Carbon dots preparation as a fluorescent sensing platform for highly efficient detection of Fe(III) ions in biological systems

Accepted Manuscript Carbon dots preparation as a fluorescent sensing platform for highly efficient detection of Fe (III) ions in biological systems Hamed Hamishehkar, Bahar Ghasemzadeh, Abdolhossein Naseri, Roya Salehi, Farzaneh Rasoulzadeh PII: DOI: Reference:

S1386-1425(15)00776-3 http://dx.doi.org/10.1016/j.saa.2015.06.061 SAA 13831

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received Date: Revised Date: Accepted Date:

7 December 2014 30 March 2015 18 June 2015

Please cite this article as: H. Hamishehkar, B. Ghasemzadeh, A. Naseri, R. Salehi, F. Rasoulzadeh, Carbon dots preparation as a fluorescent sensing platform for highly efficient detection of Fe (III) ions in biological systems, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/10.1016/j.saa. 2015.06.061

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Carbon dots preparation as a fluorescent sensing platform for highly efficient detection of Fe (III) ions in biological systems Hamed Hamishehkara, Bahar Ghasemzadehb, Abdolhossein Naserib, Roya Salehia, Farzaneh Rasoulzadeha* a

Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz 51644-14766,

Iran b

Departments of Analytical Chemistry, Faculty of Chemistry, University of Tabriz, Tabriz, 51666

16471, Iran ∗ Corresponding author. Tel.: +98 411 3363161; fax: +98 413 3363231. E-mail address: [email protected] (F. Rasoulzadeh).

Abstract

Water-soluble carbon dots (CDs) were prepared, using a facile hydrothermal oxidation route of cyclic oligosaccharide α-CD, as carbon sources, and alkali as additives. The successful synthesis of CDs was confirmed by scanning electron microscopy (SEM), dynamic light scattering (DLS), FTIR, UV-visible absorption, and emission fluorescence. The characterizations showed that the prepared CDs are spherical and well-dispersed in water with average diameters of approximately 2 nm. These water-soluble CDs have excellent photo stability towards photo bleaching during 30 days. The obtained CDs showed a strong emission at the wavelength of 450 nm, with an optimum excitation of 360 nm. The fluorescence quenching of CDs in the presence of Fe (III) ions was used as fluorescent probes for quantifying Fe (III) ions in aqueous solution. Under optimum condition, the fluorescence intensity versus Fe (III) concentration gave a linear response, according to Stern–Volmer equation. The linearity range of the calibration curve and the limit of detection were 1.60 × 10−5 to 16.6 × 10−5 mol L−1, and 6.05×10 −6 mol L−1, respectively which was in the range for serum analysis of Fe (III). It was concluded that the prepared CDs had a great potential as 1

fluorescent probes for applications in analysis of Fe (III) ions in the blood serum samples, which is hardly interfered by other ions.

Keywords:

Carbon dot, Fluorescent quenching, α-cyclodextrin ¸ Fe (III) ion, Blood serum.

Introduction Iron is the most abundant essential transition metal ion in the human body that plays an important role in biological and environmental systems. Iron is used in many proteins for oxygen transport, electron transport, and as a catalyst in oxido-reductase reactions [1, 2]. Deficiency of iron decreases oxygen delivery to cells, resulting in fatigue, poor work performance, and immunity [3]. Also, excess amounts of iron ions (Fe (III)) in a living cell can catalyze the production of reactive oxygen species (ROS) through Fenton reaction, which can damage lipids, nucleic acids, and proteins. The cellular toxicity of Fe (III) causes serious diseases, like Huntington’s, Alzheimer’s and Parkinson’s disease[4]. Therefore, clinical diagnostic of iron has importance in various therapeutic protocols. Several methods for detection of transition metal ions in various samples have been proposed, including voltammetry, atomic absorption spectrometry [5], inductively coupled plasma mass spectroscopy (ICPMS), and inductively coupled plasma atomic emission spectrometry (ICPAES). Among commonly used analytical methods, the fluorescence quenching has been one of the most successful methods employed to detect and quantify various metal ions [6, 7]. Semiconductor quantum dots (Q-dots) fabricated from lead, cadmium, and silicon as fluorescent probes are being extensively studied for optical sensing applications [8]. Such Qdots exhibited several amazing physico-chemical properties, such as, broad excitation spectrum, a narrow and tunable emission spectrum, good photochemical stability, and high 2

