Carbon dots employed for the detection of ranitidine and elaborating the detecting mechanism

doi:10.21203/rs.3.rs-4666965/v1

Download PDF

Research Article

Carbon dots employed for the detection of ranitidine and elaborating the detecting mechanism

https://doi.org/10.21203/rs.3.rs-4666965/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Carbon dots (CDs) has been widely utilized in multiple fields, especially towards kinds of drug analyses, owing to its superior optical properties and satisfactory stability. Herein, we rapidly synthesized one kind of soluble bright-blue fluorescent CDs through a facile microwave method, while disodium ethylenediaminetetraacetic acid and phosphoric acid served as the raw materials. Importantly, introducing ranitidine into these CDs resulted in its decreased fluorescence, and thus an innovative method of detecting ranitidine was successfully established, which showed the favorable selectivity and anti-interference ability. Moreover, with the optimal conditions, the standard curve of F₀/F against concentrations of ranitidine exhibited the generally satisfactory linear range of 6-2000 µM with a correlation coefficient of 0.9833, and the limit of detection (LOD) was calculated to be 4.2 µM. Meanwhile, we also explored the detecting mechanism of ranitidine by CDs, and elaborated that as the internal filtration effect. Consequently, we may broaden the avenues of detecting ranitidine on the basis of CDs.

Carbon dots

Ranitidine

Quenching of fluorescence

Internal filtration effect

Ranitidine is a potent histamine H₂ receptor antagonist that competitively blocks histamine binding to H₂ receptors on gastric wall cells, thus effectively inhibiting gastric acid secretion. It has high efficacy in gastric and duodenal ulcers with rapid and long-lasting effects ^[1], controlling the concentration of ranitidine is important in real-life applications. However, several analytical methods such as electrochemical oxidation, voltammetric determination ^{[2] [3]} and high performance liquid chromatography ^{[4] [5]} are currently utilized for the detection of ranitidine. These methods usually suffer from the lack of simplicity and the high cost of the instruments and reagents required. Therefore, the development of the fluorescence-based methods for monitoring the concentrations of ranitidine may complement the shortcomings of the existing methods.

CDs, emerging as one type of fluorescent nanomaterial basically with the diameters smaller than 10 nm ^[6], is usually prepared by the hydrothermal ^[7], electrochemical synthesis ^[8] and microwave heating ^[9]. Serving as a convenient method, microwave heating is extensively to prepare CDs, in which nitrogen, sulfur, phosphorus and other heteroatoms could be doped into the CDs, thus CDs with the satisfactory optical properties were obtained. Meaningfully, CDs has been widely applied in the aspects of photocatalysis ^[10], drug delivery ^[11] and bioimaging ^[12] based on its excellent fluorescence or phosphorescence, photo-bleaching or salt resistance, biocompatibility and beyond. Especially, CDs shows the obvious advantages towards detections by virtue of its high sensitivity, short response time and economic cost ^{[13] [14]}.

In this study, one kind of blue-fluorescence CDs with stable optical properties was successfully synthesized by the microwave heating using disodium ethylenediaminetetraacetic acid and phosphoric acid as the precursors. Importantly, an evident fluorescence quenching emerged with the addition of ranitidine hydrochloride into the CDs, which provided the possibility of developing the method for the determination of ranitidine concentrations. Additionally, we further explored the detecting mechanism of CDs responding to ranitidine, which was mainly clarified as internal filtering effect.

2.1 Chemicals and materials

All the reagents including disodium EDTA, sodium hydroxide, phosphoric acid, copper acetate, cobalt chloride, nickel sulphate, chromium chloride, lead acetate, manganese chloride, magnesium sulphate, sodium carbonate, barium chloride, strontium chloride, potassium chloride, sodium chloride, tetraethoxysilane, ethyl acetate, acetonitrile, methanol, tetrahydrofuran, N, N-dimethylformamide, ethanol, dimethylsulfoxide, formamide, pyridoxine hydrochloride, glycine, arginine, histidine, glutamate and dextrose were purchased from Chron Chemicals Co., Ltd. (Chengdu, China). Simultaneously, glutathione, isoprinosine hydrochloride, sulfadiazine sodium salt, L-lysine and tryptophan were derived from the Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China) as well as ranitidine hydrochloride, dopamine hydrochloride and isoniazid from Aladdin Reagent Co., Ltd. (Shanghai, China). The ultrapure water employed during the whole experiment was obtained from Aquapro AWL-0502-P ultrapure water system (Chongqing, China).

