Thiosemicarbazide chemical functionalized carbon dots as a fluorescent nanosensor for sensing Cu²⁺ and intracellular imaging

Abstract

Surface functionalization of nanomaterials with specific recognition units is a promising strategy to structurally novel nanosensors for target analysis and bioimaging. In this study, a fluorescent nanosensor has been successfully synthesized by conjugating thiosemicarbazide with carbon dots (CDs) through amide bonds. The functional CDs have a relatively narrow size distribution of about 1.6–4.0 nm. The CDs exhibit a highly specific recognition capability towards copper ions (Cu²⁺) over other competing metal ions, and the maximum quenching rate reaches 68.7%. A good linear correlation is also established between F₀/F with the Cu²⁺ concentration ranging from 0 to 0.4 μM and the detection limit is 3.47 nM. On account of their strong fluorescence intensity, excellent water solubility, low toxicity and good biocompatibility the nanosensor demonstrates its biolabeling potential in vivo, which might have high significance in bioanalysis and biomedical detection in the future.

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Introduction

Copper ions as the third abundant transition metal ion in the human body play extremely important roles in many physiological processes.¹ A lot of redox reactions are dependent on copper enzyme catalysis in the cytoplasm and mitochondria.^2,3 However, excessive copper ions can lead to the generation of reactive oxygen species causing some important materials such as protein, nucleic acids and fat oxidative damage. At the same time, many serious neurological diseases, such as Alzheimer's, Parkinson's disease and Wilson disease are totally caused by copper metabolic abnormalities in the human body.^4–8 In addition, they are extremely detrimental to living organisms for example algae, bacteria, fungi and viruses.⁹ Hence, selective detecting and monitoring of copper ions in biological and environmental samples are highly meaningful for human health and environmental protection. The traditional measurement methods of Cu²⁺ include colorimetric method, capillary electrophoresis, atomic absorption spectroscopy (AAS), inductively coupled plasma mass spectrometry (ICP-MS), surface-enhanced Raman scattering, polarography, chemiluminescent method, electrochemical method and so on,^10–20 but these methods require time-consuming pretreatments, tedious procedures, organic reagents, complex and expensive instrumentations, etc. Nanoparticle-based fluorescent probes as gold and silver nanoparticles, semiconductor quantum dots have also been used to measure Cu²⁺ due to their fast analysis, high sensitivity and selectivity.^21–26 However because of their high cost, complicated synthesis process, water-insolubility, poor biocompatibility and toxicity limit their applications in routine analysis. In recent years, carbon dots, a new class of carbonaceous and fluorescent nanomaterials with spherical shape and size below 10 nm,²⁷ have attracted much attention for their unique characteristics, like much more sources of raw materials, easily synthetic method, high quantum yield, excellent optical and chemical stabilities, easy surface-functionalization, low toxicity, good water-solubility and biocompatibility,^28,29 and have been applied to determine many substances, for instance, DNA, nitrite, hypochlorite, ascorbic acid, 6-mercaptopurine, superoxide anion, pH, H₂S, metal ions, etc.^30–38 But much analysis are still confined by the pristine CDs owing to lack of no special selectivity to target objects. In order to further broaden their application fields, decorating nanomaterials with highly specific recognition elements: organic molecules and biomolecules, is proved to be a worthy way towards quantitative detection, highly efficient fluorescence imaging and real-time tracking of Cu²⁺. Now, many Cu²⁺ sensors on the basis of modified CDs have been reported. Qu et al. covalently bonded water-soluble C-dots with AE-TPEA ([N-(2-aminoethyl)-N,N,N-tris(pyridin-2-ylmethyl)ethane-1,2-diamine]) for intracellular sensing and imaging of Cu²⁺.³⁹ 1,4,8,11-Tetraazacyclotetradecane-functionalized CDs (CCDs) were developed for highly selective and sensitive detection of Cu²⁺ and S²⁻ in live cells and the CCDs nanosensor exhibited reversible switching property for sensing Cu²⁺ and S²⁻.⁴⁰ Rao et al. performed a ratiometric fluorescent sensor coordinating silica-coated carbon dots with CdTe quantum dots for determination of Cu²⁺ in vegetable and fruit samples and displayed a good selectivity and low detection limit.⁴¹ Wang et al. facilely fabricated a new functionalized carbon dots for selective copper ion sensing based on the fabrication of β-amino alcohol with fluorescent carbon dots.⁴² Zhu et al. synthesized AE-TPEA acting as a specific receptor for Cu²⁺ ions and conjugated it with the responsive CdSe@CDs nanohybrid to form a carbon dots-based fluorescence probe (CdSe@CDs-TPEA probe) for imaging and biosensing of Cu²⁺ ions in living cells. The detection limit was around 1 μM,⁴³ etc. In these methods several drawbacks were existed, such as long-time raw material synthesis, complex modified processes, usage of toxic metals and much organic matters, not so low detection limits (>5 nM), etc.

