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Upconversion fluorescence resonance energy transfer—a novel approach for sensitive detection of fluoroquinolones in water samples

Abstract A novel homogeneous assay method was put forth for simultaneous sensing of fluoroquinolone derivatives (FQs) in water utilizing the upconversion fluorescence resonance energy transfer (FRET) process based on the competitive reaction between dissolved FQs and labeled AuNPs for FQs Mab connected UCPs. The prepared size-tunable β-NaLuF4:Yb,Er,Gd upconversion phosphors (UCPs) were functionalized with carboxylic acid (COOH–) and monoclonal antibody (Mab, C2F3C2) to act as a donor. The gold nanoparticles (AuNPs) labeled with the corresponding antigen (ciprofloxacin-BSA) take the role of an acceptor. Under optimized conditions, the limit of detections (LODs) for three common FQs, enrofloxacin (ENR), ciprofloxaxin (CIP), and norfloxacin (NOR), were 0.19–0.32 ng/mL based on 3σ. The recoveries were found to be in the range of 73.5–114.5% (normalized value according to the cross-reactivity of Mab) for water samples (tap water, pond water and river water). The proposed approach possesses significant advantages as follows: (i) simple procedure (only one step) without washing and separation steps, and no sample pretreatment other than filtration; (ii) fast liquid-phase kinetics with shortened incubation time; and (iii) high tolerance to various interfering substances. All of which indicated its potentiality as an efficient biosensor towards the monitoring of FQs in aquatic environments.


Introduction
Fluoroquinolones (FQs) belong to the class of synthetic antibiotics with a broad spectrum of activity against both gram-positive and gram-negative bacteria. Its potential has been appropriately centralized for the treatment of respiratory diseases and bacterial infections both in human and farm animals [1][2][3]. However, prior reports show the extensive usage of this antibiotic for pharmaceutical purposes has resulted in a widespread contamination of environmental water [4][5][6][7][8][9]. In view that the chemicals could cause adverse effect on the aquatic vertebrates and organisms, and their increased level of bacterial resistance, more and more concerns arose on the potential risk on human health and aquatic ecosystem [10][11][12][13][14]. So, some effective approaches were put forward to monitor FQs in environmental water.
Currently several analytical methods were conventionally used for the detection of FQs in water samples. Among them, the chromatographic method utilizing solid-phase extraction (SPE) as a pre-concentration step is widely adopted [2,15]. However, the drawbacks of being laborious, time-consuming and solvent intensive limited their application. As an alternative, high-throughput immunoassay is a good choice for analysis of FQs from water samples, depending on their outstanding properties, the assays could screen containments from large amounts of samples in a short period of time [16][17][18][19][20]. But the methods cannot be done with an automated instrument, and cannot carry out on-site analysis for the organic pollutants because of relatively sophisticated procedures.
To reduce the cumbersome immunoassay steps and obtain quick information from solution samples, fluorescence resonance energy transfer (FRET) was introduced, in FRET process, the energy is transferred from the donor to the acceptor through non-radiative dipole-dipole interactions [21][22][23][24][25][26][27]. In fact, before an effective FRET occurs, two conditions must be met as: (i) the fluorescent donor molecule and an acceptor molecule that are brought in a close proximity to each other; (ii) a strong overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor [28][29][30]. To obtain better sensitivity using FRET assay, the long-lived emission from Lanthanide-based upconversion nanoparticles (UCPs) was selected as a candidate of energy donors, which was because it could extend the observed lifetime of the sensitized acceptor emission, allowing energy-transfer signals to be measured in a time-resolved manner without direct acceptor emission, autofluorescence and scattered excitation [31,32]. In addition, UCPs under IR excitation possess many fascinating properties, such as low toxicity, nearly-zero background autofluorescence, high photoluminescence intensity and photostability [33][34][35]. All of which indicated that UCP has unique advantages as a potential energy donor.
However, hydrophobic UCPs cannot be directly labeled by antibodies and used in the biology detection because of low solubility in water and unfavorable surface properties, it has to be converted into biocompatible one using appropriate functional groups (-COOH, -NH 2 , -SH) through exchange ligand process or UCPs-SiO 2 core-shell structure [24,[36][37][38]. At present, the method of encapsulation with SiO 2 was widely used as a surface functionalization of hydrophobic UCPs, but this procedure was suffered from high cost, complicated procedures and difficulty in controlling the thickness and uniformity of the SiO 2 layer. Moreover, a thick layer of SiO 2 shell could deteriorate the analytical performance of FRET assay because of the increase in distance between the donor and the acceptor molecule. In order to ensure a close proximity between UCPs and fluorescent acceptor for efficient energy transfer, a direct surface modification that converts hydrophobic nanoparticles (NPs) into water-soluble ones without any coating layers was implemented [34].
In the present work, a highly monodisperse and uniform UCPs were synthesized, and the surface was modified into biocompatible by adopting the poly(acrylic acid) (PAA)-based ligand exchange strategies [36,39]. The UCPs were made as an energy donor after their surface functionalization with a group-specific Mab against FQs. At the same time, due to excellent absorption properties in the visible region [40], the AuNPs conjugated with antigens (ciprofloxacin-BSA) were chosen as acceptor molecule for the upconversion FRET measurements. A homogeneous upconversion FRET assay was developed for the simultaneous detection of FQs series in aqueous samples. The established method was evaluated with several factors that potentially influence its performance, and the accuracy of the assay also was investigated by spiked FQs in various water samples.  Table S1 (ESIelectronic supplementary information) [41].

