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Upconversion fluorescence metal-organic frameworks thermo-sensitive imprinted polymer for enrichment and sensing protein
Biosensors and Bioelectronics 79 (2016) 341–346
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Upconversion fluorescence metal-organic frameworks thermosensitive imprinted polymer for enrichment and sensing protein Ting Guo a, Qiliang Deng a,n, Guozhen Fang a, Dahai Gu b, Yukun Yang a, Shuo Wang a,n a Key Laboratory of Food Nutrition and Safety, Ministry of Education, Tianjin Key Laboratory of Food Nutrition and Safety, Tianjin University of Science and Technology, Tianjin 300457, China b Yunnan Agricultural University, Yunnan 650201, China
art ic l e i nf o
a b s t r a c t
Article history: Received 3 November 2015 Received in revised form 7 December 2015 Accepted 14 December 2015 Available online 17 December 2015
A novel fluorescence material with thermo-sensitive for the enrichment and sensing of protein was successfully prepared by combining molecular imprinting technology with upconversion nanoparticles (UCNPs) and metal-organic frameworks (MOFs). Herein, the UCNPs acted as signal reporter for composite materials because of its excellent fluorescence property and chemical stability. MOFs were introduced to molecularly imprinted polymer (MIP) due to its high specific surface area which increases the rate of mass transfer relative to that of traditional bulk MIP. The thermo-sensitive imprinted material which allows for swelling and shrinking with response to temperature changes was prepared by choosing Bovine hemoglobin (BHB) as the template, N-isopropyl acrylamide (NIPAAM) as the temperature-sensitive functional monomer and N,N-methylenebisacrylamide (MBA) as the cross-linker. The recognition characterizations of imprinted material-coated UCNPs/MOFs (UCNPs/MOFs/MIP) were evaluated, and the results showed that the fluorescence intensity of UCNPs/MOFs/MIP reduced gradually with the increase of BHB concentration. The fluorescence material was response to the temperature. The adsorption capacity was as much as 167.6 mg/g at 28 °C and 101.2 mg/g at 44 °C, which was higher than that of traditional MIP. Therefore, this new fluorescence material for enrichment and sensing protein is very promising for future applications. & 2015 Elsevier B.V. All rights reserved.
Keywords: Molecularly imprinted polymer Upconversion nanoparticles Metal-organic frameworks Thermo-sensitive Bovine hemoglobin
1. Introduction Metal-organic frameworks (MOFs) are relatively new class of highly porous crystalline materials and are prepared by metal connected by various organic ligands such as carboxylates or phosphonates (Yaghi et al., 1995; Li et al., 1999; Eddaoudi et al., 2002; Furukawa et al., 2013; Deng et al., 2012). MOFs have attracted significant interest in adsorption and separation (Bloch et al., 2010), gas storage (Chen et al., 2005), drug delivery (Imaz et al., 2010) due to high specific surface area, high porosity and tenability of MOFs. Very recently, a variety of MOF composite materials has attracted dramatic increasing interest owing to potential in biomaterial and optics of these composite materials. A few pioneering synthetic studies on the MOF composite materials including MOF@SiO2 (Rieter et al., 2008), MOF/GO (Petit et al., 2010, 2011a, 2011b), Fe3O4/MOF (Ke et al., 2012) have been reported. The MOF composite materials are widely used for adsorption and detection hazardous pollutants. A facile n
Corresponding authors. E-mail addresses: [email protected] (Q. Deng), [email protected] (S. Wang).
http://dx.doi.org/10.1016/j.bios.2015.12.040 0956-5663/& 2015 Elsevier B.V. All rights reserved.
magnetization of MOF for enrichment polycyclic aromatic hydrocarbons was also prepared (Huo and Yan, 2012). Recently, dithizone functionalized magnetic MOF was prepared and used for the determination of trace levels of lead (Wang et al., 2013). Li et al. (2014a) successfully synthesized a magnetic sphere hybrid Fe3O4/ Cu3(BTC)2/GO through a hydrothermal method for detecting methylene blue from water solution. However, these materials in application area were restricted due to lack of specificity. Recently, Upconversion nanoparticles (UCNPs) have led to intense studies due to outstanding advantages in bioimaging, biological probe and optical amplifier (Cheng et al., 2013, 2011; Yang et al., 2012; Wang et al., 2009). UCNPs, compared with traditional fluorescent materials such as fluorescence dyes and semiconductor QDs, are low toxic, lack of auto-fluorescence, long lifetimes and low photobleaching. Furthermore, UCNPs are capable of converting NIR light to UV–visible light by multiple photon absorptions or energy transfers (Lu et al., 2014; Liu et al., 2011). The multifunctional nanoparticles based on UCNPs with combined magnetic and optical characterize were used for multimodal imaging and therapy (Cheng et al., 2011). Molecular imprinting has been considered as a convenient
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technology that can create the materials with recognition sites which are spatially and chemically complementary to the template in materials (Ellen et al., 2011; Chen et al., 2011; Lofgreen et al., 2011). Compared with biological receptors, molecular imprinted polymer (MIP) possesses excellent mechanical and chemical stability and ease of preparation. However, traditional MIP bulks require crushed and sieved, leading to many limitations including irregularity in the shape of particles, low adsorption capacity and selectivity (Turner et al., 2006). The surface imprinting technology can overcome the above problem effectively. A novel surface-imprinted magnetic composite microsphere was successfully prepared for bovine serum albumin (BSA) recognition (Li et al., 2014b). Chen et al. (2014) prepared the molecularly imprinted nanoparticles by choosing the vinyl-modified silica nanoparticles as support material. Furthermore, quantum dots (QDs) also could be used as support material for surface imprinting (Tan et al., 2014; Yang et al., 2014; Li et al., 2015). Compared with traditional support materials, the advantages of MOFs include high specific surface area and porous structure, which increase the rate of mass transfer and adsorption capacity. Based on the above considerations, we propose a novel strategy combined the high selectivity of molecular imprinting technology with the excellent fluorescence of UCNPs and the high specific surface area of MOFs to prepare a new type of composite materials for the enrichment and sensing of protein. Herein, Cu3(BTC)2 (known as HKUST-1) was chosen as the representative MOF. The HKUST-1 is water-stable and contains Cu2 þ as the metal center linked to oxygen atoms from benzene tricarboxylate (H3BTC). The UCNPs were synthesized by solvothermal reaction and modified with polyacrylic acid (PAA) through ligand exchange. The UCNPs/ HKUST-1 were prepared by the reaction of copper nitrate (Cu (NO3)2) with H3BTC after dispersing the PAA-modified UCNPs in ethanol through a solvothermal method. The thermo-sensitive imprinted layer was prepared by choosing Bovine hemoglobin (BHB) as the template, N-isopropyl acrylamide (NIPAAM) as the thermo-sensitive functional monomer which allowed for swelling and shrinking with response to temperature changes, and N,Nmethylenebisacrylamide (MBA) as the cross-linker. The morphology and adsorption characterizations of the UCNPs/HKUST-1/MIP were investigated. Furthermore, the thermo-sensitive characterization of the composite material was discussed.