brightness [9, 10]. Unfortunately, the safety of Q-dots is of great concern due to their toxicity effect caused by potential leaching of precursors, such as, cadmium and/or other heavy metals from the Q-dots. This concern has significantly limited their practical applications. As such, alternative fluorescent nanomaterials, which exhibit comparable fluorescent properties as Q-dots, but of low toxicity, and low cost should be developed as fluorescent sensing probes for metal ions detection. There have been many reports and review articles on selective detection of heavy metals and transition metals using CDs as the sensing probe [13– 15]. CDs, as a new kind of fluorescent carbon nanomaterials, with the size below 10 nm have been synthesized by using carbon-based materials as carbon resources [8]. They are being successfully used, due to similar physicochemical properties of semiconductor Q-dots, but relatively low in cost, toxicity and interesting fluorescent properties [11-15]. They first obtained during purification of single-walled carbon nanotubes, through preparative electrophoresis in 2004 [16]. Several methods for fluorescent CDs synthesis have been reported by using carbon-based materials as carbon resources [16-18]. For example, laser ablation of carbon powder [17], electrochemical oxidation [19], arc discharge [20] microwave synthesis, combustion soots of candles [21, 22], thermal oxidation of carbon precursor by employing silica [23, 24] or zeolites as carriers [25, 26], commercial activated carbon [27], lampblack [28], and watermelon peel as carbon resources [29]. Most of these methods suffer from tedious processes, harsh synthetic conditions, or expensive starting materials. Cyclodextrins, which are produced from amylose fraction of starch by glucosyltransferases, are a series of cycli coligomers consisting of six, or more a-l,4-linked D-glucopyranose units [30]. Of all the cyclodextrins, the most abundant are α, β and γ- CD with six, seven, and eight glucopyranoses, respectively. They have a rigid conical molecular structure with a hydrophobic interior, and a hydrophilic exterior. The internal cavity can include a wide range of guest molecules, ranging from polar compounds, such as, alcohol, 3

acids, amines, and small inorganic anions to a polar. The driving force of the formation of inclusion complex comes from non-covalent interactions, such as, van der Waals forces, electronic effects, hydrophobic interactions, and steric factors [31]. Cyclodextrins have been widely used in the chemical, pharmaceutical or food industry, and in the catalysis of mimic enzymes. For this reason, the investigation on cyclodextrins is of remarkable significance. It was tried to develop simple, low cost and rapid synthesis method of CDs, and studied its potential application as fluorescence probes for the sensitive and selective detection of Fe (III) ions in aqueous solution. This is the first report on using α-CD, as precursor material for CDs synthesis and selectively detect Fe (III) ions in aqueous solution. This assay might provide a new pathway for the detection of Fe (III) ions, and some potential application in biological system and environment.

2. Experimental section 2.1 Materials and instrumentation α-CD, NaOH and Fe (NO3)3 were purchased from Sigma Aldrich (Poole, England). Dialysis bag was purchased from Genestar Biotechnology Co. Ltd. (Shanghai, China). The mean particle size, and distribution were measured by DLS (Nanotrac Wave, Microtrac Company) in n-hexane, and a MIRA3 FEG-SEM (Tescan Company) was used to measure the shape and size of the nanoparticles. UV–vis absorption spectra were measured on a Shimadzu 2550 UV–vis

spectrophotometer.

Fluorescence

spectra

were

recorded

on

a

FP-750

spectrofluorometer (Shimadzu, Japan). The width of both the excitation and emission slits was set at 10 nm. Fourier transform infrared spectra were performed, using a FTIR spectrometer (Shimadzu) with a KBr pellets. 2.2. Preparation of CDs The CDs were synthesized directly using α-CD aqueous solution, according to a modified 4