2.2 Instrumentation

Initially, the microwave oven from Galanz Enterprises Co. Ltd (Guangzhou, China) was utilized during the preparation of CDs, and the purified powder of CDs was acquired by a centrifuge (TG16-W) of Cence instrument Co. Ltd. (Changsha, China) and a freeze dryer of SCIENTZ-12N (Ningbo, China). Further, all the fluorescent measurements were performed with a Hitachi fluorescence spectrophotometer of F-7000 (Tokyo, Japan), while the UV-vis absorption spectra were recorded by using a Shimadzu spectrophotometer of UV-1750 (Tokyo, Japan). Again, the elemental compositions of CDs were identified by an X-ray photoelectron spectrometer (XPS) of ESCALAB 250 (New York, USA) and a Fourier transform infrared (FTIR) spectrometer of Prestige-21 (Tokyo, Japan). The acidity meter of pHS-3C was employed to determine various pH values of the solutions during the whole experimental process.

2.3 Synthesis of CDs

The synthesis of CDs was achieved by the microwave heating. Through the optimization of reaction conditions, it was determined that the CDs was synthesized as follows: 0.75 g of disodium ethylenediaminetetraacetic acid was dissolved in 4 ml of ultrapure water and stirred well in a crucible followed by introducing 1 ml of phosphoric acid. The CDs solution was obtained by heating in a microwave at medium high fire for 80 s, and its pH was adjusted to neutral with sodium hydroxide. The purified CDs solution was then obtained by centrifugation and filtration, and the final product was stored in a 4°C of refrigerator for further use.

2.4 Optimization of the detecting conditions

To achieve the optimal assay of ranitidine hydrochloride, the optimal conditions were determined by adjusting different temperature and time as well as pH values. To be specific, 200 µL of CDs solution was diluted in 800 µL of ultrapure water or PBS buffer, which served as the control. Meanwhile, 100 µL of 0.01 mol/L ranitidine hydrochloride solution was added into the paralleled CDs solutions with the same dilution ratio. The fluorescence intensity was measured immediately after two solutions of CDs without or with ranitidine were incubated with the same pH. The optimal detection pH was determined by comparing the difference of fluorescence intensity among the CDs with ranitidine at different pH values (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). Similarly, the incubation time (5, 10, 15, 20, 25, 30 min) and incubation temperature (20, 30, 40, 50, 60, 70°C) were also optimized through the same way.

3.1 Preparation of CDs

To obtain the fluorescent CDs with satisfactory optical properties, we initially optimized its synthesis conditions. As shown in Fig. 2A, the fluorescence intensity of the CDs obviously showed higher than that of the others when the dosage of disodium ethylenediaminetetraacetic acid was selected as 0.75 g. Similarly, the CDs also emitted the strongest fluorescence when the ratio of water and phosphoric acid was 4:1 (Fig. 2C). The fluorescence intensity of CDs with microwave heating time of 80 s and 90 s showed the scarce difference, and 80 s was chosen as the optimal reaction time (Fig. 2B). Finally, the medium-high fire was identified as the optimal synthesis temperature based on the corresponding fluorescence intensity (Fig. 2D).

3.2 Characterization of CDs

Subsequently, their optical properties of the synthesized CDs were characterized. As shown in Fig. 3A, the optimal excitation wavelength of the CDs was determined to be 366 nm as well as the optimal emission wavelength of 423 nm. Meanwhile, the CDs solution appeared as yellow under daylight (Fig. 3A, inset a), and emitted a blue fluorescence with UV of 365 nm (Fig. 3A, inset b). Meanwhile, the absorption spectrum of CDs was recorded (Fig. 3B). In particular, the peak of 216 nm in the absorption spectrum corresponded to the π-π* transition of C = C and C = N ^[15], while the peak at 300 nm was possibly due to the n-π* transition of C = N ^[16]. Further, the elemental composition and functional groups of CDs were investigated by Fourier Transform Infrared and X-ray Photoelectron Spectroscopy. As shown in Fig. 3C, the broad absorption peaks from 3200 cm^− 1 to 3400 cm^− 1 corresponded to the stretching vibration of -OH ^[17]; and the stretching vibration peaks of -CH- and C = N located around 3000 cm^− 1 and 1650 cm^− 1, respectively ^{[18] [19]}. Similarly, the peaks of 1260 cm^− 1 and 1110 cm^− 1 were attributed to the groups of P = O and P-O-C, while the peaks at 876 cm^− 1 and 538 cm^− 1 corresponded to P-N and PO₄³⁻, respectively.

