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Original article
03 2022
:16;
104520
doi:
10.1016/j.arabjc.2022.104520

S,O-doped carbon nitride as a fluorescence probe for the label-free detection of folic acid and targeted cancer cell imaging

, , , , , , , ,
a Clinical Laboratory, The Second Hospital of Shanxi Medical University, Taiyuan 030000, China
b College of Pharmacy, Shanxi Medical University, Taiyuan 030001, China
c School of Basic Medical Science, Shanxi Medical University, Taiyuan 030000, China
d People's Hospital of Lvliang, Lvliang 033099, China
e Bristol Chinese Christian Church, c/o Tyndale Baptist Church, 137-139 Whiteladies Road, Bristol BS8 2QG, United Kingdom

⁎Corresponding author. sxykdx_bianwei@163.com (Wei Bian)

Received: , Accepted: ,
© The Author(s) 2022
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
1 These authors contributed equally to this work.

Abstract

A novel nanoprobe based on S,O-doped carbon nitride quantum dots (S,O-CNQDs) was designed and synthesized. The as-prepared S,O-CNQDs exhibits good biocompatibility and strong fluorescence at excitation 360 nm. It is found that folic acid (FA) could efficiently quench the fluorescence of S,O-CNQDs. The obtained S,O-CNQDs is capable of acting as a sensitive and selective probe for FA detection in the range 5.0–83.3 μM with a detection limit of 90 nM. The as-prepared probe has been successfully utilized for the detection of FA in various real samples with satisfactory recoveries (98.8–107 %) and small relative standard deviation (<5%). The reaction mechanism between S,O-CNQDs and FA has been discussed. In addition, FA-S,O-CNQDs formed through a classical cross-linking reaction between FA and S,O-CNQDs easily accesses and penetrates into HepG2 cells with high folate receptors expression. FA-S,O-CNQDs with low cytotoxicity and good biocompatibility shows great potential in FA detection and targeted imaging of cancer cells.

Keywords

S, O-CNQDs
Folic acid
Fluorescence quenching
Cellular imaging
Real samples
1

1 Introduction

Folic acid (FA), a water-soluble vitamin, participates in many physiological processes as a substrate and coenzyme (Xu et al., 2022). Deficiency or overdoses of FA may lead to some diseases such as megaloblastic anemia, stomatitis and seborrheic dermatitis, etc (Shulpekova et al., 2021). Therefore, it is vital to develop highly selective and sensitive FA detection methods. Up to now, researchers have developed a variety of methods for FA determination, including microbiological methods (Iyer and Tomar, 2013), surface-enhanced Raman scattering (Sun et al., 2016), high-performance liquid chromatography (Wang et al., 2018); and electrochemical analysis (Huang et al., 2022). Although most of the above-mentioned methods present encouraging results with high sensitivity and reliability, they still suffer from some defects such as complicated operation, high cost, stringent experimental conditions (Wang et al., 2021; Massey et al., 2020; Li and Zhu, 2020). While the fluorescence method that has the advantages of simple operation, low cost, good selectivity and sensitivity has been widely utilized in the detection of drugs (Lv et al., 2022; Atchudan et al., 2020; Atchudan et al., 2019; Atchudan et al., 2018; Atchudan et al., 2017; Atchudan et al., 2016).

In the field of fluorescence detection, carbon nitride nanomaterials as emerging carbon materials have captured considerable attention from researchers due to their properties of good photostability, low cytotoxicity and easy surface functionalization, which has been widely used in chemical analysis, biosensing and bioimaging (Ding et al., 2021; Devi et al., 2021; Liu et al., 2020). Nevertheless, the drawbacks of low quantum yield (QY), unsatisfactory catalytic activity and narrow energy bandgap limit their broad applications in lots of fields (Rahman et al., 2018; Mazzanti and Savateev, 2020; Teng et al., 2017). In order to overcome these defects, researchers found that doped carbon nitride with different elements posssess more excellent chemical/optical properties and show more promising applications than non-doped carbon nitride (Zhang et al., 2021).

