Retracted Article: Amino acid derived highly luminescent, heteroatom-doped carbon dots for label-free detection of Cd²⁺/Fe³⁺, cell imaging and enhanced antibacterial activity

Abstract

A facile, economic and one-step synthesis strategy was applied for the synthesis of highly fluorescent water-soluble heteroatom doped carbon dots (CDs) from eight different amino acids viz., arginine, cysteine, glutamic acid, glutamine, aspartic acid, lysine, tyrosine, and methionine. Based on the higher quantum yield (38%) cysteine derived CDs were selected to explore their multi-functional behavior viz., sensing of metal ions, cell imaging and cytocompatibility study for MCF7 cancer cells. The cysteine derived CDs exhibit high sensitivity and selectivity toward Cd²⁺ and Fe³⁺ ions with a detection limit as low as 2.0 and 3.0 μg L⁻¹ in the linear range of 6.0–268.0 μg L⁻¹ and 6.0–250.0 μg L⁻¹, respectively. In addition, the CDs were applied for cell imaging, demonstrating their potential as excellent probes for high contrast cell imaging. Moreover, to explore an entirely different application of CDs (i.e. antibacterial and photocatalytic activity); a nanocomposite of Au/CDs was also prepared. It was observed that a very low minimum inhibition concentration value (20.0 ng mL⁻¹) was required to inhibit the growth of E. coli. Similarly, the photocatalytic activity of Au/CDs nanocomposite was also studied for H₂O₂ decomposition.

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Introduction

Carbon quantum dots (CDs) are a new class of zero-dimensional carbon nanomaterials with a size less than 10 nm.¹ CDs are not only advantageous as the traditional semiconductor quantum dots (have unique optical, photoluminescence and electrochemical properties) but also have low toxicity, water solubility, chemical inertness and environmentally friendly compared to toxic heavy metal quantum dots.² In addition, small sized CDs with its intrinsic fluorescence property are finding notable importance in biosensing, in vivo imaging,³ photocatalytic activity⁴ and other research areas. Therefore, more and more attention has been paid to the synthesis, property studies, and applications of this emerging carbon nanomaterial.

According to the literature, various synthetic methods have been developed for preparation of CDs, such as the top-down methods of laser ablation,⁵ electrochemical oxidation,^6,7 bottom-up methods of microwave assisted synthesis,^8,9 hydrothermal synthetic route¹⁰ and combustion thermal oxidation.^11,12 But these methods possess several drawbacks such as tedious synthesis steps (at high temperature), use of toxic reagents (strong acid/alkali), special synthesis equipment, and high costs, leading to their limitations for applications.¹³ Therefore, exploring new methods for synthesizing CDs are still desired.

Nowadays, several researchers have devoted their time for the preparation of fluorescent CDs in a single step using less harmful organic chemicals, solvents or natural precursors.^14,15 Zou et al. developed water-soluble fluorescent CDs using watermelon peel as a raw resource at low-temperature carbonization, which is used for live cell imaging.¹⁶ Lu and co-workers also reported a simple, economical, and hydrothermal strategy for synthesis of water-soluble, fluorescent carbon CDs with a quantum yield of ∼6.9% by using pomelo peel as a carbon source.¹⁷ Pandey's and Sharon's groups described the potential use of Indian water plant Trapa bispinosa peel for the preparation of luminescent water-soluble CDs (5–10 nm) with exceptional biocompatible against MDCK cells.¹⁸ Along with these, some luminescent CDs were also prepared by using various natural resources such as orange juice,¹⁹ Saccharum officinarum juice,²⁰ jiggery, bread, sugar,²¹ honey,²² plant soot,²³ egg membrane,²⁴ orange waste peel,²⁵ gelatine,²⁶ apple juice,²⁷ waste fry oil,²⁸ bagasse waste,²⁹ milk,³⁰ vitamin B1,³¹ Nescafe,³² and spider silk.³³ In the lieu of natural or less harmful chemicals as a precursor for carbon dot synthesis, the use of amino acid came to light. Amino acids are the unit of protein building, plentiful, cost-effective, biocompatible and eco-friendly. The amino acids have amine and carboxyl groups, in general, however, the presence of other functional groups like –SH, –phenyl etc. make them an ideal precursor for CDs synthesis. The rich content of elements other than carbon like nitrogen (N) and sulfur (S) may cause the insertion of these heteroatom in the carbon framework, resulting in the formation of heteroatom doped CDs. Initially, histidine is used as a precursor for the synthesis of CDs by Cui et al.³⁴ and Dai et al. Cui et al. have studied their chemiluminescence and photoluminescence properties. However, Dai et al. have shown the application of CDs for detection of mercury ion in the aqueous medium.³⁵ Similarly, nitrogen-doped (N-doped) photoluminescent CDs were prepared by Huang et al. using one-pot, microwave assisted hydrothermal treatment and histidine as the sole carbon source.³⁶ Other than these, Wang et al. have reported a one-step method to synthesize brightly fluorescent carbon dots using glutamic acid as a precursor compound for plant cell imaging.³⁷ Recently, Pei and his team have reported, a facile one-pot method to fabricate photoluminescent CDs by the hydrothermal treatment of three different kinds of amino acids (serine, histidine, and cystine) at mild temperatures with quantum yield 7.8%.³⁸

Along with the different synthesis procedures of CDs, their versatile applications are also very interesting. Till date, the electrochemical, cell imaging, fluorescence, luminescence, photocatalytic, sensing, bio-sensing and many more applications of CDs have been reported so far, in which the most popular one is sensing of toxic heavy metal ions viz., Fe³⁺,^22,39–41 Hg²⁺,^17,27 Cd^2+ 42 etc. in the biological and real samples. Because, the existing techniques for quantification of these two metal ions like atomic absorption/emission spectroscopy, Auger-electron spectroscopy, inductively coupled plasma mass spectrometry, ultraviolet-visible spectrometry, X-ray absorption spectroscopy, etc. requires sophisticated, expensive instrumentation and/or complicated sample preparation procedures as a result time-consuming. In contrast, fluorescent sensors offered a simple, sensitive, selective, easy to handle and low-cost approach for assaying metal ions in biological and environmental samples.

