Fluorescent Determination of Ferric Ion in vitro with Carbon Quantum Dots Prepared by L-Arginine and Citric Acid

Abstract Ferric ion (Fe3+) serves as an essential and indispensable inorganic element in various biological process. Excess Fe3+ is harmful for biological systems and human health. Herein, highly sensitive carbon quantum dots (Fe-CQDs) for ferric ions were synthesized by a high-efficiency, one-step hydrothermal route utilizing L-arginine and citric acid. The prepared Fe-CQDs exhibited good water solubility, high photostability, and bright blue florescence with relatively high quantum yields (QYs) over 21.33% and a Stokes shift exceeding 100 nm. The functionalized Fe-CQDs offer rapid, sensitive, and selective recognition of Fe3+ with a linear relationship from 0.6 to 80 μM and a low detection limit of 0.2 μM. Moreover, the synthesized Fe-CQDs illustrated strong anti-interference properties in samples and buffer solutions. The developed sensor demonstrated high biocompatibility and was successfully applied for intracellular Fe3+ determination in SHSY5Y and HT22 cells, illustrating excellent imaging application. The present work provides a promising platform for the early clinical diagnosis of Fe3+-associated diseases.


Introduction
Intracellular ferric ion (Fe 3þ ), as an essential inorganic element in protein metabolism and biological regulation in the human body, play an important role in various cell biological process, such as hemoglobin synthesis, electron transfer, cell metabolism, and blood-oxygen transport (Chua et al. 2007;Sherman et al. 2018).However, excess absorption and enrichment of Fe 3þ causes metabolic disorders and free radical overproduction in biological systems, resulting in hazards to human health, such as neurodegenerative diseases, heart diseases, metabolic disorders, and cancer (Jomova and Valko 2011).Therefore, the development of effective strategies for monitoring ferric ions in living systems may describe their contributions to human healthy and role in pathological states, which is of great meaningful and practical value to provide an effective method for early diagnosis and prevention of ferric ion-associated disease.
In recent decades, various analytical techniques have been applied to recognize ferric ion, including flame atomic absorption spectroscopy, mass spectrometry, and electrochemical (Ajlec and � Stupar 1989;Wu and Boyle 1998;Bobrowski, Nowak, and Zarebski 2005;Singh et al. 2014).Compared traditional technologies, fluorescence offers easy application, high selectivity, and good sensitivity (Zhang et al. 2019;Wang et al. 2012;Fuchs 2023;Li et al. 2016).Fluorescent carbon quantum dots (CQDs) have attracted widespread attention and high interest because of their easy synthesis, high photostability, and good water-solubility and biocompatibility (Shi et al. 2016;Lim et al. 2015).
For example, many fluorescent CQDs have been developed for metal ions and gases such as H 2 S and NO (Sonaimuthu et al. 2023;Liu et al. 2022;Singh et al. 2023).More importantly, the literature has demonstrated that CQDs may be successfully utilized for trace ferric ions (Pang and Liu 2020;Chan et al. 2019) that were synthesized using biological waste or a nitrogen-doped raw material (Atchudan et al. 2022(Atchudan et al. , 2017)).However, limitations and deficiencies include cumbersome preparation, high biotoxicity, and low selectivity.Therefore, it is of great significance to develop a novel water-soluble and highly biocompatible fluorescent nanosensor for recognizing intracellular ferric ion.
In this work, innovative Fe-CQDs were prepared with L-arginine and citric acid by one-step hydrothermal strategy.Biocompatible CQDs for tyrosinase detection have previously been successfully synthesized by our research group (Huang et al. 2020).In this study, a less toxic material was chosen to determine Fe 3þ .The selective identification from L-arginine with more additional carboxyl and amino groups has been developed for nanomolar detection of intracellular trace ferric ions.Citric acid provides carboxyl and hydroxyl to improve water solubility and reduce biotoxicity.The multifunctional Fe-CQDs offer high selectivity and fast response to Fe 3þ and was employed to determine trace Fe 3þ in SHSY5Y and HT22 cells, suggesting promising applications in early diagnosis and treatment of Fe 3þ -associated disease.