Moreover, compared to normal cells, the expression of folate receptor (FR) which is a high-affinity folate binding protein in malignant cells is higher and positively correlated with the staging of tumors (Prajapati et al., 2019). FR is generally considered as a biomarker related to cancer. Through receptor-mediated non-immunogenic endocytosis, FA/FA analogs/FA complexes can specifically bind to FR that exist on the surface of cancer cell membranes. The targeted recognition of cancer cells can be achieved through FR-mediated endocytosis (Zhang et al., 2018). Up to now, FA-modified nanomaterials have been exploited as fluorescent probes for targeted imaging of cancer cells due to FA could specifically bind to folate receptors.

In this work, S,O-CNQDs with high stability and good bio-compatibility was synthesized by using ethylenediaminetetraacetic acid disodium and thiourea as the precursors. Based on the static quenching effect, the as-prepared S,O-CNQDs as a fluorescent probe has been utilized for the detection of FA and exhibited good sensitivity and selectivity. The quenching mechanism between S,O-CNQDs and FA was preliminary explored. Moreover, S,O-CNQDs has been successfully applied for the detection of FA in real samples. FA-functionalized S,O-CNQDs was used for targeted imaging of FR overexpressed cancer cells.

2

2 Material and methods

2.1

2.1 Materials

N-Hydroxysuccinimide (C4H5NO3), FA (C19H19N7O5, >98 %) and 1-ethyl(3-dimethylaminopropyl) carbodiimide hydrochloride (C8H17N3·HCl) were purchased from Shanghai Aladdin Reagent Co., ltd. Sodium dihydrogen phosphate (NaH2PO4), disodium hydrogen phosphate (Na2HPO4) and sodium phosphate (Na3PO4) were acquired from Tianjin Beichen Founder Reagent Factory. Methyl thiazolyl tetrazolium(MTT) was obtained from Beijing Soleboard Technology Co., ltd. RPMI 1640 medium and fetal bovine serum were purchased from Hyclone Company, USA. All chemicals of analytical grade reagents were used without further purification.

2.2

2.2 Apparatus and characterization

Transmission electron microscopic (TEM) images were taken using a JEOL 2010-H TEM (Tokyo, Japan). X-ray photoelectron spectra (XPS) were carried out with an AXIS ULTRADLD X-ray photoelectron spectrometer (Kratos, Tokyo, Japan). X-ray diffraction (XRD) was performed on a D8 Advance powder X-ray diffractometer (Bremen, Germany). Fourier transform infrared spectrum (FT-IR) of S,O-CNQDs was measured on a Varian FT-IR-640 spectrometer (Agilent Technologies, Palo Alto, CA, US). Ultraviolet/visible spectra and fluorescence spectra were recorded on a Metash 6100 UV–vis spectrophotometer (Shanghai, China) and a Hitachi F-4500 spectrofluorometer (Tokyo, Japan), respectively. The fluorescence lifetimes were performed on an Edinburgh Instruments FLS920 steady-state transient fluorescence spectrometer (Livingston, UK). The cellular fluorescence images were recorded by an Olympus FV3000 confocal laser scanning microscope (Tokyo, Japan).

2.3

2.3 Preparation of S,O-CNQDs

The synthesis process of S,O-CNQDs was according to the previous work with minor modifications (Lu et al., 2015). Briefly, Ethylenediaminetetraacetic acid disodium (0.3722 g) and thiourea (0.9134 g) were ground uniformly in a mortar. Then the mixture powder was transferred to a crucible and reacted in an oven at 200℃ for 2 h. After the reaction was completed, the crucible was naturally cooled to room temperature. The brown product was washed three times with absolute ethanol. A brown–red solution was obtained after the washed product was dissolved in 10 mL ultrapure water. Then, the solution was centrifuged and filtered with a 0.22 μm microporous membrane. The filtrate was placed in a dialysis bag (with a molecular weight of 500 Da) and dialyzed with ultrapure water for 8 h. The purified S,O-CNQDs solid powder was obtained by freeze-drying dialysate.