2. Materials and methods
(Victoria, Australia) in a quartz cuvette with 1 cm path length. Fluorescence measurements were performed on an F-2500 fluorescence spectrometer (Hitachi, Japan) connected with an external 980 nm diode laser (1 W, continuous wave with 1 m fiber, Beijing Viasho Technology Co.) as the excitation source. Scanning electron microscopy (SEM) images were obtained with a SU1510 microscope (Hitachi, Japan). X-ray powder diffraction (XRD) patterns were recorded on a D8 X-ray power diffractometer at a scanning rate of 1°/min in the 2θ range from 10° to 40° (Bruker, Germany). Energy-dispersive X-ray photoelectron spectroscopic (XPS) measurements were performed on PHI-5000 Versaprobe (PHI, Japan). Thermogravimetric analysis (TGA) was performed (Meteler, Switzerland) in the temperature range from room temperature to 1000 °C with a heating rate of 10 °C/min under nitrogen atmosphere. Nitrogen adsorption–desorption analysis was performed on an autosorb-1-mp (Quantachrome, USA) with a bath temperature of 77 K. Surface areas were determined using Brunauer– Emmett–Teller (BET) theory. 2.3. Preparation of UCNPs UCNPs were synthesized according to the previous literature (Li and Zhang, 2008). To a 100 mL flask, Y (CH3COO)3 (0.78 mmol), Yb (CH3COO)3 (0.2 mmol) and Er (CH3COO)3 (0.02 mmol) were mixed with 6 mL OA and 17 mL ODE and heated to 160 °C, to form a transparent solution. The mixture solution cooled down to room temperature naturally. 10 mL methanol solution containing NaOH (2.5 mmol) and NH4F (4 mmol) was slowly dropped into the flask and stirred for 30 min. Subsequently, the solution was slowly heated for removal of methanol, degassed at 100 °C for 10 min, and then heated to 300 °C and maintained for 1 h at argon atmosphere. After the resulted solution was cooled down naturally, UCNPs were precipitated via centrifugation, and washed with ethanol for three times. PAA–UCNPs were synthesized according to the previous method with a modified procedure (Naccache, et al., 2009). PAA (300 mg) and 30 mL DEG were added into a 100 mL flask. The mixture was heated to 110 °C to form a transparent solution. 3 mL toluene solution containing 100 mg hydrophobic UCNPs was added, and kept at 110 °C for 1 h under argon protection. Then the mixed solution was heated to 240 °C and maintained for 1 h. The resulted solution was cooled down to room temperature naturally, and the PAA–UCNPs were obtained from the resulted solution with excess dilute hydrochloric aqueous solution, and washed three times with water.
2.1. Materials and chemicals 2.4. Synthesis of the UCNPs/HKUST-1 Bovine serum albumin (BSA, molecular weight (MW) 67 kDa, isoelectric point (pI) 4.9), Bovine hemoglobin (BHB, MW 66 kDa, pI 6.7) and Cytochrome c (Cyt c MW 12.4 kDa, pI 10.2) were obtained from Sangon Biotech Co. Ltd. (Shanghai, China). Y(CH3COO)3 4H2O (99.9%), Yb(CH3COO)3 4H2O (99.9%), Er(CH3COO)3 xH2O (99.9%), PAA (M¼ 1800), Cu(NO3)2 3H2O, and diethylene glycol (DEG) were purchased from Sigma Aldrich (St Louis, USA). Oleic acid (OA, 90%), 1-octadecene (ODE, 90%), H3BTC and NIPAAM were purchased from Alfa Aesar Co. Ltd. (Massachusetts, USA). MBA, ammonium perulfate (APS), N,N,N,N-tetramethylenebis (TEMED) were provided by Aladdin (Los Angeles, USA). All reagents were of the highest available purity and of analytical grade at least. Double distilled water (DDW, 18.2 MΩ cm 1) was prepared by a Water Pro water purification system (Labconco, Kansas City, USA). 2.2. Characterizations Ultraviolet absorbance at a wavelength of 400 nm was recorded on a Cary 50-Bio Ultraviolet–visible (UV–vis) spectrometer
UCNPs/HKUST-1 was synthesized according to the previous method with a modified procedure (Wang et al., 2013). To 250 mL round-bottomed flask, 0.5 g of H3BTC was mixed with 40 mL DMF and 40 mL ethanol. 20 mL ethanol solution containing 100 mg PAA-modified UCNPs was added under vigorous stirring. The mixture was refluxed for 30 min at 75 °C, followed by the addition of 1.0 g Cu(NO3)2 3H2O which was dissolved in 40 mL water. After refluxing for 4 h, the UCNPs/HKUST-1 was collected via centrifugation and washed with water and ethanol, then dried at 120 °C for 10 h. 2.5. Synthesis of the UCNPs/HKUST-1/MIP 10 mg of template protein, 50 mg of UCNPs/HKUST-1, and 10 mL of water were added in a 25 mL flask and stirred for 30 min. Then 100 mg of NIPAAM and 40 mg of MBA were added to the mixture and stirred for 1 h at room temperature. The oxygen was removed by nitrogen bubbling for 10 min. Subsequently, 10 mg of
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Fig. 1. Synthesis of molecularly imprinted fluorescence materials based on the UCNPs and MOFs.
APS and 100 μL of TEMED (5%, v/v) were added, and then polymerization was carried out at 25 °C for 5 h. The UCNPs/HKUST-1/ MIP was collected via centrifugation and washed with 0.5% Tris, which was repeated several times until no template was detected by UV–vis spectrophotometry. Finally, the non-imprinted polymer (NIP) was prepared using the same procedure except addition of the template molecule. 2.6. Characterization of the UCNPs/HKUST-1/MIP In the experiments, all fluorescence detections were performed on an F-2500 fluorescence spectrometer attached with an external 980 nm laser instead of internal excitation source, and the external diode laser was set at 1 W. 2 mg of UCNPs/HKUST-1/MIP or UCNPs/ HKUST-1/NIP and 2 mL of BHB solution with a certain concentration were added into 4 mL centrifuge tube. The mixture was shaken at room temperature for a period of time and determined quickly. The specificity experiment was performed by choosing BSA as competitive protein. 2 mg of UCNPs/HKUST-1/MIP or UCNPs/ HKUST-1/NIP and 2 mL of protein solution containing BHB and BSA were mixed and shaken at room temperature for a period of time and measured quickly.