Production of Fe-CQDs
The Fe-CQDs were synthesized by a one-step hydrothermal method utilized L-arginine and citric acid as the precursor.Briefly, a mixture of 0.174 g L-arginine and 0.21 g citric acid were added to 20 mL ethanol and heated to 180 � C for 8 h in an autoclave.The resulting mixture was cooled to room temperature, centrifuged at 12,000 rpm for 10 min, and concentrated by a rotary evaporator to remove the solvent.The oily product was dissolved in purified water with ultrasound and filtered through a microporous membrane (0.22 lM).The filtrate was dialyzed in ultrapure water (molecular weight, 500 Da) for 24 h and the brown powder was acquired by freeze drying.

Characterization of CQDs
Transmission electron microscopy (TEM, high-resolution transmission electron microscope (Hrtem; Tecnai G2 S-Twin, FEI, USA) and zeta potential analysis (Zetasizer Nano ZS Zen 3600; Malvern, UK) were utilized to characterize the particle size, hydrated size distribution, and zeta potential of the Fe-CQDs.An F-2500 spectrofluorometer (Hitachi; F-2500, JPN) was employed across the wavelength range from 300 to 600 nm.A UV-2600 spectrophotometer (Shimadzu; UV-2600, JPN) was utilized to record the absorbance spectrum from 200 to 800 nanometers.Fourier transform infrared (FT-IR) and Xray photoelectron spectra (XPS) were obtained using Nicolet iS10 and a Thermo Scientific Escalab 250Xi (CN) spectrometers, respectively.

Fluorescence titrations and selectivity assays
Various metal ions (Ba 2þ , Ca 2þ , Cu þ , Fe 2þ , Mg 2þ , Na þ , Zn 2þ , and K þ ) were employed to confirm the specificity of Fe-CQDs.An aliquot of 40 lL of 8 mM metal cation solutions were dispersed into 400 lL of Fe-CQDs solution (10 lg/mL) and equilibrated for 2 min at room temperature.Next, 40 lL of Fe 3þ solution (1 mM) were introduced.The interferences were evaluated by the fluorescence intensity using excitation at 368 nm.
In addition, 40 lL of 0-80 lM Fe 3þ were dispersed into 400 lL Fe-CQDs solution (10 lg/mL) and equilibrated at room temperature for 10 min, respectively.The fluorescence spectra were recorded using an excitation wavelength of 368 nm.

Cellular imaging and detection of Fe 31 in cellular level
The HT22 and SHSY5Y cells were prepared in laser confocal culture dish with 1.0 mL of fresh medium for 24 h.The medium was removed, and the cells were washed twice.An aliquot of 1.5 mL of media with 100 lg/mL Fe-CQDs were added at various time intervals (15 min, 30 min, 1 h, and 2 h).The cells were washed three times with phosphate buffer (PBS) to remove the residual Fe-CQDs.The cellular uptake velocity was evaluated by the fluorescence intensity of the Fe-CQDs.
Subsequently, several concentrations of ferric ion with medium were added into the confocal dish for 30 min to monitor the fluorescence changes.The influence of Fe 3þ on Fe-CQDs at the cellular level was observed by an Olympus Fv1000-ix81 laser confocal microscope.

