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DOI: 10.1039/C4NR04380D
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Yujie Ding,ab Hao Zhu,a Xiaoxia Zhang,a Jiangang Gao,b E. S. Abdel-Halim,c Liping Jiang*a and Jun-Jie Zhu*a 5
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Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x Upconversion nanophosphors (UCNPs) are extremely useful for analytical applications, since they display a high signal-to-noise ratio, and the photobleaching can be ignored. Herein, a novel upconversion nanocomposite composed of β-cyclodextrin (β-CD) derivative modified UCNPs and rhodamine B (RB)， was prepared for the detection of cholesterol (Cho). The upconversion luminescence (UCL) emission can serve as a Cho-sensing signal by an effective fluorescence resonance energy transfer (FRET) process, using UCNPs as the donor and RB as the quencher. The sensor for Cho detection in human serum shows excellent sensitivity and selectivity, which has the potential for clinical applications in the analysis of other biological and environmental samples.
1. Introduction Cho is an essential lipid for the human body and a major component of intracellular membranes in mammalian cells. The estimation of blood Cho is one of the most widely performed assays in biochemistry as Cho plays a vital role in the initiation and progression of many diseases.1, 2 The balanced quantity of Cho in human body provides durability and integrity to the cell architecture. On the one hand, excess Cho in blood serum forms plaques in the arteries of blood vessels to prevent the blood circulation, causing myxedema, atherosclerosis, diabetes milletus, nephrosis and jaundice. On the other hand, a low Cho level may result in anemia and hyperthyroidism.3, 4 Thus the levels of total Cho in serum are major parameters for diagnostic treatment. Various methods have been developed for Cho detection such as fluorescence-based assays, electrochemical means, and molecular imprinting technology. However, there still exist limitations including long time consumption and lack of a suitable internal standard in most of the current methods. Also, the detection selectivity strongly relys on the use of Cho selective enzymes and antibodies, which are expensive and prone to denaturation.5 Therefore, alternative, simple and cost-efficient methods are highly desirable. Recently, fluorescent dye based Cho-sensing methods have been developed by the competitive host-guest interaction between β-CD with the dye molecule and Cho.4, 5 However, care should be taken in order to keep the pH, ionic strength, and organic effect of the sample solutions the same for all the measurements, due to the sensitiveness of the optical properties of dye molecules to these conditions.4 In addition, under ultraviolet (UV) excitation, the autofluorescence from biological macromolecules, mainly absorbing in the UV region, will appear providing diminished signal-to-background ratio and hence decreased sensitivities.6 UCNPs are kinds of fluorophores codoped with rare-earth This journal is © The Royal Society of Chemistry [year]
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ions, capable of converting low-energy light to high-energy light through a multiphoton process, thus emitting light at the visible and near-infrared (NIR) region.7,8 They hold strong photoluminescence, high quantum yields, narrow emission bands, good chemical stability, and photostability.9,10 Under the excitation of low-energy NIR photons (normally 980 nm), the autofluorescence from organisms can be eliminated.11 All the favorable properties have indicated the great potential of UCNPs in the analysis of biological and environmental samples. Among the various lanthanide-doped UCNPs, hexagonal phase NaYF4: Yb, Er UCNPs have been recognized as the most efficient nanophosphors in which the Yb3+ ions absorb multiple NIR photons while the Er3+ ions emit visible light.12 In fact, UCNPs have been regarded as an ideal donor in various FRET-based assays. Until now, several optical sensors based on UCNPs have been developed in sensing CN-,13 Hg2+,14, 15 Cu2+,16 and so on.11, 17-19 However, to the best of our knowledge, the selective fluorescence probes based on UCNPs for Cho-sensing have not been reported. Herein we have designed an upconversion nanocomposite for FRET-based Cho-sensing and further applied in human serum.
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2.1 Chemicals All the starting materials were obtained from commercial suppliers and used as received. YCl3·6H2O (99.99%), YbCl3·6H2O (99.99%), ErCl3·6H2O (99.9%), NaOH (98%), NH4F (98%), 1-octadecene (90%), oleic acid (OA) (90%), β-CD, diethylene glycol (DEG) and citric acid were purchased from Sigma-Aldrich. Deionized water was used throughout. All other chemical reagents in analytical grade were used directly without further purification. [journal], [year], [vol], 00–00 | 1
Nanoscale Accepted Manuscript
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An Upconversion Nanocomposite for Fluorescence Resonance Energy Transfer Based Cholesterol-Sensing in Human Serum
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2.2.1 Synthesis of OA-UCNPs OA-UCNPs were prepared by a modified solvothermal process according to the reported method.20 In a typical experiment, 1 mmol RECl3·6H2O (0.78 mmol YCl3·6H2O, 0.20 mmol YbCl3·6H2O, 0.02 mmol ErCl3·6H2O) were added to a flask containing 7.5 mL OA and 17.5 mL 1-octadecene. The solution was heated to 160 oC for 30 min, and then cooled down to room temperature. Thereafter, 10 mL methanol solutions of NH4F (4.0 mmol) and NaOH (2.5 mmol) were added into the solution, and stirred for 30 min. After methanol was evaporated, the solution was heated to 300 oC under argon atmosphere for 1 h, and cooled down to room temperature. The resulting nanoparticles were precipitated by the addition of ethanol, collected by centrifugation, washed with water and ethanol for three times. 2.2.2 Synthesis of ligand-free UCNPs Ligand-free UCNPs were prepared following a protocol that had been reported previously.21,22 OA-UCNPs (100 mg) were dispersed in a 10 mL aqueous solution. The reaction was performed with stirring for 4 h while maintaining the pH at 4 by adding 0.1 M HCl solution. During this reaction, the carboxylate groups of the OA ligand were protonated (to yield OA). After the reaction was completed, the aqueous solution was mixed with diethyl ether to remove the OA by extraction with diethyl ether for three times, and the combined ether layers were re-extracted with water. Afterwards, the water layers were combined and reextracted with diethyl ether. UCNPs in the water dispersible fraction were recuperated by centrifugation after precipitation with acetone. The product was redispersed in acetone and the particles were recuperated by centrifugation. Finally the nanoparticles were dispersed in water. 