In this work, we have tried to synthesize heteroatom-doped CDs by one-step synthesis using a series of amino acids viz., arginine (Arg), cysteine (Cys), glutamic acid (Glu), glutamine (Gln), aspartic acid (Asp), lysine (Lys), tyrosine (Tyr) and methionine (Met). The prepared CDs are water-soluble with a comparatively higher quantum yield (38%) than any other reported CDs, prepared from amino acid and/or natural precursors. The synthesized CDs were characterized by UV-visible, fluorescence, Fourier Transform Infra Red spectroscopy (FT-IR), and Tunneling Electron Microscope (TEM), Energy-dispersive X-ray spectroscopy (EDAX) and X-ray Photoelectron Spectroscopy (XPS) techniques. In addition, the multifunctional behavior of CDs viz., anti-bacterial activity (towards E. coli), photocatalytic activity (towards the H₂O₂) and cancer cell imaging (MCF7) is also well explored in this work. On the other hand, the synthesized CDs are also used for quantitative estimation of Cd²⁺ and Fe³⁺ in aqueous as well as real samples. The metal ions show a direct interaction with functional groups of CDs, derived from amino acid,^35,39–41 resulting in quenching of fluorescence, which is used for estimation of metal ions. Based on the quenching behavior of metal ions in the presence of CDs, a linear concentration range of 6.0 to 260.0 μg L⁻¹ was obtained for Cd²⁺ and Fe³⁺ ions. The sensor shows the limit of detection (LOD) of 3.0 μg L⁻¹ and 2.0 μg L⁻¹ for Cd²⁺ and Fe³⁺ ions, respectively. Again, the practicability of the proposed fluorescence sensor has been validated by detecting both the metal ions in different water samples collected from various populated and industrial areas.

Experimental section

Reagents and instrumentation

All amino acids (L-Cys, L-Arg, L-Lys, L-Met, L-Asp, L-Glu, L-Tyr, L-Gln) were procured from Aldrich (Steinheim, Germany). Sulfuric acid and hydrogen peroxide (H₂O₂) were procured from Spectrochem Pvt. Ltd. (Mumbai, India) and auric(III) chloride (HAuCl₄) was purchased from Sigma-Aldrich Co. Ltd (Steinheim, Germany). Ferrous and ferric sulfate heptahydrate, magnesium sulfate (MgSO₄), mercurous chloride (Hg₂Cl₂), cadmium sulfate (CdSO₄), silver nitrate (AgNO₃), zinc chloride (ZnCl₂), lead(II) oxide (PbO), lead dioxide (PbO₂), aluminium chloride (AlCl₃), nickel sulfate (NiSO₄·6H₂O), calcium chloride (CaCl₂), were purchased from Alfa Aesar (USA) and Fluka (Steinheim, Germany). Human breast cancer cell line (MCF7) was kindly gifted by the Biotechnology department of Banaras Hindu University, Varanasi. FT-IR analysis was carried out on Varian FT/IR (USA) spectrometer. The size and morphology of prepared nanoparticles were studied using a transmission electron microscope (model Tecnai 30 G² S-Twin electron microscope) operated at 300 kV accelerating voltages by placing a drop of as-prepared sample suspension on the surface of a carbon-coated copper grid. The fluorescence spectra (FL) were recorded using a Perkin Elmer LS55 fluorescence spectrometer. UV-visible spectroscopic characterization was done on Perkin-Elmer Lambda 35 (Singapore) spectrophotometer. For cell imaging, Nikon fluorescence microscope (Eclipse Ti) under bright light illumination and fluorescence was used. All the electrochemical analyses [cyclic voltammetry (CV) and square wave voltammetry (SWV)] were performed on the CH instrument (USA, model number 660 C). All the experiments were done at room temperature (25 ± 1 °C) under ambient conditions.

Synthesis of carbon quantum dots

In a typical synthesis procedure, 0.35 g of the amino acids dissolved in 10.0 mL of water was taken in a round bottom flask, followed by the addition of 3.0 mL concentrated sulfuric acid. The mixture was refluxed at 200 °C for 8 hours, resulting in a yellow solution implying the successful conversion of amino acids to CDs.³⁸ The obtained solution was centrifuged at 15 000 rpm for 10 min and the supernatants were dialyzed against Milli-Q water with a cellulose ester membrane bag (M. Wt. = 3500) for 24 h to remove the excess precursors, if any.

Quantum yield (QY) measurement

The quantum yield of the fluorescent CDs was calculated by using established methods.⁴³ Quinine sulphate in 0.1 M H₂SO₄ was chosen as a standard reference sample which has fixed and known fluorescence quantum yield value (QY = 0.54 at 360 nm). The QY of unknown CDs was calculated according the following equation:

QY_x = QY_stdI_xA_stdn_x²/I_stdA_xn_std² (1)

where I is the measured integrated emission intensity, n is the refractive index of the solvent, and A is the optical density. The subscript “std” refers to the standard sample with known QY and “x” for the unknown samples.

Analytical applications of Cys-derived CDs

To explore the multifunctional behavior of cysteine-derived heteroatom-doped (N and S) carbon dots the cell imaging of cancer cells (MCF7) and detection of heavy metal ions i.e. Cd²⁺ and Fe³⁺ were performed. However, to study the photocatalytic activity towards H₂O₂ decomposition and antibacterial activity of CDs for E. coli, Au/CDs composite was synthesized.

Synthesis of Au/CDs nanocomposite. To explore the photocatalytic and antibacterial activity of synthesized Cys-derived CDs, their nanocomposite (Au/CDs) was synthesized with using earlier reported method.⁴ In brief, 100.0 μL HAuCl₄ solution was added to 5.0 mL CDs solution with vigorous magnetic stirring in a dark environment at room temperature. After 4 h magnetic stirring, the solution was aged overnight and it turned to orange color, implying the conjugation of Au and CDs.