3. Results and discussion 3.1. Preparation of UCNPs/HKUST-1/MIP We first prepared a novel fluorescence material for the enrichment and sensing of the protein by combing the molecular imprinting technology with UCNPs and MOFs. Here, the UCNPs were used to provide the sensing signal for composite materials. The MOFs were introduced to the MIP due to its high specific surface area which increases the rate of mass transfer and adsorption capacity relative to that of traditional bulk MIP. The general scheme for synthesis process is shown in Fig. 1. In the first step, the UCNPs were prepared through the solvothermal method in OA and ODE. The resulted UCNPs were converted into carboxyl modified UCNPs by ligand exchange of OA with PAA. Then, the UCNPs/HKUST-1 was synthesized by the coordination interaction
between Cu2 þ and the carboxyl groups on the UCNPs surface and H3BTC. Subsequently, the template protein was immobilized onto the UCNPs/HKUST-1 though non-covalent interaction. Finally, the thermo-sensitive MIP layer was prepared by the polymerization of the thermo-sensitive monomer, NIPAAM, and MBA in aqueous solution. After removal of the template protein, the recognition sites were created. The fluorescence of the imprinted materialcoated UCNPs/MOFs (UCNPs/HKUST-1/MIP) is quenched when the template protein is rebound to the sites of the MIP layer. This is probably due to a photo-induced electron transfer process (Gerhards et al., 2008; Zhong et al., 2007). Once the template protein is extracted from the composite materials, the fluorescence of the UCNPs/HKUST-1/MIP recovers. 3.2. Characterization of composite materials The SEM images of the UCNPs/HKUST-1, UCNPs/HKUST-1/MIP and UCNPs/HKUST-1/NIP are shown in Fig. 2. From Fig. 2(a), it can be seen that the UCNPs/HKUST-1 was prepared with a size of about 1 μm. Fig. 2(b) shows that UCNPs/HKUST-1 was fully coated with the MIP layer and the size of UCNPs/HKUST-1/MIP was 1.5 μm, further indicating a successful coating with MIP layer. Furthermore, there was no significant morphological difference between UCNPs/HKUST-1/MIP and UCNPs/HKUST-1/NIP (Fig. 2(c)). The XRD patterns of UCNPs/HKUST-1 and UCNPs/HKUST-1/MIP is shown in Fig. 3(a). The characteristic peaks of Cu3(BTC)2 were showed at 2θ values of about 10.31°, 11.56°, 13.39°, 14.64°, 15.01°, 16.42°, 17.44° and 19.04°, which are assigned to the (220), (222), (400), (331), (420), (422), (333) and (440) planes of Cu3(BTC)2, respectively (Li et al., 2014a; Chowdhury et al., 2009). The XRD pattern of the UCNPs/MOF showed the good agreement with the XRD pattern of HKUST-1, which indicated that the structure of MOF was maintained in the synthesis process. The XRD pattern of UCNPs/HKUST-1/MIP was lower response due to the effect of the MIP layer. To investigate the thermo-stability and solid content, TGA of UCNPs/HKUST-1 and UCNPs/HKUST-1/MIP was performed as shown in Fig. 3(b). The TGA curve of UCNPs/HKUST-1 showed two weight loss steps in the range of 25–1000 °C. The first weight loss step of about 11.83% from 25 to 118 °C was assigned to the physically and chemically absorbed water in the UCNPs/HKUST-1. The second weight loss step showed a 45.57% weight loss in the
Fig. 2. SEM images of (a) UCNPs/HKUST-1 and (b) UCNPs/HKUST-1/MIP.
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Fig. 3. (a) XRD patterns of (A) UCNPs/HKUST-1 and (B) UCNPs/HKUST-1/MIP and (b) TGA of (C) UCNPs/HKUST-1 and (D) UCNPs/HKUST-1/MIP.
temperature range 118–359 °C, which corresponded to decomposition of the UCNPs/HKUST-1 structure, the final product was attributed to CuO and rare earth oxides. In the TGA curve of UCNPs/HKUST-1/MIP, the first weight loss step, from 25 to 101 °C, corresponded to the surface adsorbed water (6.35% weight loss). The next weight loss of 67.84% was observed from 271 to 420 °C. It is worth noticing that the weight loss of the UCNPs/HKUST-1/MIP is more than that of theUCNPs/HKUST-1, due to the MIP layer coated on the surface of UCNPs/HKUST-1. To illustrate the component of UCNPs/HKUST-1 and UCNPs/ HKUST-1/MIP, the composites materials were characterized by XPS. Fig. S1(a) shows that main signals of C1s at 285 and 288 eV, O1s at 532 eV and Cu2p3 at 933 eV, which illustrated the HKUST-1 was successfully coated on the surface of the UCNPs. Fig. S1 (b) shows main signals of C1s at 285 and 288 eV, N1s at 400 eV and O1s at 532 eV, which indicated that the UCNPs/HKUST-1 was successfully coated with MIP layer. Furthermore, due to the existence of HKUST-1, the fluorescence intensity of UCNPs/HKUST-1 was lower than that of UCNPs (Fig. S2). 3.3. Thermo-sensitive property of the UCNPs/HKUST-1/MIP It is well known that the polymer based on NIPAAM presents both hydrophobic and hydrophilic state at different temperatures, meanwhile, the volume of polymer would change with the temperatures (Zhang et al., 2013). Therefore, the influence of temperature on the adsorption capacity was investigated. In this experiment, 2 mg of UCNPs/HKUST-1/MIP was mixed with 2 mL of 0.2 mg/mL BHB solution into 4 mL centrifuge tube. The mixture
was shaken at 28 °C for a period of time and measured quickly, then shaken at 44 °C for a period of time and determined quickly, subsequently the mixture was shaken again at 28 °C. This process was performed in triplicate. The fluorescence intensity of the UCNPs/HKUST-1/MIP with and without the template protein at 28 and 44 °C is shown in Figs. 4(a) and S3. It can be seen that the interaction of the UCNPs/HKUST-1/MIP with the template BHB showed a conspicuous temperature dependent on–off fluorescence intensity, which demonstrated the recognition ability of the UCNPs/HKUST-1/MIP through the changes of the temperature. After shaking at 28 and 44 °C respectively, the mixture was centrifuged and the concentration of the template protein in the supernatant was measured by a UV–vis spectrophotometer. The adsorption capacity (Q, mg/g) of UCNPs/HKUST-1/MIP for the template protein was calculated by the equation.
Q = (C0 − C )V /m
(1)
where C0 (mg/mL) represents the initial concentration of the template BHB, C (mg/mL) is the residual concentration of the BHB, V (mL) is the volume of the BHB solution and m (g) represents the mass of UCNPs/HKUST-1/MIP. Fig. 4(b) shows the influence of the temperature on the adsorption capacity of BHB on UCNPs/HKUST1/MIP. It was clearly observed that the adsorption capacity for BHB at 28 °C was lager than that at 44 °C, which was due to NIPAAM that was in the state of contraction during the change the volume of depending on temperature stimulation from the environment. Furthermore, the adsorption capacity of UCNPs/HKUST-1/MIP was much higher than that of traditional MIP (Li et al., 2014b; Zhang et al., 2012, 2005; Chen et al., 2014), which was due to high
Fig. 4. (a) Changes of the fluorescence intensity and (b) the adsorption capacity of the UCNPs/HKUST-1/MIP for BHB with a temperature swing of 28-44˚C.