2.4

2.4 Detection of FA

A 3.0 mL mixed solution was prepared by mixing S,O-CNQDs (0.15 mg/mL, 10 μL), different concentrations of FA and phosphate buffer solution (10 mM, pH 7.0). The fluorescence spectrum was captured by a fluorescence spectrometer after 5-min reaction at room temperature. The fluorescence spectra of samples were recorded at an excitation wavelength of 359 nm with slit widths of 5 nm. Due to FA was unstable when exposed to light, the experiment should be carried out in the dark.

2.5

2.5 Real samples pre-treatment and analysis

Human urine and blood samples were donated from healthy adult volunteers. After the urine samples were centrifuged at 10000 rpm for 20 min, the supernatant was diluted 100 times with 10 mM PBS (pH 6.0) for subsequence use. The human blood samples were mixed with equal volume of acetonitrile (Yang et al., 2019). The mixture was centrifuged at 10000 rpm for 10 min. The supernatant was filtered through a 0.22-μm microporous membrane. The filtrate was diluted 20 times with 10 mM PBS (pH 6.0) for further use. The ground oats and fruit juice purchased from local supermarkets were dispersed in NaOH solution. The obtained solutions were centrifuged and filtered with a 0.22 μm microporous membrane. The filtrate was diluted with phosphate buffer solution (10 mM, pH 7.0) for future use.

2.6

2.6 Assembly of folic acid onto S,O-CNQDs

A yellow solution was obtained by dissolving 20 mg FA in 8.0 mL phosphate buffer solution (10 mM, pH 7.4). Then, 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride (0.023 g) and N-hydroxy succinimide (0.028 g) were added to the above solution, respectively (Tang et al., 2022). After the solution was stirred at room temperature for 4 h, S,O-CNQDs (5.0 mL, 23 mg) was added and the reaction was continued for 24 h. Then the solution was dialyzed for another 24 h (with a molecular weight of 1000 Da). The yellow solid powder was prepared for further use after freeze-drying the solution.

2.7

2.7 Cytotoxicity test

The cytotoxicity of S,O-CNQDs on HepG2 cells was assessed through MTT assay. The HepG2 cells (5 × 103 cells per well) were planted in a 96-well plate with Dulbecco minimum essential medium (DMEM) as the medium and cultured in an incubator with an atmosphere of 5.0 % CO2 at 37℃ for 24 h. After HepG2 cells were treated with different concentrations of FA-S, O-CNQDs for 4 h, 100 μL of MTT solution was added into each well. The cells were incubated for another 4 h and the MTT solution was discarded. Finally, 150 μL of DMSO was added to each well and the absorbance was recorded by a microplate reader at 490 nm. The cell viability was calculated through Eq. (1).

(1)
C e l l v i a b i l i t y % = O D t r e a t e d / O D c o n t r o l × 100 %

2.8

2.8 Targeted fluorescence imaging of cancer cells

The targeted imaging experiments were carried out on the liver cancer cells with high FR expression (HepG2) (Cao et al., 2018; Liao et al., 2015; He et al., 2017; Zhang et al., 2013) and normal liver cells without FR expression (HL-7702) (Tang et al., 2009). Firstly, HepG2 and HL-7702 cells were inoculated into laser confocal dishes and cultured in a cell incubator for 24 h, respectively. HepG2 and HL-7702 cells were treated with FA-S,O-CNQDs (500 μg/mL) for 2 h. Then the cells were washed 3–5 times with PBS solution and added 4 % paraformaldehyde that was used to fix cells for 10–15 min. Cells were washed 3–5 times with PBS and added 300 μL PBS. The cells were observed under a laser confocal microscope.

