Development of a rapid and simple tetracycline detection system based on metal-enhanced fluorescence by europium-doped AgNP@SiO2 core–shell nanoparticles

We herein describe a rapid and selective sensing platform for tetracycline (Tc), which relies on the metal-enhanced fluorescence (MEF) effect of europium (Eu3+)-doped silver-silica core–shell nanoparticles (AgNP@SiO2). The developed assay utilizes AgNP@SiO2 as a key detection component, which is systematically optimized to have a silica shell thickness suitable for the effective MEF phenomenon. In principle, the AgNP@SiO2, which binds to Eu3+ through the electrostatic interaction, captures Tc by selective chelation with Eu3+, leading to significant fluorescence enhancement of the EuTc complex. Based on this novel strategy, we determined Tc as low as 83.1 nM with a total assay time of less than 10 min, which is comparable to or better than that of the previous fluorescence-based methods. Furthermore, the practical applicability of this strategy was successfully demonstrated by detecting Tc in tap water. This work highlights the unique features of AgNP@SiO2 for MEF-based biosensing applications.


Introduction
Tetracycline (Tc) is a versatile antibiotic against a broad range of pathogens, which is known to inhibit protein synthesis by binding to a small 30S ribosomal subunit and preventing the attachment of aminoacyl-tRNA to the ribosomal acceptor site. 1,2owever, Tc, a life-saving molecule, has been increasingly overand mis-used, which results in the emergence of antimicrobial resistance. 3In addition, ingested Tc causes side effects such as nail discoloration and onycholysis in hypersensitive individuals. 4Therefore, a great interest has been aroused for the rapid and simple detection of Tc.
Until now, various methods have been utilized to determine Tc, including liquid chromatography/mass spectrometry, highperformance liquid chromatography, 5,6 capillary electrophoresis, 7 and chemiluminescence. 8,9Especially, novel electrochemical and colorimetric detection assays based on DNA aptamers specic to Tc have been intensively attempted due to their good selectivity and sensitivity for Tc detection. 10,11owever, they entail complicated preparation steps and long assay procedures, which have limited their practical applications.
In this work, for the development of a rapid, simple, and selective sensing platform for Tc, we employed europium (Eu 3+ ) which belongs to the lanthanide ions with the unique features, including sharply spiked emission peak (<10 nm full width at half maximum), large Stokes shi (>150 nm), high quantum yield, and long decay times on the ms/ms timescale, much longer than the ns timescale of most biological auto-uorescence. 12,13Based on the fact that Eu 3+ can chelate Tc with a b-diketonate structure to form a stable uorescent complex (EuTc), [14][15][16] we adopted the metal enhanced uorescence (MEF), a phenomenon that occurs when a uorophore is at the adjacent place of the metallic surface, resulting in the increased uorescence intensity, photostability, and radiative decay rates, in order to signicantly increase the uorescence signal of EuTc complex. 17-20Specically, we synthesized AgNP@SiO 2 with the optimal silica shell thickness, which binds to Eu 3+ through the electrostatic interaction, and promotes the effective MEF phenomenon.As a result, the presence of Tc, which chelates Eu 3+ to form the stable EuTc complex on the surface of AgNP@SiO 2 , produces the signicantly increased red uorescence.With this novel strategy, we successfully analyzed Tc with the high sensitivity and selectivity even in tap water.O), arginine (L-) (Arg), Laspartic acid (Asp), L-histidine (His), L-lysine monohydrochloride (Lys), L-cysteine hydrochloride monohydrate (Cys), glutathione (GSH), L-ascorbic acid and D-(+)-glucose were purchased from Sigma-Aldrich.Silver nitrate (AgNO 3 ) and methylamine (MA, 40% solution in water) were obtained from Kojima Chemicals Co., LTD.and Junsei Chemical Co., Ltd., respectively.All of the solvents and reagents were used without any further purication.

Preparation of silver nanocore
The large AgNPs were synthesized according to the previously reported method. 17In brief, AgNPs were prepared by adding 4 mL of sodium citrate aqueous solution (38.8 mM) into 196 mL of boiling AgNO 3 (1.05mM) under vigorous stirring.The color of the solution turned from colorless to yellow, and nally turned to gray-yellow within 4 min.Aer boiling for 20 min, the reaction solution was cooled to room temperature under vigorous stirring.The as-prepared large AgNPs were stored at 4 C.In addition, the small AgNPs were synthesized according to the previously reported method. 21In brief, 5 mL of aqueous sodium citrate solution (2.5 mM), 0.25 mL of aqueous poly(sodium styrene sulphonate) (PSSS) (500 nM) and 0.3 mL of freshly prepared sodium borohydride (NaBH 4 ) (10 mM) were mixed.Then, 5 mL of AgNO 3 (0.5 mM) was added dropwise to this solution at the rate of 2 mL min À1 under vigorous stirring, which was stirred for further 3 min.The as-prepared small AgNPs were stored at 4 C.

