One-pot synthesis of carbon dot-entrenched chitosan-modified magnetic nanoparticles for fluorescence-based Cu²⁺ ion sensing and cell imaging

Abstract

In this work, a new synthetic approach is developed for the synthesis of fluorescent magnetic nanoparticles which are explored for the detection of mostly abundant transition metal Cu²⁺ ions and cell imaging. These fluorescent magnetic nanoparticles are synthesized by decoration of carbon dots (CDs) on carboxymethyl chitosan-wrapped Fe₃O₄ nanoparticles (NPs) in a one-pot method. The fluorescent magnetic nanoparticles are characterized by Fourier transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), a vibrating sample magnetometer (VSM), field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), dynamic light scattering (DLS) and photoluminescence study. Importantly, the fluorescent magnetic nanoparticles exhibit excellent selectivity for the determination of Cu²⁺ ions over other metal ions. The fluorescence intensity was found to be successfully quenched by adding different concentrations of Cu²⁺ ions. Here, the reduction of fluorescence intensity proves the detection of Cu²⁺ ions in a linear range of 0.01–200 µM, with a detection limit of 0.56 µM at a signal-to-noise ratio of 3. The fluorescent magnetic nanoparticles were successfully applied to cell imaging and subsequently conjugated with folic acid for cancer cell imaging, which suggests that the synthesized nanoparticles have great potential for diagnostic purposes.

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1. Introduction

Magnetic nanoparticles have been widely investigated due to their important role in science and technology. The unique feature of magnetic nanoparticles to be guided by an external magnetic field has been widely used in biology and medicine for imaging,¹ drug delivery,² hyperthermia,³ and bio-⁴ and chemosensing.⁵ To improve the efficacy and usefulness of magnetic nanoparticles in diverse applications like bioimaging, diagnostics, therapeutics, and biosensing, integrating magnetic properties and fluorescence into one entity is of great interest.^6,7 A large number of recent articles have addressed different methods for the synthesis of fluorescent magnetic nanoparticles.^8–10 Recently, Chekina et al. developed fluorescent magnetic nanoparticles for optical and magnetic resonance imaging using γ-Fe₂O₃ nanoparticles coated with silica or carboxymethyl chitosan and labelled with fluorescein isothiocyanate.¹¹ Wang et al. developed magnetic-fluorescent composite nanoparticles for stem cell labelling.¹² Mahmoudi et al. prepared gold-coated iron oxide nanoparticles by trapping fluorescent polymeric dyes for molecular diagnostics, imaging and therapeutic applications.¹³ Yan et al. reported the synthesis of self-assembled magnetic fluorescent polymeric micelles for magnetic resonance and optical imaging.¹⁴ Besides this, some review papers are also focused on the importance of fluorescent magnetic nanoparticles for imaging.^15–17 However, the reported synthetic procedure of fluorescent magnetic nanoparticles is not easy, because it is associated with a multi-step synthetic route. Development of simple methods for synthesizing fluorescent magnetic nanoparticles in a one-pot route is still a great challenge. Here we have designed a facile way to synthesize fluorescent magnetic nanoparticles for imaging and sensing.

Recently, carbon dots have attracted enormous research interest due to their excellent fluorescence properties.¹⁸ Fluorescent carbon dots are more superior to semiconductor quantum dots and traditional organic dyes in terms of water solubility, low toxicity and good biocompatibility. Because of these attractive merits, CDs have been widely used in biological labelling,¹⁹ imaging,¹⁹ optical sensing,²⁰ fluorescent biosensors²¹ and optoelectronic devices²² etc. Recently, major attention has been focused on developing green methods for the preparation of carbon dots using natural sources.²³ Until now, many natural materials are used as a carbon source for the synthesis of CDs.^24,25 Chitosan is the second most naturally abundant polysaccharide possessing very attractive features, such as a non-toxic nature, high biocompatibility, biodegradability and cationic properties. Recently, researchers have synthesized carbon dots form chitosan for diverse applications. Chowdhury et al. synthesized fluorescent carbon dots obtained from chitosan gel.²⁶ Gogoi et al. introduced chitosan-based carbon dots for the detection of heavy metal ions.²⁷ Xiao et al. reported the synthesis of amino-functionalized fluorescent carbon nitride dots from chitosan.²⁸ We have also reported carbon dot-embedded chitosan nanoparticles for cell imaging and drug delivery applications.²⁹ In this work, we develop a green, simple, and low cost preparative strategy toward magnetic fluorescent nanoparticles from chitosan.

