Rhodamine-Based Fluorescent Probe for Highly Selective Determination of Hg2.

The determination of mercuric ions (Hg2+) in environmental and biological samples has attracted the attention of researchers lately. In the present work, a novel turn-on Hg2+ fluorescent probe utilizing a rhodamine derivative had been constructed and prepared. The probe could highly sensitively and selectively sense Hg2+. In the presence of excessive Hg2+, the probe displayed about 52-fold fluorescence enhancement in 50% H2O/CH3CH2OH (pH, 7.24). In the meantime, the colorless solution of the probe turned pink upon adding Hg2+. Upon adding mercuric ions, the probe interacted with Hg2+ and formed a 1:1 coordination complex, which had been the basis for recognizing Hg2+. The probe displayed reversible dual colorimetric and fluorescence sensing of Hg2+ because rhodamine's spirolactam ring opened upon adding Hg2+. The analytical performances of the probe for sensing Hg2+ were also studied. When the Hg2+ concentration was altered in the range of 8.0 × 10-8 to 1.0 × 10-5 mol L-1, the fluorescence intensity showed an excellent linear correlation with Hg2+ concentration. A detection limit of 3.0 × 10-8 mol L-1 had been achieved. Moreover, Hg2+ in the water environment and A549 cells could be successfully sensed by the proposed probe.

Introduction

Recently, developing highly selective and sensitive sensors for toxic metal ions have been in demand in environmental and biological studies.[1−3] Heavy metal mercury is toxic and bio-accumulating, can be generated by both natural resources and anthropogenic activities, and ubiquitously exists in the global environment. Mercury includes elemental mercury, inorganic mercury, and organic mercury. Mercuric ions (Hg2+) are much more common than mercurous ions (Hg+), and low-dose Hg2+ in the body could cause long-standing irreversible harm to the human health.[4,5] Methylmercury is a dominant form of organic mercury and could be produced through microbiological transformation of Hg2+ in the aquatic environment. Moreover, methylmercury could bio-accumulate in the human body by biological food chain and result in a variety of illnesses including cardiovascular diseases, Minamata disease, growth retardation, dyskinesia, and so forth.[6−9] The maximum amount of Hg2+ allowed in drinking water is 2 ppb (10–8 mol L–1) according to the US Environmental Protection Agency.[10] Thus, sensing Hg2+ has high significance in environmental and medical science.

To date, many analytic approaches have been employed for sensing Hg2+ such as high-performance liquid chromatography–inductively coupled plasma mass spectrometry (HPLC–ICP-MS),[11] HPLC coupled with atomic fluorescence spectrometry (HPLC–AFS),[12] ICP-atomic emission spectrometry (ICP-AES),[13] atomic absorption spectrometry (AAS),[14] ultraviolet–visible absorption spectrometry (UV–vis),[15] fluorometry,[16,17] electrochemical methods,[18,19] and so on. Among all the methods developed, fluorometry possess remarkable advantages because of its high sensitivity, inherent simplicity, instrument operability, in situ detection, and bioimaging analysis in vivo.[16,17] Hitherto, many fluorescent probes had been constructed to determine Hg2+.[20−30] However, some analytical performances of these probes are unsatisfactory, including the slow response time,[20−23] cross interference,[24,25] poor sensitivity,[26−29] and so forth. Therefore, novel Hg2+ fluorescent probes are still desirable, which possess fast response speed, excellent selectivity, and sensitivity.

Rhodamine and rhodamine derivatives have been applied widely as fluorescent probes owing to their remarkable optical characteristics including a high fluorescence quantum yield (Φ), a large molar extinction coefficient (ε), and longer excitation and emission wavelengths.[31] So far, many fluorescent probes utilizing rhodamine derivatives had been utilized to sense different cations including Cu2+,[32] Pb2+,[33] Cr3+,[34] Fe3+,[35] Al3+,[36] Zn2+,[37] and so forth. The sensing mechanism of these probes for cations is based on the transformation from a spirocyclic form into an open cyclic structure. In the absence of cations, these probes displayed no fluorescence and colorlessness because of their spirocyclic form. After adding cations, these probes emitted a powerful fluorescence and showed a pink color because the cyclic structure of the spirolactam or spirolactone was opened through a reversible complexation or nonreversible chemical interaction. The above sensing mechanism has also been applied to construct rhodamine-based Hg2+ fluorescent probes.[38−47] However, there are still more or less limitations for some of these Hg2+ fluorescent probes including the delayed response,[38] a narrow pH range,[39] and cross interference.[40−44] Thus, developing unusual rhodamine-based fluorescent probes for Hg2+ is still very important.