In order to further prove the way that FA-S,O-CNQDs entered cells, HepG2 cells were incubated with excessive free FA (50 μg/mL) for 1 h, cleaned with PBS for 2–3 times and then incubated with FA-S,O-CNQDs (500 μg/mL) for 2 h. The cells were observed by a laser confocal microscope at the excitation wavelengths of 405 and 488 nm.

3

3 Results and discussion

3.1

3.1 Characterization of S,O-CNQDs and FA-S,O-CNQDs

Transmission electron microscopy (TEM) was used for the morphologies characterization of S,O-CNQDs. TEM and HRTEM images of S,O-CNQDs are shown in Fig. 1 and the inset of Fig. 1, respectively. The S,O-CNQDs are nearly spherical and highly dispersed with a distinct lattice fringe of 0.32 nm.

TEM image of S,O-CNQDs. The inset displays the HRTEM of S,O-CNQDs.
Fig. 1
TEM image of S,O-CNQDs. The inset displays the HRTEM of S,O-CNQDs.

Fig. 2 displays the X-ray powder diffraction pattern (XRD) of S,O-CNQDs which shows that S,O-CNQDs has two characteristic diffraction peaks at 13.7 and 27.3°. The peak at 27.3° is attributed to the interlayer superposition diffraction of graphite phase carbon nitride while the peak at 13.7° corresponds to the triazine ring structure of CNQDs (Das et al., 2017).

XRD pattern of the S,O-CNQDs.
Fig. 2
XRD pattern of the S,O-CNQDs.

XPS analysis results in Fig. 3a demonstrate that S,O-CNQDs contain four elements (C, N, O, and S) and their binding energies are located at 284.78, 399.18, 530.74, 225.90, and 161.66 eV, respectively. Fig. 3b shows the fine spectrum of C1s. It can be seen that there are absorption peaks with binding energies of 284.93, 283.90, 285.66, 284.40, 287.50, and 287.00 eV, belonging to C⚌C, C—S—C, C-OH/C—O—O, N⚌C = N, and C⚌O/COOH, respectively (Bian et al., 2016). The N1s peak in Fig. 3c is divided into three peaks which can be assigned to N-(C)3 (399.25 eV), –NH (398.50 eV), and pyridine nitrogen (399.97 eV) (Li et al., 2021). The O1s peak in Fig. 3d is split into three peaks at 530.67, 531.10 and 530.10 eV, which is corresponding to C⚌O, –OH and C-OH, respectively (Yang et al., 2018). Fig. 3e displays the S2p spectrum. The two fitted peaks at 163.95 and 168.85 eV are attributed to the C—S bond at 2p1/2 and 2p3/2, respectively (Bai et al., 2017).

(a) Full XPS of the S,O-CNQDs. Deconvoluted high-resolution XPS of (b) C1s, (c) N1s, (d) O1s, and (e) S2p.
Fig. 3
(a) Full XPS of the S,O-CNQDs. Deconvoluted high-resolution XPS of (b) C1s, (c) N1s, (d) O1s, and (e) S2p.