Copper ions (Cu²⁺) are considered as the third most abundant transition metal ion in the human body. Cu²⁺ ions play a crucial role in the electron transfer processes of many biological reactions. However, either deficiency or excess of copper will cause some diseases in human health, such as haematological manifestations, neurological problems, gastrointestinal disturbance and damage of the liver and kidneys. Therefore, it is an important issue to detect Cu²⁺ ions for the early diagnosis of these diseases. As reported in the previous literature, many techniques like atomic absorption/emission spectroscopy, inductively coupled plasma mass spectrometry, electrochemical methods and fluorometric methods have been developed. Among all these methods, fluorometric methods are mostly used due to the less complicated operational procedures, cheap instrumentation and the need for lower sample volumes. Zhao et al. developed nitrogen-doped carbon dots for label-free detection of Cu²⁺ ions.³⁰ Zong et al. used carbon dots as a fluorescent probe for off–on detection of Cu²⁺ in aqueous solution.³¹ Now, fluorescent magnetic nanoparticles are designed for the simultaneous removal and optical determination of copper ions. Liu and coworkers successfully developed europium(III) complex-functionalized magnetic nanoparticles as a chemosensor for ultra-sensitive detection and removal of copper(II) from aqueous solution.³² Recently Xie et al. reported chitosan–Fe₃O₄@CdSeS nanoparticles for simultaneous removal and optical determination of trace copper ions.³³ However, problems were involved in the above-mentioned systems including tedious multi-step processes and expensive chemicals. As far as we know, there is no report on the fabrication of carbon dots on the surface of polymer-coated magnetic nanoparticles that not only act as a copper ion sensor and separator but also serve as a bioimaging probe without complicated surface modification.

Here, we have developed newly engineered magnetic fluorescent nanoparticles by encapsulating CDs on chitosan-modified Fe₃O₄ nanoparticles in a one-pot synthesis. More importantly, the synthesized magnetic fluorescent nanoparticles can be easily separated by an external magnetic field using a small magnet. As a proof of concept, here carbon dot-embedded fluorescent magnetic nanoparticles were successfully used for the detection and separation of Cu²⁺ ions as well as for cell imaging.

2. Experimental section

2.1. Materials

Anhydrous ferric chloride (FeCl₃), ferrous sulphate (FeSO₄·7H₂O), ammonia solution (25–30%), sodium hydroxide (NaOH), monochloroacetic acid (ClCH₂COOH), isopropanol (C₃H₇OH), copper sulphate (CuSO₄) and ethanol (C₂H₅OH) were purchased from Merck, India. N-Hydroxy succinamide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were purchased from Spectrochem. Chitosan was procured from Sigma Aldrich and folic acid (FA) was obtained from TCI. The human cervix adenocarcinoma (HeLa) cell line was obtained from National Institute of Cell Science (NCCS), Pune, India. Cells were grown in DMEM medium with 10% fetal bovine serum (FBS), and penicillin/streptomycin (100 units per mL) at 37 °C and 5% CO₂. All the in vitro experiments were performed at 37 °C and at a cell density allowing exponential growth. Milli-Q water was used for all the experiments.

2.2. Preparation of O-carboxymethyl chitosan (OCMC)

1 g of chitosan was immersed in 25 mL of NaOH (50 wt%) solutions to swell and alkalise for 24 h.²⁹ Then, the swollen chitosan was washed with isopropanol. After that, 1.5 g of monochloroacetic acid was dissolved in 5 mL of isopropanol and added drop-wise into the above solution for 30 min and the reaction was continued for 12 h at the 35 °C. The product was washed with ethanol until the extra base was removed. Finally the product was vacuum dried at 50 °C for 12 h.

