Microsoft Word - 211.docx


CHEMICAL ENGINEERINGTRANSACTIONS 
 

VOL. 61, 2017 

A publication of 

 

The Italian Association 
of Chemical Engineering 
Online at www.aidic.it/cet 

Guest Editors: Petar S Varbanov, Rongxin Su, Hon Loong Lam, Xia Liu, Jiří J Klemeš 
Copyright © 2017, AIDIC Servizi S.r.l. 

ISBN978-88-95608-51-8; ISSN 2283-9216 

Highly Sensitive Ratiometric Fluorescent Probe for the 

Detection of Mercury Ions 

Yanfei Yuea, Zhao Gaoa,b, Lian Chenc, Rongxin Sua,*, Wei Qia, Zhimin Hea 

aState Key Laboratory of Chemical Engineering, Tianjin Key Laboratory of Membrane Science and Desalination 

 Technology, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China 
bCollege of Life Science, Dalian Minzu University, Dalian 116600, PR China. 
cResearch Center of Hunan Entry-Exit Inspection and Quarantine Bureau, Changsha 410001, PR China 

 surx@tju.edu.cn 

An effective dual-emission fluorescent probe combining gold nanoclusters (AuNCs) and carbon dots (CDs) 

was fabricated for the detection of mercury ions (Hg2+). CDs doped into silica nanoparticles exhibited a 

reference fluorescence signal, while the outer AuNCs layer stabilized by bovine serum albumin (BSA) was 

extremely sensitive to Hg2+. This probe selectively detected Hg2+ in a good linear range from 0.43 to 39 μM 

and a detection limit (signal-to-noise ratio is 3) of 81 nM. The increase in the Hg2+ concentration was 

accompanied by the colour change from reddish violet to blue. This method exhibited several advantages of 

facile preparation and operation and high selectivity and sensitivity. This dual-emission fluorescent probe has 

significant potential for the ‘‘naked eye’’ detection of Hg2+. 

1. Introduction 

Pollution by heavy metals leads to severe environmental and ecological problems (Chen and Bi, 2016). 

Mercury ion (Hg2+) is one of the most toxic heavy metals, which is widespread in air, water, and soil. The 

accumulation of mercury in the human body can cause severe damage to the central nervous system, 

resulting in various cognitive and motor disorders, as well as the Minamata disease. Besides, the presence of 

Hg2+ in the human body also leads to severe toxic effects on the human hematopoietic system, respiratory 

system, and the kidneys. Hence, it is imperative to develop a facile and sensitive probe for the detection of 

Hg2+. Recently, the optical methods, especially fluorescence-based analytical methods, have been employed 

for the analysis of Hg2+ in real water samples with good results (Kim et al., 2012). 

Ratiometric fluorescence method demonstrates several advantages for the measurement of the fluorescence 

intensities at two wavelengths. Compared to the typical fluorescence quenching probe based on a single 

fluorescence signal, the ratiometric fluorescent probe can decrease or eliminate the operational errors caused 

by the substrate, by the external environment, by the changes in the instrument conditions or other factors, as 

well as improve the measurement accuracy of the results (Chen et al., 2013). 

Dye-doped silica nanoparticles have been developed as ratiometric fluorescent probes, which have been 

widely used for selective determination because of their good chemical and colloidal stabilities, well-developed 

surface coupling technology, and low toxicity (Zhang et al., 2011). Zong et al. (2011) reported a nanoprobe, 

with a dye-doped silica core serving as a reference signal, thus providing a built-in correction for 

environmental effects, and a response dye was covalently grafted on the silica nanoparticle surface using a 

chelating reagent for Cu2+. 

Fluorescence carbon dots (CDs) represent a new class of carbon nanomaterials, which overcome some of the 

limitations of traditional quantum dots. Carbon dots are non-toxic and easy to implement for surface 

functionalization, in addition to their excellent optical performance and good biocompatibility (Gao et al., 2015). 

As another important fluorescent material, gold nanoclusters (AuNCs) demonstrate several advantages, e.g., 

non-toxicity, near-infrared emission, good biological compatibility, and narrow emission spectra (Xie et al., 

2010). Because of these advantages of above materials, AuNCs and CDs were combined to afford a 

                               
 
 

 

 
   

                                                  
DOI: 10.3303/CET1761253

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Please cite this article as: Yue Y., Gao Z., Chen L., Su R., Qi W., He Z., 2017, Highly sensitive ratiometric fluorescent probe for the detection 
of mercury ions, Chemical Engineering Transactions, 61, 1531-1536  DOI:10.3303/CET1761253  

1531

mailto:surx@tju.edu.cn


composite probe based on silica nanoparticles, which was applied as a ratiometric fluorescent probe for Hg2+ 

detection. 