Synthesis of silica shells on silver nanocore
The silver nanoparticles were coated with silica shells according to a modied Stöber method. 17,21Under vigorous stirring, 3.6 mL of the as-prepared AgNP solution was mixed with 32.08 mL of ethanol, 3 mL of deionized water and 200 mL of tetraethoxysilane (TEOS) at different concentrations (200 mM, 400 mM, 600 mM and 800 mM).The nal concentrations of TEOS in each suspension are 1 mM, 2 mM, 3 mM and 4 mM, respectively.The silica coating was initiated by the dropwise injection of 1.12 mL methylamine (MA, 40% in water) to the suspension.The nal concentration of MA is 0.64 mM.The solutions were stirred at room temperature for 30 minutes and then allowed to age without agitation at 4 C overnight (more than 6 hours).Each suspension of silica-coated silver nanoparticles (AgNP@SiO 2 ) was washed with distilled water (D.W.) and ethanol mixture (5 : 4) in a 50 mL tube and centrifuged at 3500 rpm for 35 minutes.The core-shell nanoparticles with the different thickness were collected from the bottom of the 50 mL tube and transferred to a 1.5 mL tube, followed by washing and centrifugation (3500 rpm, 15 minutes) twice with D.W. Finally, the mixture was suspended in D.W.
The concentration of AgNP@SiO 2 was determined according to Beer Lambert's law A ¼ 3cb (A: absorbance, 3: molar extinction coefficient, c: molar concentration, and b: path length).The absorbance spectra of 200 mL of AgNP@SiO 2 were measured in 96 well plate.The concentration of AgNP@SiO 2 was calculated by using the parameters: molar extinction coefficient of AgNP@SiO 2 is 739 Â 10 8 M À1 cm À1 (ref.22) and the path length is 0.56 cm.

Optimization of the reaction conditions
To investigate the impact of the silica shell thickness on the uorescence of EuTc, 24 mL of Eu 3+ (100 mM), 12 mL of Tc (100 mM) and 20 mL of AgNP@SiO 2 (0.133 nM) with the different thickness were added to each tube to make the nal volume of each solution to 200 mL.The mixtures were incubated for 5 minutes and the emission spectra were then recorded at the excitation wavelength of 400 nm.
The Eu 3+ concentration was optimized by adding 0 mL, 6 mL, 12 mL, 24 mL, 36 mL and 48 mL of Eu 3+ (100 mM) into the solutions containing Tc (6 mM) and AgNP@SiO 2 (0.02 nM) in a total volume of 200 mL, respectively.The mixtures were incubated for 5 minutes and the uorescent intensity at 615 nm were then recorded at the excitation wavelength of 400 nm.
The impact of AgNP@SiO 2 concentration on the uorescence of EuTc was studied by adding various concentrations of AgNP@SiO 2 (0.133 nM) in each of the 200 mL solutions containing Eu 3+ (12 mM) and Tc (6 mM).

Tc detection procedure
For the detection of Tc, 24 mL of Eu 3+ (100 mM) and 0 mL, 0.02 mL, 0.2 mL, 2 mL, 6 mL, 12 mL, 24 mL, 36 mL and 48 mL of Tc (100 mM) at different concentrations were incubated for 5 minutes, and 30 mL AgNP@SiO 2 (0.133 nM) were incubated for 4 minutes and the resulting uorescence signal was measured on 384 well plate at the excitation and emission wavelengths of 400 nm and 615 nm, respectively.