2.3. Preparation of carbon dot-embedded fluorescent magnetic nanoparticles (Fe₃O₄@OCMC@CDs)

To prepare Fe₃O₄@OCMC@CDs, firstly superparamagnetic Fe₃O₄ nanoparticles were prepared by our previously reported procedure.³⁴ In brief, 0.324 g of FeCl₃ and 0.274 g of FeSO₄·7H₂O were taken in 40 mL Millipore water under argon atmosphere. Aqueous ammonia solution (2.5 M) was added into the reaction vessel with violent stirring. The reaction was continued for 1 h at 80 °C for complete growth of the nanoparticles. After that, 500 mg of OCMC was dissolved in 50 mL of Millipore water and added drop-wise into the Fe₃O₄ reaction system and the reaction was continued for 12 h at room temperature for the preparation of OCMC-coated Fe₃O₄ nanoparticles (Fe₃O₄@OCMC). The solution temperature was enhanced to 140 °C with continuous stirring for 6 h to carbonise the OCMC. The highly fluorescent carbon dots were prepared from the free OCMC and attached to Fe₃O₄@OCMC. The carbonisation of OCMC and the formation of Fe₃O₄@OCMC@CDs were optimised at four different temperatures (120 °C, 140 °C, 160 °C, and 180 °C). The final product was magnetically separated and washed with Millipore water to remove unreacted OCMC and excess base and dried in a vacuum oven at 50 °C for 12 h.

2.4. Preparation of folic acid-conjugated Fe₃O₄@OCMC@CDs (Fe₃O₄@OCMC@CDs–FA)

Folic acid-conjugated Fe₃O₄@OCMC@CDs was prepared by activation of FA followed by addition of Fe₃O₄@OCMC@CDs via EDC/NHS chemistry according to the previously reported procedure with a slight modification. In brief, 5 mg of FA was dissolved in 10 mL of DMSO–Milli-Q water mixture (1 : 1 v/v). To the above FA solution, EDC (5 mg) and NHS (5 mg) were added. The FA activation was carried out for 4 h under dark conditions at room temperature. After that, 50 mg of an aqueous dispersion of Fe₃O₄@OCMC@CDs was added drop-wise to the activated FA solution, and the resulting mixture was stirred overnight in the dark at room temperature. Finally the FA-conjugated Fe₃O₄@OCMC@CDs was magnetically separated, washed with DMSO and water several times, and finally recovered by a magnetic concentrator.

2.5. Intracellular uptake by fluorescence microscopy studies

Cell uptake studies were carried out on two different cancer cell lines viz. cervical cancer cells (HeLa) and normal cells (NIH3T3). For this study, cells were grown on a cover slip for 24 h. After 70–80% confluence, the cells were treated with 15 µg mL⁻¹ nanoparticles for different time intervals. Then, the cover slips were washed with PBS twice to remove excess NPs and finally mounted on glass slides and imaged under green filter (Leica, Wetzlar, Germany).

2.6. Fluorescence detection of Cu²⁺ ions

The sensing of Cu²⁺ ions was performed at room temperature in Millipore water. In a typical experiment, the desired amount of Cu²⁺ ions (concentration varied from 0–200 µM) was added to 2 mL of water containing 1 mg of Fe₃O₄@OCMC@CDs. The fluorescence emission spectra were recorded after reaction for 15 min under excitation at 360 nm. The selectivity of Cu²⁺ ions was confirmed by adding other metal ion stock solutions instead of Cu²⁺ ions in a similar way. The entire experiment was executed at room temperature.

2.7. Characterisation

The functional groups of all the synthesised nanoparticles were investigated by FTIR (Agilent carry 660 instrument). The crystallographic structure of these nanoparticles was determined using powder XRD (Phillips PW 1710 X-ray diffractometer) with Cu Kα radiation. The morphological structure and the size of the Fe₃O₄@OCMC@CDs nanoparticles were determined using field emission scanning electron microscopy (FESEM, Joel JSM-6304F) and transmission electron microscopy (TEM, Phillips CM 200). The hydrodynamic size of the nanoparticles was measured by dynamic light scattering (DLS) techniques, using Malvern Instruments, UK. The characteristic emission spectra of the fluorescent magnetic nanoparticles at different excitations and the Cu²⁺ ion sensing were recorded using a Perkin Elmer LS 55 fluorescence spectrometer. The fluorescence images were captured by a fluorescence microscope (Leica, Wetzlar, Germany) for the detection of the fluorescence intensity in normal and cancer cell lines (excitation and emission wavelengths were 360 nm and 450 nm respectively).

