Of all the heavy metals that contaminate tobacco, divalent mercuric ion is one of the most dangerous. Thus, a novel label-free and enzyme-free fluorescent method, based on mercury nanoladders integrated with thiazole orange (TO), was developed for rapid, sensitive, and selective detection of Hg

 in aqueous solutions. In this study, we have determined that, in the presence of mercury ions, the thymine bases in probe 1 (P1) and probe 2 (P2) interact with Hg

-T structure, then long double helices of Hg

 nanoladders will eventually result. Subsequently, the fluorescence of the TO dye was strongly enhanced, due to the binding of TO dye to long dsDNA. The increase in fluorescence intensity was linear with Hg

 concentration in the range of 0.02–5 nM, and the limit of detection for this method was 20 pM. In addition, this method exhibits high selectivity, and experiments with tobacco samples also showed good results. We believe that the proposed system accurately and efficiently detects mercury ions within different substrates.

In recent years, because of the use of non-ferrous metal minerals, coal-fired flue gas, and solid waste incineration, a large quantity of heavy metals and toxic substances has been produced, resulting in an increased risk of excessive mercury in tobacco (1–4). Heavy metal ions in tobacco, especially mercury ions, can pose a serious health threat to smokers (5–7). Thus, it is urgent to develop accurate, sensitive, and very specific analytical methods for the determination of trace mercury ions in tobacco.

Various detection methods have been proposed to monitor trace amounts of mercury ions, including atomic absorption spectroscopy (AAS) (8,9), inductively coupled plasma–optical emission spectroscopy (ICP-OES) (10), and gas chromatography (GC) (11). Although these methods are very sensitive and accurate, they tend to be tedious and time-consuming, and depend on expensive instrumentation and professionally trained operators, thus limiting their application. At the same time, colorimetric sensors (12,13), electrochemical sensors (14,15), and fluorescence sensors (16–18) have also been developed to detect mercury ions. However, the colorimetric methodologies require modifications or additional materials to generate a signal response, and electrochemical methods have inherent disadvantages associated with modification requirements and instability of the electrodes. Therefore, fluorescence methods have been developed for mercury ion detection because of their advantages of stability, simplicity, and speed. For example, Wang and associates (16) reported a fluorescent probe based on branch migration for highly sensitive detection of mercury. Hu and associates (17) developed a DNA@MOF biosensor for successive and specific detection of Hg

. In addition, Huang and colleagues (18) developed a sensitive detection method for mercury ions by exonuclease signal amplification. However, cumbersome fluorophore labeling and enzyme-assisted signal amplification makes the above methods complicated and expensive. Thus, to solve the above problems, we have developed an enzyme-free and label-free fluorescence method, based on mercury nanoladder signal amplification and thiazole orange (TO), to detect Hg

This paper explains a simple and enzyme-free assay we have constructed for mercury ion detection based on mercury nanoladder signal amplification integrated with TO. A mercury nanoladder consists of a long double-stranded structure and relatively short sticky ends that are essentially nicked double helices of alternating copolymers (19). TO is a special asymmetric cyanine dye that has a weak fluorescence signal in aqueous solutions, but that exhibits a considerably increased fluorescence intensity when it interacts with double-stranded DNA (20,21). This occurs because when the dye binds to the nucleic acid through the positive and negative charge electrostatic attraction, the quinoline ring becomes embedded in the small groove of the double-stranded DNA molecule. Dye molecules are more planar, due to their steric hindrance, and thus exhibit increased fluorescence enhancement (22). The system discussed in the present study contains two specially designed oligonucleotide probes, probe 1 (P1) and probe 2 (P2). The interaction between thymine and Hg

-T structures, resulting in the formation of nanoladders in the presence of Hg

. In this state, TO can embed in the mercury nanoladders, producing a strong fluorescence emission. Compared with previous methods, this method has the advantages of a simple design, high sensitivity, and reduced cost, and may contribute to increased application prospects for heavy metal detection.