T. Guo et al. / Biosensors and Bioelectronics 79 (2016) 341–346
specific surface area MOF. The BET surface area of UCNPs/HKUST-1 was measured from nitrogen adsorption isotherms at 77 K. The results showed that the BET specific surface area of UCNPs/HKUST1 was calculated to be 853.52 m2 g 1. Compared with traditional support material such as silica, the UCNPs/HKUST-1 exhibited a much lager BET specific surface area. 3.4. Fluorescent optosensing of the template protein with composite materials 3.4.1. Effect of pH Due to the fact that the surface environment of the composite materials and the charge of the protein depending on pH value, the influence of the pH value on the fluorescence intensity of the UCNPs/HKUST-1/MIP was investigated in a pH range of 6–8. Fig. S4 shows the influence of pH value on recognition. It can be seen that the best imprinting effect was observed at pH 6.6. Thus, pH of 6.6 was chosen for further experiment. 3.4.2. Equilibrium binding To demonstrate the recognition ability of the UCNPs/HKUST-1/ MIP versus that of the non-imprinted material-coated UCNPs/ MOFs (UCNPs/HKUST-1/NIP), the changes of the fluorescence signal of composite material was studied at different concentrations ranging from 0 to 0.6 mg/mL. It can be seen from Fig. 5(a) that the fluorescence intensity of the UCNPs/HKUST-1/MIP was reduced gradually with the increasing concentration of the template BHB. The fluorescence was quenched mainly due to the specific interaction between the UCNPs and the template BHB when the template protein BHB close to the imprinted sites in recognition process. Inset A and B in Fig. 5 are plotted by the Stern–Volmer equation analysis for the UCNPs/HKUST-1/MIP and UCNPs/HKUST1/NIP with the template protein, respectively.
F0/F = 1 + KSV [Q ]
(2)
where F and F0 are the fluorescence intensity of UCNPs/HKUST1/MIP or UCNPs/HKUST-1/NIP in the presence and absence of BHB, respectively, Ksv represents the quenching constant of BHB, and [Q] is the concentration of BHB. It was clearly seen from the inset A and B that the decrement of the UCNPs/HKUST-1/MIP in fluorescence intensity was much larger than that of UCNPs/HKUST-1/NIP under the same template concentration. The imprinting factor (IF), which is the ratio of the KMIP and KNIP (KMIP and KNIP are the linear slopes of inset A and B, respectively), was used to evaluate the selectivity of the composite material. Under optimum conditions, the result showed that the IF (KMIP/KNIP) was 1.82, which illustrated that the UCNPs/HKUST-1/MIP with imprinted sites could recognize the template protein. In this research, to analysis the recognition kinetics performance of the UCNPs/HKUST-1/MIP and UCNPs/HKUST-1/NIP, the equilibrium binding analysis was carried out by fixing the
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concentration of BHB (0.2 mg/mL). It was found from Fig. S5 that the changes in fluorescence intensity of UCNPs/HKUST-1/MIP with the template protein was much more significant than that of UCNPs/HKUST-1/NIP, because UCNPs/HKUST-1/NIP had no imprinted sites and existed nonspecific binding in adsorption process. For UCNPs/HKUST-1/MIP, the imprinted sites with respect to the template were generated in composite material preparation process and existed nonspecific binding and specific binding during adsorption process. It was found from Fig. S5(a) that the adsorption rate had a rapid increase in 30 min and 85.5% binding was completed. The adsorption process almost reached the equilibrium within 60 min. 3.5. Specificity study In order to further investigate the performance of the UCNPs/ HKUST-1/MIP, BSA and Cyt c was chosen as competitive protein to evaluate the selectivity of UCNPs/HKUST-1/MIP. The selectivity experiment was performed for BHB, BSA and Cyt c with a same concentration of 0.2 mg/mL. It was clearly seen that the changes in fluorescence intensity of UCNPs/HKUST-1/MIP to BHB were much more obvious than that to BSA and Cyt c. However, the changes in fluorescence intensity of UCNPs/HKUST-1/NIP were not significantly different for BHB, BSA and Cyt c. The imprinted sites and cavies with complementary in size, shape and functional group to the template protein were formed in synthesis process. As the competitive protein, although BSA is similar to BHB in the molecular volume and structures, the recognition sites were not complementary to the competitive protein. Thus, the fluorescence intensity was not obviously reduced by the competitive protein. To further demonstrate the selectivity property of the UCNPs/ HKUST-1/MIP, binary competitive adsorption experiment was performed. The binary adsorption experiment was carried out by preparing a series of protein solution of BHB–BSA and fixing the concentration of BHB (0.2 mg/mL) and increasing the concentration of BSA. It can be seen from Fig. 6(b) that the intensity of fluorescence was not significant affected as the ratio of CBSA/CBHB increased, indicating excellent specificity of UCNPs/HKUST-1/MIP for the template protein. 3.6. Detection range and limit To illustrate the performance of UCNPs/HKUST-1/MIP, the detection range and limit were investigated. The UCNPs/HKUST-1/ MIP exhibited a linear in the range of 0.1–0.6 mg/mL (Table S1) with a correlation of 0.991 for BHB. The detection limit, which was calculated as the concentration of BHB that quenched three times the standard deviation of the blank signal, divided by the slope of the standard curve, was 0.062 mg/mL (Table S1). The precision for three replicate measurements of 0.2 mg/mL was 2.70% (relative standard deviation).
Fig. 5. Fluorescence emission spectra of (a) UCNPs/HKUST-1/MIP and (b) UCNPs/HKUST-1/NIP with addition of the certain concentration of protein BHB solution. Inset is the Stern–Volmer cure. F and F0 are the fluorescence intensity of composite materials in the presence and absence of BHB.
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Fig. 6. Selectivity of the UCNPs/HKUST-1/MIP on BHB under influence of the competitive protein BSA and Cyt c. The error bars were obtained from three independent experiments.
4. Conclusions In summary, we have successfully synthesized a novel fluorescence material with thermo-sensitive for the enrichment and sensing of protein through combining the high selectivity of molecular imprinting technology with the strong fluorescence of UCNPs and the high specific surface area of MOFs for the enrichment and sensing of BHB. Here, the MOFs were used due to its high specific surface area which increases the rate of mass transfer relative to that of traditional bulk MIP. The thermo-sensitive layer of imprinting which allowed for swelling and shrinking with response to temperature changes was prepared by choosing NIPAAM as the temperature-sensitive functional monomer. The results of recognition characterizations showed that the adsorption capacity of UCNPs/HKUST-1/MIP was higher than that of traditional MIP and the ability of specific recognition to the template protein can be controlled by external environment temperature benefited from thermo-sensitive. All the results indicated that the proposed enrichment and sensing materials are regarded as a promising one for future applications.