3. Results and discussion

In recent years fluorescent magnetic nanoparticles have drawn considerable attention in the research area of drug delivery, diagnosis and detection. Most representatively, either small-molecule organic fluorescent dyes or inorganic quantum dots were used to synthesise fluorescent magnetic nanoparticles. Recently Zhang et al. developed CdTe@GdS fluorescent magnetic nanoparticles for tumour-targeted dual-modal imaging.³⁵ Rosa-Romo et al. synthesized flavone-functionalized magnetic nanoparticles for Cu²⁺ ion detection.³⁶ Rittikulsittichai et al. reported the synthesis of multi-functional magnetic-fluorescent composites for bioimaging and magnetic hyperthermia therapy.³⁷ All these reported fluorescent magnetic nanoparticles hold great promise in diverse applications, but most of these methods suffer from either a complex synthetic procedure or time-consuming functionalization. The objective of this work is a simple, rapid, cost-effective, and green synthetic approach for fluorescent magnetic nanoparticles. Here a synthetic strategy is developed for highly fluorescent magnetic nanoparticles with Fe₃O₄ nanocrystals in the core and fluorescent carbon dots deposited in the shell of OCMC in a one-pot method. In this work, the synthesized fluorescent magnetic nanoparticles were employed for Cu²⁺ ion sensing and cell imaging. To extend the applications, folic acid is conjugated on the surface of the fluorescent magnetic nanoparticles for cancer cell targeting. The synthesized fluorescent magnetic nanoparticles can easily separate from the solution by magnetic separation. For a comparison study, the individual syntheses of Fe₃O₄ and the OCMC-coated Fe₃O₄ nanoparticles are elaborated in the ESI.†

3.1. FTIR analysis

The characteristic surface functional groups of the pure Fe₃O₄, Fe₃O₄@OCMC, Fe₃O₄@OCMC@CDs and Fe₃O₄@OCMC@CDs–FA were investigated by the FTIR study as shown in Fig. 1. The Fe₃O₄-only magnetic nanoparticles exhibited a strong band at 647 cm⁻¹ due to characteristic Fe–O vibrations and a broad band around 3443 cm⁻¹, which was indicative of the presence of –OH groups on the nanoparticle surface.³⁸ The FTIR spectrum of Fe₃O₄@OCMC shows a broad band at 3226 cm⁻¹ due to the stretching vibrations of amine and hydroxyl groups. Peaks at 1374 cm⁻¹ and 1606 cm⁻¹ correspond to the –C–O– stretching vibrations of the carbonyl group and N–H bending vibration of the amine group, as shown in Fig. 1.³⁸ After the formation of carbon dots, the FTIR peaks of Fe₃O₄@OCMC@CDs remain the same as those of the Fe₃O₄@OCMC nanoparticles. From FTIR analysis, it is difficult to confirm the formation of CDs on the surface of the Fe₃O₄@OCMC nanoparticles. The characteristic band at around 1400 cm⁻¹ corresponds to the phenyl ring of folic acid and the other bands are also identified in the spectrum of Fe₃O₄@OCMC@CDs–FA, confirming the successful attachment of folic acid on the Fe₃O₄@OCMC@CDs surface.

Fig. 1 FTIR spectra of Fe₃O₄, Fe₃O₄@OCMC, Fe₃O₄@OCMC@CDs, Fe₃O₄@OCMC@CDs–FA and free folic acid.