Herein, a new Hg2+ fluorescent probe 1 (Scheme ) had been constructed, which chose rhodamine as the fluorophore. Because sulfur had strong affinity for Hg2+,[39,48] we introduced a sulfur-based functional unit to the probe in the present article. To prepare an optical chemical sensor (optode) for Hg2+ in the next work, a terminal double bond was included in probe 1 to allow the probe to covalently immobilize on the activated surface of glass slides with the double bond by UV irradiation. When Hg2+ was absent, the probe existed in a spirocyclic form and exhibited no fluorescence and colorlessness. When Hg2+ was present, the probe emitted yellowish-red fluorescence, and the solution color changed to pink. The Hg2+ probe exhibited excellent sensing performances including a fast response time, excellent sensitivity and selectivity, a broad pH working range, and so forth. In addition, nearly no cytotoxic reaction was found, and the probe could be successfully utilized to image Hg2+ in A549 cells. Besides, the developed probe was also magnificently utilized to monitor Hg2+ in water environments.

Scheme 1

Preparation of Fluorescent Probe 1: (a) Hydrazine Monohydrate (85%), CH3CH2OH, Reflux, 12 h, 86%; (b) Lawesson’s Reagent, Toluene, Reflux, 4 h, 17%; (c) Allyl Bromide, Sodium Bicarbonate, Dormyl Dimethylamine, 70 °C, 48 h, 61%; and (d) CH3CH2OH, Reflux, 12 h, 75%

Results and Discussion

Spectral Characteristics of Probe 1

Figure shows a graph depicting the variation in the fluorescence spectrum of probe 1 after adding different concentrations of mercury ions in Tris–HCl buffer (CH3CH2OH/H2O, 1:1, v/v, pH 7.24). As illustrated in Figure , probe 1 emitted a very weak fluorescence when Hg2+ was not added. As the concentration of Hg2+ was increased, probe 1 exhibited enhanced fluorescence. When excess Hg2+ was added, the probe demonstrated a 52 times fluorescence increase at 586 nm. The experiment was based on these results to determine the concentration of mercury ions.

Figure 1

Fluorescence spectra of 5.0 μM probe 1 in the presence of different concentrations of Hg2+: 0, 0.03, 0.05, 0.07, 0.08, 0.10, 0.20, 0.40, 0.50, 0.60, 0.80, 1.0, 2.0, 4.0, 5.0, 6.0, 8.0, 10, 20, 40, 50, 60, 80, and 100 μM from 1 to 24. Inset: variation of fluorescence intensity of 5.0 μM probe 1 with Hg2+ concentration (λex = 520 nm; λem = 586 nm).

In order to explore the sensing mechanism of probe 1 toward Hg2+, the variation of the absorption spectrum of probe 1 with the gradual addition of mercury ions was recorded (Figure ). As demonstrated in Figure the UV–visible absorption spectrum of probe 1 showed a weak absorbance at 565 nm before the addition of mercury ions, which could be ascribed to the presence of the partial ring-opened structure of the spirolactam unit of probe 1. With the increment of Hg2+, probe 1 showed a gradual increase in absorption at 565 nm. When the Hg2+ concentration was 50 μM, the absorbance reached the maximum value. Meanwhile, the solution color change of probe 1 from colorless to pink after the addition of Hg2+ could be seen by the naked eye. The possible reason for absorption intensity enhancement at 565 nm was that the ring of the spirolactam structure of probe 1 opened upon the addition of mercury ions. The UV–visible absorption spectroscopy results also indicated that probe 1 interacted with Hg2+. Furthermore, a chemical shift of 64.23 ppm in the 13C NMR spectrum of probe 1 corresponded to the characteristic chemical shift value of the spirocyclic carbon, which also indicated that probe 1 existed in the spirolactam form before the addition of Hg2+.

