1. Introduction
China is rich in seaweed resources, among which macroalgae are abundant in various nutrients necessary for the human body and have important economic value. Macroalgae have a strong adsorption capacity for heavy metals. The heavy metal content in all kinds of macroalgae is higher than that of water, and the enrichment coefficient of heavy metal elements varies from several times to hundreds of thousands of times [
1]. Thus, macroalgae can be used not only as large nutrient reservoirs, but also as research samples for heavy metal pollution investigations. Through previous investigations, it has been found that the contents of Mn and Zn in macroalgae are very high. Mn and Zn, as essential elements, are of great significance to various physiological activities of the human body. However, excessive intake of Mn [
2] and Zn [
3] will also cause harm to the human body. Therefore, it is necessary and meaningful to establish a convenient analysis method for the detection of Mn
2+ and Zn
2+ in macroalgae.
So far, many analytical methods such as atomic absorption spectrometry (AAS), inductively coupled plasma mass spectrometry (ICP-MS), and electrochemistry have been applied to the detection of Mn
2+ and Zn
2+. However, these traditional detection methods all have the defects of complex operation and slow response time, and it is urgent to develop a method that is convenient to operate and quickly detect target elements. The fluorescent probe has attracted much attention because of its advantages of good selectivity, convenience, easy pretreatment, live-cell imaging use [
4,
5], and rapid detection of a variety of metal ions. At present, fluorescence probes commonly used in the detection of metal ions include organic small molecules, metal–organic frameworks (MOFs), aptamers, and nanomaterials. Organic fluorescent molecules, such as rhodamine, coumarin, naphthalimide, benzothiazole, pyridine, and other typical fluorescent groups, have been widely used in the detection of metal ions in food. Yu et al. [
6] designed a new coumarin-based ratiometric fluorescent probe using dithiothreitol as the recognition receptor for Hg
2+ detection based on the change in the color of the fluorescent probe from light yellow to orange. Zhou et al. [
7] designed and synthesized a novel fluorescence sensor based on fluoropyrrole and carboxyl mercaptan metal-bonded receptors for the detection of Hg
2+. The sensor was highly selective for Hg
2+, the detection limit of Hg
2+ was 5.7 nM, and the sensor responded quickly to Hg
2+ in aqueous solution in 30 s. However, poor solubility, serious photobleaching, poor bioavailability, and narrow excitation of these organic fluorescent dyes greatly limit detection and sensing. MOFs are hybrid crystalline porous materials, usually composed of inorganic nodes (metal ions or metal clusters) [
8] and functional organic linkers [
9,
10]. However, due to the instability of many MOF materials in water [
11], most current MOF-based fluorescence sensing research on metal ions is carried out in organic solvents, which are not suitable for the practical application of detection probes.
Carbon dots (CDs) have many advantages compared with traditional fluorescent materials, such as unique optical properties, excellent biocompatibility, excellent water dispersibility, low cost, high sensitivity, and easy surface functionalization [
12,
13]. These advantages make it possible to have great potential application in biomedical imaging and sensing [
14], tumor therapy [
15], light-emitting devices [
16,
17], and other fields. Much research has been conducted to synthesize CDs using chemicals [
18] or natural products [
19] as carbon sources. Xu et al. [
20] reported a facile method to prepare copper-doped carbon dots (Cu-CDs) using citric acid and cuprous chloride as precursors under hydrothermal conditions, which showed strong luminescence performance at 440 nm and with 9.81% photoluminescence quantum yield. Cu-CDs with a detection limit of 1 nM can be used for the rapid detection of Fe
3+, and can be further applied to the detection of Fe
3+ in human serum. Liu et al. [
21] reported that Bombyx mori silk was used as a raw material in the coupling of citric acid to prepare nitrogen-doped CDs using a facile one-step hydrothermal route. The as-prepared nitrogen-doped CDs emitted blue fluorescence with a quantum yield of 61.1%, which can easily bind with Fe
3+ as a consequence of fluorescence quenching, making a method for Fe
3+ detection developed with high selectivity and sensitivity. He et al. [
22] developed a hydrothermal method to synthesize Zn
2+-doped carbon quantum dots (Zn-CQDs) using zinc citrate chelate, with a fluorescence quantum yield of 48%. Zn-CQDs have stronger fluorescence sensing ability for Fe
3+ and Hg
2+. They can be used in the fields of fluorescence sensors, biological imaging, photoelectronics, and catalysis. However, most of the CDs that have been synthesized have strong emission in the blue-green electromagnetic radiation region and weak emission in the red region with a low quantum yield. Due to the interference of the inherent blue self-fluorescence in the biological matrix, it is necessary to avoid blue emission during practical sample detection. Because of the low extinction coefficient of the biological matrix in the red and near-infrared range in biosensing, red carbon dots (R-CDs) will inevitably show a large signal-to-noise ratio, which is beneficial to improve the sensitivity and accuracy of practical sample detection. Therefore, fluorescent biological probes prepared by using R-CDs can not only effectively avoid self-fluorescence interference, but also have strong penetrability to tissues [
23,
24], which makes it particularly important and urgent to prepare an R-CDs-based fluorescence probe.