3.2. XRD analysis

The phase purity and crystallinity of the pure Fe₃O₄ nanoparticles and Fe₃O₄@OCMC@CDs nanoparticles were determined by high resolution powder X-ray diffraction analysis, as shown in Fig. 2a. Fe₃O₄ shows characteristic XRD peaks indexed at different positions which correspond to the reflection plane indices.³⁹ The position of all peaks and corresponding reflection plane indices match well with the standard JCPDS pattern (card no. 85-1436). The results indicate that after the formation of the shell by the OCMC upon the Fe₃O₄ nanoparticles and the carbon dots inserting at 140 °C, there is no change of the crystalline phase of the magnetic nanoparticles. The appearance of effective broad peaks in both the Fe₃O₄ and Fe₃O₄@OCMC@CDs nanoparticles indicate that the samples are nanocrystalline in nature.

Fig. 2 (a) X-ray diffraction pattern of Fe₃O₄ and Fe₃O₄@OCMC@CDs, and (b) the magnetization as a function of applied field for Fe₃O₄ and Fe₃O₄@OCMC@CDs.

3.3. Magnetization study

The superparamagnetic property of the magnetic nanoparticles is important to ensure their application in the biomedical and bioseparation fields. The magnetization versus magnetic field curves were obtained at room temperature for both the bare Fe₃O₄ and Fe₃O₄@OCMC@CDs nanoparticles and are illustrated in Fig. 2b, using a vibration sample magnetometer (VSM). The magnetization curves of both magnetic nanoparticles show that there is zero coercivity and remenance, suggesting that the nanoparticles are superparamagnetic in nature. The saturation magnetization value of the as-synthesized bare Fe₃O₄ and Fe₃O₄@OCMC@CDs is 75.46 emu g⁻¹ and 56.87 emu g⁻¹ respectively. The difference in the magnetization value between the Fe₃O₄ nanoparticles and Fe₃O₄@OCMC@CDs nanoparticles can be attributed to the non-magnetic coating of OCMC and amorphous CDs, which can reduce the magnetization.

3.4. Surface morphology study

The actual shape and size of these nanoparticles were determined by FESEM analysis. Bare Fe₃O₄ nanoparticles are spherical in shape with a diameter ∼ 30 nm, as shown in Fig. 3a. After coating OCMC on the surface of the Fe₃O₄ nanoparticles, the particle size is increasing as illustrated Fig. 3b. So, a higher particle size and smooth morphology of the surface confirms the OCMC modification of the Fe₃O₄ NPs. When the temperature was increased to 140 °C, the carbon dots were formed in situ from the unreacted carboxymethyl chitosan and attached to the surface of the Fe₃O₄@OCMC indicated by yellow arrow, as illustrated in Fig. 3c and d. At a temperature of 140 °C, greater amounts of CDs were formed and deposited on the surface of the Fe₃O₄@OCMC nanoparticles.

Fig. 3 FESEM image of (a) Fe₃O₄, (b) Fe₃O₄@OCMC, (c) Fe₃O₄@OCMC@CDs at 120 °C, and (d) Fe₃O₄@OCMC@CDs at 140 °C.

3.5. TEM and DLS analysis

Again the microstructures of the as-synthesized Fe₃O₄, Fe₃O₄@OCMC and Fe₃O₄@OCMC@CDs particles were examined by transmission electron microscopy, as shown in Fig. 4. Fig. 4a represents the micrograph of bare Fe₃O₄ nanoparticles which were aggregated due to high magnetization. Fig. 4b displays the TEM images of the OCMC-modified Fe₃O₄ NPs. The TEM observation indicates that there is occurrence of monolayer coverage of OCMC on the surface of the Fe₃O₄ NPs. It is confirmed that the OCMC layer could be successfully formed leading to formation of core–shell NPs. The thickness of the polymeric shell is about 6–8 nm. After the formation of Fe₃O₄@OCMC, when the temperature of the reaction vessel is increased to 140 °C, the 5–10 nm carbon dots are decorated upon the Fe₃O₄ nanoparticles, as shown in Fig. 4c (indicated by arrows).

Fig. 4 TEM images of (a) Fe₃O₄, (b) Fe₃O₄@OCMC, and (c) Fe₃O₄@OCMC@CDs and the corresponding DLS spectra respectively (d–f).