In this study, a novel Cu²⁺ nanosensor was built only by conjugating thiosemicarbazide (TSC) onto the surface of CDs via amide bonds. The CDs were prepared by hydrothermal treatment taking EDTA as substrate and the surface of the as-prepared CDs are rich in –NH₂, –OH and –COOH. Thiosemicarbazide (TSC) was chosen as a Cu²⁺ identifying molecule. The synthesis process was shown in Scheme 1. The sensor displays special selectivity toward Cu²⁺ comparing with other metal ions. The good linearity is also achieved with the concentrations of Cu²⁺ from 0 to 0.4 μM and the detection limit is 3.47 nM. More importantly, the fluorescent probe also has excellent water solubility, low toxicity, good biocompatibility and cell-membrane permeability and can be employed for intracellular bioimaging and bioanalysis.

Scheme 1 Illustration of the synthesis procedures for the CDs-based nanosensor and schematic diagram of Cu²⁺ detection.

Materials and methods

Materials

EDTA was supplied by Sinopharm Chemical Reagent Co. (Shanghai, China) and used directly. Thiosemicarbazide (99%) was gotten from Aladdin Industrial Corporation (Shanghai, China). KCl, NaCl, ZnCl₂, BaCl₂, MnCl₂, AlCl₃ and AgNO₃, Mg(NO₃)₂, Cu(NO₃)₂, Ni(NO₃)₂, Co(NO₃)₂, Cd(NO₃)₂, Pb(NO₃)₂, Hg(NO₃)₂, Fe(NO₃)₃, FeSO₄ were dissolved in tris(hydroxymethyl)methyl amino methane and acetic acid buffer solution (Tris–HAc, 10.0 mM, pH = 7.4) to afford 10.0 mM cations, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxy-succinimide (NHS) were purchased from Exlen Chemistry Technology Co., Ltd. (Shanghai, China). The ultrapure water from a Milli-Q ultrapure water system (Millipore, Billerica, MA, USA) was used throughout the experiments. The other chemicals in this work were all of analytical grade and used without further purification.

Apparatus

High-resolution TEM (HRTEM) images of the CDs and TSC-CDs were taken on a JEOL JEM-2010 electron microscopy (JEOL, Japan) with an accelerating voltage of 200 kV by dropping the sample onto a carbon-coated copper grid. Fourier transform infrared spectroscopy (FT-IR) spectra were recorded by spectrum 400 F spectrometer (PerkinElmer, USA). X-ray photoelectron spectroscopy (XPS) analysis was carried out on PHI Quantera spectrometer with Al Kα excitation. Binding energy was calibrated by C 1s of 284.8 eV. UV-vis absorption spectra were collected by a TU-1810 spectrophotometer (Puxi Analytic Instrument Ltd., Beijing, China). All fluorescence emission spectra were scanned on a Cary Eclipse fluorescence spectrophotometer (VARIAN, USA) with different excitation wavelengths. The fluorescence bioimaging assays were conducted by a Leica laser confocal fluorescence microscope (TCSSP5).