The thymine-rich probe DNA sequences ([P1:5′-ATGCTTCGTTGTCTGTGCCT TCGTGTTGCA-3′] and [P2:5′-ACGGTT GCTCTTCGTTACGTTGCTTCTGAC-3′]) were obtained from Sangon Biotechnology Co., Ltd., and were dissolved in a Tris-HCl buffer (pH 7.4, 20 mM). All the synthetic DNA sequences are listed in Table I. These samples were stored at 4 oC for immediate use and long-term preservation. All other chemicals were obtained directly from commercial sources and used as received. Hg(NO

 was purchased from Sigma Aldrich, and dissolved in 0.5% HNO

. The thiazole orange (TO) was obtained from Tianjin Seans Biochemical Technology Co., Ltd. All other reagents were analytical grade and used without further purification or modification. Ultrapure water used throughout was produced from a Milli-Q water purifying system.

DNA fluorescence intensity was measured at the excitation spectra of 501 nm, using a spectrofluorophotometer (RF-5301PC, Shimadzu) where the slits were set to 5.0 and 5.0 nm for excitation and emission, respectively. The method was evaluated at a fluorescence intensity of 538 nm, which has been demonstrated to be the maximum emission intensity wavelength. Absorbance spectral measurements were carried out on a UV-2450 spectrophotometer (Shimadzu Co.). Native polyacrylamide gel electrophoresis was performed on a DYY-6C electrophoresis (Liuyi Biological Technology Co.).

Active Polyacrylamide Gel Electrophoresis

In a typical experiment, samples 1,2, and 3 included P1, P2, and P1+P2, respectively. Sample 4 was the mixture of P1, P2, and Hg

. All of the samples were incubated at 37 

C for 30 min. We used 12% PAGE gel in a 1xTBE buffer for analysis of the product, and then gel electrophoresis was run for 70 min at a stable voltage of 100 V. Finally, the gel was stained with Stain-All, and then photographed after decolorization.

The DNA probes (P1 and P2) were diluted with Tris-HCL buffer, to give the stock solutions. Then, a 250 μL reaction sample, containing 50 nM P1, 100 nM P2, 1 μM thiazole orange (TO), and different concentrations of Hg

, was prepared in a Tris-HCl buffer, followed by incubation for 30 min. The fluorescence intensity of TO was measured at 520–600 nm, where the maximum emission peak was 538 nm.

We weighed 0.5 g of a tobacco sample into a PTFE digested flask, and then 3.0 mL of concentrated nitric acid and 5.0 mL of 30% hydrogen peroxide were added. The digestate evaporated to near dryness. The residue was dissolved in 5 mL of 5% nitric acid, then the pH was adjusted to near neutral with NaOH and transferred to a 50 mL volumetric flask.


The principle of the proposed method for rapid, label-free, and enzyme-free Hg

 detection is demonstrated in Figure 1. In this study, the system contains two specially designed oligonucleotide probes: the thymine-rich probe 1 (P1), and the thymine-rich probe 2 (P2), with both including two T-rich domains. The background signal of this system is low in the absence of Hg

, owing to P1 and P2 coexisting stably and remaining single-stranded. When Hg

 was introduced into the system, the interaction between thymine and Hg

-T structures, resulting in mercury nanoladders. This cascade reaction will continue until the supply of the Hg

 ions is exhausted. In this state, TO could be embedded in the mercury nanoladders, producing a very strong fluorescent signal. The UV spectra of TO is shown in Figure 2a, and the excitation and emission spectra of TO are shown in Figure 2b, which are 501 nm and 538 nm, respectively.

To validate the feasibility of the proposed method for detecting Hg2+, the emission spectra obtained by fluorescence detection of samples containing different components were analyzed, as shown in Figure 3. From Figure 3, it was observed that, in the absence of Hg2+, the mixture composed of P1 and P2 produced relatively weak fluorescence emission. In the mixed solution of P1, P2, and Hg2+, the fluorescence intensity of TO increased significantly; this may be due to an increase of the double-chain structure in the solution.

Gel electrophoresis experiments were used to further verify the feasibility of the method and the formation of the mercury nanoladder produced by the addition of the target. As shown in Figure 4, the mercury nanoladders product was obvious in Lane 4, proving the feasibility of the amplification system.