Photocatalytic activity of Au/CDs nanocomposite. The photocatalytic activity of Au/CDs nanocomposite for H₂O₂ decomposition was tested in a three-electrode electrochemical cell with a platinum wire as the auxiliary electrode and an Ag/AgCl (saturated KCl) as the reference electrode. The working electrode was Au/CDs composites modified pencil graphite electrode (PGE), which was prepared by spreading the aqueous solution of Au/CDs composites over PGE. A 220 W UV lamp was used as a source of UV-visible light with a wavelength of λ < 365 nm to get long UV-visible light. The CV and SWV analysis were performed in both UV and visible light, in the presence of different H₂O₂ concentrations at 0.1 V s⁻¹ scan rate.

To further confirm the successful decomposition of H₂O₂ and photocatalytic activity of synthesized Au/CDs nanocomposite, a terephthalic acid photoluminescence-probing assay was performed, which detect the formation of HO˙. For this assay, terephthalic acid (THA, 0.415 g), sodium hydrogen phosphate (Na₂HPO₄, 2.2 g), and deionized water were added into a 250 mL volumetric flask to form a buffer at pH 7.5. After adding a suitable amount of catalyst into the buffer, the mixture was irradiated by visible light or no light and corresponding fluorescence of the solution was measured at pH 7.5.

Antibacterial activity of Au/CDs nanocomposite. The disc diffusion test is a very popular method to show the effect of an antibacterial agent on the growth of bacteria.⁴⁴ For the experiment, separately, 50.0 μL of E. coli suspension (100–110 CFU mL⁻¹) was cultured in Luria-Bertani (LB) agar coated disposable sterilized Petri dishes (13 mm). Then, 2.0 mm filter paper discs were impregnate into different concentration of Au/CDs (1% to 10% v/v in phosphate buffer solution, pH = 6.5) and were gently placed in the center of the bacteria growth on the LB agar Petri dishes and incubated overnight at 37 °C. If the antibacterial property of Au/CDs is sufficient to achieve inhibition of bacterial growth, a circular region of no bacterial growth develops. This is called a zone of inhibition. After incubation, bacterial growth inhibition was observed by calculating the diameter of the bacteria-free area. For the kinetic test, the culture medium of E. coli was prepared and incubated overnight at 37 °C. In the prepared medium, different concentrations of Au/CDs were added and corresponding optical density was measured at 600 nm.

To perform the minimum inhibition concentration (MIC) test, different concentrations (2000, 1000, 500, 250, 125, 75, 25, 20, 15 ng mL⁻¹) of Au/CDs were prepared in the aqueous solution. Similarly, an aqueous solution of AgNO₃ was also prepared and used for comparative tests. For the test, 5 mL of LB agar solutions was added to bacteria strain (1 × 10⁵ to 1 × 10⁶ CFU), followed by the addition of different concentrations of prepared sample solution. The as-prepared bacterial suspension was incubated at 37 °C with continuous shaking at 150 rpm and homogeneously spread on the glass plate and kept for 24 h. After the day, fresh organisms were added to the suspension and incubated for the next 24 h. The growth or no growth of bacterial colony was determined by visually observation. The lowest concentration (highest dilution) required to arrest the growth of bacteria was regarded as minimum inhibitory concentrations (MIC). For accuracy, all of the concentration values were analyzed three times and the relative standard deviation was less than 1%.

Cytocompatibility test of Cys-CDs (MTT assay). In vitro cytocompatibility of the CDs with MCF7 cells was investigated using a standard methyl thiazol tetrazolium bromide (MTT) assay. MCF-7 were seeded in a 96-well plate in a growth medium consisting Dulbecco's Modified Eagle Media (DMEM) supplemented with 10% fetal bovine serum, 1% antibiotic solution and maintained at 37 °C in a humidified 5% CO₂ atmosphere. The cells were treated with various concentrations (0–30 μg mL⁻¹) of C-dots and were cultured for another 24 h. After the incubation with CDs, 20 μL MTT was added to each well and the optical density was evaluated at 570 nm. Cell viability was calculated as follows:

Cell viability (%) = (A_sample − A_blank) × 100%/(A_control − A_blank) (2)

where A_sample is the absorbance of a well with cells, MTT solution and CDs; A_blank is the absorbance of a well with medium and MTT solution, without cells; A_control is the absorbance of a well with cells and MTT solution, without CDs. The data were expressed as the percentages of viable cells compared to the survival of a control group (untreated cells as controls of 100% viability).

Quantitative estimation of Cd²⁺ and Fe³⁺ and cross-selectivity study. For the quantitative estimation of metal ions, 10.0 mM KCl solution was taken as supporting electrolyte. In a typical assay, CDs (100.0 μL) were added into KCl solution (3.0 mL), followed by the addition of different concentrations of Cd²⁺ and Fe³⁺ and the FL spectra were recorded. The cross selectivity for Cd²⁺ and Fe³⁺ was confirmed by adding other metal ions stock solutions (Zn²⁺, Ni²⁺, Al³⁺, Pb²⁺, Pb⁴⁺, Mg²⁺, Hg²⁺, Ca²⁺, Ag⁺, Cu²⁺) instead of Cd²⁺ and Fe³⁺ in a similar way.

Result and discussions

In this paper, eight amino acids were used as a precursor to synthesize heteroatom doped CDs (Fig. 1-A). The chemical structures of all the amino acids are shown in Scheme S1.† The corresponding XPS, FT-IR, UV-visible, and fluorescence spectra were recorded. Based on the best results obtained by these three techniques one of the CDs (derived from cysteine) was selected and used for further studies.