Fig. 4 depicts the UV/visible absorption spectrum of S,O-CNQDs (curve 1). It can be seen that there are two peaks at 263 and 358 nm, which were attributed to the s-triazine ring and n-π* transition of S,O-CNQDs, respectively (Wang et al., 2018). FA has three characteristic absorption peaks at 218, 282 and 360 nm with the maximum absorption peak at 282 nm (curve 3). After FA has coupled to the surface of S,O-CNQDs, the maximum absorption peak of FA-S,O-CNQDs is blue-shifted to 278 nm (curve 2). This blue shift phenomenon can be attributed to the π-π* transition of the carbon–carbon double bond (Hai et al., 2018). S,O-CNQDs can be coupled with FA through EDC/NHS as the coupling agents. The amino group of S,O-CNQDs is linked with the carboxyl group of FA. Fig. S1a shows the FT-IR of FA, S,O-CNQDs and FA-S,O-CNQDs. The absorption peaks of S,O-CNQDs (curve 3) at 3400–3500 cm−1 are attributed to the stretching vibration of O—H/N—H. The absorption peaks of C⚌O and –COOH groups are at 1670 and 1410 cm−1, respectively. The peaks at 1180–1080 cm−1 show the existence of C⚌S/C⚌N (Li et al., 2020). The characteristic absorption of the triazine ring is at 809 cm−1 (Song et al., 2020). The IR spectrum of FA (curve 2) displays a characteristic peak at 1694 cm−1 which is ascribed to the carbonyl group of FA. The peaks at 1640 cm−1 is caused by the carbonyl stretching vibration in –CONH2. Peaks at 1605 and 1484 cm−1 belong to the characteristic absorption of phenyl and pterin rings in FA (Zhao et al., 2017). Compared to the IR spectra of FA and S,O-CNQDs, the absorption peaks of FA-S,O-CNQDs (curve 1) at 1611 and 1690 cm−1 prove the existence of amide bonds and the peaks at 1605 and 1484 cm−1 show the existence of phenyl and pterin in folic acid, indicating that FA is successfully coupled to the surface of S,O-CNQDs (Goreham et al., 2018). The fluorescence emission spectrum of S,O-CNQDs modified by FA in Fig. S1b is almost not affected by FA. The fluorescence intensity of FA-S,O-CNQDs decreases by about 20 % as compared to that of S,O-CNQDs. In addition, the surface potential of S,O-CNQDs before and after coupling FA was measured as depicted in Fig. S2. The Zeta potential of S,O-CNQDs and FA-S,O-CNQDs are −13.2 and −29.8 mV, respectively. This is due to the fact that the amino group on the surface of S,O-CNQDs has been substituted by FA and the carboxyl groups in FA has protons which reduce the Zeta potential (Chen et al., 2019). These results indicate that S,O-CNQDs and FA-S,O-CNQDs were successfully prepared.

UV–visible absorption spectra of (1) S,O-CNQDs, (2) FA-S,O-CNQDs, and (3) FA.
Fig. 4
UV–visible absorption spectra of (1) S,O-CNQDs, (2) FA-S,O-CNQDs, and (3) FA.

The stability of S,O-CNQDs under different conditions including NaCl, UV irradiation and storage was evaluated. The fluorescence intensity of S,O-CNQDs remains relatively stable even at 1.0 M NaCl as shown in Fig. 3a, indicating that S,O-CNQDs has excellent photostability in high ionic strength medium. The fluorescence intensity of S,O-CNQDs remained unchanged after UV irradiation for 180 min as depicted in Fig. S3b, inferring that S,O-CNQDs has good photostability. Fig. S3c displays that the fluorescence intensity of S,O-CNQDs remains 97 % of its initial intensity after storage for 8 days, demonstrating that S,O-CNQDs has good storage stability.

3.2

3.2 Optimization of detection conditions

Fig. S4a depicts the effect of pH (4.0–11.0) on the fluorescence intensity of S,O-CNQDs and S,O-CNQDs-FA systems, where F0 and F are the fluorescence intensities of S,O-CNQDs in the absence and presence of FA, respectively. F0 and F0/F both increase with the increase in pH (4.0–7.0) and reach the maxima at 7.0 and further increase in pH will cause the decrease in F0 and F0/F. As such, 7.0 was chosen as the optimum pH for detecting FA.

Fig. S4b shows the effect of reaction time at room temperature on detection of FA by S,O-CNQDs. The F0/F of S,O-CNQDs increases rapidly after in contact with FA, where where F0 and F are the fluorescence intensities of S,O-CNQDs in the absence and presence of FA, respectively. The fluorescence intensity reaches the highest after 5 min and then remains unchanged after 60 min. Therefore, 5 min was selected as the optimal reaction time for subsequent experiments.

