A New Dual-peak Fluorescent Probe for Water Content Detection Made From Taxus

In this paper, the leaves of Taxus were used as the sole carbon source, and two kinds of carbon dots blue and red, with different properties, were synthesized by the hydrothermal method under different conditions. The red carbon dots were quenched in the water, and the blue carbon dots had stable fluorescence properties in water environment. The bimodal fluorescence probe formed by mixing could accurately and stably measure the water content in ethanol, which was in the range of 82.5%-100%, is highly correlated with the fluorescence intensity ratio (I481/I678) of mixed carbon dots under 390 nm excitation light, with R2 = 0.995 and the detection limit as low as 0.31%. The experimental materials are environmentally friendly, low in cost, and simple to operate, as well as the water content measured by proportional fluorescence has high accuracy, which provides a new method for measuring moisture in ethanol.


Introduction
As a fluorescent nano luminescent material, carbon dots are highly sensitive and specific [1][2][3], which has received extensive attention in recent years. The main synthesis methods of carbon are various, and the main synthesis method is the hydrothermal method [4,5]. Solvothermal method [6,7] and microwave-assisted method [8][9][10][11]. Carbon has excellent selectivity in detecting trace antibiotics [12][13][14][15], heavy metal ions [5,[16][17][18], Pesticides [19][20][21][22][23], and medical imaging [24][25][26]. The aspect plays a huge role compared with a unimodal fluorescent probe, bimodal fluorescent probe has a high resolution, high precision, and strong stability [27][28][29] advantages, etc. Dried leaves of Taxus are used as biological carbon sources, and two kinds of carbon dots (481 nm and 678 nm) are synthesized by the hydrothermal method respectively. The fluorescence of red carbon dots can be quenched by water. The disadvantages are weak luminescence, large error, weak Gang Wang and Yaping Li are equally contributed to this work. sensitivity of blue carbon dots to water, and strong luminescence intensity. The dual-ratio fluorescence probe is formed by mixing two kinds of carbon dots, It can effectively make up for the shortcomings of the low sensitivity of blue carbon dots and weak luminescence of red carbon dots, enhance the stability of fluorescence data, and can be used for the detection of water content in ethanol.

Reagents
The leaves of Taxus were collected in the Taxus base of Fanghua Garden Company, which was cooperated by the Dujiangyan Campus of Sichuan Agricultural University. Silica gel, petroleum ether, ethyl acetate, ethanol, and various metal salts were purchased from Shanghai McKinley Co., Ltd., and all chemical reagents in this work are analytical pure reagents, which can be used directly without further purification.

Instruments and Characterization
The fluorescence spectrophotometer (Hitachi, F-4500, Tokyo, Japan) recorded the fluorescence spectrum and analyzed the optical properties and related activities of the products. The characterization parameters (morphology, size, etc.) of two kinds of carbon dots were detected by a high-resolution transmission electron microscope (HTEM) (JEOL 2100 F, Japan). The content and structure of elements were revealed by spectra measured by ESCALAB 250Xi photoelectron spectrometer (Thermo Scientific, USA).

The Influence of H 2 O Content on the Fluorescence Spectrum of Mixed Carbon Dots in Ethanol System
1. Fresh leaves of Taxus were dried in an oven (60 °C, 6 h), then cut into small pieces for later use, firstly synthesizing red carbon dots, weighing 1 g of dried leaves, put them in a reaction kettle, 20 ml of absolute ethanol added, left for reaction in the oven at 120 °C for 5 h, after the reaction kettle naturally cooled, absorbed the reaction solution, filtered through polyethersulfone membrane, and obtained filtrate carried out silica gel column chromatography. The eluent mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1, and the obtained chromatographic solution was concentrated by rotary evaporation, and the concentrated solution was transferred to a blank test tube (the blank test tube was weighted in advance), dried in an oven at 65 °C for 24 h, then the test tube was weighed again, and the quality of red carbon dots was obtained by differential calculation. 2. Synthesizing blue carbon dots, 1 g of dried Taxus leaves were weighed and put in a reaction kettle, 20 ml of deionized water was added, the reaction in an oven at 180 °C for 5 h, sucking the reaction solution after the reaction kettle was cooled, filtered with polyethersulfone membrane, concentrated the obtained filtrate by rotary evaporation, the concentrated solution was transferred to a blank test tube (the blank test tube was weighted in advance). After drying in an oven at 65 °C for 24 h, the test tube was weighed again, and the quality of red carbon dots was obtained by differential calculation. 3. Mixing the synthesis and fluorescence spectra of carbon dots, red carbon dots was diluted and blue carbon dots with ethanol to 100 mg/ml respectively, and mixed to form a mixed solution, setting a series of ethanol-water mixed solutions with equal water content gradient (2.5%) in the range of 82.5%-100% water content, 1 ml of red was mixed and blue carbon dots mixed solution and 1 ml of ethanol-water mixed solution with a total volume of 2 ml, The fluorescence spectra of 481 nm and 678 nm were recorded at the excitation wavelength of 390 nm, and the fluorescence intensity ratio (I 481 /I 678 ) at 481 nm and 678 nm was calculated. At the same time, 1 mM of different kinds of metal ion solutions was prepared, 100 µl of metal ion solutions were mixed with 900 µl of absolute ethyl alcohol, and 1 ml of red, blue, and carbon dots solutions were added, totaling 2 ml. The fluorescence spectra were recorded at the excitation wavelength of 390 nm and the fluorescence intensity ratio (I 481 /I 678 ) at 481 nm and 678 nm was calculated. By changing the water content in the ethanol-water mixed solution, the mixed carbon point sensing system can finally selectively detect the water content in ethanol.