Preparation of raw CDs

The CDs were gained according to a reported method with a minor change.⁴⁴ In brief, 3.00 g EDTA was added into 30.0 mL ultrapure water to form a clear solution. Then the solution was transferred to a 50 mL Teflon-equipped stainless steel autoclave and heated to 180 °C for 10 h and cooled down to room temperature naturally. The resulting solution was purified by discarding non-fluorescent deposit and large particles through centrifuging at 15 000 rpm for 20 min and filtering through 0.22 nm filter membrane, respectively. The product was dried in a vacuum drying oven at 65 °C for 24 h and stored at 4 °C.

Thiosemicarbazide modified-CDs (TSC-CDs)

Based on the literature,⁴⁵ CDs (0.20 mg) were dissolved into 20.0 mL of ultrapure water. After adding 10.0 mL EDC (20.0 mM) and 5.0 mL NHS (20.0 mM), the mixed solution was incubated at 37 °C for 30 min at 50 rpm. Then 5.0 mL thiosemicarbazide solution (50.0 g L⁻¹) was added to the reaction mixture and reacted for another 3 h. In the end the mixture was stored at 4 °C overnight to deactivate the remaining EDC–NHS. The unreacted thiosemicarbazide was discarded by dialysis through a porous cellulose membrane (MWCO 1000) against the pure water for 24 h. The purified nanosensor was dried and kept at 4 °C for further use.

Fluorescence assay of Cu²⁺

100 μL 0.01 g L⁻¹ TSC-CDs aqueous solution and a certain amount of Cu²⁺ were placed in a 1.5 mL centrifuge tube. The mixture was diluted to 1.0 mL with Tris–HAc solution and blended thoroughly. After reacting for 15 min the fluorescence spectra were recorded with an excitation at 370 nm. The sensitivity of Cu²⁺ was evaluated by introducing a series of concentrations of Cu²⁺ from 0 to 50.0 μM. The interference experiments were also explored by mixing the above metal ions in the absence and presence of Cu²⁺ under the same experimental conditions as the Cu²⁺ detection.

Cytotoxicity test

The cytotoxicity of TSC-CDs was estimated by MTT assay. HeLa cells were first seeded in microtiter plates at a density of 5 × 10⁴ cells per mL in Dulbecco's modified Eagle medium (DMEM) supported by 10% fetal bovine serum (v/v), 2 mM glutamine, 100 U mL⁻¹ penicillin, and 100 U mL⁻¹ streptomycin for attachment. After maintaining for 24 h at 37 °C in an atmosphere of 5% CO₂, the cells were cultured with different concentrations of the TSC-CDs for another 24 h. Then 20 μL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT, 5.0 g L⁻¹) was injected to every cell well and the cells were further incubated for 4 h. Removing the culture medium by 150 μL of dimethyl sulfoxide (DMSO) and shaking for 15 min at room temperature, the optical density (OD) was taken at 570 nm on a microplate reader (Biorad) and the values were compared to the control cells.

Bioimaging

E. coli and HeLa cells were selected to discuss the feasibility of TSC-CDs as bioimaging agents. After E. coli were cultured in nutrient broth at 37 °C overnight, 1.0 mL bacterial cells were centrifugated at 8000 rpm for 5 min, washed with PBS buffer solution (10.0 mM, pH = 7.4) for three times and finally resuspended in 1.0 mL of PBS solution. When the cells were treated with 1.0 mL 0.1 g L⁻¹ TSC-CDs for 8 h, the excess TSC-CDs were removed via washing three times with PBS. The fluorescence images were taken on a laser confocal fluorescence microscope.

Adding a certain amount of TSC-CDs to the HeLa cells culture the cells were incubated for 8 h and washed with PBS, the fluorescence images were captured.

After TSC-CDs uptake, the E. coli and HeLa cells were supplemented with Cu²⁺ (30.0 μM) for another 2 h and detected by confocal fluorescence microscope again.