The average hydrodynamic diameter of the individually synthesized nanoparticles and the one-pot synthesized CD-embedded magnetic nanoparticles was determined by DLS measurements as shown in Fig. 4d–f. The obtained results reveal that the bare Fe₃O₄ nanoparticles have a hydrodynamic size of around 1500 nm in aqueous medium. This may be due to the high magnetization and agglomeration. When the particles are stabilised by OCMC, the magnetization is decreased and the hydrodynamic size is reduced to less than 500 nm. After formation of carbon dots from the OCMC, the hydrodynamic size slightly increases which may be due to the carbonization or burning of the coated OCMC. Actually the DLS study was carried out in the wet state of the nanoparticles in water medium and the TEM study was in the dried form of the nanoparticles. So, the size distribution acquired from the DLS study is higher than that from the TEM study.

3.6. Photoluminescence study

Photoluminescence (PL) spectra of Fe₃O₄@OCMC@CDs were recorded with different excitations as shown Fig. 5a. The results show that the Fe₃O₄@OCMC@CDs exhibit excitation-dependent sharp emission spectra. From the emission spectra of Fe₃O₄@OCMC@CDs, it is observed that Fe₃O₄@OCMC@CDs possesses a sharp emission peak centered at 450 nm, when Fe₃O₄@OCMC@CDs was excited at 340 nm. The PL intensity of the synthesised Fe₃O₄@OCMC@CDs varied with the variation of excitation wavelength from 300 nm to 460 nm and the maximum emission spectrum was obtained at the excitation wavelength of 340 nm. Bright blue luminescence of Fe₃O₄@OCMC@CDs was observed under UV (365 nm) light in aqueous solution, as shown in Fig. 5. The excitation wavelength-dependent PL properties of the synthesised Fe₃O₄@OCMC@CDs are probably due to different emissive spectra and different sizes of the generated carbon dots on the surface of the composite.⁴⁰ For the optimization of fluorescence intensity of Fe₃O₄@OCMC@CDs, different temperatures were used to synthesize Fe₃O₄@OCMC@CDs, as shown in Fig. 5b. A high fluorescence intensity of Fe₃O₄@OCMC@CDs was observed at the synthesis temperature of 140 °C and the fluorescence property of Fe₃O₄@OCMC@CDs was decreased with further increasing the synthesis temperature. The florescence intensity is small at a lower temperature than 140 °C because OCMC is not fully carbonised and again at higher than 140 °C the as-synthesized carbon dots are agglomerated, resulting in the decrease in florescence intensity.

Fig. 5 (a) Photoluminescence spectra of the one-pot synthesized Fe₃O₄@OCMC@CDs at different excitations and (b) optimisation of fluorescence at four different temperatures (120 °C, 140 °C, 160 °C, and 180 °C).

3.7. Analytical performance for Cu²⁺ sensing

To evaluate the selectivity of the developed Fe₃O₄@OCMC@CDs toward different metal ions, a fluorescence screening experiment was carried out. Fig. 6a shows the histogram of PL response in the presence of 1 mM Cu²⁺, Al³⁺, Ca²⁺, Mg²⁺, Ni²⁺, Co²⁺, Pb²⁺, Cd²⁺, Hg²⁺, Sn²⁺, Na⁺ and K⁺ metal ion solutions. The histogram is plotted with F/F₀ against the same concentration of different metal ion solutions, where F and F₀ indicate the PL intensity at 340 nm excitation in the presence of different metal ions and the PL intensity in the absence of metal ions respectively. This plot shows that the F/F₀ ratio is small in the presence of Cu²⁺ ion solution in comparison to that with other metal ions, indicating that the synthesized sensor is highly selective for Cu²⁺ ions. The high selectivity and specificity toward Cu²⁺ ions is due to the high binding affinity between Cu²⁺ ions and the presence of amine groups on the surface of CDs compared to other metal ions. The nitrogen atom of the amine group donates an electron to a copper ion and makes surface complexation.⁴¹ The availability of a lone pair of electrons on the amino group shows its affinity to make a complex with copper ions through electron pair sharing or Lewis acid–base pairs and reduces the florescence intensity of Fe₃O₄@OCMC@CDs.