2.2.3 Preparation of cyclodextrin citrate esters (CD-Cit) CD-Cit was prepared using a semidry reaction method as reported by El-Tahlawy et al.23, 24 Firstly, 3.00 g of β-CD was mixed with 1.8 mL of water containing 1.02 g of citric acid. Then, the mixture was allowed to react in a circulating air oven at 105 °C for 3 h. The cured sample was purified by repeated washing with isopropanol to remove unreacted components and soluble fragments or byproducts. Finally, the resultant CD-Cit was dried at 60 °C for 24 h and stored in an airtight container for further use. 2.2.4 Preparation of CD-Cit-UCNPs CD-Cit-UCNPs were prepared following a modified protocol.25 15 mL DEG and 2 mL CD-Cit (2 mmol) aqueous solution were mixed in a 25 mL flask. 10 mg ligand-free UCNPs dispersed in 2 mL aqueous solution were injected into the above mixed solution, and the mixture was heated to 100 ºC for 30 min under argon atmosphere. After methanol evaporated, the solution was kept at 160 ºC for 3 h until it became clear. The resulting solution was cooled down to room temperature. The precipitates were collected by centrifugation (10, 000 rpm, 15 min) and washed for three times with ethanol and deionized water. 2.2.5 Preparation of RB-CD-Cit-UCNPs The solution of CD-Cit-UCNPs (30 mg) in 5 mL of deionized water was prepared at room temperature. After the solution became clear, a solution of RB (10 mg) in 2 mL of water was added slowly. The above solution was stirred for 24 h at room temperature, collected by centrifugation (18, 000 rpm, 10 min), and washed with water for three times. According to the amount 2 | Journal Name, [year], [vol], 00–00
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of unreacted RB and the as-prepared RB-CD-Cit-UCNPs, the percent of RB attached to the UCNPs was about 6.7%. 2.3 UCL Detection of Cho
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The as-prepared RB-CD-Cit-UCNPs were dissolved in 2 mL water, diluted with tris(hydroxymethyl)aminomethane (Tris) buffer with a pH of 7.4, and used as a stock solution. 90 µL of this solution was taken and mixed with 10 µL ethanolic solution of different concentrations of Cho. After reaction for 40 min, UCL was measured under the 980 nm excitation. Cho detection in human serum was performed using normal adult serum (provided by the Affiliated Drum Tower Hospital of Nanjing University). 0.5 mL of serum was mixed with 4.5 mL ethanol. After 10 min, the solution was centrifuged at 4000 rpm for 10 min. The different kinds of supernatant serum were taken and added with the stock solution for determination. 2.4 Characterizations UV-Vis absorption spectrum was measured using a Shimadzu UV-2550 ultraviolet-visible-near infrared spectrometer. Upconversion fluorescence spectra were recorded on a ZolixScan ZLX-UPL spectrometer using an external 1 W continuous-wave laser (980 nm) as the excitation source. X-ray diffraction (XRD) measurements were performed on a Japan Shimadzu XRD-6000 diffractometer with Cu-Kα radiation (λ= 0.15418 nm); A scanning rate of 0.05 deg s-1 was applied to record the patterns at the 2θ range of 10-800. The morphology and structure were characterized by transmission electron microscopy (TEM) and high resolution TEM (HRTEM) images using a JEOL-2100 TEM equipped with an Oxford Instrument Energy-dispersive X-ray (EDX) system operating at 200 kV. Mass spectrum (MS) was obtained on a HP 1100 LC-MS spectrometer. The fourier transform infrared (FT-IR) spectra were recorded on a Nicolet 6700 Fourier transform infrared spectrograph at the wavenumber range of 4000-500 cm-1. The X-ray photoelectron spectroscopy (XPS) spectrum was measured on an ECSALAB 250.
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The design and synthesis of a Cho-sensitive RB-quenched upconversion fluorescent probe assembled with β-CD derivative (CD-Cit) and UCNPs was performed, and the probe can be further applied in human serum. The nanosensor is based on a FRET process, using NaYF4: Yb, Er nanophosphors as the energy donor and RB as the energy acceptor. The assembly is as outlined in Scheme 1.
Scheme 1 Schematic illustration of the design and synthesis of RB-CDCit-UCNPs, and their UCL response to Cho.
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2.2 Material Synthesis
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Firstly, OA-UCNPs were treated with HCl to achieve ligand-free nanoparticles (UCNPs). Then, an aqueous solution of citric acid modified β-CD (CD-Cit), prepared by a semidry reaction method, 23, 24 was mixed with the aqueous solution of UCNPs under stirring. This as-fabricated CD-Cit coat on the nanoparticles (CD-Cit-UCNPs) through the carboxyl group of the complex coordinating with the rare-earth ion on the surface of UCNPs.14 Since the CD-Cit functionalization offers high water solubility to UCNPs, guest molecules incorporated into β-CD can be easily accessible to UCNPs.14 β-CD inclusion of RB (RB-CDCit-UCNPs) induces FRET to occur because of close contact between the donor moiety (UCNPs) and the quencher (RB) nearby. β-CD, a cyclic polysaccharide, composed of seven glucose molecules, has a hydrophilic outer surface which provides water solubility and a hydrophobic inner cavity for specific recognition of guest molecules.5, 26 It shows much stronger binding capacity towards Cho compared with RB according to the binding constant available,27, 28 due to the hydrophobic nature of Cho. FRET is switched off by virtue of the Cho-induced release of RB from the cavity of β-CD, because of the competitive host-guest interaction between CD-Cit-UCNPs with RB and Cho, resulting in the fluorescence recovery of the quenched UCNPs. The “on-off” switching of FRET with UCL changes will provide a much-improved assay of Cho.
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Fig. 1 UV-Vis absorption spectrum of RB in aqueous solution (black line) and UCL spectrum of OA-UCNPs in cyclohexane solution (green line, inset is the corresponding UCL photography).
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Fig. 2 TEM and HRTEM images of OA-UCNPs dispersed in cyclohexane solution (a and b, respectively) together with CD-Cit-UCNPs dispersed in aqueous solution (c and d, respectively).