 

 

Figure 1: Schematic for the synthesis and application of the dual-emission fluorescent probe CD@SiO2/AuNC. 

In the presence of Hg2+, the fluorescence intensity corresponding to the outside layer (AuNCs) of the hybrid 

probe decreased, whereas that of the inside layer (CDs) remained constant 

Figure 1 shows the preparation of the hybrid ratiometric probe herein and the working principle for the visual 

detection of Hg2+. Herein, CDs were prepared by the thermal pyrolysis of anhydrous citric acid and 3-

aminopropyl trimethoxysilane (APTMS). Subsequently, the CD-doped fluorescent silica core (CD@SiO2) was 

synthesized by a reverse microemulsion method. Gold nanoclusters were stabilized by bovine serum albumin 

(BSA–AuNCs), which were then covalently connected with amino-modified CD@SiO2 nanoparticles after the 

surface was activated by a carboxyl-group-based activating reagent, e.g., 1-ethyl-3-[3-(dimethylamino)propyl]- 

carbodiimide (EDC). In this dual-emission hybrid fluorescent probe, the inside CDs provided the reference 

fluorescence signal, while the outer BSA–AuNCs layer was extremely reactive to Hg2+. 

2. Materials and methods 

Citric acid, hydrochloroauric acid, APTMS, 3-aminopropyltriethoxysilane (APTES), N-hydroxysuccinimide 

(NHS), and EDC were obtained from Aladdin Reagent Company (Shanghai, China). BSA was purchased from 

Sigma-Aldrich (Beijing, China). Mercuric chloride was purchased from J&K Scientific Company (Beijing, 

China). All other chemicals were of analytical grade and were commercially obtained. Ultrafiltration centrifuge 

tubes with a molecular weight cut-off of 3,500 Da were obtained from Sigma-Aldrich (Beijing, China). 

2.1 Synthesis of fluorescent CDs 

CDs were synthesized by the one-step pyrolysis of citric acid at high temperature. In a typical synthesis 

procedure, 10 mL of APTMS was added in a 100 mL three-neck round-bottom flask. Then, the flask was 

heated to 185C using an electric heating jacket and 0.5 g of anhydrous citric acid was added into the flask. 

After 1 min, the colour of the reaction system changed to dark green. Subsequently, the flask was cooled to 

room temperature. The reaction solution was extracted five times using petroleum ether with the same 

volume. Finally, freshly prepared CDs were obtained as the dark green bottom liquid after extraction. 

2.2 Preparation of CD@SiO2 

The reverse microemulsion method was employed to synthesize fluorescent CD-doped silica nanoparticles. 

First, a W/O microemulsion solution was prepared by mixing 1.77 mL of TX-100, 7.7 mL of cyclohexane, 1.8 

mL of n-hexanol, and 380 μL of water. Second, the system was vigorously stirred for 10 min to attain stability 

and homogeneity. Third, 50 μL of TEOS was added. After mixing, 25 μL of freshly prepared CDs was added. 

Next, with gentle stirring, 200 μL of the 25 wt% ammonia solution was added, and the reaction was allowed to 

stir for 24 h at room temperature. Three times the volume of isopropyl alcohol was added, followed by 

centrifugation and washing with ethanol and water several times until no fluorescence signal was observed for 

the supernatant. Finally, the CD@SiO2 nanoparticles were dispersed in ethanol by ultrasonication. At the 

same time, silica nanoparticles were prepared without CDs as the control sample.  

2.3 Synthesis of AuNCs 

All of the glassware was washed with aqua regia (HCl/HNO3 volume ratio = 3:1) before use. In a typical 

1532



experiment, an aqueous HAuCl4 solution (1 mL, 10 mM) was first added to the BSA solution (1 mL, 50 mg/mL) 

under vigorous stirring for 2–3 min. Then, a 1 M NaOH solution was added to attain a pH of 11, and the 

reaction was allowed to stir vigorously at 40 °C for 12 h. The as-synthesized AuNCs were dialyzed in a 

membrane tubing with an MWCO of 3500 Da against 1 L of continuously stirred ultrapure water at 4 °C. After 

36 h, the tubing contents (AuNCs) were collected, and the solution was stored at 4 °C for subsequent use. 