Fig. 6 Fluorescence quenching performance of Fe₃O₄@OCMC@CDs (a) in the presence of different metal ions, and (b) in the presence of different amounts of Cu²⁺ ions. (c) Plot of F₀/F − 1 with different concentrations of Cu²⁺ ions. (d) Digital photograph image of Fe₃O₄@OCMC@CDs in water solution (1), in the presence of UV light (2), and magnetic separation of Fe₃O₄@OCMC@CDs by a magnet (3), and in the presence of UV light (4).

A continuous decrease of the luminescence property of Fe₃O₄@OCMC@CDs with different concentrations of Cu²⁺ ions was observed at a 340 nm excitation wavelength as shown in Fig. 6b. As displayed, the fluorescence quenching efficiency of the CD-attached magnetic nanoparticles is gradually decreasing with an increase in the concentration of Cu²⁺ ions. The decrease of the fluorescence intensity exhibits a linear response with the increasing Cu²⁺ ion concentration in the range of 0–55 µM, which is consistent with the photograph of the solutions under UV light. The relative intensity (F/F₀) against increasing copper concentration shows good linearity with a correlation coefficient of 0.9845 (Fig. S3†). The limit of detection (LOD) is estimated from the calibration curve and can be depicted as F₀/F = 1.049 + 0.039[Cu²⁺]. The relative standard deviation (RSD) was determined to be 0.25% through three parallel determinations (n = 3) at a fixed Cu²⁺ ion concentration of 10.00 µM. The obtained result indicates the excellent reliability of this sensor. The limit of detection was found to be 0.56 µM.

3.8. Cell imaging

For bioimaging, NIH3T3 cells were simply incubated with Fe₃O₄@OCMC@CDs at 15 µg mL⁻¹ concentrations for 30 min and 1 h. The uptake of nanoparticles was evaluated by a fluorescence study as shown in Fig. 7. Images were observed under a fluorescence microscope to determine the endocytosis of the nanoparticles. To convert the nanoparticle target-specific imaging, Fe₃O₄@OCMC@CDs is conjugated with folic acid. Folic acid is extensively used for cancer cell imaging and drug delivery due to having a high affinity towards folate receptors (FRs) in various human carcinomas.⁴² So, the appearance of folic acid on the fluorescent magnetic nanoparticles can enhance the folate receptor-mediated internalization in to FR (+) HeLa cells. Here the intracellular uptake of Fe₃O₄@OCMC@CDs and folic acid-conjugated Fe₃O₄@OCMC@CDs was studied on HeLa cells by time-dependent cellular imaging at 15 µg mL⁻¹ concentration and the images are shown in Fig. 8. As shown in the images, in the absence of FA, the cellular internalization of nanoparticles on HeLa cells is lower as compared with FA-conjugated nanoparticles. Here the nanoparticles are internalized in the cytoplasm proximity to the cell membrane up to 30 min after treatment in HeLa cells. In the case of HeLa cells, the fluorescence intensity is increased with respect to time due to the interaction with the folate receptor.

Fig. 7 Fluorescence microscopy images of Fe₃O₄@OCMC@CDs in normal (NIH3T3) cells in the time interval (a) 30 min and (b) 1 h.

Fig. 8 Fluorescence microscopy images of Fe₃O₄@OCMC@CDs–FA in cancer (HeLa) cells at two different time intervals.

4. Conclusions

In summary, we have successfully synthesized a CD-embedded magnetic nanoparticle having high fluorescence with a very good quenching response towards Cu(II) ions. The selectivity of the novel magnetic fluorescent platform to Cu²⁺ ions compared with other metal ions is evaluated. Fe₃O₄@OCMC@CDs was successfully targeted towards folate receptors of cancer cell lines by conjugating folic acid. These fluorescent magnetic nanoparticles are also efficient for cancer cell targeting. To the best of our knowledge the present approach may offer new insight for a rise in cheap, highly sensitive and selective sensors in environmental analysis and cell imaging platforms. The incorporation of Fe₃O₄ nanoparticles into carbon dot-based fluorescent materials can be used for a promising way for separation of toxic metal ions from various environments.

Acknowledgements

This work was financially supported by the DST, Government of India (SB/FT/CS-068/2013) and Indian School of Mines, Dhanbad.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra10382k

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