In this paper, R-CDs with dual-emission properties were prepared by the thermal solvent method. The surface morphology and photoluminescence characteristics of R-CDs were studied. The fluorescence probe was constructed by using the R-CDs with dual emission to detect Mn2+ and Zn2+. Its detection performance was analyzed. Finally, the constructed fluorescent probe was used to detect macroalgae produced in Fujian province, China. The accuracy of the method was verified by flame atomic absorption spectrometry (FAAS). The content of Mn and Zn elements in kelp (Laminaria Japonica) and laver (Porphyra haitanensis) samples was analyzed, and the proposed fluorescent probe could have potential application in dietary guidance and safety assessment.
2. Materials and Methods
2.1. Preparation of R-CDs
The R-CDs were prepared by the thermal solvent method, which can be described as follows: 0.6804 g reduced glutathione (Macklin, Shanghai, China) and 0.6106 g o-phenylenediamine (Merck, Darmstadt, Germany) were dissolved in 20 mL of formamide (Macklin, Shanghai, China) at room temperature. The obtained mixture was transferred into a Teflon-lined stainless steel autoclave, which was then kept in an air-circulating oven at 160 °C for 2 h. After the reaction, the autoclave was naturally cooled down to room temperature. The obtained solution was centrifuged at the speed of 1000 rpm for 20 min to remove large particles. The upper solution was diluted and dialyzed (molecular weight cut-off = 1000 Da) against ultrapure water for a week. The purified R-CDs solution was freeze-dried to obtain dark green powder for characterization. Ultrapure water (DI, >18.25 MΩ) was prepared by the Millipore Milli-Q Water Purification System (Merck, Billerica, MA, USA) for the preparation of all solutions in this work.
2.2. Structural Characterization of R-CDs
Fourier transform infrared spectroscopy (FT-IR) was obtained using a VERTEX 80V FT-IR spectrometer (Bruker, Billerica, MA, USA). FT-IR samples were prepared by mixing the powders of KBr and R-CDs in a ratio of 1:150. The obtained mixture was made into tablets. TEM (Shimadzu, Tokyo, Japan) samples were prepared by dropping the aqueous solution containing R-CDs onto carbon-coated grids and allowing the excess solvent to evaporate. The structure of the as-prepared R-CDs was characterized by XRD-6000 (Shimadzu, Tokyo, Japan). The fluorescence measurements were performed with a fluorescence spectrophotometer RF-5301PC (Shimadzu, Tokyo, Japan). The samples were excited from 360 to 420 nm, and the emission spectrum in the range of 220 to 900 nm was measured. The slit width was fixed at 10 nm for emission. The ultraviolet–visible (UV–VIS) spectra were recorded on a UV-2600 ultraviolet–visible spectrophotometer (Shimadzu, Tokyo, Japan), with scanning wavelength ranging from 200 nm to 800 nm using a xenon lamp and a tungsten lamp.
2.3. Signal-Off Detection of Mn2+
For the detection of Mn2+, 1 mL of purified R-CDs solution and varying concentrations of Mn2+ ion standard solution (Macklin, Shanghai, China) were separately added into the mixture of 980 μL of 0.1 M HEPES buffer solution (Solarbio, Beijing, China). Then, the mixture was thoroughly shaken and equilibrated at room temperature. The fluorescence spectra with 420 nm excitation wavelength and 680 nm emission wavelength were recorded and used for quantitative analysis.
2.4. Ratiometric Detection of Zn2+
For the detection of Zn2+, 1 mL of purified R-CDs solution and varying concentrations of Zn2+ ion standard solution (Macklin, Shanghai, China) were separately added into the mixture of 980 μL of 0.1 M HEPES buffer solution (Solarbio, Beijing, China). Then, the mixture was thoroughly shaken and equilibrated at room temperature. The fluorescence spectra with excitation at 420 nm and emission at 650 nm and 680 nm were recorded and used for quantitative analysis.
2.5. Sample Pretreatment
In this work, 20 different brands of macroalgae samples were purchased from supermarkets and retailers in Fujian province. Four hundred milligrams of the cleaned and dried samples was ground into powder, added with 5 mL HNO3 into the microwave digestion tank, and digested with an 800 W microwave digestion instrument. The digested sample was heated up to 140–160 °C to remove excess acid. After cooling to room temperature, approximately 1 mL of sample solution remained and was transferred to a volumetric flask with ultrapure water to 25 mL. The sample solution could be diluted with need. Blank samples were prepared according to the same method without adding macroalgae.
2.6. FAAS Determination
A series of standard concentrations of Mn solution and Zn solution were prepared. The absorbance is plotted as the function of metal element concentration to obtain a calibration curve for quantitative analysis. The pretreated sample was placed in FAAS (AA-6300C, Shimadzu, Tokyo, Japan), and the contents of Mn and Zn elements were detected according to the test parameters of different elements. Among them, the test parameters for Mn detection were lamp current 2.0 mA, wavelength 279.5 nm, slit 0.1 nm, negative high pressure −344 V, air flow 6.0 L·min−1, acetylene flow 1.0 L·min−1. The test parameters for Zn detection were lamp current 2.0 mA, wavelength 213.9 nm, slit 0.2 nm, negative high pressure −309 V, air flow 6.0 L·min−1, and acetylene flow 1.0 L·min−1.