Fig. S5 displays the effect of S,O-CNQDs concentration (0.11–0.19 mg/mL) on FA detection by plotting F0/F against CFA, where F0 and F are the fluorescence intensities of S,O-CNQDs in the absence and presence of FA, respectively and CFA is the concentration of FA. The slope of the Stern-Volmer curve increases with the increase of S,O-CNQDs concentration (0.11–0.15 mg/mL) and further increase in S,O-CNQDs concentration causes the decrease in quenching efficiency (slope of the curve). Thus, 0.15 mg/mL was chosen as the optimum concentration of S,O-CNQDs.

3.3

3.3 Detection of FA

Fig. 5. depicts the effect of FA concentration on the fluorescence spectrum of S,O-CNQDs under the optimal experimental conditions. The fluorescence intensity is gradually quenched with the increase in FA concentration. The inset of Fig. 5 shows the Lineweaver-Burk curve (Wang et al., 2020) by plotting 1/(F0-F) against 1/C, where F0 and F are the fluorescence intensities of S,O-CNQDs in the absence and presence of FA, respectively and C is the concentration of FA. The curve shows good linearity (r2 = 0.9919) in the FA range 5.0–83.3 μM. The detection limit is found to be 90 nM which is lower than other reports for FA determination as shown in Table 1. The relative standard deviation (RSD) of fluorescence intensity from 11 consecutive measurements is 2.38 %, demonstrating that the proposed probe has excellent repeatability.

Effect of FA concentration (1–19: 0.0, 5.0, 6.6, 8.3, 10.0, 11.7, 15.0, 18.3, 23.3, 28.3, 33.3, 38.3, 43.3, 48.3, 53.3, 58.3, 66.7, 76.7, and 83.3 μM) on fluorescence spectrum of S,O-CNQDs. The inset depicts the Lineweaver-Burk curve of FA concentration and S,O-CNQDs fluorescence intensity.
Fig. 5
Effect of FA concentration (1–19: 0.0, 5.0, 6.6, 8.3, 10.0, 11.7, 15.0, 18.3, 23.3, 28.3, 33.3, 38.3, 43.3, 48.3, 53.3, 58.3, 66.7, 76.7, and 83.3 μM) on fluorescence spectrum of S,O-CNQDs. The inset depicts the Lineweaver-Burk curve of FA concentration and S,O-CNQDs fluorescence intensity.
Table 1 Analytical figures of merits of various fluorescent probes for FA detection.
Fluorescent Probe LOD (nM) Linear range (μM) References
CDs 380 1.14–47.57 46
paper@CDs 280 1–300 47
HCA-CDs 490 4–100 48
Cu NCs 180 0.5–200 49
C60 FNPs 240 0–80 50
MoS2 QDs 100 0.1–125 51
S,O-CNQDs 90 5.0–83.3 This work

Table 1 summarizes the analytical figures of merits of various fluorescent probes for FA detection. It can be seen that S,O-CNQDs has lower detection limit and comparable working range for FA. S,O-CNQDs can sensitively detect FA based on the formation of hydrogen bonding between the functional groups (–OH, –COOH and –NH2) in FA and S,O-CNQDs (–OH and –COOH), leading to the quenching of fluorescence of S,O-CNQDs. In other words, the formation of hydrogen bond between S,O-CNQDs and FA improves the performance of S,O-CNQDs for FA detection.

Fig. S6 depicts the effects of common interferents such as ions, amino acids, sugars, and vitamins on the fluorescence intensity of S,O-CNQDs. These common interfering substances have no obvious effect on the detection of FA, indicating that S,O-CNQDs has the potential for detecting FA in various real samples.

3.4

3.4 Fluorescence quenching mechanism

Static quenching and dynamic quenching are two main mechanisms of fluorescence quenching. Static quenching process is involved in the formation of a non-luminescent complex between fluorescent molecule and quencher, while dynamic quenching is caused by the collision between fluorescent molecule and quencher. In the process of static quenching, the UV/visible absorption spectrum of fluorescent molecules would change with the addition of a quencher, but the fluorescence lifetime remains unchanged. In addition, the quenching constant decreases with increasing temperature. Compared to static quenching process, dynamic quenching is just opposite (Li et al., 2020).