Figure 2

UV–vis spectra of 5.0 μM probe 1 with the gradual addition of Hg2+: 0, 0.08, 0.10, 0.20, 0.40, 0.50, 0.60, 0.80, 1.0, 2.0, 4.0, 5.0, 6.0, 8.0, 10, 20, 40, 50, 60, 80, and 100 μM from 1 to 21. Inset: the variation of absorbance of 5.0 μM probe 1 with Hg2+ concentration.

Principle of Operation

To explore the linear correlation between the fluorescence intensity of probe 1 and mercury ions, the fluorescence intensity of probe 1 was studied after the addition of various concentrations of Hg2+. The fluorescence intensity of probe 1 at 580 nm linearly depended on the Hg2+ concentration when the Hg2+ concentration was varied from 8.0 × 10–8 to 1.0 × 10–5 mol L–1 (Figure ). The linear regression equation used was F = 25.1293 + 25.1931 × 106 × C (r = 0.9949); here, F is the fluorescence intensity, C represents the concentration of Hg2+, and r is the linear correlation coefficient. The detection limit was estimated to be 3.0 × 10–8 M based on 3SB/m (where SB is the standard deviation of 10 blank measurements and m is the slope of the linear regression equation),[49,50] which was more sensitive than that of formerly developed Hg2+ fluorescent probes.[26−29] Moreover, according to the UV–vis titration profile in Figure , the absorbance of probe 1 at 565 nm increased linearly with the change of the Hg2+ concentration of 8.0 × 10–8 to 2.0 × 10–5 mol L–1 (Figure S1, Supporting Information). The linear regression equation used was A = 0.0569 + 0.1253 × 106 × C (r = 0.9975), where A is the absorbance, C represents the concentration of Hg2+, and r is the linear correlation coefficient. Moreover, the detection limit was found to be 4.8 × 10–8 mol L–1 based on 3SB/m (where SB is the standard deviation of 10 blank measurements and m is the slope of the linear regression equation).[39]

Figure 3

Calibration curve between the fluorescence intensity of 5.0 μM probe 1 and the concentration of mercury ions. Inset: plot of fluorescence intensity of 5.0 μM probe 1 as a function of Hg2+ concentration in the range of 0.08–1.0 μM.

In addition, we also confirmed the stoichiometry of probe 1 with mercury ions by using Job’s plot (Figure ). When the molar fraction of the amount of Hg2+ was close to 0.5, the absorbance of probe 1 reached a maximum, indicating that probe 1 coordinated with Hg2+ through a 1:1 stoichiometry. Based on the alteration in the absorption spectrum of probe 1 in the absence and presence of mercury ions and the coordination stoichiometry between probe 1 and Hg2+, we proposed a possible structural model for the formation of complexes of probe 1 and mercury ions (Scheme ). To further support the hypothesis, the NMR titration of probe 1 in the presence of Hg2+ was carried out (Figure S2, Supporting Information). Hg2+ is a heavy metal ion and could influence the proton signals near the Hg2+ binding site.[51] From Figure S2, the 1H NMR signal of Ha belonging to probe 1 was shifted to the upfield in the presence of Hg2+, which demonstrated the binding between the oxygen atom of the hydroxyl group and Hg2+. As displayed in Figure S2, the 1H NMR signal of Hb in probe 1 also shifted upfield after the addition of Hg2+, which illustrated the coordination between the nitrogen atom of the group (−CH=N−) and Hg2+. The result of NMR titration experiment further confirmed the coordination mode between the probe and Hg2+.

Figure 4

Job’s plot for confirming the stoichiometry of probe 1 and Hg2+ in the CH3CH2OH/H2O (1:1, v/v) solution. The whole concentration of probe 1 and Hg2+ was 40 μM.

Scheme 2

Likely Complexation Style between the Proposed Probe and Hg2+

Effect of pH

For obtaining information about the pH effect, the fluorescence intensity changes of probe 1 before and after adding Hg2+ are investigated in various pH conditions (Figure ). As exhibited in Figure , the fluorescence intensity of probe 1 is substantially unchanged in the range of pH 4.50 to 12.00. However, in the presence of Hg2+, the probe exhibited a strong fluorescence in the range of 4.50–8.50. The above results demonstrated that the fluorescent probe was not affected in the pH range of 4.50–8.50 and thus could be exploited to sense Hg2+ in the actual samples. When the pH was less than 4.50, probe 1 emitted enhanced fluorescence with reduced pH values, which could be attributed to the ring-opened structure of probe 1 under acidic conditions. Considering the response speed, sensitivity, and practical application, the pH 7.24 Tris–HCl buffer was used in this experiment.

