Enhanced performance of Fe³⁺ detection via fluorescence resonance energy transfer between carbon quantum dots and Rhodamine B

Abstract

Carbon quantum dots (CQDs) were prepared by a facile hydrothermal method and emitted a broad fluorescence covering the entire blue and green light wavelength scope. Because their emission spectra overlaped with the absoprtion spectrum of Rhodamine B (RhB) molecules, fluorescent resonance energy transfer (FRET) phenomenon between CQDs as the energy donors and RhB as the energy acceptors was observed when CQDs were mixed with RhB in a solution. To obtain the optimal FRET efficiency, the concentrations of CQDs and RhB should be adjusted to 0.559 mg mL⁻¹ and 1.25 μM, respectively, at pH = 6.2. None of the metal ions except for Fe³⁺ hindered this FRET process as well as deactivated the electronic excitation energy of RhB molecules through migration, resulting in an enhancement of fluorescence quenching rates. Therefore, the developed system allowed enhancing the selectivity and sensitivity of Fe³⁺ detection via the FRET effects, and could be used for accurate measurements of time-dependent conformational changes and monitoring the corrosion processes of iron materials over an extended period.

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1. Introduction

Fluorescence resonance energy transfer (FRET) is a process which involves the non-radiative transfer of excitation energy from an excited donor to an acceptor.^1–3 It has been employed to enhance the selectivity and sensitivity of a fluorescence probe. At present, traditional quantum dots (such as CdTe and CdSe) were generally used as the excited state energy donor in a FRET system.^4–9 However, they have certain limitations because they contain toxic elements. Carbon quantum dots (CQDs), a new kind of fluorescent probe, have exhibited many advantages, including photoluminescence tunability, long fluorescence lifetimes and high photostability without incurring the limitations of intrinsic toxicity or elemental scarcity.^10–19 Therefore, many efforts were made to develop CQDs for applications in detection systems. Several successful examples have been reported; for instance, some metal ions (such as Cu²⁺, Hg²⁺ and Ag⁺) were recognized through fluorescence changes of CQDs.^20–25 However, few reports indicated that CQDs acted as a component of a FRET system to recognize the metal ions.

Rhodamine B (RhB) belongs to the group of xanthene dyes that are commonly used in FRET systems as the excited state energy acceptor because of its many advantages, such as good solubility, excellent photostability, high extinction coefficient and high fluorescence quantum yields.^26,27 It had been utilized in FRET systems to detect DNA, metal ions, small molecules, and other analytes.^4,5,28,29 In the present work, the FRET effect between CQDs and RhB was observed and used for a fluorescence probe to detect Fe³⁺ ions. The FRET system of CQDs and RhB (RhB@CQDs) showed significantly higher selectivity and sensitivity towards Fe³⁺ ions compared to RhB and CQDs, respectively.

2. Experiments

Materials and apparatus

Ethylene glycol and Rhodamine B were purchased from Aladdin (Shanghai, China). A variety of cations were introduced by addition of soluble metal salts, such as Fe(NO₃)₃, Al(NO₃)₃, MnCl₂, Pb(NO₃)₂, NiCl₂, CaCl₂, CuCl₂, ZnCl₂, HgCl₂, Mg(NO₃)₂, Fe(SO₄)₂, Na₂CO₃, KCl, Ba(NO₃)₂ and CdCl₂. Absorption and FL emission was measured by Shimadzu UV-2550 UV/Vis spectrometer and Hitachi F4500 fluorescence spectrophotometer, respectively. Transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) images were obtained using an FEI Tecnai G2 F20 microscope with a field-emission gun operating at 200 kV. The infrared spectra were obtained on a Thermo Nicolet 360 FT-IR spectrophotometer.