2.4 Synthesis of dual-emission fluorescent probe (CD@SiO2/AuNC) 

The first step involves the amination of CD@SiO2. First, the as-prepared CD@SiO2 nanoparticles were 

dispersed in 20 mL of ethanol with a final concentration of 0.1 mg/mL. Second, 100 μL of APTES was rapidly 

added. Next, the resulting solution was heated to the reflux temperature and stirred for 24 h. Finally, the 

nanoparticles thus obtained were separated by centrifugation and dispersed in a phosphate buffer (10 mM, pH 

7.0). The second step involves the grafting of AuNCs on CD-doped silica nanoparticles. 0.1 mL of the as-

prepared BSA–AuNCs solution was diluted to 1 mL using a phosphate buffer (10 mM, pH 7.0). Then, 0.5 mL 

of EDC (100 mM) and 0.5 mL of an NHS (400 mM) solution were added and stirred for 0.5 h at room 

temperature. After that, 1 mL of the as-prepared CD@SiO2 was added in the mixed solution and stirred for 12 

h under 4°C. 

2.5 Fluorescence detection of mercury ions 

To detect Hg2+, different solutions with increasing Hg2+ concentration were added into 3 mL of the 

CD@SiO2/AuNC solution. Then, fluorescence spectra were recorded at an excitation wavelength of 380 nm 

with an excitation or emission slit width of 10 nm. The selectivity for Hg2+ detection was investigated by the 

addition of other interferences, following the same procedure. All experiments were performed at room 

temperature and repeated at least three times. 

3. Results and discussion 

3.1 Structural and optical characterization of CD@SiO2 

As shown in the UV–vis spectrum of CD@SiO2 in Figure 2a, a weak absorption peak was observed at 342 

nm, indicating that CDs are successfully coated inside of the SiO2 nanoparticles. Next, the fluorescence 

properties of the as-prepared CD@SiO2 were further examined (Figure 2b). The maximum emission peaks 

corresponding to fluorescent CD@SiO2 blue-shifted to 455 nm as compared with that observed for CDs (Zhao 

et al., 2004). Figure 2c and 2d show the typical SEM and TEM images of as-prepared CD@SiO2, respectively. 

The size distribution of CD@SiO2 was 40–70 nm. After doping with SiO2 nanoparticles, CD@SiO2 could be 

dispersed in water and ethanol and still exhibited blue fluorescence under UV light (Figure 2c, inset). Both 

FTIR spectra and time-correlated single photon counting (TCSPC) were used to characterized for CD@SiO2 

and SiO2. As compared to only CDs, the CD@SiO2 complex exhibited a shorter lifetime. 

 

 

Figure 2: UV–vis (a) and fluorescence emission spectra (b) of CDs and CD@SiO2. SEM (c) and TEM images 

(d) of CD@SiO2. Inset in (c) shows the size distributions of CD@SiO2 according to the SEM image 

1533



3.2 Structural and optical characterization of CD@SiO2/AuNC 

Figure 3 shows the TEM and HRTEM images of the hybrid probe, i.e., CD@SiO2/AuNC. After the formation of 

the outer BSA–AuNCs layer, the CD@SiO2/AuNC surface was rougher than the CD@SiO2 surface, with a 

size of approximately 1 nm. The optical characteristics of CD@SiO2/AuNC were further examined by UV–vis 

and fluorescence spectroscopy. As shown in Figure 3c, BSA–AuNCs exhibited a characteristic absorption 

peak at 280 nm, corresponding to protein adsorption, whereas CD@SiO2 exhibited a peak at 342 nm, 

corresponding to CDs. CD@SiO2/AuNC exhibited peaks at 280 nm and 342 nm, indicating that BSA–AuNCs 

are decorated on the CD@SiO2 surface. With irradiation using a UV lamp at a wavelength of 365 nm, the 

aqueous solution of CD@SiO2/AuNC exhibited red luminescence (inset photograph of Figure 3d). The 

CD@SiO2/AuNC nanohybrids emitted two fluorescence peaks at 455 nm and 650 nm, with different colors, 

under a single wavelength of 380 nm. The red fluorescence corresponded to BSA–AuNCs (Tao et al., 2013), 

with sensitive quenching in the response to Hg2+, while the blue fluorescence corresponded to the CDs, acting 

as the internal reference. 