Characterizations of the Fe-CQDs
The preparation of Fe-CQDs is illustrated in Scheme 1.Briefly, the Fe-CQDs were synthesized from an aqueous mixture of L-arginine and citric acid by one-step hydrothermal method.After 8 h of reaction at 180 � C, the Fe-CQDs were purified by filtering with a microporous membrane and dialyzing through a dialysis membrane.The dark brown solid powder sample was obtained through vacuum freeze drying.
The features of Fe-CQDs were examined by TEM, ultraviolet-visible (UV-vis) spectroscopy, fluorescence spectroscopy (FL), FT-IR spectroscopy, Raman spectroscopy, and X-ray photoelectron spectroscopy (XPS).The morphology of Fe-CQDs were characterized by TEM (Figure 1) and includes uniformly spheres and excellent monodispersity.The nanoparticles were smooth with a diameter of 4 nm.
The absorption spectrum includes a typical peak at 220 nm due to the p-p � transition of aromatic bonds in the carbon core (Figure S1).The weak peak from 360 to 410 nm is due to the n-p � transition of C-O/C-N bonds.The fluorescence emission was recorded at 450 nm with excitation at 368 nm, which shows blue fluorescence from functional groups on the surface of carbon core (Figure 1(C)).The quantum yield (QY) of Fe-CQDs in aqueous solution was determined to be 21.33% utilizing quinoline sulfate as reference material, which confirmed the high sensitivity and potential for practical analysis.The aqueous Fe-CQDs solution was vitreous and clear under natural light, while bright and providing strong blue fluorescence under 365 nm excitation (Scheme 1, inset).
The emission spectra of Fe-CQDs illustrated typical characteristics of Fe-CQDs (Figure 1(D)).As the excitation wavelength increased from 320 to 370 nm, the fluorescence emission peak was red-shifted.The fluorescence intensity decreased with the emission wavelength.This dependence upon the excitation wavelength is due to the size of the CQDs or various surface light-emitting sites (Zhu et al. 2015).
The functional groups of Fe-CQDs were characterized by FT-IR spectroscopy (Figure 1(E)).Hydroxyl groups and primary amines were present on the surface of Fe-CQDs, showing the flexural vibration of oxygen-hydrogen (O-H) at 1384 cm À 1 and the symmetric stretching vibration of primary amino group at 3392 cm À 1 .Additionally, the intense peak at 2982 cm À 1 was due to the stretching vibration of carbon-hydrogen (C-H).The robust peak from 3400 to 2970 cm À 1 was caused by the stretching of oxygenhydrogen (O-H), and the sharp peak at 1707 cm À 1 by stretching of the carbonyl group (C¼O) on the surface of Fe-CQDs.The presence of primary amines, hydroxyl, and carboxyl groups on the surface of Fe-CQDs ascertained their good hydrophilic properties and water solubility.
The Raman spectrum exhibited a strong G-band (1572 cm À 1 ) and a weak D-band (1353 cm À 1 ), which represent in-plane vibration of sp 2 carbon atoms and defects in the sp 2 carbons (Figure 1 2D band around 2700 cm À 1 infers that the inner core of CQDs may not be a single graphene layer.
XPS was utilized to further characterize the surface groups and elemental compositions of the Fe-CQDs (Figure 2).The full XPS spectrum contains three conspicuous peaks at 284.5, 398.8 and 532 eV (Figure 2

Stability of the Fe-CQDs
The stability and photostability of Fe-CQDs were evaluated by their fluorescence change during different periods in several buffer solutions.The fluorescence intensity of Fe-CQDs was consistent after for 7 and 14 d in various media, including 0.9% sodium chloride, pH 7.2 PBS, and Dulbecco's modified eagle medium (DMEM) with 10% serum.These results demonstrate that Fe-CQDs possessed robust fluorescence stability with storage time and in several buffer solutions (Figure S2(A-C)).Interestingly, the fluorescence intensity of Fe-CQDs remained nearly constant after 60 days of storage (Figure S2(D)).

Specificity evaluation for Fe 31
To determine the practicality of Fe-CQDs as fluorescent nanosensors, cations found in biological systems were utilized to evaluate the selectivity and anti-interference ability of the Fe-CQDs (Figure 3).The fluorescence intensity nearly remained invariant (<4% decrease) in the presence of Na þ , K þ , Mg 2þ , Mn 2þ , Fe 2þ , Zn 2þ , Ba 2þ , and Ca 2þ (Figure 3(A)).However, it was dramatically reduced (> 86% decrease) after the addition of Fe 3þ (Figure 3(B)).
These experiments show that most ions and biological materials may coexist with the Fe-CQDs without interference to ferric ion determination, illustrating potential for the analysis of complex samples.
Photographs of Fe-CQDs solutions containing different metal ions were taken under UV and sunlight exposure (Figure 3(C)).The photographs of 80 lM Fe-CQDs solutions containing different metal ions were obtained at 368 nm, revealing the fluorescence quenching of Fe-CQDs in the presence of Fe 3þ .Conversely, the other metal ions exhibited constant fluorescence.Similar quenching was observed in the presence of Fe 3þ in sunlight.
Moreover, the Fe-CQDs exhibited a prominent absorption peak at 368 nm, which underwent substantial attenuation upon the incorporation of exogenous Fe 3þ (Figure 4(A)).Additionally, the zeta potential of Fe-CQDs was determined to be À 5.6 mV.However, the zeta potential underwent a significant increase to 11 mV after introduction of exogenous Fe 3þ , which indicated a negative migration (Figure 4(B)).These distinguishing features are indicative to the quenching by the Fe-CQDs.
Furthermore, the specific recognition of Fe-CQDs to Fe 3þ was evaluated.The fluorescence intensity gradually quenched with the concentration of Fe 3þ , which was nearly complete when the ferric ion concentration was 80 lM (Figure 4(C)).
The fluorescence intensity changed with the Fe 3þ concentration from 0.6 to 80 lM which illustrated enhanced quenching.Notably, the quenching efficiency FL 450 /FL 368 exhibited a linear correlation (R 2 ¼ 0.993) with the concentration of Fe 3þ from 0.6 to 80 lM as shown in Figure 4(D).The fitted linear equation was FL 450 /FL 368 ¼ 0.0222[Fe 3þ ] þ 1.1205, where FL 450 and FL 368 are the fluorescence intensities of Fe-CQDs in the presence and absence of Fe 3þ , respectively.The limit of detection (LOD) from this plot was 0.2 lM according to the 3r signal-to-noise criterion, which was considerably lower than the Fe 3þ levels in drinking water based upon the literature.
Table S1 compares the CQDs/Fe-CQDs with literature carbon precursors for Fe 3þ determination.The LOD (0.2 lM) was favorable compared with the literature.The linear relationship and low detection limit illustrated potential for the Fe-CQDs to provide selective and sensitive determination of trace Fe 3þ in biological systems.