Preparation of RhB@CQDs

The CQDs were prepared by hydrothermal treatment of ethylene glycol as previously reported by us.³⁰ Briefly, 25 mL of ethylene glycol was transferred into a 50 mL Teflon equipped stainless steel autoclave and then placed in a drying oven at 200 °C for 5 h. After cooling to room temperature, the transparent solution containing CQDs were obtained. Subsequently, it was mixed with 125 μL of RhB (0.5 mM). After stirring for more than 2 h at room temperature, the complex of RhB@CQDs was finally obtained. The pH of the samples was adjusted by adding HCl and NaOH.

3. Results

As shown in Fig. 1A, the TEM image of the CQDs reveals that they are uniformly dispersed with size of several nanometers. The typical HRTEM image shows lattice fringes with interplanar spacings similar to a graphitic structure. The FTIR spectrum (Fig. 1B) further suggests that some O-containing groups, including C=O, C–O–C and OH groups, are present in the CQDs. Such surface groups impart excellent water solubility and suitability to the CQDs for subsequent functionalization with various organic and inorganic species via covalent or noncovalent bonds.

Fig. 1 (A) TEM and HRTEM images of CQDs; (B) FTIR spectrum of CQDs.

FRET generally requires a nonzero integral of the spectral overlap between donor emission and acceptor absorption.^1–3 Therefore, the normalized fluorescence (FL) emission spectrum of the CQDs and the absorption spectrum of the RhB molecules are shown in Fig. 2A. It can be seen that the spectral overlap is present in the shadow area. Fig. 2B shows the FL emission comparisons between CQDs, RhB and RhB@CQDs. RhB@CQDs exhibits two FL peaks at 427 and 581 nm. The FL peak at 427 nm from RhB@CQDs becomes weaker than that from only CQDs, while the FL peak from RhB@CQDs at 581 nm is significantly stronger than that from only RhB molecules at the same excitation wavelength. This suggests that the excited CQDs have transferred energy into RhB molecules, i.e. FRET occurs. However, the FL emission obtained at the excitation wavelength of 365 nm significantly quenched, when Fe³⁺ ions were added into the RhB@CQDs system (Fig. 2C). Fig. 2D gives the FL emission behaviors of the RhB@CQDs excited at a wavelength of 554 nm, which is the maximum absorption peak for the RhB molecules, under the conditions of the absence and presence of Fe³⁺ ions. It can be noted that the CQDs hardly emit at the excitation wavelength of 554 nm, thus the emission obtained at 554 nm excitation is attributed to the RhB molecules. According to Fig. 2D, Fe³⁺ ions also quenched the FL emission of RhB molecules. However, different quenching degrees were obtained for the same content of Fe³⁺ ions, when 365 and 554 nm excitation wavelengths were employed, respectively (Fig. 2C and D, respectively). Furthermore, Fe³⁺ ions also caused the FL emission peak at 581 nm to have a small red-shift irrespective of the employed excitation wavelength.

Fig. 2 (A) The normalized FL emission spectrum of CQDs and absorption spectra of RhB; (B) the FL emission spectra of CQDs, RhB and RhB@CQDs at the excitation wavelength of 365 nm; (C and D) the FL emission spectra of RhB@CQDs in the absence and presence of 500 μM of Fe³⁺ at the excitation wavelength of 365 and 554 nm, respectively.

Fig. 3 shows the comparisons of the emission quenching rates of the FL emission peaks at 427 and 581 nm between CQDs, RhB and RhB@CQDs under the same content of Fe³⁺ ions. The emission quenching rate of RhB@CQDs is 2.5 times higher than those of CQDs and RhB, suggesting that RhB@CQDs has a high efficiency to detect Fe³⁺ ions.

Fig. 3 The quenching rate comparisons of FL emission peaks at 427 and 581 nm, respectively, between CQDs, RhB and RhB@CQDs in the presence of 500 μM Fe³⁺ ions. Here, F₀ and F are the FL emission intensities of CQDs, RhB and RhB@CQDs without and with Fe³⁺ ions, respectively.