 

Figure 3: TEM (a) and HRTEM (b) images of the CD@SiO2/AuNC nanohybrid. (c) UV–vis spectra of the 

CD@SiO2, BSA–AuNCs, and ratiometric hybrid probe particles. (d) Normalized fluorescence emission spectra 

of the CD@SiO2 (I), BSA–AuNCs (II), and ratiometric hybrid particles (III). Inset: photographs of the three 

samples under a 365 nm UV lamp 

 

Figure 4: Stabilities of the fluorescence intensities of CD@SiO2 at 455 nm excited by 350 nm (a), 

CD@SiO2/AuNCs at 455 nm and 650 nm excited by 380 nm (b) 

The stability of CD@SiO2 was tested by measuring the fluorescence emission intensity at 455 nm under an 

excitation light irradiation of 350 nm. As shown in Figure 4a, with prolonged illumination, the fluorescence 

intensity of CD@SiO2 remained unchanged within 2h, indicating high stability and anti-fluorescence 

photobleaching ability. The fluorescence intensity of the CD@SiO2/AuNC hybrid probe remained unchanged 

under continuous illumination by 455 nm and 650 nm for up to 2 h (Figure 4b). The change in the fluorescence 

intensity was less than 5 % within testing for 2 h, indicative the good stability of the ratiometric fluorescent 

probes for promising applications in quantitative determination. 

1534



3.3 Fluorescence detection of mercury ions 

The developed dual-emission hybrid ratiometric probe was used to detect Hg2+ utilizing fluorescence emission 

intensities. Figure 5 shows the recorded response. The results confirm the effective fluorescence quenching of 

AuNCs by Hg2+, related to high-affinity metallophilic interactions. The probe exhibited two emission peaks at 

455 nm and 650 nm under a single wavelength excitation of 380 nm, which are emitted from the CDs on the 

surface and AuNCs embedded within the silica nanoparticles. With the addition of Hg2+, the intensity of the red 

emission at 650 nm (Fr) was gradually decreased, while that of the blue emission at 455 nm (Fb) remained 

constant, acting as an internal reference. This process was accompanied by an obvious colour change from 

reddish violet to blue (Figure 5a, inset). The ratios of fluorescence intensities at 455 and 650 nm (Fr/Fb) 

exhibited good linearity with increasing Hg2+ concentrations in the range from 0.43 to 39 μM (Figure 5b). The 

linear regression equation was Fr/Fb = 0.01894C + 0.9333, and the detection limit was 81 nM (signal-to-

noise ratio is 3). To examine probe reproducibility, 10 parallel measurements using a solution with a certain 

Hg2+ concentration were conducted, and a relative standard deviation of 2.67 % was obtained, suggesting that 

this probe is suitable for quantitative determination. 

 

 

Figure 5:  Fluorescence spectra (a, c) and the corresponding working curve (b, d) of the ratiometric probe (a, b) 

and pure BSA–AuNCs (c, d) upon the addition of different Hg2+ concentrations. Inset: optical images for the 

fluorescence color of the ratiometric probe in the presence of Hg2+. Fluorescence photographs were recorded 

under a UV lamp (365 nm) 

Blank Hg(II) Ag(I) Cu(II) Zn(II) Mg(II) K(I) Na(I) Ni(II) Mn(II)Fe(III) Cd(II) Co(II) Ca(II) Pb(II)

0.4

0.6

0.8

1.0

 

F
r/

F
b

 

 

 

Figure 6: Ratiometric fluorescence responses of the CD@SiO2/AuNC sensor to various substances (1 mM 

concentration) 

As a contrast experiment, BSA-AuNCs were separately used as the fluorescent probe for Hg2+ detection 

(Figure 5). For a Hg2+ concentration range of 0.24–16.8 μM, the fluorescence intensity of AuNCs and Hg2+ 

concentration exhibited good linearity (R2 = 0.998), with a detection limit of 52 nM. The detection range of 

dual-emission hybrid ratiometric probe was broader than the individual BSA-AuNCs detection. Besides, Chen 

1535



et al. (2014) have used a chitosan-functionalized AuNP for colorimetric detection and obtained a detection 

range of 9–50 μM. Lee et al. (2013) have utilized the fluorescence of rhodamine hydrazone derivatives and 

obtained a detection range of 0.28–0.8 μM. The sensitivity of the developed method is comparable or superior 

to those of the above-mentioned methods for the determination of Hg2+ in solutions. Hence, the dual-emission 

fluorescent probe exhibited a higher detection limit and a wider linear range, and it can be suitable for naked 

eye visual inspection. 