Furthermore, the XPS spectra revealed that the CDs we prepared was mainly composed of four elements including C, N, O and P (Fig. 3D). Specifically, the spectrum of C 1s was divided as three peaks, which were originated from C-C/C = C (284.5 eV), C-P/C-N (285.8 eV) and C-O (286.8 eV), respectively ^[20] (Fig. 4A). Meanwhile, the N 1s spectrum was fitted as two peaks for C-N = C at 399.6 eV and N-(C)₃/N-P at 400.8 eV (Fig. 4B). Again, three peaks obtained from the O 1s fitting were O = P at 530.6 eV, C-OH/C-O-C at 531.8 eV and O-P at 532.3 eV ^[21] (Fig. 4C). Moreover, two peaks of P 2p were responsible for P-C/P-N (132.8 eV) and P-O (133.2 eV), respectively (Fig. 4D). Taken together, these findings suggested that there existed abundant functional groups on the surface of CDs we prepared here.

3.3 Fluorescence stability of CDs

To investigate the optical stability of CDs, we recorded its fluorescence with varying conditions by altering the UV irradiation time, pH, temperature, concentrations of sodium chloride, organic reagents and metal ions. As shown in Fig. 5A, the fluorescence intensity of CDs slightly decreased with the increase of UV irradiation time, and still maintained 83% of the initial fluorescence intensity after 90 minutes of irradiation, indicating its generally acceptable photostability. However, a significant decrease of the fluorescence intensity was observed with increasing pH, demonstrating that pH showed the obvious influence on the optical properties of CDs (Fig. 5B). In the temperature variation range of 20°C to 80°C, the fluorescence of CDs scarcely exhibited the change (Fig. 5C). Meanwhile, the addition of different concentrations of sodium chloride hardly effected the fluorescence intensity of CDs, proving the favorable salt resistance of CDs (Fig. 5D). As shown in Fig. 5E and 5F, the fluorescence stability of CDs was examined by adding the routine organic solvents and metal ions. The results demonstrated that CDs basically maintained its fluorescence intensity in presence of organic solvents or metal ions.

3.4 Establishing the assay of ranitidine by CDs

Considering that CDs could function as a fluorescent probe to detect the potential target on the basis of its satisfactory optical properties, we addressed whether the CDs described here could serve as a probe. Consequently, we explored the fluorescent response of CDs to ranitidine hydrochloride. As shown in Fig. 6A, the fluorescence intensity was accordingly quenched after the addition of two concentrations of ranitidine to the CDs, demonstrating its potential of a fluorescent probe for detecting ranitidine.

3.5 Optimizing the detecting conditions of ranitidine by CDs

To acquire the optimal conditions of the CDs detecting ranitidine, a series of experiments was performed. As shown in Fig. 6B, the fluorescence quenching was most pronounced at pH = 4, which was determined to be the optimal pH for the assay. Similarly, the decreased fluorescence of CDs with ranitidine reached the maximum when the incubation time was 20 minutes (Fig. 6C). Again, the optimal incubation temperature was identified as 70°C by comparing the fluorescence intensities of CDs and ranitidine with different incubation temperature (Fig. 6D). Therefore, the optimized detecting conditions of pH = 4, 20 min and 70°C were selected. Furthermore, the fluorescence intensity of CDs gradually decreased with the concentration of ranitidine increasing from 6 µM to 2000 µM (Fig. 6E), and the corresponding linear equation was fitted as F₀/F = 0.0005C + 1.0041 with R² of 0.9833 (Fig. 6F).