Because the excited CQDs transfer their excitation energy to the proximal ground state of the RhB molecules through long-range dipole–dipole interactions based on the FRET mechanism,^1,3 the FL emission of CQDs reduces with the increase of the FL emission of RhB. Thus, the intensity ratio between the FL peaks at 581 nm (F_a) and 427 nm (F_d) should reflect a FRET efficiency at the same concentrations of CQDs and RhB in the system of RhB@CQDs.

Fig. 4A shows the effects of the pH value on the ratio of F_a/F_d. The values of F_a/F_d reach the maximum and are maintained almost constant at pH = 6–8, suggesting a higher FRET efficiency in and around the neutral environment. The pH values can modulate the electrostatic interactions between the CQDs and the RhB molecules. As a consequence, the pH variation could influence the absorption ability between the CQDs and RhB, which mainly relies on an electrostatic reaction, thus change the distance between them. This could result in a change in the FRET efficiency on the basis of the FRET mechanism. Fig. 4B and C show the effects of the concentrations of CQDs and RhB on FRET efficiency. It can be seen that there is not a linear increase with the concentrations of CQDs and RhB. The optimal concentrations of CQDs and RhB are 0.559 mg mL⁻¹ and 1.25 μM, respectively. Under the condition of the optimal concentrations of CQDs and RhB, the effects of the pH values on the quenching rates of the emission peak at 581 nm from RhB@CQDs, in the presence of 500 μM Fe³⁺ ions are given in Fig. 4D. The largest quenching rate can be obtained at pH = 6.2.

Fig. 4 (A) The effects of the pH values on FRET efficiency of RhB@CQDs, where F_a and F_d are the intensities of FL emission peaks at 581 and 427 nm, respectively. (B) The effects of the concentrations of CQDs on the FRET efficiency of RhB@CQDs. Herein, the concentration increase of CQDs ranges from a to e (a–e: 0.14, 0.279, 0.419, 0.559, and 0.698 mg mL⁻¹), RhB = 1.25 μM, pH = 6.20; (C) the effects of the RhB concentrations on the FRET of RhB@CQDs. Herein, the concentration increase of RhB ranges from a to f (a–f: 0.25, 0.5, 0.75, 1, 1.25, and 1.5 μM), CQDs = 0.559 mg mL⁻¹; pH = 6.20; (D) the relationship between FL emission quenching rates of RhB@CQDs in the presence of 500 μM iron ions and pH values under the conditions; CQDs = 0.559 mg mL⁻¹ and RhB = 1.25 μM, where F₀ and F are the FL emission intensities of RhB@CQDs at 581 nm without and with Fe³⁺ ions, respectively.

Fe³⁺ ions in varying concentrations were introduced to the system of RhB@CQDs that contained the optimal concentrations of CQDs and RhB at pH = 6.2. Consequently, the emission peak at 581 nm was gradually quenched with the increase of the Fe³⁺ ion concentrations (Fig. 5A). The plot of emission intensity versus Fe³⁺ ion concentrations shows a linear relationship in a wide range with the correlation equation of F₀/F = 1.0939 + 0.00911 × C (Fe³⁺) (Fig. 5B). The limit of Fe³⁺ ions, based on 3σ/slope, was estimated to be about 30 nM.

Fig. 5 (A) The FL emission spectra changes of RhB@CQDs with Fe³⁺ concentrations, C (from top to bottom: 0, 25, 50, 75, 100, 200, 300, 400 and 500 μM); (B) the plot of F₀/F against the concentrations of Fe³⁺, C, under the conditions: CQDs = 0.559 mg mL⁻¹, RhB = 1.25 μM, pH = 6.20.