procedure as previously described [32]. Briefly, 1 g of α-CD and 0.2 g of sodium hydroxide were transferred to 25 ml of water with vigorous stirring. The mixture solution was heated at a constant temperature of 160 0C for at least 4 h in oil bath until the yellowish solution was obtained. Then, the solution was cooled at room temperature. Then, it was isolated by centrifuging at a speed of 10,000 rpm for 25 min to remove the deposit. The results were neutralized by 1 mol L-1 hydrochloric acid solution, and extensively dialyzed against distilled water through a dialysis membrane, with a molecular weight cutoff of 12,000 to remove salt and impurities. After drying, the sample was kept in a dark and cool place for further study. 2.3. Metal ions effects on fluorescence intensity Stock aqueous solutions of metal ions (Al3+, Fe3+, Sn2+, Ca2+, Cd2+, Hg2+, Co2+, Cu2+, Mg2+, Zn2+, Ni2+, Pb2+, Na+, K+ and Li+) were prepared in distilled water. To prepare 10 -4 mol L-1, or 10-6 mol L-1 of metal ions, an appropriate amount of standard metal ions solution was added to water, and the mixture was stirred at room temperature. The fluorescent intensity of mixed solution was measured with fluorescence spectrofluorometer, with the following settings of the spectrofluorometer: excitation wavelength (λex), 360 nm; excitation slit (EX), 10 nm; emission slit (EM), 10 nm. 2.4. Real samples To evaluate the capability of the proposed method in real sample analysis, it was easily applied to the determination of Fe (III) in blood plasma samples. To precipitate protein, 2 mL of human serum added to 10 mL of HClO4 acid. The mixture was centrifuged for 10 min to remove protein, and minimize nonspecific interactions of the CDs with proteins. Supernatant liquid was transferred to a fresh test tube, and then pH was adjusted to ∼3, and the resulting solution was spiked with different amounts of Fe (III). The solution was then analyzed, according to the given procedure. The obtained results are given in Table 2. 3. Results and discussion 5

3.1. Characterization of CDs 3.1.1. Morphological study The CDs aqueous solution is yellowish, transparent, and clear under daylight. Different spectroscopic and analysis methods were used to characterize the prepared CDs. SEM was performed to confirm CDs extraction, and to investigate their morphology. The CDs prepared by this method from α-CD are spherical and well-dispersed (Fig. 1a). The size distribution histograms by DLS suggest that the particle size of CDs is in the range of 1–5 nm (Fig. 1b).

a b

Fig. 1. a) SEM micrographs and b) Particle size distribution of CDs.

3.1.2. Spectroscopic measurements The UV–vis spectra of the CDs, in water are shown in Fig. 2. The CDs exhibit UV–vis absorption from 200 nm to 600 nm, showing a maximum peak at 260 nm. Fig. 3 shows the fluorescence spectra of the CDs under the excitation of different excitation wavelengths. With increasing the excitation wavelength, the CDs emit at longer wavelength, showing similar luminescent property with the CDs from glucose and sucrose. So, the results show that the CDs exhibit an excitation-dependent emission phenomenon [16, 17, 26, 33]. Further, the emission intensity shows the highest value at 450 nm for CDs under the excitation of 360 6

nm. Therefore, 360 nm was selected as the excitation wavelength for the following experiments. The quantum confinement of the CDs surface energy trap is thought to be the reason for the strong emission. The exact mechanism of the CDs fluorescence is not completely clear and further efforts are needed to explain the phenomenon.

Fig.2. UV–vis absorption spectra 0.5 ml of the CDs in water.

Ex 360 Ex 340 Ex 320 Ex 300

Fig.3. Emission spectra of the CDs at different excitation wavelengths.

7

Further, the comparison of fluorescence spectra was depicted after 30 days. The fluorescent intensity of CDs remains unchanged for nearly a month, suggesting the stable fluorescent properties of CDs (Fig. 4). 2 days 30 days

Fig. 4. The stability of CDs with the comparison of fluorescence spectra after 2 and 30 days.

FT-IR spectra in Fig. 5 show broad and intense peaks around 3,410 cm-1 correspond to the OH stretching vibration, indicating the existence of large numbers of hydroxyl groups. The peaks at around 1670–1690 cm-1 are attributed to carboxyl groups [29]. Another broad peak with increased intensity at 1400 cm-1 indicates the existence of C–H bonds, while the strongest peak ranged between 1200–1000 cm-1 is due to the C–O stretching vibration. The existence of carboxyl groups on the CDs surface offers good water solubility, and provide potential platform for various surface modification [34].

8

Fig.5. FT-IR spectra of CDs.