3.6 Anti-interference and selectivity for CDs detecting ranitidine

To investigate the specificity of CDs assaying ranitidine, equal amounts of other drugs were separately introduced into the CDs. As shown in Fig. 7A, only ranitidine exhibited the obvious fluorescence quenching effect on CDs. To further verify the anti-interference ability of CDs during detecting ranitidine, several common medicines (Fig. 7B), chemical compounds (Fig. 7C) and metal ions (Fig. 7D) were separately introduced into the paralleled mixtures of CDs with ranitidine. Through comparing their fluorescence variations, it was found that the fluorescence intensity of CDs with ranitidine in the presence of interfering substances scarcely varied, proving the practicability of the proposed assay for CDs detecting ranitidine.

3.7 Detection mechanism of ranitidine by CDs

To deeply understand the quenching mechanism of ranitidine on the fluorescence intensity of CDs, we initially recorded the UV-Vis spectrum of ranitidine and fluorescence spectra of CDs, and there was no overlapping between the UV absorption of ranitidine and fluorescence emission of CDs (Fig. 8A), thus ruling out the fluorescence quench mechanism of fluorescent resonance energy transfer (FRET) ^[22]. Meanwhile, we also found that the UV absorption peak partially overlapped with the excitation peak of CDs, thus hypothesizing that the internal filtration effect (IFE) may be the main mechanism of this fluorescence quenching ^[23]. In addition, by comparing the UV absorption spectra (Fig. 8B) and infrared spectra (Fig. 8C) of CDs, ranitidine and CDs in presence of ranitidine, we found that the UV absorption and infrared spectra of CDs hardly changed with and without ranitidine. This result revealed that there was no reaction occurring between ranitidine and CDs to produce new compounds ^[24], thus the fluorescence static quenching effect (SQE) ^[25] and photoelectron transfer (PET) ^[26] were excluded. Finally, the fluorescence lifetime of CDs scarcely varied before and after adding ranitidine (Fig. 8D), which ruled out the fluorescence quenching caused by dynamic quenching effect (DQE) ^[27]. Thereby, it was concluded that the internal filtration effect (IFE) was mainly responsible for the fluorescence quenching described here ^[28].

In summary, we have rapidly synthesized CDs with bright-blue fluorescence by using the microwave-heating method, while disodium ethylenediaminetetraacetic acid played the role of the precursor. Through regulating the different environmental aspects, it was determined that the CDs prepared here exhibited the satisfactory optical properties as well as high stability. Meaningfully, we further developed a strategy of detecting ranitidine based on the fluorescence response of CDs to ranitidine, and this assay showed the favorable selectivity and anti-interference ability during CDs determining ranitidine. Finally, we elucidated that the internal filtration effect (IFE) resulted in the fluorescence quenching of CDs once ranitidine was introduced, thus successfully establishing a new analytical method for assaying ranitidine.

Acknowledgments

This work was financially supported by the Opening Research Project from Key Laboratory (Deyang) of Chemical Drug Quality Research of Sichuan Provincial Drug Administration (DHY-001).

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

Mei Zhang: Methodology, Data processing, Review & editing, Investigation. Jingwen Zhao: Software, Formal analysis, Original draft. Yingying Long: Resources, Validation. Changsong Li: Writing - review & editing, Supervision. Xiaoming Yang: Conceptualization, Writing-review & editing, Supervision, Project administration, Funding acquisition.