The selectivity of RhB@CQDs towards Fe³⁺ was also evaluated according to the effects several cations, including Fe³⁺, Zn²⁺, Pb²⁺, Ni²⁺, Na⁺, Mn²⁺, Mg²⁺, K⁺, Hg²⁺, Fe²⁺, Ca²⁺, Cu²⁺, Cd²⁺, Ba²⁺, and Al³⁺, on the emission response of RhB@CQDs as shown in Fig. 6. Almost all the metal ions had no effect on the emission peak at 581 nm of RhB@CQDs, except Fe²⁺ which could slightly quench the emission. It is noted that the higher quenching rate can only be obtained from the system of RhB@CQDs, even though the emission of CQDs and RhB molecules can also be quenched by Fe³⁺ cations (Fig. 6). Therefore, the RhB@CQDs sensor exhibited better performance than only CQDs or RhB molecules to serve as a sensor for the detection of Fe³⁺ ions.

Fig. 6 Histograms of F₀/F, where F₀ and F are the FL emission intensities of RhB@CQDs (black), CQDs (red) and RhB molecules (blue) in the absence and presence of 500 μM of different metal ions, respectively, at the excitation wavelength of 365 nm. The inset contains the photos of RhB@CQDs solutions (CQDs = 0.559 mg mL⁻¹, RhB = 1.25 μM, pH = 6.20) with and without metal ions (H₂O).

In addition, dozens of interfering cations with unknown concentrations and with known Fe³⁺ concentration were added into the detection system in order to further observe the selectivity of RhB@CQDs towards the Fe³⁺ ions in practical conditions. Results showed that the known amount of Fe³⁺ ions added and the Fe³⁺ value obtained from the above correlation equation had error range of only 3–5%.

4. Discussion

According to the FRET principle, the donor CQDs initially absorbed the energy of the incident light and then directly transferred the excited state energy to the nearby acceptor RhB molecules without emitting photons (Fig. 7A). The energy transfer resulted in the decrease or quenching of the fluorescence of CQDs, also accompanied by the enhancement in RhB fluorescence intensity. In addition to the overlapping emission and absorption spectra of the CQDs and RhB molecules, FRET also could occur between CQDs and RhB molecules that were separated by considerably larger distances than the sum of their van der Waals radii (1–10 nm)³¹ and was governed by the Förster mechanism. According to the Förster model,²⁸ FRET efficiency (E) can be defined as follows:where r is the separated distance between the CQDs and RhB, n is the average number of RhB molecules interacting with one carbon quantum dot, and R₀ is the Förster radius, which can be expressed as follows:²⁸where N_A, Q_D and n_D are Avogadro's number, the quantum yield of CQDs, and the refractive index of the medium, respectively. I is the spectral overlap integral. The quantum yield of CQDs (Q_D) is a constant, at the given incident light. However, n and I depend on the concentration ratio between CQDs and RhB molecules, thus causing the changes in the FRET efficiency. The optimal concentrations of CQDs and RhB molecules are provided in the abovementioned experimental results (Fig. 3B and C).

Fig. 7 Schematic illustration of the FRET process (A), and FL quenching mechanism of Fe³⁺ ions in RhB@CQDs (B) LUMO and HOMO represent the lowest unoccupied molecular orbital and highest occupied molecular orbital, respectively.

When Fe³⁺ ions were added to the RhB@CQDs system, they could be adsorbed on the surface groups of CQDs and RhB molecules through the Brownian movement. According to the above mentioned characterizations, CQDs with graphitic structure contained various types of oxidized carbon groups on their surface. RhB also presents O or N-contained groups in its molecular structure. Such active groups could specially combine with paramagnetic Fe³⁺ ions rather than other cations through coordinating or chelating interactions, which have been widely used for color reactions in traditional organic chemistry.^32–34 Therefore, the excitation energy of CQDs preferentially migrates to the nearer Fe³⁺ ions rather than the farther RhB molecules by the FRET process (Fig. 7B). Because Fe³⁺ ions have the propensity to deactivate the excited state electrons by relaxation,^32,33 they act as the quenching centers. Fe³⁺ ions diffuse in a random-walk manner. Those excited CQDs and RhB molecules near Fe³⁺ ions predominantly relax by direct energy migration; those more distant from Fe³⁺ ions, however, must first diffuse into the vicinity of CQDs and RhB molecules before relaxation occurs. On the basis of diffusion limited energy transfer, the rate of excitation density of CQDs can be expressed as follows:³⁵

In eqn (3), the first term on the right represents the diffusion rate of Fe³⁺ ions, where D is the diffusion constant; the second and third term represent the probability of energy transfer and the intrinsic decay probability of CQDs, respectively.