The ratio of probes P1 to P2 that affects the fluorescence intensity of the product was optimized. The concentration ratio of probes P1 and P2 was experimentally measured from 1:1 to 1:3, as shown in Figure 6. It can be seen that a ratio of P1:P2 = 1:2 affords the best signal-to-background difference (F/F

 represent the fluorescence intensity of the solution in the presence and absence of Hg

, respectively, and the fluorescence was strongest at 1:2. Therefore, it was determined that at a 1:2 concentration ratio of the probes P1 and P2, the optimum concentration ratio is obtained.

It is worth noting that using TO as the dye agent plays a very important role in the detection of fluorescent signals, and, therefore, it is necessary to optimize the concentration of TO. As shown in Figure 5b, when the concentration of TO is increased from 0.25 μM to 2 μM, a fluorescence emission was observed for detection at a maximum of 1 μM. Therefore, 1 μM is taken as the optimum concentration of TO.

 can promote DNA hybridization and requires optimization of the Mg

 concentration increases, it is observed that the binding efficiency of P1 and P2 is higher, and F/F

-1 is gradually increased. However, at higher concentrations, the background signal is high and F/F

-1 gradually decreases. Figure 5c shows that the fluorescence intensity ratio F/F

-1 reaches a maximum at 1 mM. Therefore, 1 mM of Mg

 was used as the optimum concentration in the assay.

Temperature is an important factor in the reaction, and the intensity of the fluorescence signal is different at different temperatures. As shown in Figure 6a, the fluorescence signal is highest at 37 

C so the overall system reaction is optimized at this temperature.

Incubation time is another important factor affecting the strength of the fluorescence signal. It can be concluded from Figure 6b that a platform is reached by the intensity of the fluorescence signal after more than 30 min of incubation time. Therefore, 30 min pours can be used as the best time for reaction.

In order to understand the sensitivity of this method, the change in fluorescence intensity of Hg

 concentration. From the Figure 7a, the fluorescence signal can be enhanced with the increase of Hg

 concentration under optimal conditions. As shown in Figure 7b, when the Hg

 concentration was from 0.02 nM to 5 nM, the linear regression equation was ∆F = 12.23C + 4.998, and the correlation coefficient is 

represent the fluorescence intensity difference and the Hg

 concentration, respectively. The detection limit of Hg

 detection limits previously reported for various methods are listed in Table II.

To overcome the interference of other ions with Hg

, it is valuable to measure the selectivity of Hg

 by changing different metal ion targets under the same conditions, such as Ni

, respectively. As shown in Figure 8, 1 μM Hg

 significantly altered the intensity of the fluorescent signal in the selective experiment. By comparison, it is found that the interfering metal ions (including Ni

) had no positive effect on the fluorescent signal. At the same time, when other interfering ions and mercury ions are mixed, the change in fluorescence intensity is not much different from that in the presence of mercury ions alone. This result indicates that the present strategy exhibits excellent selectivity for the detection of Hg

, and is not easily interfered by competing ions, resulting in more accurate results.

The proposed method has been successfully applied to the determination of mercury in tap water and tobacco. The recovery of mercury ion by adding different concentrations of mercury ion in a sample was carried out, and the results are shown in Table III. The data in the Table III reveals that the proposed methodology offers great potential for the specific detection of Hg

We have successfully described a sensitive and selective detection method for mercury ions by using mercury nano-ladder signal amplification. P1 and P2 formed a long double-stranded complex after adding mercury ions, also named mercury nanoladders, that had the ability to combine with TO to produce a strong fluorescence emission. Consequently, this approach possesses a significant advantage of avoiding cumbersome labeling procedures and enzymes. At the same time, the method also exhibits excellent sensitivity and selectivity with a limit of detection of 20 pM. Moreover, the system can be practically applied for the detection of Hg

 in tobacco samples, showing an extensive application prospect. The success of the system suggests that it can be employed as a universal sensor for more substrates.