Detection of Tc in real samples
The tap water was obtained in the lab and used without further purication.Tc at varying concentrations was spiked into the tap water, which was subsequently analyzed using the same procedure to detect Tc as described above. 23

Instruments
Transmission electron microscopy (TEM) images were recorded with a Cs-corrected STEM (JEM-ARM200F).The EuTc-doped AgNP@SiO 2 samples were cast onto the copper grids (300 mesh) with a lacey carbon lm (LC300-CU, Electron Microscopy Sciences) and dried at room temperature overnight.The average shell thickness and the distribution of the SiO 2 shell thickness were analyzed based on the TEM images.The nanoparticle size distribution was analyzed based on at least 150 nanoparticles by using ImageJ soware.Energy dispersive X-ray spectroscopy (EDS) images and element mapping data were also obtained during the TEM measurements.The UV-vis absorption spectra and uorescence spectra were obtained with an Innite M200 PRO Multi-Detection Microplate Reader (Tecan).

Results and discussion
The basic principle of the new strategy to determine Tc is illustrated in Scheme 1, wherein Eu 3+ -doped AgNP@SiO 2 is utilized as the key detection component.In the absence of Tc, Eu 3+ -doped AgNP@SiO 2 emits negligible uorescence upon excitation at 400 nm.On the contrary, in the presence of Tc, Eu 3+ on the surface of AgNP@SiO 2 chelates Tc to form a stable EuTc complex.When excited at 400 nm, the EuTc complex emits the signicantly increased red uorescence at 615 nm through the effective energy transfer from Tc to Eu 3+ and the MEF effect of AgNP@SiO 2 .As a result, Tc could be sensitively determined by measuring the uorescence signal of the samples.
First, we synthesized the AgNP@SiO 2 by utilizing silica shell as the distance adjusting layer between the uorescent EuTc complex and the silver core.The core-shell AgNP@SiO 2 was prepared by following a modied Stöber method (see materials and methods for details).The synthesized AgNP@SiO 2 were characterized by transmission electron microscopy (TEM).As shown in Fig. 1(a), the silica shell is homogeneously formed around the silver core and Eu 3+ was doped on the surface of the silica shell, which was conrmed by the energy-dispersive X-ray spectroscopy (EDS) mapping (Fig. 1(b)).
Next, we veried the feasibility of the developed strategy by measuring the uorescence emission spectra from different samples.As illustrated in Fig. 2(a), the negligible uorescence signal was generated from the EuTc complex (green curve) due to the uorescence quenching by the coordinated water molecules, 24 while the uorescence intensity of the EuTc complex was enhanced in the presence of AgNP (blue curve), which is attributed to the replacement of the coordinated water molecules by the citrate ions on the surface of AgNPs and the MEF effect by AgNPs.However, the signal enhancement, which was dened as the uorescence intensities of samples divided by uorescence intensities of EuTc, was only 3-fold, which was because the distance between AgNPs and EuTc was too close to effectively induce the MEF effect.Most importantly, the maximum signal enhancement up to 18-fold was obtained when AgNP@SiO 2 was added to (yellow curve), which provides the optimal distance between AgNP and EuTc complex for the effective MEF.It is also noteworthy that the absorbance spectra of AgNP@SiO 2 overlapped with the excitation spectra of EuTc-doped AgNP@SiO 2 , which indicates that AgNP@SiO 2 is suitable for the MEF effect to enhance the uorescence signal of EuTc (Fig. 2(b)).In addition, AgNP@SiO 2 with the different silica shell thickness from 13 nm to 44.36 nm (Table S1 †) were prepared to nd the optimal thickness for the effective MEF.As shown in Fig. S1, † when the shell thickness was around 30 nm, the highest uorescence signal resulting from the most effective MEF was obtained.Overall, these results clearly conrmed that the silica shell on the surface of AgNP does not only provide a doping site for EuTc complex but also creates a proper distance for the MEF effect. 17e then investigated the optimal conditions required for the efficient analysis of Tc.The results of experiments, in which the concentrations of Eu 3+ (Fig. S2(a) †) and the AgNP@SiO 2 (Fig. S2(b) †) were varied, demonstrate that 12 mM of Eu 3+ and 0.02 nM of AgNP@SiO 2 , are ideal for the efficient analysis of Tc.Under the optimal conditions, we determined the detection sensitivity by measuring the uorescent intensity at 615 nm (F 615 ) as a function of Tc concentration.As shown in Fig. 3(a) and S3, † the uorescence intensities increased with increasing Scheme 1 Schematic illustration the detection of Tc using Eu 3+doped AgNP@SiO 2 .