OA-NaYF4: Yb, Er UCNPs, prepared by a modified cothermolysis process with OA as the surface ligand, have a hydrophobic surface.20 The XRD pattern of OA-UCNPs could be attributed to hexagonal phase NaYF4 (JCPDS NO. 28-1192) (Fig. S1a). The particle size was calculated to be 35.5 nm according to Debye-Scherrer formula.30 EDX analysis showed that the nanoparticles were mainly composed of Na, Y, F, Yb and a small amount of Er element (Fig. S1b). TEM images displayed that OA-UCNPs with polyhedral morphologies were well-dispersed in cyclohexane solution, and had an average diameter of 36 nm (Fig. 2a), which was in agreement with the XRD results. The asprepared CD-Cit was characterized by MS (Fig. S2). Major peak at 1330.8 can be assigned to the target compound (CD-Cit), and other one at 1156.9 is attributed to the unreacted β-CD. After coating CD-Cit onto the ligand-free UCNPs, the hybrid could be easily dispersed in water, and the size and shape of the UCNPs remained essentially unchanged (Fig. 2c). HRTEM images proved the highly crystalline nature of the nanoparticles (Fig. 2b and 2d).
Under 980 nm excitation, OA-UCNPs give dual emission bands in green and red parts of the visible region, as shown in Fig. 1, whose inset shows the photography of the strong eyevisible green light from the cyclohexane solution of the nanoparticles. The emission bands at around 522 nm, 541 nm and 655 nm can be assigned to 2H11/2 - 4I15/2, 4S3/2 - 4I15/2, 4F9/2 - 4I15/2 transitions of Er3+, respectively.29 RB has a broad absorption band centered at 554 nm, which can overlap well with the green emissions of UCNPs, implying a FRET process through the donor and quencher nearby. It is reasonable that the degree of energy transfer between UCNPs and RB can be modulated by the concentration of Cho (Scheme 1). In addition, to achieve ratiometric UCL detection, The NIR UCL emission at 655 nm was chosen as the reference. Fig. 3 FT-IR spectra of the as-prepared OA-UCNPs, UCNPs, CD-CitUCNPs and CD-Cit, respectively.
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Nanoscale Accepted Manuscript
DOI: 10.1039/C4NR04380D
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Fig. 4 UCL intensity (a) and the corresponding photos ((b), from left to right) of OA-UCNPs in cyclohexane solution (black line) and UCNPs (red line), CD-Cit-UCNPs (blue line), RB-CD-Cit-UCNPs (green line), RB-CD-Cit-UCNPs+ 80 µM Cho (pink line) in the Tris-HCl buffer solutions (pH= 7.4), respectively.
The UCL emission intensity of the modified nanomaterials was characterized as shown in Fig. 4. Both the disappearance of OA molecule and the coating of CD-Cit decreased the luminescent intensity, because the organic compound and the water molecule with their vibration frequencies could quench the emission of the rare earth ions to a great extent.31 RB could make FRET process occur, leading to the reduced UCL intensity. Yet, after 80 µM Cho was added to the Tris-HCl buffer solutions of RB-CD-CitUCNPs, the green UCL emission intensity increased while the red part remained unaffected, because Cho replaced RB to enter the lipophilic cavity of β-CD inhibiting the FRET process.5 It could be found that the UCL intensity of CD-Cit-UCNPs was decreased dramatically by RB within a short period of time, 4 | Journal Name, [year], [vol], 00–00
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suggesting that the inclusion of RB into the β-CD occurred rapidly. After the addition of the competitive binder Cho, the UCL intensity gradually increased by increasing the reaction time, reaching a plateau after 25 min (Fig. S4), which was much longer than that time for RB inclusion into β-CD to reach equilibrium. Taking the sensitivity into consideration, we set a time of 40 min for the reaction between Cho and RB-CD-CitUCNPs.
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Fig.5 UCL spectra of 0.5 mg/mL RB-CD-Cit-UCNPs in the Tris-HCl buffer solutions (pH= 7.4) upon gradual addition of Cho (0-200 µM). Inset is GRR (UCL515-565/UCL640-685, UCLA-B is the integrated emission intensity from A to B nm) as a function of Cho concentration.
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Fig. 6 Linear plot of the relative UCL increasing (GRR) as a function of Cho concentration (GRR denotes the relative fluorescence intensity UCL515-565/UCL640-685).
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To show the sensitivity of the nanoprobe, the changes in the UCL spectra of RB-CD-Cit-UCNPs was monitored in the presence of different concentrations of Cho, as shown in Fig. 5. Addition of Cho into the solution increased the green UCL intensity and the enhancive fluorescence was directly related to the amount of the added Cho. The reason is that upon more and more Cho competitive inclusion into the cavity of β-CD, the release of more and more RB from the inside resulted in the greater decay of the FRET effect. The UCL intensity in green emission increased while the red part remained unchanged with the increase of Cho concentration from 0 to 200 µM, as can be observed in the calibration curve of the green-to-red ratio (GRR) versus concentration of Cho in the inset of Fig. 5. GRR increased linearly with the concentration of Cho in the range of 10~110 µM This journal is © The Royal Society of Chemistry [year]
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The assembling processes were further confirmed by FT-IR spectra (Fig. 3). In the case of OA-UCNPs, The peaks at 2921, 2854, 1554 and 1462 cm–1 could confirm the existence of OA molecule, which limited the growth of the nanoparticles and prevented their agglomeration. The above characteristic peaks disappeared in ligand-free UCNPs, indicating that the OA had been fully wiped off. The peaks in the spectrum of CD-Cit at 1732 and 1202 cm-1 could be assigned to β-CD linkage to 23, 24 citrate. Furthermore, it was obvious that the spectrum of the CD-Cit-UCNPs showed a drastic change as compared to that of UCNPs, showing that CD-Cit could not be removed from the solid materials even by extensive washing with deionized water. When RB was assembled with CD-Cit-UCNPs, the colour of the as-prepared nanocomposite changed from colourless to pink even through repeated washings, which suggested that RB and CD-Cit-UCNPs had been combined together in the final products. The XPS experiment further verified this result, by virtue of simultaneous observation of the N, Na, Y, F, Yb, Er, C and O elements (Fig. S3). The above characterization confirmed the inorganic-organic hybrid nanophosphors had been prepared as expected.