Instrumentations
Fluorescence spectra of UCPs were recorded using a Lumina Fluorescence Spectrometer (Thermo Fisher Scientific) and modified with an external 980 nm laser (Beijing Hi-Tech Optoelectronic Co., China). The morphology of the synthesized UCPs was studied using a transmission electron microscope (TEM, H-800, Hitachi Ltd, Japan). X-ray powder diffraction patterns were obtained at a D8 advance X-ray diffractometer (Bruker Instruments Inc., Billerica, MA). The Fourier transform infrared spectroscopy (FT-IR) was performed on a Perkin-Elmer GX2000.

Synthesis of the β-NaLuF4:Yb,Er,Gd NPs
Oil-dispersible β-NaLu 0.68 F 4 :Yb 0.18 , Er 0.02 ,Gd 0.12 UCPs have been successfully synthesized according to the earlier reported procedures with slight modifications [39,[42][43][44]. Typically, (i) Lu 2 O 3 (3.4 mmol), Gd 2 O 3 (0.6 mmol), Yb 2 O 3 (0.9 mmol), and Er 2 O 3 (0.1 mmol) were added to 30 mL of hydrochloric acid solution (20:10; v/v of H 2 O/concentrated HCl). The mixture was heated at 70°C for 1 h under reflux condenser. Then the mixture was evaporated to dryness using a vacuum evaporator. The obtained powder was dissolved in 50 mL of methanol, as a RECl 3 precursor. (ii) 5 mL of RECl 3 precursor, OA (8 mL) and ODE (18 mL) were added to a three-necked flask that is fitted with a condenser, and heated to 160°C under vigorous stirring (500 rpm) for 30 min. The solution obtained was then cooled to room temperature (RT) and the solution turned transparent yellow. To this mixture was added 10 mL of methanolic solution containing NH 4 F (4 mmol) and NaOH (2.5 mmol) and the entire mixture was stirred at room temperature for 90 min, followed by heating at 300°C under nitrogen atmosphere for 90 min. The solution was brought down to RT. The final product was collected by centrifuging and subsequent washing with ethanol. The collected powder was dried at 60°C for 12 h under vacuum atmosphere.