Results and discussion

Characterization of TSC-CDs

In this work, the pristine CDs were obtained by the hydrothermal method using EDTA as the carbon source to get the carboxyl capped CDs. Then the thiosemicarbazide-functionalized CDs were synthesized through the reaction of carboxylic groups of the prepared CDs with amine groups of thiosemicarbazide. The as-synthesized TSC-CDs are highly water soluble. The particle size and morphology of the TSC-CDs were examined by HRTEM (Fig. 1). The images show that the TSC-CDs have good monodispersity and narrow size distribution in the range of 1.6 to 4.0 nm with the average size of 2.63 nm (Fig. 1b2) which have a little change in contrast to the pristine CDs (2.38 nm, Fig. 1a2). The characterization certifies the successful conjugation of thiosemicarbazide on the surface of CDs. The conjugated thiosemicarbazide devoted 0.25 nm to the overall diameter.

Fig. 1 Transmission electron microscopy images of the synthesized CDs (a1) and the nanosensor (b1). Inset: the HRTEM images of the CDs and the nanosensor, respectively. Corresponding size distributions of the CDs (a2) and the nanosensor (b2).

The surface functional groups of the CDs, TSC-CDs and TSC were monitored by FT-IR spectrum. The results were displayed in Fig. 2. The broad bands at 3426 cm⁻¹ and 1622 cm⁻¹ are assigned to the stretching vibration of N–H and C=O respectively (curve b), the bands at 3055 cm⁻¹ and 1324 cm⁻¹ are from the stretching vibration and deformation peak of O–H, which suggest that there are many amine groups, hydroxyl groups and carboxyl groups on the surface of the prepared CDs which made the CDs excellent water-solubility. Compared to the CDs, in the FT-IR spectra of TSC-CDs (curve c) the peaks at 3368 cm⁻¹ (ν_N–H) and 1622 cm⁻¹ (ν_C=O) are also existent, but the peak at 3055 cm⁻¹ (ν_O–H) is absence, these mean that the carboxyl groups of the prepared CDs are converted into amide groups. The result is verified by the emerging peak at 1288 cm⁻¹ (ν_–NHC=O). In addition, the peaks at 3263 cm⁻¹ and 3179 cm⁻¹ originated from thiosemicarbazide (curve a) are also observed in the spectra of the TSC-CDs. The peaks at 1398 cm⁻¹ and 1001 cm⁻¹ are assigned to C=S bending vibration and stretching vibration, weak C–S stretching vibration is detected at 801 cm⁻¹. The FT-IR analysis confirms the successful decoration of thiosemicarbazide onto the surface of CDs.

Fig. 2 FT-IR spectra of TSC (a), CDs (b) and TSC-CDs (c).

XPS spectra were acquired to illustrate the composition of TSC-CDs. The XPS survey spectrum (Fig. 3a) of TSC-CDs shows three dominant peaks at 529.5 eV, 397.6 eV, and 282.6 eV which are ascribed to O 1s, N 1s and C 1s respectively. The other two weak peaks at 226.6 eV and 159.4 eV are assigned to S 2s and S 2p. For the high-resolution C 1s spectrum (Fig. 3c) five peaks at 284.0, 284.7, 286.0, 287.6 and 288.5 eV are belonged to the C–C/C=C, C–O, C–S, C=O/C=S and C–N groups, respectively.^46,47 The spectrum of O 1s (Fig. 3d) indicate two peaks at 531.3 eV and 532.8 eV, which are corresponded to C=O and O–H. The peaks at 161.7 and 163.1 eV in the S 2p pattern (Fig. 3b) are close to the 2p_3/2 and 2p_1/2 positions of the –C–S bonds,⁴⁸ which are in good accord with the FT-IR analysis. Furthermore, the TSC-CDs contain 69.1%, 8.3%, 20.2%, 2.4% of C, N, O and S, respectively, testifying that thiosemicarbazide has been modified on the surface of the TSC-CDs.

Fig. 3 Full survey XPS spectra of the obtained TSC-CDs (a) and high-resolution XPS spectra of S 2p (b), C 1s (c) and O 1s (d) of TSC-CDs.