Data Availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Norouzi P., Ganjali M.R., Daneshgar P. (2007) A novel method for fast determination of Ranitidine in its pharmaceutical formulations by fast continuous cyclic voltammetry. Pharmacol Toxicol. Methods. 55 (3):289-296. https://doi.org/10.1016/j.vascn.2006.09.001.
Borges P.H.S., Catto A.C., Longo E. (2020) Nossol Electrochemical synthesis of reduced graphene oxide/ruthenium oxide hexacyanoferrate nanocomposite film and its application for ranitidine detection. Electroanal Chem. 878: 114558. https://doi.org/10.1016/j.jelechem.2020.114558.
Talay Pınar P., Yardım Y., Şentürk Z. (2018) Electrochemical oxidation of ranitidine at poly(dopamine) modified carbon paste electrode: Its voltammetric determination in pharmaceutical and biological samples based on the enhancement effect of anionic surfactant. Sensors Actuators, B Chem. 273:1463-1473. https://doi.org/10.1016/j.snb.2018.07.068.
Kiszkiel-Taudul I., Starczewska B. (2019) Single drop microextraction coupled with liquid chromatography-tandem mass spectrometry (SDME-LC-MS/MS) for determination of ranitidine in water samples. Microchem. 145:936-941. https://doi.org/10.1016/j.microc.2018.12.015.
Ashiru D.A.I., Patel R., Basit A.W. (2007) Simple and universal HPLC-UV method to determine cimetidine, ranitidine, famotidine and nizatidine in urine: Application to the analysis of ranitidine and its metabolites in human volunteers. Chromatogr B Anal. Technol. Biomed Life Sci. 860 (2):235-240. https://doi.org/10.1016/j.jchromb.2007.10.029.
Xu Q., Kuang T., Liu Y. et al. (2016) Heteroatom-doped carbon dots: synthesis, characterization, properties, photoluminescence mechanism and biological applications. Mater Chem B. 4 (45):7204–7219. https://doi.org/10.1039/c6tb02131j.
Ma Y., Chen Y., Liu J., et al. (2018) Ratiometric fluorescent detection of chromium(VI) in real samples based on dual emissive carbon dots. Talanta. 185:249–257. https://doi.org/10.1016/j.talanta.2018.03.081.
Li H., Huang J., Song Y. et al. (2018) Degradable carbon dots with broad-spectrum antibacterial activity. ACS Appl Mater Interfaces. 10:26936–26946. https://doi.org/10.1021/acsami.8b08832.
Zhang L., Wang D., Huang H. et al. (2016) Preparation of gold–carbon dots and ratiometric fluorescence cellular imaging. ACS Appl Mater Interfaces.8:6646–6655. https://doi.org/10.1021/acsami.5b12084.
Xue Z., Gao H., Li X. (2018) A Green and lower-temperature synthesis of two-color fluorescent nitrogen dopedgraphene quantum dots. Dyes Pigm. 156:379–385. https://doi.org/10.1016/j.dyepig.2018.04.032.
Matai I., Sachdev A., Gopinath P. (2015) Self-assembled hybrids of fluorescent carbon dots and PAMAM dendrimers for epirubicin delivery and intracellular imaging. ACS Appl Mater Interfaces. 7:11423–11435. https://doi.org/10.1021/acsami.5b02095.
Vandarkuzhali SAA., Jeyalakshmi V., Sivaraman G. et al. (2017) Highly fluorescent carbon dots from Pseudo-stem of banana plant: applications as nanosensor and bio-imaging agents. Sensors Actuators B-Chem. 252:894–900. https://doi.org/10.1016/j.snb.2017.06.088.
Li D., Wang S., Azad F. et al. (2020) Single-step synthesis of polychromatic carbon quantum dots for macroscopic detection of Hg²⁺. Ecotoxicology Environmental Safety. 190:110141. https://doi.org/10.1016/j.ecoenv.2019.110141.
Tafreshi F.A., Fatahi Z., Ghasemi S.F. et al. (2020) Ultrasensitive fluorescent detection of pesticides in real sample by using green carbon dots. PLoS ONE. 15(3): 0230646. https://doi.org/10.1371/journal.pone.0230646.
Zhu S., Zhao X., Song Y. et al. (2016) Beyond bottom-up carbon nanodots: citric-acid derived organic molecules. Nano Today. 11:128-132. https://doi.org/10.1016/j.nantod.2015.09.002.
Qu D., Zheng M., Li J. et al. (2015) Tailoring color emissions from N-doped graphene quantum dots for bioimaging applications. Light: Sci. Appl. 4:364. https://doi.org/ 10.1038/lsa.2015.137.