Fig. S7 shows the effect of FA concentration (0.0–80 μM) on the UV–visible absorption spectrum of S,O-CNQDs. It is obvious that when FA is added, the absorption of S,O-CNQDs increases and the spectrum is slightly red-shifted, indicating that FA and S,O-CNQDs possibly form complexes. Fig. 6 depicts the effect of FA concentration (0.0–80 μM) on the fluorescence lifetime of S,O-CNQDs. The lifetime of S,O-CNQDs does not change in the presence of FA. In addition, Fig. S8 displays the effect of temperature on the Stern-Volmer plot of S,O-CNQDs-FA reaction system at various temperatures. The results show that the slope of the curve (quenching constant) decreases with the increase in temperature. As such, the interaction of S,O-CNQDs with FA is possibly governed by static quenching.

Effect of FA concentration (1–5: 0.0, 20, 40, 60, and 80 μM) on fluorescence lifetime of S,O-CNQDs.
Fig. 6
Effect of FA concentration (1–5: 0.0, 20, 40, 60, and 80 μM) on fluorescence lifetime of S,O-CNQDs.

3.5

3.5 Analysis of FA in real samples

To verify the feasibility of S,O-CNQDs for detection of FA in real samples such as urine, blood, oats, fruit juice, the recoveries were studied and display in Table 2. The recoveries of FA in real samples are 98.8–107.0 % with RSD ≤ 4.1 %, indicating that our proposed probe is reliable and accurate for FA analysis in real samples.

Table 2 Detection and recovery of FA in various samples (n = 3).
Samples Measured (μM) Added (μM) Found (μM) Recovery (%) RSD (%)

Urine		 0.00 10.00 9.88 98.8 3.2
40.00 40.3 100.8 2.4
70.00 69.92 99.9 3.6
Blood 10.00 16.14 99.1 1.9
6.23 40.00 46.31 100.2 2.4
70.00 76.43 100.3 2.8

Oat		 10.00 16.15 107.0 4.1
5.45 40.00 46.69 103.1 2.6
70.00 75.34 99.8 3.5
Fruit juice 10.00 16.11 101.0 2.1
6.01 40.00 45.83 99.6 2.9
70.00 76.74 101.0 4.0

3.6

3.6 Targeted fluorescence imaging of cancer cells

MTT method was utilized to evaluate the cytotoxicity of FA-S,O-CNQDs as shown in Fig. S9. The cell viability is not affected after being treated with different concentrations of FA-S,O-CNQDs for 24 h and the cell survival rate is above 85 %. These results show that FA-S,O-CNQDs has good biocompatibility and can be used as a fluorescent probe for cell imaging.

Studies have confirmed that FRs are widely expressed on the surface of various tumor cells, while only small amounts of FRs are expressed in normal cells (Cao et al., 2018; Liao et al., 2015; He et al., 2017; Zhang et al., 2013). FRs have a high affinity for FA and are able to internalize FA or FA conjugates through receptor-mediated endocytosis (Liu et al., 2022). In order to explore the possibility of FA-S,O-CNQDs for targeted imaging of cancer cells, FR-overexpressed HepG2 cells and FR-not-overexpressed HL-7702 cells were selected as the experimental objects. Figs. 7 and 8 shows the fluorescence microscopy images of HepG2 and HL-7702 cells treated with PBS and FA-S,O-CNQDs at excitation 405 and 488 nm, respectively. HepG2 cells show blue (excitation 405 nm) and green emissions (excitation 488 nm), while HL-7702 cells do not have emission. The reason for this difference is that a specific affinity between FR and FA in HepG2 cells has taken place. In essence, FA-S,O-CNQDs can readily enter the cancer cells rather than the normal cells. Thus, FA-S,O-CNQDs can be applied for fluorescence targeted imaging of cancer cells.