The mechanism of the detection of Fe 31 with Fe-CQDs
The mechanism of the interaction between Fe 3þ and Fe-CQDs was investigated.According to the literature (Li et al. 2013), it was believed that the quenching after Fe-CQDs were treated with Fe 3þ involved hydroxyl groups or amino groups on the quantum dot surface (Scheme 1).
The fluorescent quenching of Fe-CQDs by Fe 3þ was investigated using the Stern-Volmer equation (Liu et al. 2018).A linear correlation was observed between FL 368 /FL 450 and the concentration of Fe 3þ (Figures 4(C) and 5(D)).The correlation coefficient was 1.2, indicating that the quenching mechanism is static (Li et al. 2019).

Fe 31 determination in serum and cells
Human serum was analyzed to characterize the suitability of the developed protocol for biological samples.The Fe-CQDs were mixed with standard and prepared Fe 3þ samples in fresh human serum.The fluorescence intensity was measured to determine the concentration of Fe 3þ according to the calibration relationship.The recoveries and relative standard deviation (RSD) values are summarized (n ¼ 3) in Table S2.The results show that the determined concentrations were consistent with the added values.The recovery was between 98.6% and 101.9% with acceptable relative standard deviations, demonstrating trace Fe 3þ was determined by the Fe-CQDs.These results demonstrate the applicability of Fe-CQDs for Fe 3þ with potential for clinical analysis.
In addition, the SHSY5Y and HT22 cells were utilized to investigate the ingestion rate of the Fe-CQDs.Figure 5(A,B) shows that the fluorescence of Fe-CQDs was observed inside the cell after 0.25 h co-incubation by confocal laser scanning microscopy.The fluorescence intensity increased after 2 h of incubation (Figure 5(C)), indicating that the synthesized Fe-CQDs were gradually taken up by the cells via a timedependent endocytosis process.
The confocal fluorescence cellular imaging for intracellular Fe 3þ was performed to further characterize the developed assay.The confocal images of HT22 and SHSY5Y cells were obtained after 2-h of incubation with 15 lg/mL Fe-CQDs.
The Fe-CQDs solution was incubated with SHSY5Y and HT22 cells for 2 h and washed with pH 7.2 PBS three times to remove external Fe-CQDs.Freshly prepared culture medium containing exogenous Fe 3þ ions (0.5 lM) was added and incubated for 0.5 h.As exhibited in Figure 6(A), the blue fluorescence of the Fe-CQDs in the cells significantly decreased after treatment with Fe 3þ (Figure 6(B)), indicating potential for cellular Fe 3þ determination.Notably, the images in the bright field also showed that Fe-CQDs did not change the cellular morphology in the presence or absence of Fe 3þ .Consequently, these experiments demonstrate that the Fe-CQDs are suitable for intracellular Fe 3þ detection which may allow early diagnosis for associated diseases.
Scheme 1. Schematic diagram of the preparation of the nanosensor and the fluorescence quenching mechanism in the presence of Fe 3þ .

Figure 5 .
Figure 5. (A,B) Confocal images of SHSY5Y and HT22 cells incubated in culture medium with Fe-CQDs (100 mg/mL) for 0.25 h, 0.5, 1, and 2 h using the blue channel.(C) Linear relationship between fluorescence intensity as a function of time.(D) Viability of SHSY5Y and HT22 cells cultured with different concentrations of Fe-CQDs for 24 h.