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(R2= 0.9884) (Fig. 6). A concentration of Cho as low as 3.0 µM could be readily detected on the basis of the formula of detection limit.13 The sensitivity test was carried out for three times, and the same linear range (10~110 µM) and similar detection limit (2.7 µM, 3.0 µM, 3.1 µM, respectively) were achieved even though the values of the UCL intensity and GRRs could change slightly to some extent. The average value and absolute average deviation of the detection limit were 2.9 µM and 0.17, respectively. The UCL analysis supported the idea that the signal enhancement was attributed to the formation of an inclusion complex of the Cho moiety in β-CD, resulting in separation of RB from UCNPs.4 This phenomenon is explained by the guestinduced location change of RB from inside to outside of the cavity, suggesting that the upconversion nanocomposite RB-CDCit-UCNPs is an effective fluorescent probe for Cho-sensing. The sensitivity can be comparable to most of the existing Cho detection methods. 4, 5 To confirm the necessity of the CD-Cit functionalization, a control experiment was carried out. The aqueous solution of the same amount of RB was directly mixed with UCNPs and without CD-Cit), then incubated with different concentrations of Cho. In this case, the UCL intensity changed little and the GRRs were hardly altered compared with that using CD-Cit, as shown in Fig. 7.
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Fig. 8 UCL spectra (a) and GRR (b) of 0.5 mg/mL RB-CD-Cit-UCNPs in the Tris-HCl buffer solutions (pH= 7.4) before and after addition of 80 µM Cho or other 4 mM interfering substances (1. NaCl, KCl, MgCl2, CaCl2, ZnCl2; 2. Glycine + glucose; 3. Glutathione + lecithin; 4. Vitamin C; 5. Boracic acid + sucrose; 6. Bilirubin + sorbitol + mannitol; 7. Troptophan), respectively.
To examine the selectivity of this assembly system in serum samples, we carried out studies with the main constituents normally found in serum. The common substances present in serum with 4 mM concentration had been tested. It could be found that their UCL spectra (Fig. 8a）and the GRRs (Fig. 8b) were both similar to pure RB-CD-Cit-UCNPs, except for 80 µM Cho added. The above results suggest that the determination is free from interference from constituents of serum and the method can be extended for Cho detection in human blood. Table 1 Comparison of assay results of human serum samples for the clinical test (the enzymatic analysis) and the proposed method
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Fig.7 The GRR of UCL intensity upon gradual addition of different concentrations of Cho with and without CD-Cit modification, respectively.
Serum samples (adults) 1 2 3 4 5 6
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This journal is © The Royal Society of Chemistry [year]
The clinical test The proposed method (µM) (µM) 17.2 16.8 25.6 25.2 39.3 39.7 58.7 59.1 73.6 73.0 98.5 97.3
RDs (%) −2.3 −1.6 1.0 0.7 −0.8 −1.2
In order to evaluate the feasibility of the proposed method for clinical application, the real human blood serum test was also performed. According to the above-mentioned procedure, the detected Cho content in serum, derived from the standard curve and the regression equation, was listed in Table 1. In comparison with the enzymatic analysis that has been widely used in the clinical test, the relative deviations (RDs) in the proposed method show acceptable practicability for the detection of Cho. This demonstrates that the developed nanosensor with high sensitivity and selectivity, has great application potentials in clinical diagnosis in the future. In the end, to decrease the influence of some other substances in practical applications, a linear relationship from real serum sample should be found. It can be obtained through the addition of different serum with known concentration of Cho into Journal Name, [year], [vol], 00–00 | 5
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DOI: 10.1039/C4NR04380D
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the sensor and monitoring the changes in the UCL spectra. This method can reduce the interference from other constituents of serum and increase sensitivity and accuracy.
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4. Conclusions
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In summary, the design and synthesis of a novel upconversion nanocomposite that is composed of RB and β-CD derivative modified UCNPs was described. Furthermore, we presented its utility as an UCL ratiometric probe for Cho detection in human serum with high sensitivity and selectivity. The probe is based on a highly efficient FRET process using UCNPs as an energy donor and RB as a quencher. The proposed detection method is simple and cost-efficient as it doesn’t need use of Cho selective enzyme or antibody. UCNPs, as a kind of excellent emitters with their low autofluorescence and high penetration depth to biosamples, can endow this system with further application potentials in biological and analytical fields. Moreover, we have also provided an original design strategy of general nanoprobes for sensing studies, which can be as a beneficial reference for other fluorescent sensing system.
Acknowledgements
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The authors gratefully appreciate the support from National Basic Research Program (2011CB933502) of China, and National Natural Science Foundation (21121091, 21405001, 51303002), Program for New Century Excellent Talents in University (NCET-12-0256), and the State Key Laboratory of Analytical Chemistry for Life science (SKLACLS1307). The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding the work through the research group project (RGP-VPP-029).
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Notes and References a
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State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210093, China. E-mail: [email protected], [email protected] b College of Biochemical Engineering, Anhui Polytechnic University, Wuhu, 241000, China. c
Petrochemical Research Chair, Department of Chemistry, College of Science, King Saud University, Riyadh 11451P.O. Box 2455, Kingdom of Saudi Arabia.