Figure 5

Fluorescence intensity variation of 5.0 μM probe 1 with pH before (open circles) and after (solid circles) adding Hg2+.

Selectivity

In order to ensure that probe 1 could be used in a wide variety of environments, we examined the selectivity of probe 1 for Hg2+ (Figure ). It can be found from the black histogram in Figure that the solution of Li+, Na+, K+, Mg2+, Ca2+, Fe3+, Al3+, Cd2+, Zn2+, Ag+, Mg2+, Pb2+, Ba2+, and Cr3+ ions had almost no effect on the fluorescence intensity of probe 1 at a concentration of 10 times the Hg2+ concentration. In addition, when the concentrations of Cu2+, Co2+, Ni2+, Mn2+, and Hg2+ were equal, probe 1 displayed a notable fluorescence increase only upon adding Hg2+. The color change, fluorescence change, and UV–vis response of the probe in the absence and presence of those different ions mentioned above were also studied (Figure S3, Supporting Information). From Figures and S3, the probe showed high selectivity for Hg2+ by fluorometry. However, the detection of Hg2+ was interfered by Cu2+ by ultraviolet–visible absorption spectrometry. Moreover, competition experiments were executed in which the developed probe was used to sense Hg2+ (1.0 × 10–5 mol L–1) in the coexistence solution of Hg2+ and other metal ions (white histogram in Figure ). The experimental results showed that the relative error of usual interference including alkaline earth, alkali, and transition-metal ions was lower than ±5%, which was thought to be acceptable. These experiments demonstrated that the coexistence of different metal cations and mercury ions did not affect the determination of Hg2+, making the probe more likely to be used to determine actual samples.

Figure 6

Metal-ion selectivity of 5.0 μM probe 1. The concentration of ions was 1.0 × 10–5 M for Hg2+, Co2+, Cu2+, Ni2+, and Mn2+ and 1.0 × 10–4 M for all other ions. Black bars: various metal ions were provided. White bars: various metal ions in the presence of Hg2+ were provided.

For comparison, the complexation study with Hg2+ had been carried out using compound 2 (Figure S4, Supporting Information) or compound 3 (Figure S5, Supporting Information) in Tris–HCl buffer (CH3CH2OH/H2O, 1:1, v/v, pH 7.24). As demonstrated in Figure S4, in Tris–HCl buffer (CH3CH2OH/H2O, 1:1, v/v, pH 7.24), compound 2 showed fluorescence enhancement only in the presence of Cu2+. From Figure S5, compound 3 demonstrated a strong fluorescence increase and a pink color only in the presence of Cu2+ or Hg2+ (Figure S5 in the revised Supporting Information). Thus, probe 1 possessed higher selectivity for Hg2+ compared with compound 2 and compound 3.

Reversibility and Response Time

It was apparent to all that reversibility is a significant factor to prepare an excellent chemical sensor. We tested the reversibility of probe 1 by adding an EDTA solution (Figure ). As demonstrated in Figure , the fluorescence intensity of probe 1 drastically increased after providing mercury ions, and the solution color transformed from colorlessness to pink. However, after the EDTA solution was added to the above pink solution, the fluorescence intensity decreased rapidly, and the solution color was altered from pink to colorlessness. These experimental consequences explained that the complexation process of probe 1 and Hg2+ was reversible.

Figure 7

Reversibility of probe 1 for Hg2+ in Tris–HCl buffer (CH3CH2OH/H2O, 1:1, v/v, pH 7.24). ···:5.0 μM probe 1; −:5.0 μM probe 1 with 10 μM Hg2+; —:5.0 μM probe 1 with 10 μM Hg2+; and following the addition of 40 μM EDTA 2Na.

At the same time, we also studied the time response of probes to mercury ions (Figure ). Hg2+ (1.0 × 10–5 mol L–1) were added to probe 1 solution, and the change in fluorescence intensity from 0.0 to 5.0 min was recorded to examine the time response of probe 1 for Hg2+. From Figure , it can be observed that the complexation speed of probe 1 and Hg2+ was very rapid, and the fluorescence intensity could reach the maximum within 60 s. The response time of the proposed probe for Hg2+ was shorter than that of formerly reported fluorescent probes for Hg2+.[20−23] Therefore, the fluorescent probe would be utilized for real-time Hg2+ detection.

