Development and utilization of a fluorescence sensor for Zn2+ detection

doi:10.21203/rs.3.rs-4970970/v1

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Development and utilization of a fluorescence sensor for Zn²⁺ detection

https://doi.org/10.21203/rs.3.rs-4970970/v1

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published 05 Nov, 2024

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A fluorescence probe (Probe-Zn) was designed and synthesized for the rapid detection of Zn²⁺ based on the excited state intramolecular proton transfer (ESIPT) mechanism. The specific recognition ability of Probe-Zn to Zn²⁺ was tested using UV-VIS and fluorescence emission spectroscopy. The results demonstrated a rapid response within 40s upon addition of an appropriate amount of Zn²⁺ into the mixed solution of the target probe molecules (DMSO/PBS = 5/5). Under 365 nm ultraviolet light irradiation, the colorless solution changed to yellow-green fluorescence with a 150-folds increase in intensity. Furthermore, the detection limit for specific recognition of Zn²⁺ by the probe molecule is only 17.3 nmol/L, indicating high sensitivity. The practical application potential of the probe molecules was enhanced by employing on information storage and conducting cell imaging experiments.

Zn2+

Fluorescent probe

Designed and synthesized

Application

The original sentence has been polished to: "Organic fluorescence probes are extensively utilized in the realms of analytical chemistry, medicine, biology, and environmental studies due to their exceptional sensitivity, selectivity, simplicity and rapid response [1–6]. In the last two decades, there has been rapid progress in the development of fluorescent probes or chemical sensors designed for the selective recognition of metal ions. Zn²⁺ is a crucial trace element in organisms and is widely distributed [7–9]. It plays a vital regulatory role in physiological processes such as enzyme catalysis, protein sequencing, gene expression, and children's growth and development [8, 9]. However, excessive Zn²⁺ can not only inhibit enzyme activity but also antagonize other trace elements, leading to hematopoietic mechanism disorders and cholesterol metabolism disorders in the body which result in decreased resistance among other issues. Therefore, our primary focus lies on developing chemical sensors capable of identifying zinc ions in environmental and biological samples. In recent years, Zn²⁺ fluorescence sensors based on various fluorescent parent structures such as quinoline [5], fluorescein [6], coumarin [7], and pyrene [8] have garnered significant attention. In comparison to traditional fluorophores (e.g., fluorescein, Rhodamine), the fluorescence chromophores with excited state intramolecular proton transfer (ESIPT) properties exhibit a large Stokes shift, effectively avoiding molecular self-absorption, demonstrating good biological activity, and serving as an important reference for the design of organic fluorescence probes. Furthermore, their incorporation of heteroatoms like N, O, and S enables them to form stable complexes with certain metal ions [9]. For instance, Schiff base-metal ion complexes possess notable biological activities in bacteriostatic and bactericidal effects, anti-tumor properties, as well as fluorescence detection capabilities [10–13], indicating their broad potential applications. Nevertheless, challenges persist in the design and synthesis of organic fluorescence probe molecules, including intricate synthesis methods, prolonged response times, and high detection limits, all of which significantly impede their practical application [14–16]. Leveraging the adaptability of organic synthesis technology, a novel fluorescence probe named Probe-Zn was developed and synthesized based on the ESIPT mechanism for rapid Zn²⁺ detection. This probe offers advantages such as readily available raw materials, straightforward synthesis procedures, specific recognition capabilities, and swift response times. It holds potential for real-time monitoring of Zn²⁺ in actual environmental settings.

2.1. Materials

The photophysical experiments were conducted using the F-320 fluorescence spectrophotometer (Tianjin Gangdong Technology Company) and UV-2600 ultraviolet-visible spectrophotometer (Shimadzu Company, Japan). The molecular structure was determined by the Bruker400 nuclear magnetic resonance hydrogen spectrometer (Bruker) and the Bruker100 nuclear magnetic resonance carbon spectrometer (Bruker). Analytically pure chemical reagents including o-hydroxybenzaldehyde (Innochem Company), 3-Amino-N,N-diethyl-4-methoxybenzenesulfonamide (McLean Company), ultra-dry ethanol (Innochem Company), PBS buffer (Innochem Company), etc., were utilized, along with distilled water for testing purposes.