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†Electronic Supplementary DOI:10.1039/b000000x/
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H. G. Li, M. H. El-Dakdouki, D. C. Zhu, G. S. Abela and X. F. Huang, Chem. Commun., 2012, 48, 3385. 2. V. Raj, R. Jaime, D. Astruc and K. Sreenivasan, Biosensors & Bioelectronics, 2011, 27, 197. 3. S. R. Cao, L. Zhang, Y. Q. Chai and R. Yuan, Biosens. Bioelectron., 2013, 42, 532. 4. N. Zhang, Y. Y. Liu, L. L. Tong, K. H. Xu, L. H. Zhuo and B. Tang, Analyst, 2008, 133, 1176. 5. A. Mondal and N. R. Jana, Chem. Commun., 2012, 48, 7316. 6. Y. J. Ding, X. X. Zhang, H. Zhu and J. J. Zhu, J. Mater. Chem. C, 2014, 2, 946. 7. Q. C. Sun, H. Mundoor, J. C. Ribot, V. Singh, Smalyukh, II and P. Nagpal, Nano Lett., 2014, 14, 101. 8. H. L. Qiu, G. Y. Chen, R. W. Fan, L. M. Yang, C. Liu, S. W. Hao, M. J. Sailor, H. Agren, C. H. Yang and P. N. Prasad, Nanoscale, 2014, 6, 753. 9. Y. J. Ding, X. Teng, H. Zhu, L. L. Wang, W. B. Pei, J. J. Zhu, L. Huang and W. Huang, Nanoscale, 2013, 5, 11928. 10. Z. W. Wei, L. N. Sun, J. L. Liu, J. Z. Zhang, H. R. Yang, Y. Yang and L. Y. Shi, Biomaterials, 2014, 35, 387. 11. Y. H. Wang, Z. J. Wu and Z. H. Liu, Anal. Chem., 2013, 85, 258.
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DOI: 10.1039/C4NR04380D
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Yujie Ding,ab Hao Zhu,a Xiaoxia Zhang,a Jiangang Gao,b E. S. Abdel-Halim,c Liping Jiang*a and Jun-Jie Zhu*a 5
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Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x Upconversion nanophosphors (UCNPs) are extremely useful for analytical applications, since they display a high signal-to-noise ratio, and the photobleaching can be ignored. Herein, a novel upconversion nanocomposite composed of β-cyclodextrin (β-CD) derivative modified UCNPs and rhodamine B (RB)， was prepared for the detection of cholesterol (Cho). The upconversion luminescence (UCL) emission can serve as a Cho-sensing signal by an effective fluorescence resonance energy transfer (FRET) process, using UCNPs as the donor and RB as the quencher. The sensor for Cho detection in human serum shows excellent sensitivity and selectivity, which has the potential for clinical applications in the analysis of other biological and environmental samples.
1. Introduction Cho is an essential lipid for the human body and a major component of intracellular membranes in mammalian cells. The estimation of blood Cho is one of the most widely performed assays in biochemistry as Cho plays a vital role in the initiation and progression of many diseases.1, 2 The balanced quantity of Cho in human body provides durability and integrity to the cell architecture. On the one hand, excess Cho in blood serum forms plaques in the arteries of blood vessels to prevent the blood circulation, causing myxedema, atherosclerosis, diabetes milletus, nephrosis and jaundice. On the other hand, a low Cho level may result in anemia and hyperthyroidism.3, 4 Thus the levels of total Cho in serum are major parameters for diagnostic treatment. Various methods have been developed for Cho detection such as fluorescence-based assays, electrochemical means, and molecular imprinting technology. However, there still exist limitations including long time consumption and lack of a suitable internal standard in most of the current methods. Also, the detection selectivity strongly relys on the use of Cho selective enzymes and antibodies, which are expensive and prone to denaturation.5 Therefore, alternative, simple and cost-efficient methods are highly desirable. Recently, fluorescent dye based Cho-sensing methods have been developed by the competitive host-guest interaction between β-CD with the dye molecule and Cho.4, 5 However, care should be taken in order to keep the pH, ionic strength, and organic effect of the sample solutions the same for all the measurements, due to the sensitiveness of the optical properties of dye molecules to these conditions.4 In addition, under ultraviolet (UV) excitation, the autofluorescence from biological macromolecules, mainly absorbing in the UV region, will appear providing diminished signal-to-background ratio and hence decreased sensitivities.6 UCNPs are kinds of fluorophores codoped with rare-earth This journal is © The Royal Society of Chemistry [year]
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ions, capable of converting low-energy light to high-energy light through a multiphoton process, thus emitting light at the visible and near-infrared (NIR) region.7,8 They hold strong photoluminescence, high quantum yields, narrow emission bands, good chemical stability, and photostability.9,10 Under the excitation of low-energy NIR photons (normally 980 nm), the autofluorescence from organisms can be eliminated.11 All the favorable properties have indicated the great potential of UCNPs in the analysis of biological and environmental samples. Among the various lanthanide-doped UCNPs, hexagonal phase NaYF4: Yb, Er UCNPs have been recognized as the most efficient nanophosphors in which the Yb3+ ions absorb multiple NIR photons while the Er3+ ions emit visible light.12 In fact, UCNPs have been regarded as an ideal donor in various FRET-based assays. Until now, several optical sensors based on UCNPs have been developed in sensing CN-,13 Hg2+,14, 15 Cu2+,16 and so on.11, 17-19 However, to the best of our knowledge, the selective fluorescence probes based on UCNPs for Cho-sensing have not been reported. Herein we have designed an upconversion nanocomposite for FRET-based Cho-sensing and further applied in human serum.