Conversion of hydrophobic UCPs into water-soluble ones
The method was mainly based on the previously reported works [39,42]. Typically, a mixture of PAA (0.5 g) and DEG (10 mL) was heated at 110°C under vigorous stirring (500 rpm) for 10 min. To the heated mixture, was added 5 mL toluene solution containing 30 mg UCP and stirred at 1800 rpm in order to speed up the evaporation of toluene. When the temperature had reached 230°C, the exchange ligand process occurred violently, and so the stirring speed was reduced to 1000 rpm in order to prevent the expulsion of the un-exchanged particles. This step was maintained for an hour and carried out in an inert atmosphere. The mixture was further cooled to RT followed by addition of dilute hydrochloric acid (pH 4-5). The final product PAA-UCPs were collected by centrifugation process at 12,000 rpm for 15 min and washed thrice with water/ethanol mixture (1:1, v/v).

Preparation of Mab-conjugated β-NaLuF4:Yb, Er, Gd UCPs
Primarily, 5 mg of PAA-UCPs was dissolved in 2 mL of MES buffer (0.195 g MES was dissolved in 100 mL ultrapure water and the pH was adjusted to 5.5 using NaOH). The solution was filtered through a 0.22 μm micropore membranes, sterilized and preserved at 4°C. It was then incubated in a shaker (30°C, 160 rpm) for 2 h after the addition of 4 mM EDC and 10 mM NHS. On centrifugation the precipitate was obtained which was then washed thrice with distilled water. The obtained precipitate and 0.5 mg FQs-antibody were added to 2 mL of HEPES buffer (0.238 g HEPES was dissolved in 100 mL ultrapure water, and pH adjusted to 7.2 using NaOH), followed by subsequent shaking for about 2 h (30°C, 160 rpm). Any excess NHS was deactivated by the addition of 15 mg bovine serum albumin (BSA). The entire content was centrifuged to obtain the final precipitate of Ab-UCNP. The precipitate was washed thrice with distilled water and dispersed in 1 mL HEPES buffer for further usage [39].

Synthesis of Au NPs and preparation of antigen (CIP-BSA)-conjugated AuNPs
AuNPs were synthesized and conjugated with antigen following prior reports [29,45,46]. Briefly, 1 mL of HAuCl 4 (1%) was dissolved in 50 mL deionized water and heated to boiling. To it was added 1 mL of 2% sodium citrate solution and maintained at the same temperature for 15 min along with stirring. On cooling the mixture (final product) to RT, 50 mL of distilled water was added for further dilution. Subsequently, 0.5 mL of 8 mg mL −1 antigen was added into 800 mL Au NP solution (the pH was adjusted to 8.0 using borate buffer) under magnetic stirring in an ice bath for 1 h. To the mixture was added 0.1 mL of 10% BSA solution under stirring for another 1 h. The solution was then centrifuged at 6000 rpm for 15 min at 4°C, and resultant sediment was washed with borate buffer solution for several times. After this step, the antigen (CIP-BSA)-conjugated AuNPs were stored in Tris-HCl buffer for further use.
Both Mab labeled UCPs and antigen conjugated AuNPs were stable for more 6 months and played a role as two components of FRET assay.

FRET assay
A simple approach was adopted and is detailed as follows. Typically, Mab labeled UCPs (prepared in the assay buffer) (100 μL, 12.5 μg mL −1 ), 72 μL standard solutions or water samples and 28 μL (0.125 μg mL −1 ) antigen conjugated AuNPs (dissolved in Tris-HCl buffer) were pipetted out into a series of fluorescence cuvettes. It was incubated for 1 h at room temperature with slow shaking. The experiments were repeated for 5 times. Analyses were carried out in triplicates and a sigmoidal standard curve was fitted to the mean values using Origin7.5 (Microcal, Studio City, CA).