This work was supported by the Basic Research Foundation of Yunnan Province (Nos. 2016CP021, 2017FD236), the Basic Research Foundation of China Tobacco Yunnan Industrial Co., Ltd. (No. 2019XY01, 2018JC04), the Project Foundation of Yunnan Key Laboratory of Tobacco Chemistry (KCFZ-2017-1096) and the Technology talent and platform program of Yunnan province (No. 2019HB079).

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(25) T. Bao, W. Wen, X. Zhang, Q. Xia, and S. Wang, 

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Xiaoxi Si, Juan Li, Yuanxing Duan, Yuting Yang, Yuandong Li, Chunbo Liu, Yang Zhao, Fengmei Zhang, Jianjun Xi, Qinpeng Shen, 

are with the Yunnan Key Laboratory of Tobacco Chemistry, at China Tobacco Yunnan Industrial Co., Ltd., in Kunming, in the People's Republic of China. 

is with the Key Laboratory for Green Organic Synthesis and Application of Hunan Province, at the Key Laboratory of Environmentally Friendly Chemistry and Application of Ministry of Education, at the College of Chemistry of Xiangtan University, in Xiangtan in the People's Republic of China. Direct correspondence to: ashb345@126.com

Articles

An Ultrasensitive Fluorescence Method for Mercury Ion Detection Based on Mercury Nanoladder Signal Amplification and Thiazole Orange 

A Judd-Ofelt analysis, quantifying the optical intensities of the rare-earth ions’ 4

 manifolds, is conducted in detail, based on the absorption spectrum from the visible to infrared spectral range for a Pr:YLF crystal. The values of key parameters, such as radiation lifetime, radiation transition probability, and branching ratio, are obtained accordingly. Additionally, the emission spectra are measured with precaution against reabsorption, and the emission cross sections are calculated using the Füchtbauer-Ladenburg formula, applied frequently in precise emission spectrum analysis, with the radiation lifetime derived from the Judd-Ofelt model. The results obtained regarding the orange and deep red spectral range are higher than those from previous reports, providing further insights into the lasing mechanism at the concerned wavelengths.

Highly-efficient visible lasers have attracted substantial research interest in recent years (1–5). Praseodymium (Pr), thanks to its energy structure (providing a direct down-conversion mechanism pumped by the booming blue diodes coming out recently) has become the most promising laser gain ion in the visible range (6). As for the host material, yttrium lithium fluoride (YLF), a positive uniaxial with tetragonal structure of scheelite type (7), is well-known for its superior physical and optical properties. The Mohs hardness of YLF is 4~5 (8), with a relatively high thermal conductivity (6 W·m

-axis [8]), facilitating its wide application in high-power lasers (9,10) and the Kerr lens mode-locking technique (11). In particular, Pr-doped YLF (Pr:YLF), as a fluoride crystal, is characterized by its low phonon energy (460 cm

), compared with its oxide counter- parts ( 550 cm

for praseodymium-doped yttrium aluminum perovskite (Pr:YAP) with the formula Pr:AlYO

, leading to a weaker non-radiative multi-phonon relaxation, and thus better lasing performance.

The spectroscopic characteristics of the crystal used in a laser being critical to laser performance, and involve both the absorption and emission spectra, respectively. The former can be derived through a Judd-Ofelt analysis, and the latter by employing the Füchtbauer-Ladenburg formula (14), both of which are analytical calculation procedures. Judd-Ofelt theory, which was proposed independently by B. R. Judd (12) and G. S. Ofelt (13) in 1962, describes the possibilities of radiative emissions in the 4f

 configuration, and is extensively adopted to determine the radiative lifetime and branching ratio of each emitting level for trivalent rare-earth-doped laser materials (15–24). On the other hand, the Füchtbauer-Ladenburg formula was derived from the fluorescence analysis correlated with an electronic transition (25), and has gained wide application in the determination of emission cross sections of laser gain media (22–24). To optimize the analytical procedure for characterizing Pr:YLF crystal, various improvements have been made in both theory and experiment. For example, Dunina and associates modified the Judd-Ofelt theory by considering the finite 4

 energy (26), Kornienko and colleagues introduced third order perturbation theory into the line intensity calculation (27,28), Goldner and associates adopted a normalized least-squares method in the fitting process to obtain a stable output (29), and Quimby and colleagues used the experimental branching ratios, other than theoretical ones, in deriving emission cross sections (30).