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2.1 Chemicals All the starting materials were obtained from commercial suppliers and used as received. YCl3·6H2O (99.99%), YbCl3·6H2O (99.99%), ErCl3·6H2O (99.9%), NaOH (98%), NH4F (98%), 1-octadecene (90%), oleic acid (OA) (90%), β-CD, diethylene glycol (DEG) and citric acid were purchased from Sigma-Aldrich. Deionized water was used throughout. All other chemical reagents in analytical grade were used directly without further purification. [journal], [year], [vol], 00–00 | 1
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An Upconversion Nanocomposite for Fluorescence Resonance Energy Transfer Based Cholesterol-Sensing in Human Serum
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2.2.1 Synthesis of OA-UCNPs OA-UCNPs were prepared by a modified solvothermal process according to the reported method.20 In a typical experiment, 1 mmol RECl3·6H2O (0.78 mmol YCl3·6H2O, 0.20 mmol YbCl3·6H2O, 0.02 mmol ErCl3·6H2O) were added to a flask containing 7.5 mL OA and 17.5 mL 1-octadecene. The solution was heated to 160 oC for 30 min, and then cooled down to room temperature. Thereafter, 10 mL methanol solutions of NH4F (4.0 mmol) and NaOH (2.5 mmol) were added into the solution, and stirred for 30 min. After methanol was evaporated, the solution was heated to 300 oC under argon atmosphere for 1 h, and cooled down to room temperature. The resulting nanoparticles were precipitated by the addition of ethanol, collected by centrifugation, washed with water and ethanol for three times. 2.2.2 Synthesis of ligand-free UCNPs Ligand-free UCNPs were prepared following a protocol that had been reported previously.21,22 OA-UCNPs (100 mg) were dispersed in a 10 mL aqueous solution. The reaction was performed with stirring for 4 h while maintaining the pH at 4 by adding 0.1 M HCl solution. During this reaction, the carboxylate groups of the OA ligand were protonated (to yield OA). After the reaction was completed, the aqueous solution was mixed with diethyl ether to remove the OA by extraction with diethyl ether for three times, and the combined ether layers were re-extracted with water. Afterwards, the water layers were combined and reextracted with diethyl ether. UCNPs in the water dispersible fraction were recuperated by centrifugation after precipitation with acetone. The product was redispersed in acetone and the particles were recuperated by centrifugation. Finally the nanoparticles were dispersed in water. 2.2.3 Preparation of cyclodextrin citrate esters (CD-Cit) CD-Cit was prepared using a semidry reaction method as reported by El-Tahlawy et al.23, 24 Firstly, 3.00 g of β-CD was mixed with 1.8 mL of water containing 1.02 g of citric acid. Then, the mixture was allowed to react in a circulating air oven at 105 °C for 3 h. The cured sample was purified by repeated washing with isopropanol to remove unreacted components and soluble fragments or byproducts. Finally, the resultant CD-Cit was dried at 60 °C for 24 h and stored in an airtight container for further use. 2.2.4 Preparation of CD-Cit-UCNPs CD-Cit-UCNPs were prepared following a modified protocol.25 15 mL DEG and 2 mL CD-Cit (2 mmol) aqueous solution were mixed in a 25 mL flask. 10 mg ligand-free UCNPs dispersed in 2 mL aqueous solution were injected into the above mixed solution, and the mixture was heated to 100 ºC for 30 min under argon atmosphere. After methanol evaporated, the solution was kept at 160 ºC for 3 h until it became clear. The resulting solution was cooled down to room temperature. The precipitates were collected by centrifugation (10, 000 rpm, 15 min) and washed for three times with ethanol and deionized water. 2.2.5 Preparation of RB-CD-Cit-UCNPs The solution of CD-Cit-UCNPs (30 mg) in 5 mL of deionized water was prepared at room temperature. After the solution became clear, a solution of RB (10 mg) in 2 mL of water was added slowly. The above solution was stirred for 24 h at room temperature, collected by centrifugation (18, 000 rpm, 10 min), and washed with water for three times. According to the amount 2 | Journal Name, [year], [vol], 00–00
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of unreacted RB and the as-prepared RB-CD-Cit-UCNPs, the percent of RB attached to the UCNPs was about 6.7%. 2.3 UCL Detection of Cho
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The as-prepared RB-CD-Cit-UCNPs were dissolved in 2 mL water, diluted with tris(hydroxymethyl)aminomethane (Tris) buffer with a pH of 7.4, and used as a stock solution. 90 µL of this solution was taken and mixed with 10 µL ethanolic solution of different concentrations of Cho. After reaction for 40 min, UCL was measured under the 980 nm excitation. Cho detection in human serum was performed using normal adult serum (provided by the Affiliated Drum Tower Hospital of Nanjing University). 0.5 mL of serum was mixed with 4.5 mL ethanol. After 10 min, the solution was centrifuged at 4000 rpm for 10 min. The different kinds of supernatant serum were taken and added with the stock solution for determination. 2.4 Characterizations UV-Vis absorption spectrum was measured using a Shimadzu UV-2550 ultraviolet-visible-near infrared spectrometer. Upconversion fluorescence spectra were recorded on a ZolixScan ZLX-UPL spectrometer using an external 1 W continuous-wave laser (980 nm) as the excitation source. X-ray diffraction (XRD) measurements were performed on a Japan Shimadzu XRD-6000 diffractometer with Cu-Kα radiation (λ= 0.15418 nm); A scanning rate of 0.05 deg s-1 was applied to record the patterns at the 2θ range of 10-800. The morphology and structure were characterized by transmission electron microscopy (TEM) and high resolution TEM (HRTEM) images using a JEOL-2100 TEM equipped with an Oxford Instrument Energy-dispersive X-ray (EDX) system operating at 200 kV. Mass spectrum (MS) was obtained on a HP 1100 LC-MS spectrometer. The fourier transform infrared (FT-IR) spectra were recorded on a Nicolet 6700 Fourier transform infrared spectrograph at the wavenumber range of 4000-500 cm-1. The X-ray photoelectron spectroscopy (XPS) spectrum was measured on an ECSALAB 250.
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The design and synthesis of a Cho-sensitive RB-quenched upconversion fluorescent probe assembled with β-CD derivative (CD-Cit) and UCNPs was performed, and the probe can be further applied in human serum. The nanosensor is based on a FRET process, using NaYF4: Yb, Er nanophosphors as the energy donor and RB as the energy acceptor. The assembly is as outlined in Scheme 1.
Scheme 1 Schematic illustration of the design and synthesis of RB-CDCit-UCNPs, and their UCL response to Cho.
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Firstly, OA-UCNPs were treated with HCl to achieve ligand-free nanoparticles (UCNPs). Then, an aqueous solution of citric acid modified β-CD (CD-Cit), prepared by a semidry reaction method, 23, 24 was mixed with the aqueous solution of UCNPs under stirring. This as-fabricated CD-Cit coat on the nanoparticles (CD-Cit-UCNPs) through the carboxyl group of the complex coordinating with the rare-earth ion on the surface of UCNPs.14 Since the CD-Cit functionalization offers high water solubility to UCNPs, guest molecules incorporated into β-CD can be easily accessible to UCNPs.14 β-CD inclusion of RB (RB-CDCit-UCNPs) induces FRET to occur because of close contact between the donor moiety (UCNPs) and the quencher (RB) nearby. β-CD, a cyclic polysaccharide, composed of seven glucose molecules, has a hydrophilic outer surface which provides water solubility and a hydrophobic inner cavity for specific recognition of guest molecules.5, 26 It shows much stronger binding capacity towards Cho compared with RB according to the binding constant available,27, 28 due to the hydrophobic nature of Cho. FRET is switched off by virtue of the Cho-induced release of RB from the cavity of β-CD, because of the competitive host-guest interaction between CD-Cit-UCNPs with RB and Cho, resulting in the fluorescence recovery of the quenched UCNPs. The “on-off” switching of FRET with UCL changes will provide a much-improved assay of Cho.