Fluorescence microscopy images of HepG2 treated with PBS(b) and FA-S, O-CNQDs(e: blue channel & h: green channel) for 2 h and their corresponding bright-field (a, d & g) and merged images (c, f & i).
Fig. 7
Fluorescence microscopy images of HepG2 treated with PBS(b) and FA-S, O-CNQDs(e: blue channel & h: green channel) for 2 h and their corresponding bright-field (a, d & g) and merged images (c, f & i).
Fluorescence microscopy images of HL-7702 treated with PBS(b) and FA-S, O-CNQDs(e: blue channel & h: green channel) for 2 h and their corresponding bright-field (a, d & g) and merged images(c, f & i).
Fig. 8
Fluorescence microscopy images of HL-7702 treated with PBS(b) and FA-S, O-CNQDs(e: blue channel & h: green channel) for 2 h and their corresponding bright-field (a, d & g) and merged images(c, f & i).

In order to illustrate FA-S,O-CNQDs to enter cells, further experiments were conducted. Fig. 9 depicts the laser confocal microscopy of HepG2 cells incubated with excessive FA and treated with FA-S,O-CNQDs (pretreated with an excess of FA for FRs saturation) at excitation 405 and 488 nm, respectively. HepG2 cells that were co-incubated with excessive FA do not show fluorescence, while these cells that were cultured with FA-S, O-CNQDs have very weak fluorescence. Again, this difference is due the fact that FR on the surface of HepG2 cells has reached saturation state by co-incubation of FA so that FA-S,O-CNQDs is longer to combine with FR to enter the HepG2 cells. These observations indicate the internalization of FA-S,O-CNQDs into cancer cells through receptor-mediated endocytosis and is depicted in Scheme 1.

Laser confocal microscopy of HepG2 cells incubated with excessive FA (b: blue channel & e: green channel), treated with FA-S,O-CNQDs (pretreated with an excess of FA for FRs saturation) (h: blue channel & k: green channel) and their corresponding bright-field (a, d, g & h) and merged images(c, f, i & l).
Fig. 9
Laser confocal microscopy of HepG2 cells incubated with excessive FA (b: blue channel & e: green channel), treated with FA-S,O-CNQDs (pretreated with an excess of FA for FRs saturation) (h: blue channel & k: green channel) and their corresponding bright-field (a, d, g & h) and merged images(c, f, i & l).
FA-S,O-CNQDs for targeted fluorescence imaging of cancer cells.
Scheme 1
FA-S,O-CNQDs for targeted fluorescence imaging of cancer cells.

4

4 Conclusion

In this work, S,O-CNQDs with good water solubility and biocompatibility was prepared by using ethylenediaminetetraacetic acid disodium and thiourea as the precursors. A fluorescence probe to detect FA was constructed which is based on the fluorescence quenching of S,O-CNQDs by FA. Under the optimal conditions, S,O-CNQDs has been used for FA detection with a concentration range 5.0–83.3 μM and a detection limit of 90 nM. The interaction between FA and S, O-CNQDs is possibly governed by static quenching. The fluorescent probe has successfully applied to detect FA in urine, blood, oats, and fruit juice samples with satisfactory recoveries of 98.8–107.0 % (RSD < 5 %). Through the specific binding between FA and FR, the recognition of cancer cells is realized by utilizing FA-S,O-CNQDs as a probe. The as-prepared fluorescence probe shows promising applications in biological sample analyses as well as diagnoses of cancer cells.

Acknowledgement

This work was supported by the Natural Science Foundation of China (21874087), Natural Science Foundation of Shanxi Province (201901D111210), Special Project of Lvliang for Introduced High-Level Science and Technology Talents (2021RC-2-1), Key Research Project of Science and Technology in JinZhong-Social Development Projects (Y213003), and Research start-up Fund of Shanxi Medical University (TPJS2019004).

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Appendix A

Supplementary material

Supplementary data to this article can be found online at https://doi.org/10.1016/j.arabjc.2022.104520.

Appendix A

Supplementary material

The following are the Supplementary data to this article:

Supplementary data 1

Supplementary data 1


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