In this paper, the spectroscopic properties of a Pr:YLF laser crystal (31) were studied by analyzing the polarization-dependent absorption and emission spectra, accordingly. Radiation transition probabilities, theoretical branching ratios, and intrinsic radiation lifetimes were obtained from the absorption spectrum analysis. Experimental branching ratios were demonstrated by emission spectrum analysis. In addition, by introducing the intrinsic radiation lifetimes derived from Judd-Ofelt analysis and experimental branching ratios into the Füchtbauer-Ladenburg formula, emission cross sections are also reported.

For the absorption spectrum measurement, to reduce the absorption effect of the crystals, a relatively low doping concentration of 0.3 atomic percent (at.%) was adopted. The absorption spectrum was measured using a Lambda 1050 spectrophotometer (PerkinElmer). The data sampling interval was set at 0.05 nm. Polarizing prisms were placed in the sample path and the reference path, to measure absorption spectra in both polarizations.

The absorption coefficients for different wavelengths (

) were calculated from the absorption spectrum using equation 1:

 are the light intensities of the sample light path and the reference light path, respectively. The crystal absorption cross sections can then be calculated by equation 2:

 in the crystal. The calculated absorption cross sections are shown in Figure 1.

Table I lists the peak wavelengths, absorption cross sections, and line widths of major transitions in the visible spectral range. There are three absorption bands in the blue spectral range, corresponding to transitions 

, peaking at 479.2 nm, possesses the largest absorption cross section (2.16 × 10-19 cm2) with the narrowest line width (0.5 nm). Transition 

 (443.9 nm) has a relatively large absorption cross section (9.0 × 10

) and line width (1.8 nm). In addition, there are four major absorption bands in the infrared spectral region, 

 at 2.3 μm, respectively. From the calculation results ranging from the visible to the infrared regime, it can be concluded that the absorption cross sections in π polarization are generally larger than their σ counterparts.

According to the Judd-Ofelt theory (12,13), the experimental line intensity can be expressed as:

 are the total angular momentum quantum numbers of the initial and final energy levels, respectively, 

 is the ›integral absorption cross sections. The values of the polarization-dependent line intensities are listed in Table II, in which λ is the average of measured wavelength weighted by the accordance absorption cross sections.

The mean experimental line intensity is defined as

 can be achieved using the least squares fitting, performed as

 are doubly reduced matrix elements in accordance with specific rare-earth ions. In our case, we use matrix elements evaluated and given by Weber for Pr

 configuration is very close to that of the 4

 configuration. The line intensity from the ground-state level to the highest emitting level is larger than the calculated value, so the hypersensitive transition 

 is always neglected in the fitting procedure (33). The results of the intensity parameters are Ω

. Because the line intensity parameters Ω

 are material dependent, the calculated Ω

 values are applicable to all transitions of the same material. Considering that the magnetic dipole component for Pr

 could be neglected (34), the calculated line intensity S

 is given by the electric dipole line intensity, shown as below:

The experimental and calculated values of line intensities, and doubly reduced matrix elements of absorption transitions, are shown in Table III. 

To verify the calculation accuracy, the root-mean-square (RMS) error is calculated, and its expression is given by:

 is the number of required parameters and 

 is the number of spectral bands to be analyzed. Combined with our calculation, where 

 = 12%, which is within the normal scope (5%~25%) (35).