Acknowledgments The authors are grateful for the financial support provided by the Ministry of Science and Technology of China (Project no. 2013AA102202) and the National Natural Science Foundation of China (Project no. 21375094 and 31225021) and Innovation Talents of Science and Technology Plan of Yunnan Province (Project no. 2012HA009).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.12.040.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Upconversion fluorescence metal-organic frameworks thermosensitive imprinted polymer for enrichment and sensing protein Ting Guo a, Qiliang Deng a,n, Guozhen Fang a, Dahai Gu b, Yukun Yang a, Shuo Wang a,n a Key Laboratory of Food Nutrition and Safety, Ministry of Education, Tianjin Key Laboratory of Food Nutrition and Safety, Tianjin University of Science and Technology, Tianjin 300457, China b Yunnan Agricultural University, Yunnan 650201, China
art ic l e i nf o
a b s t r a c t
Article history: Received 3 November 2015 Received in revised form 7 December 2015 Accepted 14 December 2015 Available online 17 December 2015
A novel fluorescence material with thermo-sensitive for the enrichment and sensing of protein was successfully prepared by combining molecular imprinting technology with upconversion nanoparticles (UCNPs) and metal-organic frameworks (MOFs). Herein, the UCNPs acted as signal reporter for composite materials because of its excellent fluorescence property and chemical stability. MOFs were introduced to molecularly imprinted polymer (MIP) due to its high specific surface area which increases the rate of mass transfer relative to that of traditional bulk MIP. The thermo-sensitive imprinted material which allows for swelling and shrinking with response to temperature changes was prepared by choosing Bovine hemoglobin (BHB) as the template, N-isopropyl acrylamide (NIPAAM) as the temperature-sensitive functional monomer and N,N-methylenebisacrylamide (MBA) as the cross-linker. The recognition characterizations of imprinted material-coated UCNPs/MOFs (UCNPs/MOFs/MIP) were evaluated, and the results showed that the fluorescence intensity of UCNPs/MOFs/MIP reduced gradually with the increase of BHB concentration. The fluorescence material was response to the temperature. The adsorption capacity was as much as 167.6 mg/g at 28 °C and 101.2 mg/g at 44 °C, which was higher than that of traditional MIP. Therefore, this new fluorescence material for enrichment and sensing protein is very promising for future applications. & 2015 Elsevier B.V. All rights reserved.
Keywords: Molecularly imprinted polymer Upconversion nanoparticles Metal-organic frameworks Thermo-sensitive Bovine hemoglobin
1. Introduction Metal-organic frameworks (MOFs) are relatively new class of highly porous crystalline materials and are prepared by metal connected by various organic ligands such as carboxylates or phosphonates (Yaghi et al., 1995; Li et al., 1999; Eddaoudi et al., 2002; Furukawa et al., 2013; Deng et al., 2012). MOFs have attracted significant interest in adsorption and separation (Bloch et al., 2010), gas storage (Chen et al., 2005), drug delivery (Imaz et al., 2010) due to high specific surface area, high porosity and tenability of MOFs. Very recently, a variety of MOF composite materials has attracted dramatic increasing interest owing to potential in biomaterial and optics of these composite materials. A few pioneering synthetic studies on the MOF composite materials including MOF@SiO2 (Rieter et al., 2008), MOF/GO (Petit et al., 2010, 2011a, 2011b), Fe3O4/MOF (Ke et al., 2012) have been reported. The MOF composite materials are widely used for adsorption and detection hazardous pollutants. A facile n
Corresponding authors. E-mail addresses: [email protected] (Q. Deng), [email protected] (S. Wang).
http://dx.doi.org/10.1016/j.bios.2015.12.040 0956-5663/& 2015 Elsevier B.V. All rights reserved.
magnetization of MOF for enrichment polycyclic aromatic hydrocarbons was also prepared (Huo and Yan, 2012). Recently, dithizone functionalized magnetic MOF was prepared and used for the determination of trace levels of lead (Wang et al., 2013). Li et al. (2014a) successfully synthesized a magnetic sphere hybrid Fe3O4/ Cu3(BTC)2/GO through a hydrothermal method for detecting methylene blue from water solution. However, these materials in application area were restricted due to lack of specificity. Recently, Upconversion nanoparticles (UCNPs) have led to intense studies due to outstanding advantages in bioimaging, biological probe and optical amplifier (Cheng et al., 2013, 2011; Yang et al., 2012; Wang et al., 2009). UCNPs, compared with traditional fluorescent materials such as fluorescence dyes and semiconductor QDs, are low toxic, lack of auto-fluorescence, long lifetimes and low photobleaching. Furthermore, UCNPs are capable of converting NIR light to UV–visible light by multiple photon absorptions or energy transfers (Lu et al., 2014; Liu et al., 2011). The multifunctional nanoparticles based on UCNPs with combined magnetic and optical characterize were used for multimodal imaging and therapy (Cheng et al., 2011). Molecular imprinting has been considered as a convenient
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technology that can create the materials with recognition sites which are spatially and chemically complementary to the template in materials (Ellen et al., 2011; Chen et al., 2011; Lofgreen et al., 2011). Compared with biological receptors, molecular imprinted polymer (MIP) possesses excellent mechanical and chemical stability and ease of preparation. However, traditional MIP bulks require crushed and sieved, leading to many limitations including irregularity in the shape of particles, low adsorption capacity and selectivity (Turner et al., 2006). The surface imprinting technology can overcome the above problem effectively. A novel surface-imprinted magnetic composite microsphere was successfully prepared for bovine serum albumin (BSA) recognition (Li et al., 2014b). Chen et al. (2014) prepared the molecularly imprinted nanoparticles by choosing the vinyl-modified silica nanoparticles as support material. Furthermore, quantum dots (QDs) also could be used as support material for surface imprinting (Tan et al., 2014; Yang et al., 2014; Li et al., 2015). Compared with traditional support materials, the advantages of MOFs include high specific surface area and porous structure, which increase the rate of mass transfer and adsorption capacity. Based on the above considerations, we propose a novel strategy combined the high selectivity of molecular imprinting technology with the excellent fluorescence of UCNPs and the high specific surface area of MOFs to prepare a new type of composite materials for the enrichment and sensing of protein. Herein, Cu3(BTC)2 (known as HKUST-1) was chosen as the representative MOF. The HKUST-1 is water-stable and contains Cu2 þ as the metal center linked to oxygen atoms from benzene tricarboxylate (H3BTC). The UCNPs were synthesized by solvothermal reaction and modified with polyacrylic acid (PAA) through ligand exchange. The UCNPs/ HKUST-1 were prepared by the reaction of copper nitrate (Cu (NO3)2) with H3BTC after dispersing the PAA-modified UCNPs in ethanol through a solvothermal method. The thermo-sensitive imprinted layer was prepared by choosing Bovine hemoglobin (BHB) as the template, N-isopropyl acrylamide (NIPAAM) as the thermo-sensitive functional monomer which allowed for swelling and shrinking with response to temperature changes, and N,Nmethylenebisacrylamide (MBA) as the cross-linker. The morphology and adsorption characterizations of the UCNPs/HKUST-1/MIP were investigated. Furthermore, the thermo-sensitive characterization of the composite material was discussed.