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Fig. 1 UV-Vis absorption spectrum of RB in aqueous solution (black line) and UCL spectrum of OA-UCNPs in cyclohexane solution (green line, inset is the corresponding UCL photography).
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Fig. 2 TEM and HRTEM images of OA-UCNPs dispersed in cyclohexane solution (a and b, respectively) together with CD-Cit-UCNPs dispersed in aqueous solution (c and d, respectively).
OA-NaYF4: Yb, Er UCNPs, prepared by a modified cothermolysis process with OA as the surface ligand, have a hydrophobic surface.20 The XRD pattern of OA-UCNPs could be attributed to hexagonal phase NaYF4 (JCPDS NO. 28-1192) (Fig. S1a). The particle size was calculated to be 35.5 nm according to Debye-Scherrer formula.30 EDX analysis showed that the nanoparticles were mainly composed of Na, Y, F, Yb and a small amount of Er element (Fig. S1b). TEM images displayed that OA-UCNPs with polyhedral morphologies were well-dispersed in cyclohexane solution, and had an average diameter of 36 nm (Fig. 2a), which was in agreement with the XRD results. The asprepared CD-Cit was characterized by MS (Fig. S2). Major peak at 1330.8 can be assigned to the target compound (CD-Cit), and other one at 1156.9 is attributed to the unreacted β-CD. After coating CD-Cit onto the ligand-free UCNPs, the hybrid could be easily dispersed in water, and the size and shape of the UCNPs remained essentially unchanged (Fig. 2c). HRTEM images proved the highly crystalline nature of the nanoparticles (Fig. 2b and 2d).
Under 980 nm excitation, OA-UCNPs give dual emission bands in green and red parts of the visible region, as shown in Fig. 1, whose inset shows the photography of the strong eyevisible green light from the cyclohexane solution of the nanoparticles. The emission bands at around 522 nm, 541 nm and 655 nm can be assigned to 2H11/2 - 4I15/2, 4S3/2 - 4I15/2, 4F9/2 - 4I15/2 transitions of Er3+, respectively.29 RB has a broad absorption band centered at 554 nm, which can overlap well with the green emissions of UCNPs, implying a FRET process through the donor and quencher nearby. It is reasonable that the degree of energy transfer between UCNPs and RB can be modulated by the concentration of Cho (Scheme 1). In addition, to achieve ratiometric UCL detection, The NIR UCL emission at 655 nm was chosen as the reference. Fig. 3 FT-IR spectra of the as-prepared OA-UCNPs, UCNPs, CD-CitUCNPs and CD-Cit, respectively.
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DOI: 10.1039/C4NR04380D
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Fig. 4 UCL intensity (a) and the corresponding photos ((b), from left to right) of OA-UCNPs in cyclohexane solution (black line) and UCNPs (red line), CD-Cit-UCNPs (blue line), RB-CD-Cit-UCNPs (green line), RB-CD-Cit-UCNPs+ 80 µM Cho (pink line) in the Tris-HCl buffer solutions (pH= 7.4), respectively.
The UCL emission intensity of the modified nanomaterials was characterized as shown in Fig. 4. Both the disappearance of OA molecule and the coating of CD-Cit decreased the luminescent intensity, because the organic compound and the water molecule with their vibration frequencies could quench the emission of the rare earth ions to a great extent.31 RB could make FRET process occur, leading to the reduced UCL intensity. Yet, after 80 µM Cho was added to the Tris-HCl buffer solutions of RB-CD-CitUCNPs, the green UCL emission intensity increased while the red part remained unaffected, because Cho replaced RB to enter the lipophilic cavity of β-CD inhibiting the FRET process.5 It could be found that the UCL intensity of CD-Cit-UCNPs was decreased dramatically by RB within a short period of time, 4 | Journal Name, [year], [vol], 00–00
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suggesting that the inclusion of RB into the β-CD occurred rapidly. After the addition of the competitive binder Cho, the UCL intensity gradually increased by increasing the reaction time, reaching a plateau after 25 min (Fig. S4), which was much longer than that time for RB inclusion into β-CD to reach equilibrium. Taking the sensitivity into consideration, we set a time of 40 min for the reaction between Cho and RB-CD-CitUCNPs.
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Fig.5 UCL spectra of 0.5 mg/mL RB-CD-Cit-UCNPs in the Tris-HCl buffer solutions (pH= 7.4) upon gradual addition of Cho (0-200 µM). Inset is GRR (UCL515-565/UCL640-685, UCLA-B is the integrated emission intensity from A to B nm) as a function of Cho concentration.
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Fig. 6 Linear plot of the relative UCL increasing (GRR) as a function of Cho concentration (GRR denotes the relative fluorescence intensity UCL515-565/UCL640-685).
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To show the sensitivity of the nanoprobe, the changes in the UCL spectra of RB-CD-Cit-UCNPs was monitored in the presence of different concentrations of Cho, as shown in Fig. 5. Addition of Cho into the solution increased the green UCL intensity and the enhancive fluorescence was directly related to the amount of the added Cho. The reason is that upon more and more Cho competitive inclusion into the cavity of β-CD, the release of more and more RB from the inside resulted in the greater decay of the FRET effect. The UCL intensity in green emission increased while the red part remained unchanged with the increase of Cho concentration from 0 to 200 µM, as can be observed in the calibration curve of the green-to-red ratio (GRR) versus concentration of Cho in the inset of Fig. 5. GRR increased linearly with the concentration of Cho in the range of 10~110 µM This journal is © The Royal Society of Chemistry [year]
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The assembling processes were further confirmed by FT-IR spectra (Fig. 3). In the case of OA-UCNPs, The peaks at 2921, 2854, 1554 and 1462 cm–1 could confirm the existence of OA molecule, which limited the growth of the nanoparticles and prevented their agglomeration. The above characteristic peaks disappeared in ligand-free UCNPs, indicating that the OA had been fully wiped off. The peaks in the spectrum of CD-Cit at 1732 and 1202 cm-1 could be assigned to β-CD linkage to 23, 24 citrate. Furthermore, it was obvious that the spectrum of the CD-Cit-UCNPs showed a drastic change as compared to that of UCNPs, showing that CD-Cit could not be removed from the solid materials even by extensive washing with deionized water. When RB was assembled with CD-Cit-UCNPs, the colour of the as-prepared nanocomposite changed from colourless to pink even through repeated washings, which suggested that RB and CD-Cit-UCNPs had been combined together in the final products. The XPS experiment further verified this result, by virtue of simultaneous observation of the N, Na, Y, F, Yb, Er, C and O elements (Fig. S3). The above characterization confirmed the inorganic-organic hybrid nanophosphors had been prepared as expected.