In addition to the sensitivity requirement, high specificity is also important in a majority of detection scenarios, 

especially for actual sample detection. To evaluate the specificity of this ratiometric probe, interference 

experiments in the presence of common metallic ions, e.g., K+, Na+, Ca2+, Mg2+, Zn2+, Cu2+ and Fe3+, were 

carried out under the same conditions. Marked fluorescence changes were observed after the addition of 30 

μM metallic ion solutions. As shown in Figure 6, only Hg2+ clearly affected the fluorescence intensity range of 

the CD@SiO2/AuNC sensor, indicating that the probe demonstrates excellent selectivity for Hg2+. 

4. Conclusions 

In summary, siloxane-functionalized carbon quantum dots were successfully synthesized using long-chain 

amino siloxane. Then, the functionalized carbon quantum dots formed carbon dot–silica composite particles, 

with an average particle size of approximately 60 nm. A facile dual-emission fluorescent probe was 

constructed by the hybridization of gold nanoclusters and carbon-dot-based silica particles and utilized for the 

visual detection of Hg2+. The CD@SiO2/AuNC hybrid probe can be used for the quantification of Hg2+ in 

solutions on the basis of the ratiometric fluorescence measurement. The probe exhibited facile operation, high 

selectivity, and sensitivity and provided potential for the detection of Hg2+ by the naked eye. 

Acknowledgments  

This study was supported by the Ministry of Science and Technology of China (No. 2012YQ090194), the 

Natural Science Foundation of China (No. 51473115), and the Natural Science Foundation of Tianjin (No. 

16JCZDJC37900). 

References 

Chen J., Bi W., 2016, Monitoring Technology of Trace Heavy Metals in Seawater Based on 

Spectrophotometry, Chemical Engineering Transactions, 55, 313-318. 

Chen T., Hu Y., Cen Y., Chu X., Lu Y., 2013, A Dual-Emission Fluorescent Nanocomplex of Gold-Cluster-

Decorated Silica Particles for Live Cell Imaging of Highly Reactive Oxygen Species, Journal of the 

American Chemical Society, 135(31), 11595-11602. 

Chen Z., Zhang C., Tan Y., Zhou T., Ma H., Wan C., Lin Y., Li K., 2014, Chitosan-Functionalized Gold 

Nanoparticles for Colorimetric Detection of Mercury Ions Based on Chelation-Induced Aggregation, 

Microchimica Acta, 182(3-4), 611-616. 

Gao Z., Wang L., Su R., Huang R., Qi W., He Z., 2015, A Carbon Dot-Based "Off-On" Fluorescent Probe for 

Highly Selective and Sensitive Detection of Phytic Acid, Biosensors & Bioelectronics, 70, 232-238. 

Kim H.N., Ren W.X., Kim J.S., Yoon J., 2012, Fluorescent and Colorimetric Sensors for Detection of Lead, 

Cadmium, and Mercury Ions, Chemical Society Reviews, 41(8), 3210-3244. 

Lee H.Y., Swamy K.M.K., Jung J.Y., Kim G., Yoon J., 2013, Rhodamine Hydrazone Derivatives Based 

Selective Fluorescent and Colorimetric Chemodosimeters for Hg2+ and Selective Colorimetric 

Chemosensor for Cu2+, Sensors and Actuators B: Chemical, 182, 530-537. 

Tao Y., Lin Y., Ren J., Qu X., 2013, A Dual Fluorometric and Colorimetric Sensor for Dopamine Based on 

BSA-Stabilized Au Nanoclusters, Biosensors & Bioelectronics, 42, 41-46. 

Zhao X., Bagwe R.P., Tan W., 2004, Development of Organic-Dye-Doped Silica Nanoparticles in a Reverse 

Microemulsion, Advanced Materials, 16, 173-176. 

Xie J., Zheng Y., Ying J.Y., 2010, Highly Selective and Ultrasensitive Detection of Hg2+ Based on 

Fluorescence Quenching of Au Nanoclusters by Hg2+-Au+ Interactions, Chemical Communications, 46(6), 

961-963. 

Zhang K., Zhou H., Mei Q., Wang S., Guan G., Liu R., Zhang J., Zhang Z., 2011, Instant Visual Detection of 

Trinitrotoluene Particulates on Various Surfaces by Ratiometric Fluorescence of Dual-Emission Quantum 

Dots Hybrid. Journal of the American Chemical Society, 133(22), 8424-8427. 

Zong C., Ai K., Zhang G., Li H., Lu L., 2011, Dual-Emission Fluorescent Silica Nanoparticle-Based Probe for 

Ultrasensitive Detection of Cu2+. Analytical Chemistry, 83(8), 3126-3132. 

1536