Tolerance evaluation and accuracy verification
Interferences may arise due to matrix effects and variations in assay conditions and thus, cascading to unspecific reactions. Hence, the current method was evaluated for possible application using the water samples containing a series of heavy metals (Cu 2+ , Hg 2+ , Pb 2+ , Cr 2+ ), common ions (Ca 2+ , Mg 2+ , K + and Na + ) at varying pH values. The accuracy of the method was evaluated using recoveries obtained from water samples (tap water, pond water and river water) spiked with typical FQs (NOR, ENR, and CIP). Tap water was collected from a lab in the school of environmental science, Jiangsu University. Pond water and river water samples were obtained from Neijiang River and adjacent rural areas. All the samples were filtered to remove particles larger than 0.22 μm so that no FQs were present in the samples prior to FRET analysis.

Characterization of the donor
In the present work, NaLuF 4 was chosen as the host material, Yb 3+ as the sensitizer and Er 3+ as the activator. It is well known that NaLuF 4 exists in two phases namely the hexagonal and cubic. The fluorescent intensity of hexagonal phase of NaLuF 4 is higher than that of the cubic phase. The hexagonal phase NaLuF 4 is often synthesized in organic solvents under high temperature or by using suitable dopants. Gd 3+ was added to the system in order to increase the upconversion luminescence and for the reduction of particle size [36,42,44,47]. The shapes of the particles are highly influenced by the crystallographic phase of the initial seeds that have been formed during the nucleation process. The formation of a stable phase is highly dependent on the environmental factors, and especially on temperature. The reaction temperature dependence on size and fluorescent intensity of NaLuF 4 are shown in Fig. S1 (ESIelectronic supplementary information). The small size and high fluorescent intensity of NaLuF 4 UCPs were obtained using 18% mol of Gd 3+ at 300°C. This temperature was chosen as an optimal reaction temperature.
The phase transition of NPs was also controlled by reaction time and hence, it was considered as an important factor in the synthesis of UCPs. The cubic phase does not seem to represent the state of equilibrium when the physical dimension of the initial particle increases to critical size of the phase transition. The reaction time is prolonged to cause a rapid dissolution of cubic phase seeds followed by the release of monomers, and thus resulting in a sudden nucleation of hexagonal phase seeds [48,49]. Both the cubic phase and hexagonal phase existed when the reaction was performed for duration of 30 min at 300°C. The phase transition from cubic phase to hexagonal phase started only after 60 min of the reaction at 300°C. This phase transition was confirmed by XRD analysis, as shown in Fig. S2 (ESI). When the reaction was carried out for 90 min, the size of the obtained nanoparticles was found to be~25 nm and the shape was near hexagonal. Further prolongation of reaction time led to an increase in the crystal size (~160 nm after 10 h) and therefore, resulted in a hexagonal shape, and is shown in the TEM images (Fig. S3 (ESI)).
Under the optimized conditions (reaction temperature, 300°C; reaction time, 90 min), β-NaLuF 4 :Yb,Er,Gd UCPs have a well dispersed, uniform, and a narrow size distribution with an average diameter of~25 nm, as shown in Fig. 1a. With the high resolution transmission electron microscopy image (Fig. 1b), the interplanar distances are found to be~0.51 and 0.3 nm, which correspond to the d-spacing value of ð1010Þ and ð1120Þ planes of β-NaLuF 4 :Yb, Er, Gd. The inset of Fig. 1b shows the selected area electron diffraction (SAED) pattern of the UCP sample. Further confirmation of the above results was carried out using the powder X-ray diffraction (XRD) analysis (Fig. 1c). All the diffraction peaks could be indexed to the hexagonal phase (JCPDS No. 27-0726). The dominant hexagonal phase suggested that Gd 3 + helps for the phase transition from cubic to hexagonal. The ionic radius of Gd 3 + ion is larger than that of the Lu 3 + ion in NaLuF 4 . Therefore, NaLuF 4 codoped with a high concentration of Gd 3+ tends to promote the phase transition from cubic phase to hexagonal phase because of the larger ionic radius of the doped ions that exhibit a high tendency towards the electron cloud distortion and thus, leading to an increased dipole polarizability [50].
The OA-capped hydrophobic UCPs were functionalized using PAA-based ligand exchange strategy instead of the conventional SiO 2 encapsulation of hydrophobic nanocrystals [39,51]. The role of PAA was to replace monodentate OA shell on UCP surfaces by its multidentate binding sites. Fig. 2a confirmed the well dispersal of the surface modified UCPs in water and this may be ascribed to the presence of -COOH group on the surface. No obvious variation in fluorescence emission peaks and crystal structure was observed after the PAA exchange except for a slight decrease in emission intensity ( Fig. 2a and b). This can probably be attributed to the quenching of water molecules after the introduction of the carboxyl groups [52]. Fourier transform infrared (FT-IR) spectroscopy was performed to verify the functional groups present on the surface of the NPs after the ligand exchange. As shown in Fig. 2c, the oleic acid exhibited a broadband around 3400 cm − 1 , corresponding to stretching vibration of a hydroxyl group. The peaks at approximately 2920 and 2850 cm − 1 can be assigned to the asymmetric and symmetric stretching vibration of methylene (-CH 2 ) in the long alkyl chain. Two strong bonds were observed at 1418 and 1558 cm − 1 , respectively and it can be ascribed to the asymmetric and symmetric vibration modes of the -COO − group of the bound oleate ligand through the bidentate bonds [51,53,54]. After surface modification using PAA exchange, a strong bond was observed at 1728 cm − 1 . This peak is assigned to the C=O stretching vibration of the -COOH groups of the PAA. The shoulder observed at 2955 cm − 1 associated with the asymmetrical stretching mode of -CH 3 groups became inconspicuous after ligand exchange [42,51,55,56]. Based on FT-IR spectra and the good dispersal of UCPs in water after PAA exchange, they can be considered as the direct evidences for the exchange ligand process. After functionalization of the UCP surface with -COOH group, it was labeled with the FQs Mabs using cross-linkers (EDC), which activated the carboxy groups, and thus amide bonds were formed with the antibodies.