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(R2= 0.9884) (Fig. 6). A concentration of Cho as low as 3.0 µM could be readily detected on the basis of the formula of detection limit.13 The sensitivity test was carried out for three times, and the same linear range (10~110 µM) and similar detection limit (2.7 µM, 3.0 µM, 3.1 µM, respectively) were achieved even though the values of the UCL intensity and GRRs could change slightly to some extent. The average value and absolute average deviation of the detection limit were 2.9 µM and 0.17, respectively. The UCL analysis supported the idea that the signal enhancement was attributed to the formation of an inclusion complex of the Cho moiety in β-CD, resulting in separation of RB from UCNPs.4 This phenomenon is explained by the guestinduced location change of RB from inside to outside of the cavity, suggesting that the upconversion nanocomposite RB-CDCit-UCNPs is an effective fluorescent probe for Cho-sensing. The sensitivity can be comparable to most of the existing Cho detection methods. 4, 5 To confirm the necessity of the CD-Cit functionalization, a control experiment was carried out. The aqueous solution of the same amount of RB was directly mixed with UCNPs and without CD-Cit), then incubated with different concentrations of Cho. In this case, the UCL intensity changed little and the GRRs were hardly altered compared with that using CD-Cit, as shown in Fig. 7.
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Fig. 8 UCL spectra (a) and GRR (b) of 0.5 mg/mL RB-CD-Cit-UCNPs in the Tris-HCl buffer solutions (pH= 7.4) before and after addition of 80 µM Cho or other 4 mM interfering substances (1. NaCl, KCl, MgCl2, CaCl2, ZnCl2; 2. Glycine + glucose; 3. Glutathione + lecithin; 4. Vitamin C; 5. Boracic acid + sucrose; 6. Bilirubin + sorbitol + mannitol; 7. Troptophan), respectively.
To examine the selectivity of this assembly system in serum samples, we carried out studies with the main constituents normally found in serum. The common substances present in serum with 4 mM concentration had been tested. It could be found that their UCL spectra (Fig. 8a）and the GRRs (Fig. 8b) were both similar to pure RB-CD-Cit-UCNPs, except for 80 µM Cho added. The above results suggest that the determination is free from interference from constituents of serum and the method can be extended for Cho detection in human blood. Table 1 Comparison of assay results of human serum samples for the clinical test (the enzymatic analysis) and the proposed method
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Fig.7 The GRR of UCL intensity upon gradual addition of different concentrations of Cho with and without CD-Cit modification, respectively.
Serum samples (adults) 1 2 3 4 5 6
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This journal is © The Royal Society of Chemistry [year]
The clinical test The proposed method (µM) (µM) 17.2 16.8 25.6 25.2 39.3 39.7 58.7 59.1 73.6 73.0 98.5 97.3
RDs (%) −2.3 −1.6 1.0 0.7 −0.8 −1.2
In order to evaluate the feasibility of the proposed method for clinical application, the real human blood serum test was also performed. According to the above-mentioned procedure, the detected Cho content in serum, derived from the standard curve and the regression equation, was listed in Table 1. In comparison with the enzymatic analysis that has been widely used in the clinical test, the relative deviations (RDs) in the proposed method show acceptable practicability for the detection of Cho. This demonstrates that the developed nanosensor with high sensitivity and selectivity, has great application potentials in clinical diagnosis in the future. In the end, to decrease the influence of some other substances in practical applications, a linear relationship from real serum sample should be found. It can be obtained through the addition of different serum with known concentration of Cho into Journal Name, [year], [vol], 00–00 | 5
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the sensor and monitoring the changes in the UCL spectra. This method can reduce the interference from other constituents of serum and increase sensitivity and accuracy.
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4. Conclusions
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In summary, the design and synthesis of a novel upconversion nanocomposite that is composed of RB and β-CD derivative modified UCNPs was described. Furthermore, we presented its utility as an UCL ratiometric probe for Cho detection in human serum with high sensitivity and selectivity. The probe is based on a highly efficient FRET process using UCNPs as an energy donor and RB as a quencher. The proposed detection method is simple and cost-efficient as it doesn’t need use of Cho selective enzyme or antibody. UCNPs, as a kind of excellent emitters with their low autofluorescence and high penetration depth to biosamples, can endow this system with further application potentials in biological and analytical fields. Moreover, we have also provided an original design strategy of general nanoprobes for sensing studies, which can be as a beneficial reference for other fluorescent sensing system.
Acknowledgements
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The authors gratefully appreciate the support from National Basic Research Program (2011CB933502) of China, and National Natural Science Foundation (21121091, 21405001, 51303002), Program for New Century Excellent Talents in University (NCET-12-0256), and the State Key Laboratory of Analytical Chemistry for Life science (SKLACLS1307). The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding the work through the research group project (RGP-VPP-029).
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Notes and References a
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State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210093, China. E-mail: [email protected], [email protected] b College of Biochemical Engineering, Anhui Polytechnic University, Wuhu, 241000, China. c
Petrochemical Research Chair, Department of Chemistry, College of Science, King Saud University, Riyadh 11451P.O. Box 2455, Kingdom of Saudi Arabia.
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†Electronic Supplementary DOI:10.1039/b000000x/
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DOI: 10.1039/C4NR04380D