Fig. 1 (A) Camera image of all the prepared carbon dots in UV-light with their respective PL intensity and quantum yield values. UV-visible (B and C) and fluorescence (D and E) spectra of amino acid derived carbon dots.

Spectroscopic analysis of carbon dots

UV-visible and photoluminescence study. In order to explore the optical properties of the amino acid generated CDs, their UV-vis absorption and photoluminescence emission spectra were recorded (Fig. 1-B and C). As depicted in Fig. 1-B, the UV-vis absorption spectrum showed a peak at 270 nm. According to the literature, the CDs typically show optical absorption in the UV region with a tail extending to the visible range attributed to the π–π* transition of the C=C bonds and the n–π* transition of C=O bonds.^22,38,45 The peak position is constant in all the spectra, but they have a variation in their intensity value. In all the spectra, cysteine derived CDs have highest peak intensity and tyrosine derived CDs have the lowest one.

The photoluminescence (PL) of the amino acid generated CDs are shown in Fig. 1-C and D. It was noted that a strong emission spectrum at 425 nm is observed in all amino acids when it is excited at 350 nm, indicating the fluorescent nature of prepared CDs. The full width at half maximum (FWHM) is ca. 80 nm, which is approximately equal to that of most reported C-dots.⁴⁶ Contrast to the others, PL spectra of methionine and aspartic acid shows one additional peak at around 480 nm. This behavior indicates that there are at least two types of excitation energy trapped on the surface of heteroatom-doped CDs.⁴⁷ Fig. 1-A shows the optical images of the CDs under the illumination of UV light (365 nm). The brightest illumination was observed for Cys derived CDs, which is strong enough to be easily seen with the naked eyes. The corresponding PL intensity was also shown in the Fig. 1-A. Herein, again the maximum PL intensity was found for Cys derived CDs and minimum for tyrosine derived CDs.

The high PL intensity of CDs is still a matter of debate, but is probably caused by the emissive traps, the quantum confinement effect, aromatic structures, oxygen-containing groups, free zigzag sites, and edge defects.^45,48 Besides this, the origin of the PL of CDs has been assigned to several other reasons also: optical selection of differently sized nanoparticles (quantum effect), defects and surface states, surface groups, surface passivation, fluorophores with different degrees of p-conjugation, and the recombination of electron–hole pairs localized within small sp2 carbon clusters embedded within a sp3 matrix.⁴⁵ Along with these, the incorporation of heteroatom may result in higher PL properties.⁴⁹ Using quinine sulfate as a reference, the quantum yield (QY) of each CDs was also calculated and portrayed in Fig. 1-A. The maximum quantum yield of 38% was found for Cys derived CDs and minimum for tyrosine CDs (10%). Therefore, Cys derived CDs was selected for further studies.

FT-IR study. To understand the surface functional groups FT-IR study was performed and it was observed that carboxylic acid and amide groups were well-preserved on the surface of the carbon dot and constantly present in all the spectra (Fig. S1†). Amide I is the most intense absorption band in amino acids derived CDs, which is primarily governed by the stretching vibrations of the –C=O group (1632 cm⁻¹). The band for amide II derives mainly from in plane NH bending appears in the range of 1510 to1580 cm⁻¹. However, the other major groups of amino acids viz., amide III or C–N stretching vibrations (1365 cm⁻¹), –COOH, –NH₂, –NH₃⁺ (3432, 3216, 3130 cm⁻¹) also visible in the spectra of amino acids derived CDs. Along with this, the characteristic bands of each amino acids are also clearly present in the spectra viz., –SH band of cysteine (2551 cm⁻¹). From the study is can be easily concluded that the synthesized CDs retain the characteristic functional group of their respective amino acids.

X-ray photoelectron spectroscopy (XPS) and EDAX analysis. X-ray photoelectron spectroscopy (XPS) was applied to analyze the chemical states of the elements in the Cys-CDs. The XPS survey spectra of Cys-CDs show four typical peaks of C1s (around 285 eV), N1s (around 398.5 eV), O1s (around 531 eV) and inconspicuous S2p (around 164 eV) (Fig. 2-A). The deconvolutions of the C1s spectra (Fig. 2-B) are fitted by three peaks, which are assigned to C–C (284.6 eV), C–N (289.4 eV) and C–S (294.2 eV).^48,49 The N1s spectra (Fig. 2-C) reveal the presence of two nitrogen species of C–N (399.4 eV) and N–H (400 eV).^49,50 The O1s peak can be de-convolved into three components (Fig. 2-D) attributed to the presence of C=O (536 eV), C–O (532 eV), and C–OH (526 eV) groups.⁵¹ Moreover, the spectrum shown for sulfur atom consists of two peaks, positioned at 166.9 eV and 168.6 eV, suggesting the presence of two forms of the sulfur atom (Fig. 2-E). The two peaks could be attributed to the 2p3/2 and 2p1/2, positions of the –C–S– covalent bond of the cysteine, owing to spin–orbit couplings.^49,50

Fig. 2 (A) XPS survey spectra of Cys-CDs. The detailed (high-resolution) scans for (B) C1s, (C) N1s, (D) O1s, and (E) S2p species. (F) EDAX spectra of Cys-derived carbon dots.

To further confirm the elemental composition of cysteine derived carbon dots, EDAX analysis was also performed (Fig. 2-F). The spectra show three peaks for ‘C’, ‘O’ and ‘S’ with 61.0, 23.0 and 14.0 percentage. These analyses suggest the successful synthesis of Cys-derived carbon dot with ‘C’, ‘O’, ‘N’ and ‘S’ elements.

Morphological studies of cysteine derived carbon dots (Cys-CDs)

Fig. 3-A shows TEM images of the cysteine derived carbon dots. As evident from the TEM images, mono-dispersed spherical particles of size 2–3 nm is observed. These CDs show the average particle size of nearly 2 nm. Fig. 3-B shows the corresponding HR-TEM images of CDs. The imaged lattice spacing 2.16 Å corresponds to the (110) planes of the hexagonal primitive structure of carbon with space group P6_3mc having cell parameters as a = b = 2.49 Å, c = 4.14 Å. This result confirms the formation of carbon dots during the synthesis. The corresponding particle size distribution (PSD) is represented in Fig. 3-E.