Figure 8

Time response of probe 1 (5.0 μM) for 10 μM Hg2+. Time points are 0, 30, 60, 90, 120, 150, 180, 210, 240, 270, and 300 s; λex = 520 nm. Inset: visual fluorescence color of probe 1 (5.0 μM) in the absence (left) and presence (right) of Hg2+ for 60 s (UV lamp, 365 nm).

Compared with other probes for Hg2+ based on rhodamine derivatives (as displayed in Table S1, Supporting Information), this probe possessed many advantages including high sensitivity and specificity, fast response time, a wide pH working range, and so forth.

Preliminary Analytical Application

For verifying the application of probe 1 in actual sample analysis, probe 1 was utilized for the sensing of mercury ions in tap water and river water samples. The river water and tap water were directly used after being filtered by a 0.45 μm filter. The river water and tap water were measured by probe 1 for mercury ion content, which contained no Hg2+, and then the standard solutions of different concentrations of Hg2+ were separately added for the determination of the recovery rate (Table ). As found from Table , the fluorescent probe has a satisfactory determination result of Hg2+ recovery rate in the tap water and river water. Thus, the probe could be effectively used to sense Hg2+ in the actual samples.

Table 1

Sensing Hg2+ in Tap and River Water Samples with Probe 1

	sample	Hg2+ spiked (mol L–1)	Hg2+ recovered (mol L–1)	recovery (%)
river water	1	0	not detected
	2	5.00 × 10–6	(5.15a± 0.10b)	103.0
	3	1.00 × 10–5	×10–6	102.0
			(1.02a± 0.02b) × 10–5
tap water	1	0	not detected
	2	5.00 × 10–6	(5.18a± 0.12b)	103.6
	3	1.00 × 10–5	×10–6	98.0
			(0.98a± 0.03b) × 10–5

Mean values of three determinations.

Standard deviation.

We also conducted a bio-imaging application research of probe 1. To assess the biocompatibility of probe 1, we performed a cytotoxicity assay using the MTT colorimetric assay (Figure S6, Supporting Information). From Figure S1, the cell viability of A549 cells was found to be higher than 90% when probe 1 was present, indicating that probe 1 was almost not cytotoxic. Next, we verified whether probe 1 could be employed for sensing Hg2+ in living cells by laser confocal fluorescence imaging experiments (Figure ). As can be seen from Figure a, after culturing A549 cells in medium containing probe 1 (5.0 μM) for 30 min, the cells showed substantially no fluorescence. However, when A549 cells were cultured for 30 min with 1.0 μM Hg2+ under the same conditions, the cells exhibited a strong fluorescence (Figure d). According to the research results, probe 1 could be applied to fluorescence imaging of Hg2+ in living cells.

Figure 9

Images of A549 cells incubated with the developed probe 1. (a) Fluorescence image of A549 cells attached to 5.0 μM probe 1 for 30 min at 37 °C; (b) bright-field transmission image of cells demonstrated in (a); (c) overlap image of (a,b); (d) fluorescence image of A549 cells attached to 5.0 μM probe 1 for 30 min, washed three times, and further treated with 1.0 μM Hg2+ for 30 min; (e) bright-field transmission image of cells displayed in (d); and (f) overlap image of (d,e).

Conclusions

On the whole, a new fluorescent probe has been planned and prepared to quantify Hg2+, which used rhodamine as the fluorophore. After providing Hg2+, the probe displayed a strong fluorescence emission and a pink color due to its open-cycle structure of the corresponding spirolactam via a reversible coordination. The fluorescent probe exhibited excellent sensing abilities, including excellent sensitivity and specificity, rapid response, a wide pH working range, and so forth. The fluorescent probe had been applied to sense Hg2+ in both tap and river water samples and demonstrated acceptable consequences. Furthermore, the probe also demonstrated superior biocompatibility, which permitted us to obtain fluorescence imaging of Hg2+ in A549 cells.

Experimental Section

Materials and Instruments

Lawesson’s reagent (97%) and allyl bromide were purchased from Aldrich. Rhodamine B and hydrazine monohydrate (85%) were bought from Shanghai Sinopharm Group Company. Before toluene was utilized, it was freshly distilled through adding sodium. Unless otherwise stated, other chemical reagents used in this work were of analytical grade and could be applied directly. Water used in all assays was ultrapure water.