concentrations of Tc up to 18 mM, but reached a plateau at concentrations higher than 18 mM.][27] To evaluate the specicity of the present strategy towards Tc, the effect of the potential interfering species including amino acids, ascorbic acid, and glucose to induce the uorescence enhancement was then examined.As shown in Fig. 3(b) and S4, † the strong uorescence signal was observed only in the presence of Tc, but other interfering species led to negligible uorescent signals, conrming the high selectivity of the developed system for Tc detection.Importantly, it should be noted that the developed system is quite simple and rapid with the signal-on uorescence response, allowing the convenient and cost-effective determination of Tc.
It has been known that the uorescence intensities of uorescent dye or complex on the surface of AgNP@SiO 2 become strongly enhanced only when the spectra between the uorescent dye or complex and the localized surface plasmon resonance (LSPR) of the metal nanoparticles are overlapped.3][34] Since MEF is the ability of the metal nanoparticle to scatter the coupled plasma, larger metal nanoparticles are regarded as the ideal materials for the effective MEF. 18In addition, it has been reported that almost all electrons in the nearly spherical AgNPs with the diameter of 10-50 nm experience the same phase of the incident electromagnetic eld, which leads to the excitation of a dipole resonance. 35s the particle size increases over 50 nm, the excitation of higher plasmon modes such as a quadrupole resonance can be achieved in addition to the dipole resonance (Fig. S6 and S7 †). 36n terms of the shape effect on the MEF, anisotropic nanoparticles such as silver nanoprisms and fractal-like structures, which provides the large scattering cross-section, have been known to enhance the local eld more effectively than spherical nanoparticles. 14,28Specically, the tips of the anisotropic nanoparticles can act as antennas, which effectively emit radiation when coupled to uorophores.Thus, thicker metal structures with larger surface areas are preferred for the signicant uorescence enhancement through MEF. 37n addition, the silica shell thickness on the surface of AgNPs can also affect the MEF.9][40] In principle, as the silica shell thickness increases over 20 nm, the radiative energy transfer becomes dominant as compared to the non-radiative energy transfer.Thus, 35 nm silica shell used in our work leads to the signicant uorescence enhancement through the radiative energy transfer from the excited AgNPs to the lanthanide complex (EuTc). 39g. 2 (a) Feasibility of the developed system for Tc.(b) Spectral overlap between the absorbance spectra of AgNP@SiO 2 and the excitation spectra of EuTc-doped AgNP@SiO 2 .The excitation wavelength is set at 400 nm.Fig. 3 Sensitivity and selectivity of the developed system for Tc.(a) Fluorescent intensity at 615 nm (F 615 ) of Eu 3+ -doped AgNP@SiO 2 with various concentrations of Tc (0-24 mM).Insert: a linear relationship between F 615 and the concentration of Tc (0-6 mM).(b) Fluorescent intensity at 615 nm (F 615 ) of Eu 3+ -doped AgNP@SiO 2 in the presence of Tc, amino acids, ascorbic acid, and glucose (the concentrations of Tc, amino acids, ascorbic acids, and glucose are all 10 mM).The excitation wavelength is set at 400 nm.
Finally, the practical applicability of the developed strategy was veried by determining Tc in tap water.As shown in Fig. S5, † the uorescence intensities in tap water increased as the concentration of Tc increased, which was almost comparable to that from the distilled water (Fig. 3), indicating that the proposed method is robust to the interfering substances present in the tap water.Then, the F 615 from unknown samples were measured to determine the Tc concentration in tap water (Table 1).The results in Table 1 show that the reproducibility and precision of the developed method were quite good, yielding the Relative Standard Deviation (RSD) less than 5% and recovery rates of 89.3-105.9%.These observations clearly indicate that the developed strategy can be used to reliably determine Tc in real samples.

Conclusions
In summary, we have developed a rapid, convenient and costeffective method for the detection of Tc using the Eu 3+ -doped AgNP@SiO 2 as a key detection component.By adjusting the shell thickness of AgNP@SiO 2 that imparts the MEF effect, the most signicant uorescence enhancement in the presence of Tc was achieved, which was successfully utilized for the sensitive and selective determination of Tc.Importantly, this strategy does not require any expensive reagents or sophisticated instruments, which effectively overcomes the drawbacks such as the high cost and complexity in the previous Tc detection methods.Due to these outstanding features, the proposed MEF-based sensing method is expected to pave the way for the development of other chemical and biological sensing platforms.

Table 1
41tection of Tc in tap water Mean of the three measurements.bStandard deviation of three measurements.cRelative Standard Deviation (RSD) ¼ SD/mean Â 100.dMeasured value/added value Â 100.41 a