FRET assay
On excitation at 980 nm, the UCPs showed a strong fluorescence (FL) emission at 543 nm. This emission band was overlap with absorption band of the AuNPs (average diameter of 13 mm - Fig. S4 (ESI)), thus UCPs and AuNPs can function as a FRET donor and acceptor in the FRET pair, respectively (Fig. 3) [40,57]. Based on the above-mentioned donor and acceptor, a single-step homogeneous method had been developed and elaborated in Scheme 1. To better explain the principle of the method, it was divided into two major parts: firstly, the dissolved FQs and antigen conjugated AuNPs were competitively bound with the Mab labeled UCPs (the categories of FQs were determined by the cross-reactivities of the Mab, as seen in Table S1 (ESI)). The specific recognition between antibody and antigen makes both the NPs to attain a suitable spacing for the occurrence of FRET. Next, under an excitation of 980 nm, the emitted green light of the UCPs was quenched quickly by the acceptor. As shown in Fig. 4a, the green fluorescence intensity is decreased gradually with increasing amount of antigen labeled AuNPs.
To obtain better performance, the quantity of labeled UCPs and AuNPs (connected with NOR-OVA) to be added in the assay was optimized using sensitivity as the key criterion, where three typical FQs (ENR, CIP and NOR) were selected for evaluation of the analytical performance of FRET assay. The standard calibration curves were constructed using various concentrations of FQs under the optimized conditions (donor; 100 μL 12.5 μg/mL, acceptor; 28 μL 0.125 μg/mL). Fig. 4b illustrates the constructed calibration curve for ENR as an example. The LODs for NOR, ENR and CIP were calculated as 0.32 ng/mL, 0.20 ng/mL, and 0.19 ng/mL, respectively, based on the mean of zero calibrators (3σ).
Without an expensive or sophisticated instrumentations, extensive sample preparation, and more skilled person required, the LOD values obtained by present method are the same order with those of other methods for the detection of FQs (see Table 1). Therefore, it can be deemed as a good biosensor for on-line monitoring the FQs in environmental water samples.