Fig. 3 (A) TEM, (B) HR-TEM images (with inset showing zoom image of HR-TEM) of Cys-derived carbon dots. Camera images of antibacterial activity of (C) Cys-derived Au/CDs and (D) AgNO₃ by the disc diffusion method; (E) particle size distribution images of Cys-derived carbon dots; (F) bacterial growth curve with or without Cys-derived Au/CDs (concentration from 1.0 to 20.0 μg mL⁻¹).

Stability of Cys-derived CDs

Prior to exploring the multi-functional behaviour of Cys-derived CDs, their stability was studied. As shown in Fig. S2,† the photoluminescence intensity of CDs did not change with ion strength (various concentrations of NaCl), indicating that the aqueous solution of CDs is stable in the presence of higher concentration of ions (0.5 M). Unlike the earlier reported CDs,⁵² the photoluminescence intensity of the Cys-derived CDs are pH independent too. The CDs are stable enough at highly acidic i.e. pH = 2, extreme basic i.e. pH = 12 and physiological pH, i.e. 7 (Fig. S3†). Along with these, the CDs show excellent photostability, as the photoluminescence intensity did not change even after continuous irradiation at a wavelength of 350 nm for 45 min (Fig. S4†).

Multifunctional behaviour of cysteine derived carbon dots

Photocatalytic activity. The photocatalytic activity of CDs towards H₂O₂ decomposition was studied by Au/CDs nanocomposites. Before experiment, PL spectrum of CDs and Au/CDs were also recorded and shown as Fig. 4-A(a and b). As depicted, only CDs (curve a), give higher PL intensity than the Au/CDs nanocomposite (curve b). The PL intensity is effectively quenched in case of Au/CDs nanocomposite, which is attributed to an ultrafast electron transfer process in the composite.⁴ The photocatalytic activity was studied in three-electrode electrochemical cell containing 200 μL H₂O₂ (30%) in the normal and UV light by CV and SWV techniques. As shown in Fig. 4-B and C, the bare PGE shows no obvious electrochemical response while the Au/CDs-modified PGE exhibits catalytic ability for H₂O₂ decomposition. Under visible-light irradiation, the response of Au/CDs-modified electrode for H₂O₂ decomposition is stronger than that without light irradiation. The experiments suggested the UV light-promoted the catalytic activity of Au/CDs composites.

Fig. 4 (A) PL spectra (485 nm excitation) of Cys-CDs (curve a) and Au/CDs (curve b); (B) cyclic and (C) square wave voltammograms of bare and Au/CDs-modified pencil graphite electrode in the presence of 30% H₂O₂ with or without UV-light. (D) PL spectra of terephthalic acid fluorescent isomer in the presence (curve a) and absence (curve b) of light. (E) Time dependence of the fluorescence intensity of the supernatant liquid of Au/CDs composites in the absence and presence of visible light. (F) Scheme for reaction between hydroxyl radicals and terephthalate yielding one intensely fluorescent mono-hydroxylated isomer.

To confirm the photocatalytic activity of Au/CDs towards H₂O₂, a terephthalic acid-probing assay was used. It was assumed that photocatalytic decomposition of H₂O₂ would produce HO˙ as main active oxygen species. In the same solution if terephthalic acid was added, it reacts with hydroxyl radicals and forms one intensely fluorescent isomer (Fig. 4-F). The corresponding PL spectra of fluorescent isomer in the absence (curve a) and presence (curve b) of light is shown in the Fig. 4-D. As shown in the figure, the PL intensity is much higher in the presence of light, due to formation of large number of fluorescent isomer and/or HO˙ radical. Fig. 4-E shows the variation in PL intensities of the supernatant solution containing fluorescent isomer, after Au/CDs composites removal in the presence or absence of visible-light with time decay. The study confirms that Au/CDs catalyst successfully participated in the photocatalytic decomposition of H₂O₂, resulting in the formation of a larger number of HO˙ radical.

Antibacterial activity. The antibacterial properties of Au/CDs nanocomposite against Gram-negative (E. coli) bacteria were confirmed using Kirby–Bauer disk diffusion method. AgNO₃ was used as a model compound for the comparison of activity. As shown in Fig. 3, the disks with Au/CDs were surrounded by a larger diameter of inhibition zone (4.8 mm, Fig. 3-C) compared to AgNO₃ (0.88 mm, Fig. 3-D) for E. coli strain. The zone of inhibition of Au/CDs was higher than that of AgNO₃ for 10 h incubation. The diameter of inhibition zone increases with the increase in concentration of Au/CDs (1% to 10% v/v in phosphate buffer solution, pH = 6.5). Similarly, a bacterial inhibition growth curve was used to study the growth kinetics of E. coli with prepared bacterial strain (Fig. 3-F). The optical density at 600 nm was measured to monitor bacterial growth. As shown in the figure, the bacterial growth was delayed as the concentration of Au/CDs increased as compared to the control (without Au/CDs). The growth of E. coli was completely inhibited by the CDs when the concentration is more than 5.0 μg mL⁻¹.

For the evaluation of minimum concentration of Au/CDs nanocomposite required for inhibition of bacterial growth MIC test was performed. It was found that, initially (for high concentration of nanocomposite or AgNO₃), growth of E. coli is totally inhibited for 24 and 48 h (Table S1†). It was also found that for similar concentrations (25 ng mL⁻¹), the bacterial growth is visible in the plate containing AgNO₃, and however, growth is inhibited in the Au/CDs nanocomposite coated plate. It was also found that MIC values at 48 h were higher than those at 24 h because of the addition of new organisms for type of samples. A very low MIC value (20.0 ng mL⁻¹) was observed to inhibit the growth of E. coli, which suggests the good antibacterial property of proposed Au/CDs nanocomposite.