The optical properties of thiosemicarbazide, CDs, and TSC-CDs were investigated using UV-vis absorption spectra. The results (Fig. 4) expressing a new absorption of the TSC-CDs at 289 nm except the original absorption of the CDs reveal a representative combination of thiosemicarbazide and the synthesized CDs.

Fig. 4 UV-vis absorption of CDs, TSC-CDs and TSC.

The fluorescence property of TSC-CDs was also surveyed. The fluorescence emission is excitation-dependent (Fig. 5) in the wavelength range from 290 to 510 nm. Increasing the excitation wavelength, the fluorescence peak was red shifted and the fluorescence intensity changed. The strong fluorescence emission is received at 450 nm under the 370 nm maximum excitation wavelength (Fig. 6). The excitation-dependent PL behavior may result from the multiple C-, N-, O- and S-containing functional groups on the surface of the TSC-CDs, thus these groups result in various surface states with different energy levels and emissive traps. Exciting with a 365 nm lamp the TSC-CDs aqueous solution emits intense blue light (Fig. 6, inset).

Fig. 5 Fluorescence emission spectra of the TSC-CDs under different excitation wavelengths.

Fig. 6 Fluorescence excitation (a) and emission spectra (b) of TSC-CDs in aqueous solution. Inset: photograph of TSC-CDs under UV light (365 nm).

pH, buffer systems and ionic strength effects on the fluorescence of TSC-CDs

As different pH may significantly affect the particle surface states of TSC-CDs and further impact on the fluorescence intensity, the experiments were carried out to seek for the optimal pH value by adding 0.1 M NaOH and HCl to change pH values of TSC-CDs solutions. The effects were given in Fig. 7a, when pH increases from 3 to 10, only a small decrease in the fluorescent intensity is noticed. This variation can be the consequence of the functionalization by TSC, which has a high pK_a value (pK_a = 13) and its existence is in the undissociated form, so the fluorescence emission of TSC-CDs remains stable. As copper ions easily hydrolyze in the higher pH, pH = 7.4 is selected as the optimum pH. In order to improve the detection of Cu²⁺, buffer systems effects on fluorescence intensity were estimated at the same pH (pH = 7.4) using PBS buffer, Tris–HCl buffer, Tris–HAc buffer, HAc–NaAc buffer and citric acid–sodium citrate (CA–SC) buffer. The maximal FL intensity is tested in Tris–HAc buffer (Fig. 7b). The fluorescence intensity also remains constant at various concentrations of NaCl (between 0 and 1.0 M) (Fig. 7c). These stabilities are beneficial for applicating the nanosensor in practical sensing and bioimaging.

Fig. 7 pH (a), buffer systems (b) and concentration of NaCl (c) effects on fluorescence intensity of the TSC-CDs at 370 nm excitation wavelength.

Detection strategy

Adding Cu²⁺ ions in the unfunctionalized CDs only small fluorescence changes happened at the excitation of 370 nm (Fig. 8a), but when the same concentration of Cu²⁺ ions were added in the functionalized CDs the emission intensity of TSC-CDs notably decreased (Fig. 8b). It is well known that nitrogen atoms have strong coordinating ability with copper ions and C=S/C=O is also an effective coordination site for heavy metal ions.⁴⁹ Attaching TSC on the surface of CDs may play a key role in the Cu²⁺ sensing. The complexation of Cu²⁺ with the TSC-CDs may lead to the obvious fluorescence quenching of CDs possibly due to the inner filter effect (Scheme 1). Just as the literature reported,⁴⁹ Shi succeeded in designing a copper probe through bonding thiosemicarbazide with fluorescent material (anthraquinone) and used it for the copper ions determination in biological living cells.

Fig. 8 Fluorescence quenching of CDs (a) and TSC-CDs (b) at the same concentration of Cu²⁺ (30.0 μM) at 370 nm excitation wavelength.