Carbon Point Characterization
HTEM analysis of the red and blue carbon dots and their mixed carbon dots (Fig. 1) shows that the red carbon dots are clustered, with a size of 37.8 ~ 64.8 nm, the lattice width of 0.32 ~ 0.36 nm, the blue carbon dots are spherical, with the size of 2.7 ~ 4.1 nm and the lattice width of 0.33 ~ 0.45 nm. After the red and blue carbon dots were mixed, the blue carbon dots gathered around the red carbon dots to form a new aggregate, forming a dual-signal fluorescent solution (Fig. 2).

Optical Properties of Mixed Carbon Points
The ethanol solution of mixed carbon dots is pale yellow or yellow under natural light, which indicates that the prepared carbon dots have good solubility in organic solvents. The fluorescence spectra of blue carbon dots, red carbon dots, and their mixed carbon dots were studied at room temperature. Separately different excitation conditions study in blue carbon dots with fixed concentrations, and the excitation wavelength ranges from 360 to 410 nm, as shown in Fig. 3. When the excitation wavelength is 360 nm, a blue fluorescence peak was observed at about 450 nm. With the increase of the excitation wavelength, the fluorescence peak moves to the far-infrared wavelength end, and the intensity of the fluorescence peak gradually decreases. The fluorescence spectra of red fluorescent carbon dots with different excitation wavelengths were studied separately. When the excitation wavelength was 360 nm, the red fluorescence peak was observed at about 678 nm. With the increase of excitation light wavelength, the fluorescence intensity gradually increased, and the fluorescence peak did not move. After the mixing of the blue-red carbon dot solution, two peaks were formed at 450 nm and 678 nm, which together form a dual emissivity w system for moisture detection. In order to determine the best mixing ratio of two kinds of carbon dots, 6 kinds of mixed carbon dot solutions were prepared and carried out for fluorescence detection. It can be seen from the figure that the blue band fluorescence intensity decreases with the increase of excitation wavelength, while the red band fluorescence intensity increases with the increase of excitation wavelength (Figs. 3 and 4). Finally, considering comprehensively, it is determined that the excitation wavelength is 390 nm, and the mixing ratio of blue and red carbon dots is 1:1, so as to complete the final construction of the dual-ratio fluorescent probe (Figs. 5 and 6).

Sensitivity of H 2 O
The fluorescence quenching effect was observed by adding different water contents into a mixed carbon point ethanol solution (Fig. 7). The mixed fluorescence spectra showed that with the increase of water content in the sample, the fluorescence intensity at 481 nm remained unchanged, the quenching effect at 678 nm was obvious, and the fluorescence intensity decreased gradually. The ratio of fluorescence intensity (I 481 /I 678 ) increased with the increase in water content. It shows that the fluorescence intensity ratio of mixed carbon dots (I 481 /I 678 ) is highly correlated with water content. The fluorescence intensity ratio I 481 /I 678 and water content (R 2 = 0.995) fit well in the range of 82.5%-100% (Fig. 8). The detection limit is obtained by the ratio of triple blank standard deviation to the curve slope, and its value is as low as 0.31%.

Selectivity of Mixed Carbon Points
In order to test the anti-interference performance of the dualratio fluorescent probe, in the presence of 8 kinds of metal ions with a concentration of 100 μM, the mixed solution of ethanol and carbon dots was excited at 390 nm, and the fluorescence intensity ratio (I 481 /I 678 ) of the solution with only fluorescent mixed carbon dots was set as F 0 , and the fluorescence intensity ratio (I 481 /I 678 ) of other metal ions was set as follows The F 0 /F produced by the quenching phenomenon was observed and calculated. These results show that even if there are excessive metal ions coexisting with the solution,   (Fig. 9).

The Analysis of H 2 O with Standard Addition
In order to evaluate the practical application ability of this method, H 2 O was spiked and recovered, and three kinds of water contents 85%, 90%, and 95%, were spiked and recovered respectively, and then the spiked analysis table was made based on its fluorescence analysis. It can be seen from the Table 1 that the average recovery rate of H 2 O added in ethanol is 99.36%-100.96%, and the relative standard deviation is less than 1%, which can be used for quantitative analysis of samples.

Conclusions
In this study, the branches and leaves of Taxus were used as raw materials, and two kinds of carbon dots with different properties were synthesized by the hydrothermal method. After mixing them, based on the quenching effect of H 2 O on the mixed carbon dots, a double-emission fluorescence system was prepared, and a new proportional fluorescence determination method of H 2 O was proposed. The developed method has a linear range of 82.5%-100% and a low detection limit of 0.31%. H 2 O in ethanol was determined, The result is satisfactory, which shows that the sensing system has good sensitivity, high selectivity, and effective feasibility.