In a different study, effect of Au/CDs nanocomposite solution pH on the antibacterial study was also studied and the results are portrayed in the Table S2.† According to the results, MIC values of Au/CDs nanocomposite does not altered with variation in the pH of the solution. Approximately similar MIC value was observed for the solution having pH from 6.0 to 8.5. Generally, nanoparticles show good antibacterial properties arising from their large surface area to volume ratio providing desirable contact with bacterial cell.⁵³ Based on the literature, the reactive oxygen species (ROS) are generated, due to the interaction of nanoparticles with bacterial cell wall, resulting in oxidation of membrane lipids leading to their membrane dysfunction, and lastly to cell death.⁵⁴ These results confirmed that the Cys-derived CDs could be successfully used as good antibacterial as well as photocatalytic nanomaterial.

Carbon dots as a fluorescent agent for cell imaging. In order to evaluate the cell permeability and cytocompatibility of Cys-derived CDs live-cell imaging and MTT assay were carried out. After the incubation with the CDs at 37 °C for 3 h, the MCF-7 cells under living conditions became brightly illuminated when imaged under the microscope. The obtained images clearly visualize the bright field (Fig. 5-A) and high contrast, fluorescence images (Fig. 5-B) of CDs distributed around the cytoplasm of MCF-7 cells, displaying that the CDs can label both the cell membrane and the nucleus of MCF-7 cells. Besides the strong fluorescence and good stability in the physiological conditions, the CDs also show quite low cytotoxicity as shown in Fig. 5-C. Evaluation of in vitro toxicity of the CDs was conducted using MCF7 cells as representative cell lines. It was found that more than 97% of the cells were viable when incubated with 30.0 μg mL⁻¹ or lesser CDs. The efficient cellular uptake, non-toxicity, and strong fluorescence show that the Cys-derived CDs can be used as excellent probes for high contrast cell imaging.

Fig. 5 (A) Bright field and (B) fluorescence images of MCF7 cancer cells after incubation with Cys-derived CDs; (C) cell viability of MCF7 cancer cells after 24 h treatment with Cys-derived CDs calculated from MTT assay.

Label-free, selective and sensitive estimation of metal ions (Cd²⁺ and Fe³⁺). The high quantum yield renders Cys-derived CDs a possible type of promising fluorescence probe for sensing of heavy metal ions. To study the sensitivity of CDs for Fe³⁺ detection, different concentrations of Fe³⁺ were added to the aqueous solution of CDs and the fluorescence intensities were measured. Fig. 6-A and B shows the PL spectra of CDs in the presence of different concentrations of Fe³⁺ and Cd²⁺, respectively. It was observed that the CDs solution in the absence of these metal ions, exhibits a strong PL peak at 450 nm (Fig. 6-A and B, inset), which continuously decreases with an increase in concentration of metal ions. This indicates the CDs-metal ion interaction resulting in quenching of PL intensity.²² The fluorescence quenching efficiency was calculated by Stern–Volmer equation [eqn (3)]. Linear relationship could be expressed by [(I₀/I) − 1] in the concentration of these two metal ions (μg L⁻¹).

(I₀/I) − 1 = K_svC (3)

where I₀ is the initial fluorescence intensity in the absence of analyte, I is the fluorescence intensity in the related concentrations of metal, K_sv (L μg⁻¹) is the Stern–Volmer quenching constant, and C (μg L⁻¹) is the concentration of metal ions. The limit of detection (LOD) was calculated as three times the standard deviation of the blank measurement in the absence of metal, divided by the slope of the calibration plot between [(I₀/I) − 1] and metal concentration (Fig. 6-C). The fluorescence quenching efficiency exhibits a good linear response towards Cd²⁺ (curve a) and Fe³⁺ (curve b) in the range of 6.0–268.0 μg L⁻¹ and 6.0–250.0 μg L⁻¹, respectively, and the corresponding calibration equation is given below:

(1) For Cd²⁺: [(I₀/I) − 1] = (0.0060 ± 0.0003) × C + (6.1386 ± 0.0625); R² = 0.99; LOD = 2.0 μg L⁻¹

(2) For Fe³⁺: [(I₀/I) − 1] = (0.0059 ± 0.0003) × C + (4.8100 ± 0.0497); R² = 0.99; LOD = 3.0 μg L⁻¹

Fig. 6 Fluorescent emission spectra of the Cys-CDs aqueous solutions upon addition of different concentrations of Cd²⁺ (A) and Fe³⁺ (B). Insets in (a) and (b) show the intensity of Cys-CDs PL spectra in absence of metal ions; (C) calibration curves for Cd²⁺ (a) and Fe³⁺ (b); (D) selective PL response of aqueous CDs solution towards various metal ions (Mn⁺ = 100 μg L⁻¹). Here, F₀ and F are the fluorescence intensities of CDs in the absence and presence of metal ions, respectively.

To demonstrate the selectivity of the proposed method, we investigate the fluorescence response of CDs towards Cd²⁺ and Fe³⁺ in the presence of some other metal ions (i.e. Zn²⁺, Ni²⁺, Al³⁺, Pb²⁺, Pb⁴⁺, Mg²⁺, Hg²⁺, Ca²⁺, Ag⁺, Cu²⁺). Fig. 6-D shows the changes in the relative PL intensity (F/F₀) of the CDs occurred with representative metal ions under the similar conditions including interfering metal ions. It is seen that no observable decrease was observed with the addition of other ions into the CDs solution. In contrast, a much lower PL intensity was observed for CDs upon addition of Cd²⁺ and Fe³⁺. The outstanding selectivity and specificity can be probably attributed to the strong affinity of Cd²⁺ and Fe³⁺ and the carboxylic group of CDs surface than other metal ions. From the above observations, it can be easily concluded that the present fluorescent sensing probe exhibits high selectivity and sensitivity toward Cd²⁺ and Fe³⁺ metal ions.