), the radiation probability of different transitions 

For transitions from the same excited state, the theoretical branching ratios β

 is defined as the reciprocal of the sum of all radiation transition probabilities from the same energy level, shown as equation 10. The radiation transition probabilities, theoretical branching ratios, and intrinsic radiation lifetimes of 3P0, 3P1, and 1I6 level are listed in Table IV.

 levels obey the Boltzmann distribution, the radiation lifetimes of those levels must be considered simultaneously. The effective radiation lifetime of the upper level is the average of the intrinsic radiation lifetimes of 

 level, according to the distribution of the number of electrons (36,37), given by

) is the degeneracy of corresponding energy level, being 1, 13, and 3, respectively, and Δ

By substituting the radiation transition rates listed in Table IV into equation 11, the effective radiation life- time of the upper energy level was calculated to be 49 μs, with a derivation of ±10%, derived from the error of Judd-Ofelt theory. The effective radiation lifetime of the upper level τ

 is the non-radiative transition rate, including multi-phonon transition (

 [38,39]), and so on. It could be deduced that τ

, which could be reduced by lowering the doping concentration of rare earth ions.

In terms of the emission spectrum, the measurement device is shown in Figure 2. Using an InGaN laser diode (LD) with a wavelength of 444 nm as the pump source, the driving current was set at 100 mA, and the corresponding output power was about 30 mW. The pump light is focused into the sample, whose emitting spectrum was converged into the slit of the monochromator through a lens with large aperture and long focal length. A photo-multiplier (PMT, Hamamatsu R3896) was employed. The phase-locked amplification technique, implemented with a chopper, was utilized to extract the small signal in the noise. A Mercury vapor lamp (color temperature 2900 K) was used for wavelength correction and calculation of spectrometer transfer function. The experiments were conducted at room temperature.

The emission spectrum, showing multiple transitions in the visible range, is shown in Figure 3. The main transitions of 

 correspond to the peak wavelengths of 479.4, 522.6, 607.2, 639.5, 697.7, and 720.7 nm. Blue 

 (720.7 nm) are mainly π-polarized, while red light 

A stimulated emission cross section is usually treated as a parameter for evaluating the gain performance of the laser material. The methods for calculating the stimulated emission cross section mainly include the reciprocity method (40), the Füchtbauer-Ladenburg formula (14), and the reverse of the laser performance (41). The first method is applicable to quasi-three-level transitions with clarified ground-state level distribution, the second method is suitable to either quasi-three-level or four- level transitions with a highly accurate emission spectrum, and the third method is strongly dependent to the measurements of laser experiments. Based on the emission measurement results in the 450–750 nm spectral range, the Füchtbauer-Ladenburg formula was employed to calculate the stimulated emission cross section.

For a uniaxial laser crystal with two polarization directions, the Füchtbauer-Ladenburg formula can be written as (42):

 was employed, instead of the theoretical value calculated based on Judd-Ofelt theory, owing to the small energy differences between the 4

where the denominator is an integral over the entire fluorescence spectrum. The calculation results are shown in Table V.

By substituting equation 14 into equation 13, the numerator of 

 can be eliminated; therefore, equation 13 could be simplified to

The results are displayed in Figure 4, with the emission cross sections and line widths of the main transitions listed in Table VI.

Note that the emission cross sections in 

 polarization are generally larger than those in σ polarization. The σ-polarized 640 nm transition has the largest emission cross section, reaching 22.3 x 10

It should be noted that some results different from those of previous reports (43,44) were demonstrated. For the orange transition at 607 nm, a slightly larger value was achieved (1.57×10

 [43]), thanks to the precaution to avoid the ground-state reabsorption process 

. In terms of the transitions in the deep red spectral region, the emission cross sections of 698 and 721 nm were 1.07×10

, about 2x larger than those in (43) (0.5×10

). Because emission cross sections are proportional to the corresponding laser thresholds, further investigations in the deep red range laser thresholds can be used to verify the accuracy of the emission cross sections.

The spectroscopic properties of a Pr:YLF laser crystal were studied theoretically and experimentally. The polarization-dependent ab- sorption cross sections at room temperature from the visible to infrared spectral range were demonstrated. The maximum absorption of 21.7×10

 peaked at 478 nm, with the narrowest line-width of 0.5 nm. In the framework of Judd-Ofelt theory, intensity parameters Ω

 were obtained when the hypersensitive transition 

 was excluded from the fitting procedure. For the emission spectrum, the experimental branching ratio was introduced to calculate the emission cross section using the Füchtbauer-Ladenburg formula in the visible spectral range. Results different from those seen in previous reports were obtained in both the orange and deep red spectral regions. This investigation provides insight into the spectroscopic analysis of Pr:YLF crystals used in lasers.