When the concentration of Fe³⁺ ions is low, only a small fraction of the total number of excited CQDs and RhB molecules are within the critical migration distance of Fe³⁺ ions. In this limit, the principle excited state energy of CQDs can be still transferred to the nearby RhB molecules. As the concentration of Fe³⁺ ions is increased, a larger fraction of CQDs and RhB molecules are surrounded by Fe³⁺ ions and within the critical interaction range of the energy migration. The FRET process is completely limited and the intrinsic FL emission of RhB molecules is affected due to the migration of their excited state energy to Fe³⁺ ions. This accounts for the quenching of fluorescence emissions by the Fe³⁺ ions from the RhB@CQDs system.

5. Application

To investigate the potential use of this sensor, an attempt was made to monitor the Fe³⁺ concentration on the surface of an iron sheet and on stainless steel for the process of metal corrosion. Iron and stainless steel sheets were polished with sandpaper until the surface of the metal was very smooth. They were then washed several times with distilled water and ethanol, and dried in a vacuum oven at 60 °C for 2 h. RhB@CQDs were added into aqueous solutions of polyvinyl alcohol (PVA) (5% w/v), that were prepared by dissolving PVA powders in deionized water and then used for obtaining the RhB@CQDs–PVA sol that was employed to coat on the prepared iron and stainless steel sheets.

Fig. 8 shows the images of RhB@CQDs–PVA films on iron (I) and stainless steel (II) sheets under the visible light and UV lamp. It can be seen that the emission intensity of the RhB@CQDs–PVA films on iron sheets reduces with time, under the UV lamp. While, this phenomenon does not take place on the stainless steel sheet. The above results could be attributed to the differences in the corrosion-resistant ability between iron and stainless steel in ambient conditions. Because these differences can be hardly distinguished by the naked eyes, our proposed RhB@CQDs sensor will have potential in the application of fast monitoring of the iron metal corrosion process, helping us avoid sudden failure of devices and equipments.

Fig. 8 Images of RhB@CQDs–PVA film on iron (I) and stainless steel (II) sheets with time (0, 15 and 30 min) under the visible light (A, C and E) and UV lamp (B, D and F).

6. Conclusions

CQDs that could detect Fe³⁺ ions were prepared by hydrothermal treatment of ethylene glycol and they were then mixed with RhB molecules. As an energy donor, CQDs can transfer the excited state energy to RhB acceptors to enhance the FL emission of RhB molecules. Fe³⁺ ions can be adsorbed on the surface of CQDs and RhB molecules. The excited state energy from CQDs and RhB molecules preferentially migrates to Fe³⁺ ions to relax, thus blocking the FRET process and quenches some intrinsic FL emission of RhB molecules. Therefore, the RhB@CQDs sensor combined the abilities of both CQDs and RhB towards Fe³⁺ detection. Both higher sensitivity and selectivity were obtained. The RhB@CQDs sensor could have potential in the application of fast monitoring of the corrosion process of iron devices and equipments.

Acknowledgements

We thank the financial support from the National Natural Science Foundation of China (no. 51272301, 51172214, 51172120), China Postdoctoral Science Foundation funded project (no. 2012M510788, 2013T60269), Shanxi Province Science Foundation for Youths (2014021008), 131 Talent Plan of Higher Learning Institutions of Shanxi, and State Key Laboratory of New Ceramic and Fine Processing Tsinghua University.

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Footnote

† These authors contributed equally.

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