2. Materials and methods
(Victoria, Australia) in a quartz cuvette with 1 cm path length. Fluorescence measurements were performed on an F-2500 fluorescence spectrometer (Hitachi, Japan) connected with an external 980 nm diode laser (1 W, continuous wave with 1 m fiber, Beijing Viasho Technology Co.) as the excitation source. Scanning electron microscopy (SEM) images were obtained with a SU1510 microscope (Hitachi, Japan). X-ray powder diffraction (XRD) patterns were recorded on a D8 X-ray power diffractometer at a scanning rate of 1°/min in the 2θ range from 10° to 40° (Bruker, Germany). Energy-dispersive X-ray photoelectron spectroscopic (XPS) measurements were performed on PHI-5000 Versaprobe (PHI, Japan). Thermogravimetric analysis (TGA) was performed (Meteler, Switzerland) in the temperature range from room temperature to 1000 °C with a heating rate of 10 °C/min under nitrogen atmosphere. Nitrogen adsorption–desorption analysis was performed on an autosorb-1-mp (Quantachrome, USA) with a bath temperature of 77 K. Surface areas were determined using Brunauer– Emmett–Teller (BET) theory. 2.3. Preparation of UCNPs UCNPs were synthesized according to the previous literature (Li and Zhang, 2008). To a 100 mL flask, Y (CH3COO)3 (0.78 mmol), Yb (CH3COO)3 (0.2 mmol) and Er (CH3COO)3 (0.02 mmol) were mixed with 6 mL OA and 17 mL ODE and heated to 160 °C, to form a transparent solution. The mixture solution cooled down to room temperature naturally. 10 mL methanol solution containing NaOH (2.5 mmol) and NH4F (4 mmol) was slowly dropped into the flask and stirred for 30 min. Subsequently, the solution was slowly heated for removal of methanol, degassed at 100 °C for 10 min, and then heated to 300 °C and maintained for 1 h at argon atmosphere. After the resulted solution was cooled down naturally, UCNPs were precipitated via centrifugation, and washed with ethanol for three times. PAA–UCNPs were synthesized according to the previous method with a modified procedure (Naccache, et al., 2009). PAA (300 mg) and 30 mL DEG were added into a 100 mL flask. The mixture was heated to 110 °C to form a transparent solution. 3 mL toluene solution containing 100 mg hydrophobic UCNPs was added, and kept at 110 °C for 1 h under argon protection. Then the mixed solution was heated to 240 °C and maintained for 1 h. The resulted solution was cooled down to room temperature naturally, and the PAA–UCNPs were obtained from the resulted solution with excess dilute hydrochloric aqueous solution, and washed three times with water.
2.1. Materials and chemicals 2.4. Synthesis of the UCNPs/HKUST-1 Bovine serum albumin (BSA, molecular weight (MW) 67 kDa, isoelectric point (pI) 4.9), Bovine hemoglobin (BHB, MW 66 kDa, pI 6.7) and Cytochrome c (Cyt c MW 12.4 kDa, pI 10.2) were obtained from Sangon Biotech Co. Ltd. (Shanghai, China). Y(CH3COO)3 4H2O (99.9%), Yb(CH3COO)3 4H2O (99.9%), Er(CH3COO)3 xH2O (99.9%), PAA (M¼ 1800), Cu(NO3)2 3H2O, and diethylene glycol (DEG) were purchased from Sigma Aldrich (St Louis, USA). Oleic acid (OA, 90%), 1-octadecene (ODE, 90%), H3BTC and NIPAAM were purchased from Alfa Aesar Co. Ltd. (Massachusetts, USA). MBA, ammonium perulfate (APS), N,N,N,N-tetramethylenebis (TEMED) were provided by Aladdin (Los Angeles, USA). All reagents were of the highest available purity and of analytical grade at least. Double distilled water (DDW, 18.2 MΩ cm 1) was prepared by a Water Pro water purification system (Labconco, Kansas City, USA). 2.2. Characterizations Ultraviolet absorbance at a wavelength of 400 nm was recorded on a Cary 50-Bio Ultraviolet–visible (UV–vis) spectrometer
UCNPs/HKUST-1 was synthesized according to the previous method with a modified procedure (Wang et al., 2013). To 250 mL round-bottomed flask, 0.5 g of H3BTC was mixed with 40 mL DMF and 40 mL ethanol. 20 mL ethanol solution containing 100 mg PAA-modified UCNPs was added under vigorous stirring. The mixture was refluxed for 30 min at 75 °C, followed by the addition of 1.0 g Cu(NO3)2 3H2O which was dissolved in 40 mL water. After refluxing for 4 h, the UCNPs/HKUST-1 was collected via centrifugation and washed with water and ethanol, then dried at 120 °C for 10 h. 2.5. Synthesis of the UCNPs/HKUST-1/MIP 10 mg of template protein, 50 mg of UCNPs/HKUST-1, and 10 mL of water were added in a 25 mL flask and stirred for 30 min. Then 100 mg of NIPAAM and 40 mg of MBA were added to the mixture and stirred for 1 h at room temperature. The oxygen was removed by nitrogen bubbling for 10 min. Subsequently, 10 mg of
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Fig. 1. Synthesis of molecularly imprinted fluorescence materials based on the UCNPs and MOFs.
APS and 100 μL of TEMED (5%, v/v) were added, and then polymerization was carried out at 25 °C for 5 h. The UCNPs/HKUST-1/ MIP was collected via centrifugation and washed with 0.5% Tris, which was repeated several times until no template was detected by UV–vis spectrophotometry. Finally, the non-imprinted polymer (NIP) was prepared using the same procedure except addition of the template molecule. 2.6. Characterization of the UCNPs/HKUST-1/MIP In the experiments, all fluorescence detections were performed on an F-2500 fluorescence spectrometer attached with an external 980 nm laser instead of internal excitation source, and the external diode laser was set at 1 W. 2 mg of UCNPs/HKUST-1/MIP or UCNPs/ HKUST-1/NIP and 2 mL of BHB solution with a certain concentration were added into 4 mL centrifuge tube. The mixture was shaken at room temperature for a period of time and determined quickly. The specificity experiment was performed by choosing BSA as competitive protein. 2 mg of UCNPs/HKUST-1/MIP or UCNPs/ HKUST-1/NIP and 2 mL of protein solution containing BHB and BSA were mixed and shaken at room temperature for a period of time and measured quickly.
