of the PMA-PSS system towards Arginine and Lysine, we have evaluated the response of our sensing platform towards various amino acids and the results have been presented in Fig. 15. As can be seen from the figure, Arginine and Lysine displays the highest response whereas relatively insignificant response was noted for the other tested amino acids. As discussed earlier, Lysine and Arginine being positively charged in nature, interacts electrostatically with the sulfonate groups of PSS, whereas other amino acids, due to the lack of positive charge, fails to undergo any significant complexation with PSS. Note that Arginine registers a marginally higher response than Lysine which can be ascribed to the differences in the side chains associated with Arginine and Lysine and their subsequent strength of interaction with the sulfonate groups of PSS which is also supported by relatively

Fig. 15. Response plot of PMA-PSS system for (△I376/480) for various amino acids and sodium chloride at a concentration of 2 mM.

higher binding affinity of Arginine as compared to Lysine. Using SternVolmer analysis, the binding constants have been calculated to be 8.68 × 102 M−1 for Arginine and 6.98 × 102 M−1 for Lysine (Figure S7-S9, ESI). The difference in the strength of electrostatic interaction of the side chains of Arginine and Lysine can be rationalized in terms of Pearson’s theory which states that the interaction between a bulky soft base and a bulky soft acid is stronger as compared to that between a bulky soft base and a small hard acid and vice verse [59,63]. Thus, according to this theory, the guanidinium group (bulky soft acid) of Arginine, in comparison to ammonium group (small hard acid) of Lysine, will undergo stronger electrostatic interaction with the sulfonate groups (bulky soft base) of PSS. Overall, these results indicated that the present system shows a reasonably high selectivity towards the basic amino acids. We have also tested the response of the present sensing system towards various anions and cations (Figure S10 – S13, ESI) and the observed results suggest that the effect of the tested anions and metal cations are significantly less as compared to that observed for Arginine and Lysine. To explore the practical utility of our current sensing platform, we have also attempted to evaluate the response of the PMA-PSS system towards Arginine in human serum samples and the results have been presented in Fig. 16. As evident from the figure, PMA-PSS system shows a reasonable response towards Arginine even in human serum samples. The ratiometric response was found to be linear over a much wider 8

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Kapoor for their constant encouragement and support during the course of this work. S.P.P. thanks Human Resource Development Division, Bhabha Atomic Research Centre, for approving the project internship. The authors also thank Mr. Gaurav Singh and Ms. Vidya R. Singh for their help with serum experiments. S.P.P. also thanks Department of Biotechnology, Viva College, Mumbai for approving her M. Sc. research project to be carried out at Bhabha Atomic Research Centre, Mumbai, India. The funding from the Department of Atomic Energy, India is gratefully acknowledged. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.snb.2019.127182. References

Fig. 16. Steady–state fluorescence spectra of PMA (35 μM)-PSS (0.3 μM) complex in 1% Human serum matrix (λexc = 340 nm) in presence of various concentrations of Arginine (in mM) (1) 0 (2) 1.14 (3) 3.36 (4) 5.21 (5) 8.66 (6) 11.61. Inset: Variation of ratio of emission at 376 to 480 nm with increasing concentration of Arginine. The blue circles represent the data points, whereas the solid red line represents the linear fit to the data. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

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concentration range of 0–12 mM. The linear regression was found to be I376/I480 = 0.45[Arg/mM]+1.33 (R2 = 0.996) with a limit of detection of 22 μM. To demonstrate the performance of our sensor system in a more competitive condition, we have tested the response of our sensor system in human serum sample with its content as high as 30 percentage where the ratiometric response was found to be linear over a Arginine concentration range of 0–110 mM (Figure S14, ESI). These results thus implicate that our sensor system can be possibly applied in real life samples. 4. Conclusions In conclusion, we have developed a fluorescence based ratiometric sensor for the detection of basic amino acids which is sensitive, selective, simple, rapid, and assembled from commercially available components. The sensing scheme is based on the Arginine/Lysine induced dissociation of the aggregates of a cationic pyrene fluorophore assembled on the surface of polystyrene sulfonate. The preferential interaction of the basic amino acids with the PSS leads to weakening of the interaction of PMA aggregates with PSS and causes a significant modulation in the monomer-aggregate equilibrium which yields a useful ratiometric response for the sensitive and selective detection of Arginine and Lysine. One of the prime features for the current sensing system is its ratiometric response which is known to be technologically advantageous and much more robust towards small variation in experimental conditions as opposed to the ones which operate through single wavelength based measurements. Another noteworthy advantage with this sensing system is the inexpensive commercial availability of the probe molecule which avoids complicated and time-consuming synthetic protocols associated with most of the previously reported sensors for basic amino acids. Importantly, our sensor system also shows very good response in real samples of human serum which suggests that our sensing system may find potential application in real–life scenario. Declaration of Competing Interest The authors declare no competing financial interest. Acknowledgments The authors acknowledge Dr. S. Adhikari, Dr. H. Pal and Dr. S. 9

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Shrishti P Pandey is currently a Masters student of Biotechnology at Department of Biotechnology, Viva College, Mumbai, India. Her research interest involves developing fluorescence based sensors for various important bio-analytes which includes amino acids, protein aggregates, Heparin etc. Prabhat K. Singh is currently working as a scientist at Radiation and Photochemistry Division of Bhabha Atomic Research Centre, India. He is also an Assistant Proferssor at Homi Bhabha National Institute, Mumbai, India. His research interests include crafting of self-assembled materials and their usage in designing optical sensors for bio-sensing applications.

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