This work is supported by NSAF (No. U1830123), the National Natural Science Foundation of China (No. 61627802), the Natural Science Foundation of Jiangsu Province (No. BK20180460), and the High-Level Educational Innovation Team Introduction Plan of Jiangsu, China.

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A Full Spectroscopic Study of Pr:YLF Crystals Used in Lasers

A new indole-based chemosensor, called FL ((E)-N’-(3-chloro-2-hydroxybenzylidene)-1H-indole- 2-carbohydrazide) capable of recognizing F

 ions and exhibiting a turn-on fluorescence response, was synthesized and tested. FL showed a high fluorescence enhancement in the presence of F

CN-phosphate and buffer solution (PBS) (99:1, v/v) with a significant color change (from colorless to yellow). FL had a good linear relationship with F

 from 0 to 11.85 μM and a detection limit up to 3.2 nM. The Job plot indicated that FL formed a compound with F

 ions in a 2:1 ratio and the binding constant was calculated to 3.6 × 10

. The proposed bonding mechanism between FL and the F

 ion was speculated as hydrogen bonding by O-H∙∙∙F. Results of fluoride ion detection in water samples and cell imaging demonstrated that FL has the potential application to investigate biological processes involving F

Fluoride ion, one of the most important nucleophilic anions, has attracted special attention because of its extensive application in oral health and clinical treatment of osteoporosis (1–5). A moderate amount of fluoride ions can help the normal development of bones and teeth (6). It can also improve the resistance of tooth enamel to acid corrosion by bacteria, and it has an obvious effect on preventing dental caries. However, excessive intake of fluoride ions may cause a variety of diseases, such as gastric disease, kidney disease, dental fluorosis, skeletal fluorosis, urolithiasis, and even death, because of its toxicity (7–9). When the fluoride ion content in drinking water exceeds 4 ppm, people are prone to develop skeletal fluorosis, which leads to bone marrow deformity. Therefore, the detection, evaluation, and prevention of excessive fluoride ions is important because of its impact on human health.

There are many methods that have been widely used to detect fluoride ions. These methods include ion selective electrodes (10–16), ion chromatography (17–19), colorimetry (20), and high-performance liquid chromatography (HPLC) (21–22). These common methods can detect fluoride ions quickly and accurately, but they have several shortcomings, which include high cost, poor selectivity, and supplementary sample preparation. With the rapid development of supramolecular chemistry and the deepening of research on anion recognition, chemical sensors based on fluoride ion recognition have become a current research priority (23–25).

One of these chemical sensors is known as the 

. Coumarin derivatives are designed as fluoride ion chemical sensors because of their high quantum yield and large stock shift (26,27). Indole, which is a hydrogen bond donor, is favored by researchers as a chemosensor for fluoride ions. In a recent study, Bu’s group synthesized a series of indole-based fluoride ion chemosensors with 2,3-diindol-3-yl diketone (28–30). By connecting 2,3-diindol-3-yl diketone with diaminomaleonitrile, the novel chemosensor displayed a highly selective response to F- in dimethyl sulfoxide, and the color of the solution changed from yellow to red-orange (29). After connecting with 1,2,3,4,5,6-hexaaminobenzene, a visible chemosensor based on photoinduced electron transfer (PET) and intramolecular charge transfer (ICT) mechanisms was formed with a 42 nm red shift of fluorescence emission (30). These chemosensors are characterized using -NH and -OH as hydrogen bond donors to form hydrogen bonds with fluoride ion for identification.

This article discusses how a chemosensor, referred to as “FL”, containing indole and salicylaldehyde moieties (FL) and based on hydrogen bonding for fluoride ion sensing was designed and characterized (Figure 1). FL exhibited a selective and sensitive emission response to the F

 ion, and a significant color change (from colorless to yellow) is visible in the presence of the F

 ion in a CH3CN:PBS (99:1, v/v) solution. Moreover, its ability to image F

 ions in cervical cancer cells (Hela cells) has been demonstrated using confocal microscopy.

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