2.2. Synthesis of Probe-Zn.

The synthesis of the Probe-Zn involved adding salicylaldehyde (0.733g, 6 mmol) and 3-Amino-N,N-diethyl-4-methoxybenzenesulfonamide (0.646g, 5 mmol) to a round-bottom flask, followed by the addition of 40 mL dry anhydrous ethanol and 3–5 drops of ice acetic acid after dissolution under N₂ protection. The mixture was then subjected to reflux reaction with stirring for 4 hours, followed by TLC detection. A light yellow powder solid was obtained with a yield of 78.6%, Melting point: 131.5℃-133.1℃. ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 13.35 (s, -OH, 1H), 9.01 (s, -CH = N, 1H), 7.81–7.66 (m, Ar-H, 3H), 7.43 (t, J = 7.9 Hz, Ar-H ,1H), 7.30 (d, J = 8.7 Hz, Ar-H ,1H), 7.04–6.91 (m, Ar-H, 2H), 3.94 (s, -CH₃, 3H), 3.17 (d, J = 7.1 Hz, -CH₂, 4H), 1.06 (t, J = 7.1 Hz, -CH₃, 6H). ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 164.93, 161.11, 156.03, 137.57, 134.08, 133.25, 132.33, 127.34, 119.78, 119.49, 118.13, 117.18, 112.86, 56.84, 42.44, 14.72. MS (ESI): m/z [M + H] + calcd for C₁₈H₂₂N₂O₄S, 363.22; found, 363.16.

The detailed characterization of the final molecules Probe-Zn is provided in the Supplementary Information (S1-S2)

3.1. Study of selectivity

The ability to selectively recognize specific substances is a fundamental requirement for fluorescence sensors. The same equivalent substance was introduced into the solution containing Probe-Zn (DMSO/PBS = 5/5) encompassing Zn²⁺, Na⁺, Mn²⁺, Cr³⁺, Cu²⁺, Fe³⁺, Cd²⁺, HCO₃⁻, NO₂⁻, Mg²⁺, Ca²⁺, Hg²⁺, CO₃²⁻, SO₄²⁻, NH₄⁺, CH₃COO⁻ and SH⁻. Subsequent to acquiring the fluorescence emission spectra (Fig. 1a), it was observed that upon addition of Mg²⁺, the ESIPT peak at 550nm vanished, giving rise to a new fluorescence emission peak at 520nm with an approximately 150-fold increase in fluorescence intensity. Upon irradiation with a 365nm ultraviolet lamp, a rapid color change from colorless to yellowish green was perceptible to the ‘naked eye’ in the mixed solution, which did not occur following addition of other competitive substances. The UV-VIS spectra of different ions (Fig. 1b) also reveal the emergence of a new peak at 415 nm following the addition of Zn²⁺, attributed to the chelation of Zn²⁺ with the probe molecule. This interaction restricts the isomerization of the C = N bond and enhances the conjugation of the probe molecule. It is evident that the target molecule Probe-Zn exhibits exceptional selectivity for Zn²⁺ and can be visually distinguished.

3.2. Detection Limits

By measuring the fluorescence intensity of various concentrations of Zn²⁺ added to the target probe solution, it is observed that within a specific range (0 ~ 100µmol/L), there exists a strong linear relationship between the fluorescence emission intensity and the concentration of Zn²⁺ (y=-473.27778 + 995.5x, R² = 0.99824) as depicted in Fig. 2. Based on the formula DL = 3δ/S, the detection limit (DL) of the probe molecule is determined to be 17.3 nmol/L, indicating excellent quantitative detection capability for Zn²⁺. Furthermore, it is evident that despite a blue shift from 550nm to 520nm with increasing Zn²⁺ concentration, the maximum emission peak of Probe-Zn remains unchanged while the fluorescence intensity continues to increase until reaching stability.

3.3. Study of response time

Rapid recognition ability serves as another key indicator for assessing the effectiveness of the probe. The fluorescence intensity of Probe-Zn at the maximum emission wavelength (520 nm) can be observed over time in the diagram. Upon addition of varying concentrations of Zn²⁺, the fluorescence intensity of the mixed solution increases rapidly and stabilizes after approximately 40 seconds. The addition of a high concentration of Zn²⁺ leads to a significant increase in both fluorescence response rate and intensity, as depicted in Fig. 3. It is evident that the chelation between the target probe molecule and Zn²⁺ occurs almost instantaneously. The correlation between response behavior and time validates the potential of Probe-Zn as a fluorescence sensor for rapid detection of Zn²⁺.

3.4. Stability investigation

The stability of Probe-Zn was assessed by measuring the precision (intra-day and inter-day) and reproducibility of the probe molecules. Analysis of Table 1 reveals that the intra-day RSD ranges from 1.29–3.78%, while the inter-day RSD ranges from 1.32–3.45%. Additionally, the RSDs of different batches of probes range from 2.78–3.59%. These test results demonstrate that Probe-Zn exhibits excellent precision and reproducibility, indicating good stability which enhances the practical application potential of probe molecules.