Tolerance evaluation studies
The real environmental water samples were collected from various aquatic resources, such as tap, pond, and river water, which potentially possess various matrix effects. Various environmentally relevant parameters in aquatic system have to be considered such as salinity, water hardness, varied pH and heavy metals, as these could influence the specific immunoreactions or result in coagulation of NPs and thus, cause a negative impact on the FRET performance. Hence, the evaluation of the tolerance of the proposed method becomes vital in variation to these parameters.   Considering the case or real samples, where the pH is found to be in the range of pH 6.0-9.0, the established FRET assay has a good tolerance, as shown in Fig. S5a (ESI). This may be correlated to the high concentration of buffer used in the study. The effects of salinity were evaluated using KCl and NaCl as model salts. Fig. S5b (ESI) indicates that the method has high tolerance to both K + and Na + ions (0-10%, w/v). However, a relatively poor tolerance was observed after the introduction of Ca 2+ and Mg 2+ ions to the water samples. When the concentration of Ca 2+ and Mg 2+ exceeded 1.25% (w/v), coagulation occurred in the system, and the interference cannot be eliminated by the addition of the complexing reagent (EDTA and oxalate, shown in Fig. S5c and d (ESI)). This can be ascribed to the aggregation of AuNPs [58]. By using EDTA for the removal of Ca 2+ and Mg 2+ , there occurred the formation of new complexes at higher concentration of Ca 2+ and Mg 2+ and thus led to the change in color, as shown in Fig. S5c and S5d (ESI). Owing to low amounts of Ca 2+ and Mg 2+ present in surface water (b1%, w/v), their effect on the FQs determination is negligible.
The effect of heavy metals was considered to cause the denaturation of the antibodies and antigens, and then subsequently lead to the disappearance of FRET. The following ions, namely Cu 2+ , Hg 2+ , Pb 2+ , and Cr 2+ , were chosen as potential interferes. Our results (data not shown) demonstrated that no obvious variations in the signal were noted after the addition of the heavy metal ions to the water samples (0-1 mg/L). These results are in agreement with our previous report [20].

Accuracy verification
The accuracy and precision of the proposed method were evaluated using different sources of water samples containing 2 μg/L for each one of ENR, CIP and NOR (three typical FQs). As shown in Table S2 (ESI), the intra-assay coefficient of variation (CV) values were 4.7-9.7%, and the recoveries were within 73.5-114.5% (normalized value according to the cross-reactivity of Mab). The higher recovery values might have been originated from the calibration. The proposed method relies on calibration with suitable standards in solvent. In the case of real samples, the matrix effect is usually corrected by performing matrixmatched standards calibration or isotope dilution. Unfortunately, these techniques are found to be difficult in application to real time environmental analysis because of the higher variability in samples from different origin [59,60]. Nevertheless, the obtained recoveries were quite satisfactory and made a strong implication of the proposed approach to real sample analysis [2].

Conclusions
The current study demonstrates the multi-facets of the synthesized β-NaLuF 4 :Yb,Er,Gd UCPs with near hexagonal shapes, narrow size distribution, good mono-dispersibility, and high efficiency. The direct surface modification using PAA-based ligand exchange strategy showed an excellent dispersibility in water after the covalent linkage of the -COOH group. The FRET approach had been developed using UCPs as donors and AuNPs as acceptors and successfully applied for the detection of trace amounts of FQs in environmental waters. The proposed homogeneous method was accurate with a high tolerance limit against various potential interferes. It involves a simple one step process involving only filtration and without any sample pretreatment, separation, or washing steps. Therefore, the immense potency of the method using high cross-reactivity FQs Mab, toward the on-line monitoring of total amount of a class of compounds in environmental waters can be well established.