3. Results and discussion 3.1. Preparation of UCNPs/HKUST-1/MIP We first prepared a novel fluorescence material for the enrichment and sensing of the protein by combing the molecular imprinting technology with UCNPs and MOFs. Here, the UCNPs were used to provide the sensing signal for composite materials. The MOFs were introduced to the MIP due to its high specific surface area which increases the rate of mass transfer and adsorption capacity relative to that of traditional bulk MIP. The general scheme for synthesis process is shown in Fig. 1. In the first step, the UCNPs were prepared through the solvothermal method in OA and ODE. The resulted UCNPs were converted into carboxyl modified UCNPs by ligand exchange of OA with PAA. Then, the UCNPs/HKUST-1 was synthesized by the coordination interaction
between Cu2 þ and the carboxyl groups on the UCNPs surface and H3BTC. Subsequently, the template protein was immobilized onto the UCNPs/HKUST-1 though non-covalent interaction. Finally, the thermo-sensitive MIP layer was prepared by the polymerization of the thermo-sensitive monomer, NIPAAM, and MBA in aqueous solution. After removal of the template protein, the recognition sites were created. The fluorescence of the imprinted materialcoated UCNPs/MOFs (UCNPs/HKUST-1/MIP) is quenched when the template protein is rebound to the sites of the MIP layer. This is probably due to a photo-induced electron transfer process (Gerhards et al., 2008; Zhong et al., 2007). Once the template protein is extracted from the composite materials, the fluorescence of the UCNPs/HKUST-1/MIP recovers. 3.2. Characterization of composite materials The SEM images of the UCNPs/HKUST-1, UCNPs/HKUST-1/MIP and UCNPs/HKUST-1/NIP are shown in Fig. 2. From Fig. 2(a), it can be seen that the UCNPs/HKUST-1 was prepared with a size of about 1 μm. Fig. 2(b) shows that UCNPs/HKUST-1 was fully coated with the MIP layer and the size of UCNPs/HKUST-1/MIP was 1.5 μm, further indicating a successful coating with MIP layer. Furthermore, there was no significant morphological difference between UCNPs/HKUST-1/MIP and UCNPs/HKUST-1/NIP (Fig. 2(c)). The XRD patterns of UCNPs/HKUST-1 and UCNPs/HKUST-1/MIP is shown in Fig. 3(a). The characteristic peaks of Cu3(BTC)2 were showed at 2θ values of about 10.31°, 11.56°, 13.39°, 14.64°, 15.01°, 16.42°, 17.44° and 19.04°, which are assigned to the (220), (222), (400), (331), (420), (422), (333) and (440) planes of Cu3(BTC)2, respectively (Li et al., 2014a; Chowdhury et al., 2009). The XRD pattern of the UCNPs/MOF showed the good agreement with the XRD pattern of HKUST-1, which indicated that the structure of MOF was maintained in the synthesis process. The XRD pattern of UCNPs/HKUST-1/MIP was lower response due to the effect of the MIP layer. To investigate the thermo-stability and solid content, TGA of UCNPs/HKUST-1 and UCNPs/HKUST-1/MIP was performed as shown in Fig. 3(b). The TGA curve of UCNPs/HKUST-1 showed two weight loss steps in the range of 25–1000 °C. The first weight loss step of about 11.83% from 25 to 118 °C was assigned to the physically and chemically absorbed water in the UCNPs/HKUST-1. The second weight loss step showed a 45.57% weight loss in the
Fig. 2. SEM images of (a) UCNPs/HKUST-1 and (b) UCNPs/HKUST-1/MIP.
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Fig. 3. (a) XRD patterns of (A) UCNPs/HKUST-1 and (B) UCNPs/HKUST-1/MIP and (b) TGA of (C) UCNPs/HKUST-1 and (D) UCNPs/HKUST-1/MIP.
temperature range 118–359 °C, which corresponded to decomposition of the UCNPs/HKUST-1 structure, the final product was attributed to CuO and rare earth oxides. In the TGA curve of UCNPs/HKUST-1/MIP, the first weight loss step, from 25 to 101 °C, corresponded to the surface adsorbed water (6.35% weight loss). The next weight loss of 67.84% was observed from 271 to 420 °C. It is worth noticing that the weight loss of the UCNPs/HKUST-1/MIP is more than that of theUCNPs/HKUST-1, due to the MIP layer coated on the surface of UCNPs/HKUST-1. To illustrate the component of UCNPs/HKUST-1 and UCNPs/ HKUST-1/MIP, the composites materials were characterized by XPS. Fig. S1(a) shows that main signals of C1s at 285 and 288 eV, O1s at 532 eV and Cu2p3 at 933 eV, which illustrated the HKUST-1 was successfully coated on the surface of the UCNPs. Fig. S1 (b) shows main signals of C1s at 285 and 288 eV, N1s at 400 eV and O1s at 532 eV, which indicated that the UCNPs/HKUST-1 was successfully coated with MIP layer. Furthermore, due to the existence of HKUST-1, the fluorescence intensity of UCNPs/HKUST-1 was lower than that of UCNPs (Fig. S2). 3.3. Thermo-sensitive property of the UCNPs/HKUST-1/MIP It is well known that the polymer based on NIPAAM presents both hydrophobic and hydrophilic state at different temperatures, meanwhile, the volume of polymer would change with the temperatures (Zhang et al., 2013). Therefore, the influence of temperature on the adsorption capacity was investigated. In this experiment, 2 mg of UCNPs/HKUST-1/MIP was mixed with 2 mL of 0.2 mg/mL BHB solution into 4 mL centrifuge tube. The mixture
was shaken at 28 °C for a period of time and measured quickly, then shaken at 44 °C for a period of time and determined quickly, subsequently the mixture was shaken again at 28 °C. This process was performed in triplicate. The fluorescence intensity of the UCNPs/HKUST-1/MIP with and without the template protein at 28 and 44 °C is shown in Figs. 4(a) and S3. It can be seen that the interaction of the UCNPs/HKUST-1/MIP with the template BHB showed a conspicuous temperature dependent on–off fluorescence intensity, which demonstrated the recognition ability of the UCNPs/HKUST-1/MIP through the changes of the temperature. After shaking at 28 and 44 °C respectively, the mixture was centrifuged and the concentration of the template protein in the supernatant was measured by a UV–vis spectrophotometer. The adsorption capacity (Q, mg/g) of UCNPs/HKUST-1/MIP for the template protein was calculated by the equation.
Q = (C0 − C )V /m
(1)
where C0 (mg/mL) represents the initial concentration of the template BHB, C (mg/mL) is the residual concentration of the BHB, V (mL) is the volume of the BHB solution and m (g) represents the mass of UCNPs/HKUST-1/MIP. Fig. 4(b) shows the influence of the temperature on the adsorption capacity of BHB on UCNPs/HKUST1/MIP. It was clearly observed that the adsorption capacity for BHB at 28 °C was lager than that at 44 °C, which was due to NIPAAM that was in the state of contraction during the change the volume of depending on temperature stimulation from the environment. Furthermore, the adsorption capacity of UCNPs/HKUST-1/MIP was much higher than that of traditional MIP (Li et al., 2014b; Zhang et al., 2012, 2005; Chen et al., 2014), which was due to high
Fig. 4. (a) Changes of the fluorescence intensity and (b) the adsorption capacity of the UCNPs/HKUST-1/MIP for BHB with a temperature swing of 28-44˚C.