Real sample analysis

To investigate the practical applicability of proposed sensor, seven different water samples were collected from differentially populated areas viz., industrial, coal-mine and normal. The collected water samples were added into the sample vial having Cys-CDs solution. The corresponding PL intensity was measured. It was found that the concentration of heavy metal ion (Cd²⁺ and Fe³⁺) is very high in the water collected from coal mine and industrial areas (Table 1). The contamination order is as follows:

Coal mine area > waste water > industrial area water

whereas, the heavy metal ions concentration are below the LOD in the common living area water samples like our institute drinking water, distilled water, tap water and river water. Furthermore, the samples were divided in three parts and a fixed and known concentration of metal ions stock solution of Cd²⁺ and Fe³⁺ was directly spiked in the each one and corresponding concentrations, standard deviation and recoveries were calculated. The analytical results were portrayed in Table 1. The good recoveries ranging from 98.0% to 100.9% with an RSD of around 1.3% to 1.7% were observed in each sample. The remarkable photoluminescence sensitivity of the cysteine derived CDs towards Fe³⁺ and Cd²⁺ can be ascribed to the heteroatom-doping induced modulation of the chemical and electronic characteristics and the easy formation of complexes between the CDs and Fe³⁺ and/or Cd²⁺.⁵⁰ The high selectivity of these CDs for Fe³⁺ and Cd²⁺ might be due to the faster chelating process of these metal ions with CDs through N, O and S in comparison with other metal ions.⁵⁰ The results demonstrate the accuracy and reliability of the present fluorescence method for detecting the Cd²⁺ and Fe³⁺ in practical applications.

Table 1 Analytical results for determination of Cd²⁺ and Fe³⁺ in various water sample using Cys-derived CDs as fluorescence probe

S. no.	Name of samples	Concentration of Cd^2+ (μg L^−1)		Recovery (%)	^bRSD (%)	Concentration of Fe^3+ (μg L^−1)		Recovery (%)	^bRSD (%)
		Added	Determined^a ± S.D.			Added	Determined^a ± S.D.
a S.D. = standard deviation for three replicate measurements.b RSD = relative standard deviation.
1	Drinking water	—	—	—	—	—	—	—	—
	Sample 1	20.0	19.60 ± 0.23	98.0	1.2	20.0	19.90 ± 0.25	99.5	1.3
	Sample 2	20.0	19.93 ± 0.25	99.6	1.3	20.0	19.90 ± 0.25	99.5	1.3
	Sample 3	20.0	19.89 ± 0.27	99.4	1.4	20.0	19.85 ± 0.29	99.2	1.5
2	Tap water	—	—	—		—	7.00	—	—
	Sample 1	20.0	19.91 ± 0.35	99.5	1.7	20.0	20.10 ± 0.39	100.5	1.5
	Sample 2	20.0	19.85 ± 0.35	99.2	1.7	20.0	20.12 ± 0.37	100.6	1.4
	Sample 3	20.0	19.86 ± 0.31	99.3	1.6	20.0	20.01 ± 0.42	100.0	1.6
3	River water	—	—	—	—	—	8.0	—	—
	Sample 1	20.0	19.90 ± 0.25	99.5	1.3	20.0	20.19 ± 0.34	100.9	1.5
	Sample 2	20.0	19.80 ± 0.23	99.0	1.2	20.0	20.17 ± 0.38	100.8	1.7
	Sample 3	20.0	19.80 ± 0.23	99.0	1.2	20.0	20.17 ± 0.38	100.8	1.7
4	Waste water	—	9.0	—	—	—	10.0	—	—
	Sample 1	20.0	20.1 ± 0.38	100.5	1.3	20.0	20.01 ± 0.38	100.5	1.3
	Sample 2	20.0	20.13 ± 0.44	100.6	1.5	20.0	20.01 ± 0.38	100.5	1.3
	Sample 3	20.0	20.01 ± 0.44	100.0	1.5	20.0	20.13 ± 0.35	100.6	1.2
5	Distilled water	—	—	—	—	—	—	—	—
	Sample 1	20.0	19.90 ± 0.21	99.5	1.1	20.0	19.85 ± 0.29	99.2	1.5
	Sample 2	20.0	19.90 ± 0.27	99.5	1.4	20.0	19.70 ± 0.31	98.5	1.6
	Sample 3	20.0	19.90 ± 0.23	99.5	1.2	20.0	19.95 ± 0.33	99.7	1.7
4	Industrial water	—	8.00	—	—	—	9.00	—	—
	Sample 1	20.0	20.15 ± 0.46	100.7	1.7	20.0	20.18 ± 0.43	100.9	1.5
	Sample 2	20.0	20.16 ± 0.49	100.8	1.8	20.0	20.09 ± 0.40	100.4	1.4
	Sample 3	20.0	20.15 ± 0.46	100.7	1.7	20.0	20.19 ± 0.40	100.9	1.4
5	Coal-mine area	—	10.00	—	—	—	12.0	—	—
	Sample 1	20.0	20.18 ± 0.35	100.9	1.2	20.0	20.15 ± 0.38	100.7	1.2
	Sample 2	20.0	20.18 ± 0.38	100.9	1.3	20.0	20.16 ± 0.41	100.8	1.3
	Sample 3	20.0	20.07 ± 0.41	100.3	1.4	20.0	20.16 ± 0.41	100.8	1.3
6	Our institute	—	—		—	—	—	—	—
	Sample 1	20.0	19.79 ± 0.29	99.0	1.5	20.0	19.90 ± 0.31	99.5	1.6
	Sample 2	20.0	19.80 ± 0.29	99.0	1.5	20.0	19.90 ± 0.31	99.7	1.6
	Sample 3	20.0	19.9 ± 0.27	99.5	1.4	20.0	19.80 ± 0.27	99.0	1.4

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Comparative study with earlier reported methods

According to the literature, many research works have been already published for synthesis of carbon dots from natural precursors.^{16–20,22–38} The comparison of CDs derived from natural resources and their applications in various fields is summed in Table 2. The normal problem comes with these CDs is low quantum yield (0.7–31.6%). In this work, we have reported the high quantum yield CDs derived from amino acids. Additionally, a multi-functional aspect of CDs is more clearly described in this work.