3.2. Influence of pH on fluorescence of CDs The CDs were dispersed in aqueous solution, and the effect of pH as one of the major variables altering the intensity of fluorescent species was evaluated. The effect of pH in a range between 2.00 and 11.00 was studied to select the optimum conditions for next experiments.

a

b

Fig. 6. Effect of pH a) on the fluorescence intensity of CDs only, and b) Degree of quenching of CDs in the presence of Fe (III).

9

As shown in Fig. 6 (a), pH influences the fluorescence intensity of CDs. The results showed that the fluorescence intensity of CDs is higher at neutral solutions, while it decreases upon changing to acidic, or basic solutions. At lower pH, decreased fluorescence intensities could be due to the formation of hydrogen bonding between carboxylic acid moieties on the surface of CDs. Besides exchange of various ligands on the surface of CDs with the highly concentrated protons cause the particles aggregation that make to decrease fluorescence intensities [35].

Also, at higher pH, the lower solubility of CDs and deprotonation of

carboxylic groups would occur that lead to buildup negative charges on the surface of CDs [36]. The presence of large bundles of COO-group at the surface of CDs would result in the formation of negatively charged double layers. Such double layers would interfere with the fluorescence origin, especially in the promotion of photo-induced electron transfer (PET) mechanism during which electrons from the double layers could relax back to the ground state of CDs by replacing the initial relaxation process that gave rise to fluorescence [37]. Further, to study the effect of pH to alteration the detection performance of the CDs towards the Fe (III) ions, an additional test has been done by studying the quenching of the CDs with Fe (III) ions of the same concentration under different pH conditions (Fig. 6(b)). The results showed high degree of quenching in the acidic region of pH=2.3, but the degree of quenching dropped by increasing the pH from 2.30 to 9.23. This trend shows the increment of the solubility of Fe (III) ions under hydroxide rich solution. As the pH is recorded to be higher, more hydroxide ions will be available to form complex with the Fe (III)) ions causing less interaction with the CDs. However, at higher pH, the solubility of the Fe (III) hydroxide increases, due to the formation of charged complex, and can promote quenching of CDs. Although the sensitivity can be improved at pH=2.3, it is not practical to adopt this condition

10

in real systems. Thus, the pH has been controlled at the neutral range about 7 for the subsequent studies [38]. 3.3. Metal ions effects on fluorescence intensity To assess the selectivity of the proposed method, the effect of several metal ions (Cu 2+, Al3+, Zn2+, Ca2+, Mg2+, Ni+, Co 2+, Pb2+, Cd 2+, Hg2+, Li+, Sn2+, Na+ and K+ ) in two levels of concentration (10 -4 and 10-6 M) on emission intensity of CDs were investigated. A significant quenching effect compared to other ions was observed for Fe (III) ions, indicating CDs could potentially be used for the detection of Fe (III) ions with high sensitivity (Fig. 7).

Fig. 7. Effect of types of interfering ions on fluorescence intensity of CDs in water.

It is speculated that the observed higher sensitivity of CDs towards Fe (III) ions could be due to their high number of valence electrons, which causes stronger interaction with the CDs, and induced effective quenching. The proposed mechanism is based on the complex formation of metal ions on the CDs surface that disturbs the initial energy transition emitting fluorescence. In this case, Fe (III) ions are considerably good oxidizing agents with their oxidation number can be converted to +2. Thus, it is speculated that the complex formation of ions on the CDs surfaces can promote electron transfer quenching during the oxidation 11

process of Fe ions from +3 to +2 states. Potential interfering ions were found to be very poor quenchers for CDs in water.

3.4. Effect of Fe (III) on the spectra of the CDs Fig. 8(a) shows the emission spectra of CDs at different Fe (III) ions concentrations. It was observed that the fluorescence intensity of CDs gradually decreased in the presence of amounts of Fe (III) (1.66 to 16.6) × 10 -5 M. To consider the significant quenching of fluorescence intensity, the possibility of developing sensitive methods for Fe (III) based on spectrofluorometry has been evaluated. In this study, the CDs were observed with no significant shift in the emission peak that centered around 450 nm, which implies most possible mechanism is attributed to electron transfer. Experimental data were fitted to the Stern-Volmer relationship as shown in Eq. (1) (Fig. 8(b)).