Table 1

Reproducibility of the synthesized probe.
[Zn²⁺] /(µmol/L)	RSD%(n = 7)
[Zn²⁺] /(µmol/L)	Intra-day	Inter-day	Different batches of Probe
1.0	1.29	1.32	2.78
5.0	3.13	2.89	3.11
10.0	3.78	3.45	3.59

3.4. Application in information storage

Considering the good recognition ability of the target fluorescent molecule Probe-Zn to Zn²⁺, we designed a portable Zn²⁺ detection invisible ink (Fig. 4) to evaluate the practical application ability of Probe-Zn. Using a 10 µM solution of Zn(CH₃COO)₂, we inscribed the letter ‘M’ onto a filter paper strip treated with Probe-Zn (DMSO/PBS = 5/5), which exhibited minimal changes when exposed to sunlight but fluoresced brightly under 365nm UV light. These findings indicate that Probe-Zn can serve as a highly efficient portable sensor for secure personal data storage, functioning as an invisible ink with the added benefits of convenience, real-time analysis, and cost-effectiveness.

3.5. Application of Probe-Zn in living cells

Cell imaging experiments further investigated the potential biological applications of the target probe molecule. The cytotoxicity of Probe-Zn was assessed using a CCK-8 assay in HeLa cells initially (Fig. S4, SI). At a concentration of 10 µM, the cell viability was found to be > 90% after 1 hour, indicating low-toxic cytotoxicity of Probe-Zn. The probe was utilized in fluorescence imaging experiments on Hela cells as depicted in Fig. 5. Initially, Hela cells were exposed to Probe-Zn with 1% DMSO at a temperature of 37°C for half an hour; however, no discernible fluorescence was detected under fluorescent microscopy. Nevertheless, under identical conditions following exposure to Probe-Zn for half an hour and Zn²⁺ for another half an hour resulted in noticeable fluorescence signals. These findings indicate that Probe-Zn exhibits favorable cell permeability and can effectively monitor changes in cellular Zn²⁺ levels under physiological conditions.

3.6. Sensing mechanism

The response mechanism of the target probe molecule, Probe-Zn to Zn²⁺ is widely believed to involve the presence of -N and -OH groups in the probe molecule, which contain lone pair electrons. Transition metal ions often possess empty orbitals. When the empty orbital of the metal ion and the lone pair electron of the probe molecule coincide, chelation can typically occur to form conjugated large π bonds. Consequently, fluorescence is significantly enhanced [17], as depicted in Fig. 6. Zn²⁺, Hg²⁺, and Cd²⁺ belong to the same main group and share similar chemical structures and properties. However, only Zn²⁺ elicits a response from the probe molecule, possibly due to its smaller size; hence, other metal ions' empty orbitals cannot perfectly match those of Zn²⁺, leading to ineffective identification.

In this study, we developed a novel fluorescence probe for the rapid detection of Zn²⁺. The designed fluorescence probe features a straightforward molecular synthesis method, gentle reaction conditions, and low production cost. Results from UV-VIS absorption spectrum and fluorescence emission spectrum demonstrate that the target fluorescent probe molecules exhibit unique selectivity and high sensitivity to Zn²⁺. Information storage and live cell imaging experiments have significantly broadened the practical application potential of the target fluorescent probe molecules. This work holds significant reference value for the design and construction of specific metal ion fluorescence sensors.

Acknowledgment

This work was supported by the projects ZR2020QB180 and ZR2020QB161 supported by Shandong Provincial Natural Science Foundation.

Author’s Declarations

Funding: Researchers supporting project number (ZR2020QB180, ZR2020QB161) Shandong Provincial Natural Science Foundation.

Conflicts of interest/Competing interests: The authors declare that they have no conflict of interest.

Availability of data and material: All data available.

Code availability: Not applicable.

Author’s contributions:

Xiujuan Li: Investigation, Formal analysis, Writing - original draft.

Tinghuan Gao: Investigation and Formal analysis

Shoucheng Wang: Investigation

Qing Zhang: Investigation

Siyu Chen: Investigation

Qianqian Liu: Investigation, Writing - review & editing.

Ethics approval: Not applicable

Consent to participate: Informed consent obtained from all individual participants included in the study.

Consent for publication: Not applicable

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Scheme 1 is available in the Supplementary Files section.

No competing interests reported.

Supportinginformation.docx
scheme1.png
Scheme 1. Synthesis of Probe-Zn.

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You are reading this latest preprint version

Development and utilization of a fluorescence sensor for Zn²⁺ detection

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Journal Publication

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Abstract

Figures

1. Introduction

2. Experimental section

2.1. Materials

2.2. Synthesis of Probe-Zn.

3. Results and discussion

3.1. Study of selectivity

3.2. Detection Limits

3.3. Study of response time

3.4. Stability investigation

3.4. Application in information storage

3.5. Application of Probe-Zn in living cells

3.6. Sensing mechanism

4. Conclusion

Declarations

References

Scheme 1

Additional Declarations

Supplementary Files

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