Sensitivity and selectivity of the fluorescence probe

The quenching was analyzed by Stern–Volmer plots. From Fig. 9a it can be seen the FL intensities reduce regularly with increasing the concentrations of Cu²⁺ from 0 to 50.0 μM which demonstrates that Cu²⁺ can effectively bond with TSC on the surface of TSC-CDs and the quenching rate is up to 68.7%. Meanwhile a good linear response is gained under the concentrations of Cu²⁺ in the range of 0–0.4 μM (Fig. 9b inset). The Stern–Volmer equation is F₀/F = 0.9989 + 1.098[Cu²⁺] (R² = 0.9998, F₀ and F are the fluorescence intensities in the absence and presence of Cu²⁺, respectively.), the quenching constant was 1.10 × 10⁵ L mol⁻¹. The detection limit (LOD) is calculated to be 3.47 nM (LOD = 3σ/s) which is much lower than the upper limit of copper ions in drinking water (20 μM) by the U.S Environmental Protection agency⁵⁰ and other carbon dots-based Cu²⁺ sensors (Table 1).

Fig. 9 (a) Fluorescence responses of TSC-CDs upon adding different concentrations of Cu²⁺ (0, 0.03, 0.10, 0.15, 0.20, 0.25, 0.40, 5.0, 10.0, 30.0, 50.0 μM) (from top to bottom). (b) The dependence of FL intensity on the Cu²⁺ concentration in the range of 0 to 50.0 μM, inset shows the linear correlation between the F₀/F and the concentration of Cu²⁺. TSC-CDs: 0.01 g L⁻¹; λ_ex = 370 nm.

Table 1 Comparison of different carbon dots-based methods for Cu²⁺ detection

Material	Modified compounds	Detection limit	Ref.
N-CDs	Polyethyleneimine	0.090 μM	51
N-CDs	Prawn shells	5.0 nM	52
TPEA-CDs	AE-TPEA	10.0 nM	39
N,S-CDs	[C4mim][Cys]	0.18 μM	53
CD@SiO2@CdTe	CdTe	0.096 μM	41
APTES-CDs	3-(Aminopropyl)-triethoxysilane	0.30 μM	54
CCDs	1,4,8,11-Tetraazacyclo tetradecane	0.13 μM	40
BPEI-CDs	Branched poly(ethyleneimine)	6.0 nM	55
CdSe@CDs-TPEA	AE-TPEA	1.0 μM	42
BSA-CDs	Bovine serum albumin and lysine	1.3 ppm	45
TSC-CDs	Thiosemicarbazide	3.47 nM	This work

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In order to assess the possibility of our approach, the selectivity of TSC-CDs probe toward Cu²⁺ ions were researched. First, 30.0 μM different metal ions such as Na⁺, K⁺, Ag⁺, Zn²⁺, Mg²⁺, Ba²⁺, Ni²⁺, Co²⁺, Mn²⁺, Pb²⁺, Cu²⁺, Cd²⁺, Fe²⁺, Hg²⁺, Al³⁺, and Fe³⁺ ions were separately added into the TSC-CDs solutions and the FL signals were collected in Fig. 10a. The figure depicts that a distinct quenching effect is observed after the addition of Cu²⁺, the other metal ions only show a slight FL signal change and the influences are almost negligible except Ag⁺. But the quenching of Ag⁺ is only one sixth as much as that of Cu²⁺ suggesting that the nanosensor responds selectively towards Cu²⁺. In another experiment, 30.0 μM of the above-mentioned metal ions were added into the TSC-CDs solutions containing 30.0 μM Cu²⁺ respectively at the same experimental conditions, then the fluorescence intensity was measured. The results in Fig. 10b manifest the negligible influence from the other coexisting metal ions in comparison with the efficient quenching effect of Cu²⁺. These observations indicate that the TSC-CDs sensor is more sensitive and selective towards Cu²⁺ than the other metal ions.