F0 F

= 1+ K sv [Q] = 1+ k q τ0 [Q]

Eq (1)

Fig. 8. (a) Emission spectra of CDs in the presence of Fe (III) (from up to down, the concentration of Fe (III) is 0, 1.66, 3.33,…16.6µM, respectively b) The Stern–Volmer plots for the quenching of CDs by Fe(III) at 298K

12

Where, Fo and F are the fluorescence intensities recorded in absence and presence of Fe (III) ions, respectively, [Q] is the concentration of Fe (III) ions and Ksv is the Stern-Volmer quenching constant. Linear relationship, based on the Stern-Volmer equation, was obtained for Fe (III) ions concentration at lower concentration. Under optimum conditions, calculation was made based on 3σ/s (σ is the Standard Deviation of the corrected blank signals of the CDs (n=11), and s is the slope of the calibration curve). It could be seen that the CDs lowest LOD for Fe (III) ions can be achieved to 6.05 µM implying the CDs can be used as a new kind of Fe (III) ions sensing probe, with practically feasibility for Fe (III) detection in real Samples. Table 1 compares the analytical characteristics of the proposed method with those of previously published analytical methods to determine Fe (III). Concerning LOD, the findings are completely competitive to other results, indicative the proposed method has a good sensitivity and extractability. Table.1. Analytical characteristics of the proposed method and comparison with some other analytical methods.

Method Spectroflourimetric

LOD (µM) 6.05

Reference This work

mass spectrometry

10

[37]

Spectrophotometric

1.42

[38]

Spectrophotometric

6.6

[39]

Quenching of the fluorescence may occur by energy transfer [39], charge diverting [40], surface absorption [41], the quenching mechanisms can be classified as dynamic quenching, and static quenching [42]. The results showed that within the range of the investigated concentrations (0 –16.6) × 10-5 M, at higher concentration, there is a deviation towards the yaxis, clearly indicating both dynamic quenching and static quenching is predominant.

13

3.5. Analysis of real samples A standard addition method was applied for the detection of Fe (III) in blood serum samples. A series of mixtures of the diluted supernatants (25 µL) and CDs (500 µL) were spiked with the standard solution of Fe (III) (0–100 µM) that had been diluted in double-distilled water, where double-distilled water was added to make their final volumes of 3 mL. Recovery experiments were performed to evaluate the accuracy of this method. The obtained results were summarized in Table 2. Statistical analysis of these results using Student t-test showed that there are no significant differences between added and analyzed values. Table.2. Results for the determination of Fe (III) in real samples.

a b

Sample

Added (µg L−1)

Blood serum

0.00 3.33 8.33 16.66 33.33

Found (µg L−1) a 52.00±1.76 54.30±1.30 61.10±2.31 69.79±2.20 83.53±1.69

Recovery (%)

t-Statistic b

98.12 101.27 101.64 97.88

2.10 0.77 1.04 1.80

Mean of three determinations ± standard deviation. t-critical=3.18 for n=3

4. Conclusions A novel and convenient technique for Fe (III) analysis has been developed, based on the quenching of the fluorescence of CDs. The preparation is simple and cheap. Under optimum conditions, CDs can be used to detect Fe (III) ions with good selectivity and sensitivity in an aqueous solution. Also, it is highly selective and hardly interfered by other metal ions, upon excitation at 360 nm in aqueous solution. A good linear relationship between fluorescence intensity of the system, and concentration of Fe (III) in the range from (0 –16.6) × 10-5 mol L−1 can be achieved. This technique indicates that CDs could be selectively applied to detect of Fe (III) ions in the biological samples of which may contain the residual Fe (III). 14

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Graphical abstract In the present study, Carbon dots have been prepared and used as a fluorescent sensing platform

Fluorescence intensity(a.u)

for highly efficient detection of Fe (III) ions in biological systems.

380

SEM micrographs of CDs

430

480λ (nm)530

580

Emission spectra of CDs in the presence of Fe (III)

Highlights

•

Simple and cheap method for preparation of CDs from α-cyclodextrin.

•

High sensitivity and selectivity of CDs for the detection of Fe (III) ions in the present of interfering ions.

•

A good linear relationship between fluorescence intensity of the system and concentration of Fe (III).

•

Great potential as fluorescent probes for the applications in analysis of Fe (III) ions in the blood serum.

24