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specific surface area MOF. The BET surface area of UCNPs/HKUST-1 was measured from nitrogen adsorption isotherms at 77 K. The results showed that the BET specific surface area of UCNPs/HKUST1 was calculated to be 853.52 m2 g 1. Compared with traditional support material such as silica, the UCNPs/HKUST-1 exhibited a much lager BET specific surface area. 3.4. Fluorescent optosensing of the template protein with composite materials 3.4.1. Effect of pH Due to the fact that the surface environment of the composite materials and the charge of the protein depending on pH value, the influence of the pH value on the fluorescence intensity of the UCNPs/HKUST-1/MIP was investigated in a pH range of 6–8. Fig. S4 shows the influence of pH value on recognition. It can be seen that the best imprinting effect was observed at pH 6.6. Thus, pH of 6.6 was chosen for further experiment. 3.4.2. Equilibrium binding To demonstrate the recognition ability of the UCNPs/HKUST-1/ MIP versus that of the non-imprinted material-coated UCNPs/ MOFs (UCNPs/HKUST-1/NIP), the changes of the fluorescence signal of composite material was studied at different concentrations ranging from 0 to 0.6 mg/mL. It can be seen from Fig. 5(a) that the fluorescence intensity of the UCNPs/HKUST-1/MIP was reduced gradually with the increasing concentration of the template BHB. The fluorescence was quenched mainly due to the specific interaction between the UCNPs and the template BHB when the template protein BHB close to the imprinted sites in recognition process. Inset A and B in Fig. 5 are plotted by the Stern–Volmer equation analysis for the UCNPs/HKUST-1/MIP and UCNPs/HKUST1/NIP with the template protein, respectively.
F0/F = 1 + KSV [Q ]
(2)
where F and F0 are the fluorescence intensity of UCNPs/HKUST1/MIP or UCNPs/HKUST-1/NIP in the presence and absence of BHB, respectively, Ksv represents the quenching constant of BHB, and [Q] is the concentration of BHB. It was clearly seen from the inset A and B that the decrement of the UCNPs/HKUST-1/MIP in fluorescence intensity was much larger than that of UCNPs/HKUST-1/NIP under the same template concentration. The imprinting factor (IF), which is the ratio of the KMIP and KNIP (KMIP and KNIP are the linear slopes of inset A and B, respectively), was used to evaluate the selectivity of the composite material. Under optimum conditions, the result showed that the IF (KMIP/KNIP) was 1.82, which illustrated that the UCNPs/HKUST-1/MIP with imprinted sites could recognize the template protein. In this research, to analysis the recognition kinetics performance of the UCNPs/HKUST-1/MIP and UCNPs/HKUST-1/NIP, the equilibrium binding analysis was carried out by fixing the
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concentration of BHB (0.2 mg/mL). It was found from Fig. S5 that the changes in fluorescence intensity of UCNPs/HKUST-1/MIP with the template protein was much more significant than that of UCNPs/HKUST-1/NIP, because UCNPs/HKUST-1/NIP had no imprinted sites and existed nonspecific binding in adsorption process. For UCNPs/HKUST-1/MIP, the imprinted sites with respect to the template were generated in composite material preparation process and existed nonspecific binding and specific binding during adsorption process. It was found from Fig. S5(a) that the adsorption rate had a rapid increase in 30 min and 85.5% binding was completed. The adsorption process almost reached the equilibrium within 60 min. 3.5. Specificity study In order to further investigate the performance of the UCNPs/ HKUST-1/MIP, BSA and Cyt c was chosen as competitive protein to evaluate the selectivity of UCNPs/HKUST-1/MIP. The selectivity experiment was performed for BHB, BSA and Cyt c with a same concentration of 0.2 mg/mL. It was clearly seen that the changes in fluorescence intensity of UCNPs/HKUST-1/MIP to BHB were much more obvious than that to BSA and Cyt c. However, the changes in fluorescence intensity of UCNPs/HKUST-1/NIP were not significantly different for BHB, BSA and Cyt c. The imprinted sites and cavies with complementary in size, shape and functional group to the template protein were formed in synthesis process. As the competitive protein, although BSA is similar to BHB in the molecular volume and structures, the recognition sites were not complementary to the competitive protein. Thus, the fluorescence intensity was not obviously reduced by the competitive protein. To further demonstrate the selectivity property of the UCNPs/ HKUST-1/MIP, binary competitive adsorption experiment was performed. The binary adsorption experiment was carried out by preparing a series of protein solution of BHB–BSA and fixing the concentration of BHB (0.2 mg/mL) and increasing the concentration of BSA. It can be seen from Fig. 6(b) that the intensity of fluorescence was not significant affected as the ratio of CBSA/CBHB increased, indicating excellent specificity of UCNPs/HKUST-1/MIP for the template protein. 3.6. Detection range and limit To illustrate the performance of UCNPs/HKUST-1/MIP, the detection range and limit were investigated. The UCNPs/HKUST-1/ MIP exhibited a linear in the range of 0.1–0.6 mg/mL (Table S1) with a correlation of 0.991 for BHB. The detection limit, which was calculated as the concentration of BHB that quenched three times the standard deviation of the blank signal, divided by the slope of the standard curve, was 0.062 mg/mL (Table S1). The precision for three replicate measurements of 0.2 mg/mL was 2.70% (relative standard deviation).
Fig. 5. Fluorescence emission spectra of (a) UCNPs/HKUST-1/MIP and (b) UCNPs/HKUST-1/NIP with addition of the certain concentration of protein BHB solution. Inset is the Stern–Volmer cure. F and F0 are the fluorescence intensity of composite materials in the presence and absence of BHB.
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Fig. 6. Selectivity of the UCNPs/HKUST-1/MIP on BHB under influence of the competitive protein BSA and Cyt c. The error bars were obtained from three independent experiments.
4. Conclusions In summary, we have successfully synthesized a novel fluorescence material with thermo-sensitive for the enrichment and sensing of protein through combining the high selectivity of molecular imprinting technology with the strong fluorescence of UCNPs and the high specific surface area of MOFs for the enrichment and sensing of BHB. Here, the MOFs were used due to its high specific surface area which increases the rate of mass transfer relative to that of traditional bulk MIP. The thermo-sensitive layer of imprinting which allowed for swelling and shrinking with response to temperature changes was prepared by choosing NIPAAM as the temperature-sensitive functional monomer. The results of recognition characterizations showed that the adsorption capacity of UCNPs/HKUST-1/MIP was higher than that of traditional MIP and the ability of specific recognition to the template protein can be controlled by external environment temperature benefited from thermo-sensitive. All the results indicated that the proposed enrichment and sensing materials are regarded as a promising one for future applications.
Acknowledgments The authors are grateful for the financial support provided by the Ministry of Science and Technology of China (Project no. 2013AA102202) and the National Natural Science Foundation of China (Project no. 21375094 and 31225021) and Innovation Talents of Science and Technology Plan of Yunnan Province (Project no. 2012HA009).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.12.040.
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