Table 2 Comparative study of previously reported carbon dots derived from natural precursors and amino acids

S. no.	Precursors	^aQY	Synthesis procedure	Applications of synthesized carbon dots	Reference
a QY = quantum yield.
1	Flour	5.4%	Microwave treatment	Selective detection of mercury(II)	8
2	Watermelon peel	7.1%	Carbonization at 220 °C	Live cell imaging & optical imaging probes	16
3	Pomelo peel	6.9%	Hydrothermal treatment	Hg^2+ detection in lake water	17
4	Trapa bispinosa	1.2%	Normal heating at 90 °C	Biocompatible against MDCK cells	18
5	Orange juice	26%	Hydrothermal treatment	Bio-imaging	19
6	Sugar cane	5.76%	Hydrothermal treatment	Cell imaging of Escherichia coli and yeast	20
7	Honey	19.8%	Hydrothermal treatment	Detection of Fe^3+ and cell imaging	22
8	Plant soot	0.72%	Normal reflux	Cellular and fish imaging	23
9	Egg membrane	14%	Microwave treatment	Sensitive probe for glutathione	24
10	Orange peels	12.3%	Hydrothermal treatment	Photocatalyst for dye degradation	25
11	Gelatin	31.6%	Hydrothermal treatment	Live cell imaging	26
12	Apple juice	6.4%	Hydrothermal treatment	Detection of Hg^2+	27
13	Waste frying oil	3.66%	Carbonization	Cell imaging	28
14	Bagasse wastes	9.3%	Hydrothermal carbonization	Biolabeling and bioimaging in cancer cells	29
15	Milk	12%	Hydrothermal	Imaging of U87 cell line	30
16	Vitamin B1	76%	Carbonization	Fluorescent imaging probes	31
17	Nescafe	5.5%	Normal heating	Cells and fish imaging	32
18	Spider silk	17.6%	Hydrothermal treatment	Cancer cell imaging	33
19	Histidine	—	Microwave synthesis	Intense photoluminescence	34
20	Histidine	10.7%	Hydrothermal treatment	Melamine detection	35
21	Histidine	8.9%	Microwave synthesis	Sensing and bioimaging	36
21	Glutamic acid	30.7%	Hydrothermal treatment	Plant cell imaging	37
22	Serine, histidine, & cystine	7.5%	Hydrothermal approach	Only photoluminescence property	38
23	Cysteine	38%	Hydrothermal carbonization	Antibacterial, photocatalytic activity, MCF7 cell imaging, cytocompatibility and label free sensing of Cd^2+ and Fe^3+	This work

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We have also compared the proposed method with earlier reported work for estimation of Cd²⁺ and Fe³⁺ (Table S3†). As shown in the table, the earlier reported methods have very high detection limit for estimation of Cd²⁺ and Fe³⁺, except, Yang et al. who have reported the detection of Fe³⁺ using honey derived CDs, with very low detection limit (0.095 μg L⁻¹). Based on our knowledge, for Cd²⁺ detection no such type of method was reported with as much low detection limit as reported in the proposed work. Henceforth, the results and studied demonstrated that Cys-derived CDs could serve for label free detection of Cd²⁺ and Fe³⁺, with a very good limit of detection.

Conclusion

Herein, we creatively synthesize a series of environment-friendly CDs using amino acid precursors via a simple, one-step synthesis procedure. It is found that the structure of starting materials can effectively influence the optical properties of the resulting CDs such as PL intensity and the quantum yields. Among all of the eight amino acids derived CDs, Cys-CDs were used throughout the work to explore multi-functional behaviour of CDs. The Cys-derived CDs possess anti-bacterial towards activity E. coli, photocatalytic activity towards H₂O₂ and good cytocompatibility with MCF7 cell line. It also shows a good fluorescence in vitro cell imaging. Additionally, combining their high fluorescent property and low cytotoxicity, these synthesized CDs were employed as a label-free sensor for the quantification of Cd²⁺ and Fe³⁺ metal ions in aqueous as well as real samples. Therefore, such CDs may pave a new way for designing of multi-directional materials which have potential applications in cell imaging, sensing, anti-bacterial and photocatalytic activities.

Acknowledgements

Authors are thankful to Department of Science and Technology, Government of India for sanction of the Fast Track Research Project for Young Scientists to Dr Rashmi Madhuri (Ref. No. SB/FT/CS-155/2012) and Dr Prashant K. Sharma (Ref. No. SR/FTP/PS-157/2011). Dr Sharma (FRS/34/2012-2013/APH) and Dr Madhuri (FRS/43/2013-2014/AC) are also thankful to Indian School of Mines, Dhanbad for grant of Major Research Project under the Faculty Research Scheme. We are also thankful to Board of Research in Nuclear Sciences (BRNS), Department of Atomic Energy, Government of India for major research project. The authors are also grateful to Dr R. J. Choudhary, UGC–DAE Consortium for Scientific Research, Indore for the XPS analysis.

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Footnote

† Electronic supplementary information (ESI) available: Chemical formula of all the amino acids used for carbon dots synthesis, FT-IR spectra of glutamine, methionine, cysteine, aspartic acid, arginine, lysine, tyrosine and glutamic acid-derived carbon dots, effect of NaCl, pH, light irradiation on the stability of Cys-derived carbon dots, minimum inhibitory concentration tests of Au/CDs nanocomposite and AgNO₃ for E. coli, MIC values of Cys-CDs against E. coli at different pH values for 24 h, comparative study for detection of metal ions by cysteine derived carbon dots and other reported methods. See DOI: 10.1039/c5ra09525e

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