UV–vis and fluorescence spectra were measured on a UV-2600 spectrometer and a Hitachi F-7000 spectrophotometer, respectively. A Bruker DRX-500 NMR spectrometer was utilized to measure the NMR spectra. Fluorescence images of living cells were acquired through an Olympus FV1200-MPE multiphoton laser scanning confocal microscope with a 40× objective lens. The pH of the solution was measured with a Mettler Toledo Delta 320 pH meter. SigmaPlot software was used to perform the data processing.

Syntheses

The method of preparation of fluorescent probe 1 is demonstrated in Scheme . During the process, product 2 was obtained through the interaction of hydrazine monohydrate (85%) and rhodamine B, as reported in the previous literature.[52] Compound 3 was obtained by the chemical reaction between Lawesson’s reagent and compound 2 following an earlier developed method.[52] 4-Formyl-3-hydroxyphenyl allyl ether (compound 4) was synthesized from 2,4-dihydroxybenzaldehyde and allyl bromide following a formerly developed procedure.[53]

Synthesis of Compound 1

Compound 3 (0.19 g, 0.40 mmol) and compound 4 (0.096 g, 0.54 mmol) were put in 50 mL CH3CH2OH. The mixture was warmed to refluxing for 12 h. After the solvents were cleaned in decompression, purification of the crude product was done through column chromatography (CH3COOCH2CH3/petroleum ether = 1:10, v/v) to produce probe 1 (0.19 g, 75%) as a pale yellow solid. 1H NMR (500 MHz, CDCl3): δ(ppm) 11.50 (1H, s), 8.62 (1H, s), 8.09 (1H, dd, J = 5.8 Hz, 3.2 Hz), 7.41 (2H, dd, J = 5.8 Hz, 3.2 Hz), 7.19 (1H, d, J = 8.5 Hz), 7.13–7.11 (1H, m), 6.74 (2H, d, J = 8.8 Hz), 6.49–6.42 (2H, m), 6.32–6.27 (4H, m), 6.05–5.97 (1H, m), 5.39 (1H, dd, J = 17.2 Hz, 1.4 Hz), 5.27 (1H, dd, J = 10.5 Hz, 1.2 Hz), 4.51 (2H, d, J = 5.3 Hz), 3.31 (8H, q, J = 7.0 Hz), 1.14 (12H, t, J = 7.0 Hz) (Figure S7, Supporting Information). 13C NMR (125 MHz, CDCl3): δ(ppm) 170.13, 162.38, 161.97, 161.07, 155.13, 151.93, 148.33, 135.19, 133.02, 132.67, 132.16, 130.11, 127.91, 127.12, 122.21, 117.99, 111.75, 110.17, 108.28, 107.63, 101.92, 97.53, 68.83, 64.23, 44.37, 12.60 (Figure S8, Supporting Information). MS (ESI) m/z: 633.2902 (M + H)+ (Figure S9, Supporting Information).

Measurement Procedures of Fluorescence Intensity

The preparation of 1.0 × 10–5 mol L–1 probe 1 was done by adding the needed quantity of probe 1 to CH3CH2OH. 8 × 10–7–1.0 × 10–3 mol L–1 Hg2+ stock solution was obtained by gradually diluting 1.0 × 10–2 mol L–1 mercury nitrate solution with 0.05 mol L–1 Tris–HCl buffer (pH, 7.24). The 0.05 mol L–1 Tris–HCl solution was adjusted by adding HCl or NaOH solution to obtain a range of pH buffer solutions. 12.50 mL of 1.0 × 10–5 mol L–1 probe 1 and 2.50 mL of different concentrations of Hg2+ solution were put into a 25 mL volumetric flask and then it was made up to 25 mL using 0.05 mol L–1 Tris–HCl buffer. A solution containing 5 × 10–6 mol L–1 probe 1 and 8 × 10–8–1×10–4 mol L–1 Hg2+ was obtained. The same procedure without adding Hg2+ was used to obtain the blank solution of probe 1.

All solutions were kept at 4 °C in the dark. The excited wavelength was 520 nm, and both the entrance and exit slits were of 2.5 nm, and the fluorescence intensity was recorded in the range of 540–650 nm.