Fig. 10 Fluorescence responses of the nanosensor to different metal ions (30.0 μM) in the absence (a) and presence (b) of the same concentration of Cu²⁺, respectively. TSC-CDs: 0.01 g L⁻¹; λ_ex = 370 nm.

Cytotoxicity and bioimaging

For effective bioimaging, the fluorescent marker must have both optical merits and low cytotoxicity. To evaluate the cytotoxicity of TSC-CDs, the MTT assay was conducted selecting HeLa cells as representative cell lines which were treated with different concentrations of TSC-CDs. From Fig. 11 we can see that the viability remains greater than 93.9% when the cells were exposed with TSC-CDs at a concentration of 0.5 g L⁻¹ for 24 h. Increasing the concentrations of TSC-CDs from 1.0 g L⁻¹ to 2.0 g L⁻¹ the cells viability only decreased to 87.9%, 87.2% and 86.4% respectively, most cells are still alive verifying that TSC-CDs are scarcely toxic in nature.

Fig. 11 Cell viability values (%) estimated by MTT assays after incubation with different concentrations of the nanosensor (0.1, 0.2, 0.5, 1.0, 1.5 and 2.0 g L⁻¹).

Considering of its small size, low cytotoxicity and good water-solubility, the performance as fluorescent cell labels were investigated by HeLa cells and E. coli incubating with TSC-CDs for 8 h. After incubating with TSC-CDs the HeLa cells and E. coli become brightly illuminated with blue (Fig. 12c and h) and green colors (Fig. 12d and i) at excitation wavelengths of 405 and 488 nm respectively, imaging on a confocal fluorescence microscope. However, no emission signals from the cells (without TSC-CDs) are observed (Fig. 12b and g) at the same condition, which suggest that a large amount of the TSC-CDs have been internalized into the HeLa cells and E. coli and they have good biocompatibility. It is also clear that the nanosensor can easily cross the cell membrane and accumulate in the cells. But further incubating with 30.0 μM Cu²⁺ ions for another 2 h the green fluorescence images from the TSC-CDs became dimmer (Fig. 12e and j). These results indicate that the TSC-CDs can be acted as a promising candidate for imaging with multicolour and biosensing.

Fig. 12 Confocal fluorescence microscopic images of HeLa cells (a–e) and E. coli cells (f–j): control (b and g) and cells treated with TSC-CDs before (a, c, d and f, h, i) and after (e and j) incubating with Cu²⁺ under bright field (a and f), 405 nm (c and h) and 488 nm excitation (d, i, e and j), respectively.

Conclusions

In this work, a special sensitive and selective fluorescent nanosensor has been successfully developed via carbodiimide chemistry with CDs as fluorescent material and thiosemicarbazide as recognition unit for the first time. The synthesized TSC-CDs have a narrow size and strong fluorescence intensity. It is suitable for simple, highly sensitive and selective detection of trace Cu²⁺ in aqueous media and the detection limit is as low as 3.47 nM in a wide range of pH values and ionic strengths. The detection mechanism is also proposed that it may be caused by the complexation of Cu²⁺ with the TSC-CDs leading to the obvious fluorescence quenching of CDs due to the inner filter effect. Possessing excellent water solubility, low toxicity and perfect biocompatibility the nanosensor can be acted as optical imaging agents to HeLa cells and E. coli. The imaging results imply that they have great potentials in bioanalysis and biomedical detection, which might be high important in clinical diagnosis in the future. Moreover, this method is valuable to design specific CDs-based nanosensors adopting other recognition elements and enlarge its application fields.

Acknowledgements

We gratefully acknowledge the financial support of the Nature Science Foundation of China (30970696 and 21172056), Key Programs of Henan for Science and Technology Development (142102310273), PCSIRT (IRT1061), Key Project of Henan Ministry of Education (14A150018), the Natural Science Foundation of Henan Province (No. 132300410139) and Pillar Project of postdoctor (2015M582188), the Program for Innovative Research Team in University of Henan Province (2012IRTSTHN006), the analysis support from the Instrumental Analysis Center of Tsinghua University.

Notes and references

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