Wang C., Xu J., Li H. et al. (2020) Tunable multicolour S/N co-doped carbon quantum dots synthesized from waste foam and application to detection of Cr³⁺ ions. Luminescence. 35 (20):1373-1383. https://doi.org/10.1002/bio.3901.
Hagiwara K., Horikoshi S., Serpone N. et al. (2021) Luminescent monodispersed carbon quantum dots by a microwave solvothermal method toward bioimaging applications. J Photochem Photobiol A. 415:113310. https://doi.org/10.1016/j.jphotochem.2021.113310.
Wang M., Liu Y., Ren G. et al. (2018) Bioinspired carbon quantum dots for sensitive fluorescent detection of vitamin B₁₂ in cell system. Anal Chim Acta. 1032:154–162. https://doi.org/10.1016/j.aca.2018.05.057.
Shen T.-Y., Jia P.-Y., Chen D.-S.et al. (2021) Hydrothermal synthesis of N-doped carbon quantum dots and their application in ion-detection and cell-imaging. Spectrochim Acta Part A Mol Biomol Spectrosc. 248:119282. https://doi.org/10.1016/j.saa.2020.119282.
Li W., Huang S., Wen H. et al. (2020) Fluorescent recognition and selective detection of nitrite ions with carbon quantum dots. Anal Bioanal Chem. 412:993–1002. https://doi.org/10.1007/s00216-019-02325-9.
Wang Q., Zhang Z., Yang T. et al. (2022) Multiple fluorescence quenching effects mediated fluorescent sensing of captopril Based on amino Acids-Derivative carbon nanodots. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 269:1386-1425. https://doi.org/10.1016/j.saa.2021.120742.
Carneiro S.V., Oliveira J.J.P., Rodrigues V.S.F. et al. (2022) Doped carbon quantum dots/PVA nanocomposite as a platform to sense nitrite ions in meat. ACS Appl. Mater. Interfaces. 14 (38):43597-43611. https://doi.org/10.1021/acsami.2c09197.
Hu P., Huang R., Ning J. et al. (2024) Aptamer functionalized fluorescent probe for detection of thrombin in human serum via static quenching. Arabian Journal of Chemistry.17 (2):105088. https://doi.org/10.1016/j.arabjc.2023.105508.
Zhang Q., He S., Zheng K. et al. (2022) Green synthesis of mustard seeds carbon dots and study on fluorescence quenching mechanism of Fe³⁺ ions. Inorganic Chemistry Communications.146:110034. https://doi.org/10.1016/j.inoche.2022.110034.
Huang M., Lv C. Huang Q. et al. (2019) A novel and fast responsive turn-on fluorescent probe for the highly selective detection of Cd²⁺ based on photo-induced electron transfer. RSC Advances.62:36011-36019. https://doi.org/10.1039/c9ra06356k.
Yang Y., Zou T., Wang Z. et al. (2019) The fluorescent quenching mechanism of N and S Co-doped graphene quantum dots with Fe³⁺ and Hg²⁺ ions and their application as a novel fluorescent sensor. Nanomaterials.9 (5):738. https://doi.org/10.3390/nano9050738.
Zhang R., Zhang L., Yu R. et al. (2023) Rapid and sensitive detection of methyl parathion in rice based on carbon quantum dots nano-fluorescence probe and inner filter effect. Food Chemistry. 413:135679. https://doi.org/10.1016/j.foodchem.2023.135679.

No competing interests reported.

Download PDF

Editorial decision: Revision requested
24 Jul, 2024
Reviews received at journal
22 Jul, 2024
Reviewers agreed at journal
22 Jul, 2024
Reviewers invited by journal
18 Jul, 2024
Editor assigned by journal
09 Jul, 2024
Submission checks completed at journal
09 Jul, 2024
First submitted to journal
01 Jul, 2024

You are reading this latest preprint version

Carbon dots employed for the detection of ranitidine and elaborating the detecting mechanism

Status:

Version 1

Abstract

Figures

1 Introuduction

2 Experimental Section

2.1 Chemicals and materials

2.2 Instrumentation

2.3 Synthesis of CDs

2.4 Optimization of the detecting conditions

3 Results and discussion

3.1 Preparation of CDs

3.2 Characterization of CDs

3.3 Fluorescence stability of CDs

3.4 Establishing the assay of ranitidine by CDs

3.5 Optimizing the detecting conditions of ranitidine by CDs

3.6 Anti-interference and selectivity for CDs detecting ranitidine

3.7 Detection mechanism of ranitidine by CDs

4. Conclusion

Declarations

References

Additional Declarations

Status:

Version 1