Biosensors and Bioelectronics 74 (2015) 498–503
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Molecularly imprinted upconversion nanoparticles for highly selective and sensitive sensing of Cytochrome c Ting Guo 1, Qiliang Deng 1, Guozhen Fang, Cuicui Liu, Xuan Huang, Shuo Wang n 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
art ic l e i nf o
a b s t r a c t
Article history: Received 6 March 2015 Received in revised form 18 June 2015 Accepted 25 June 2015 Available online 30 June 2015
A novel method combined the high selectivity of molecular imprinting technology with the strong fluorescence property of upconversion nanoparticles (UCNPs) for sensing of Cytochrome c (Cyt c) was proposed. The molecularly imprinted material-coated upconversion nanoparticles (UCNPs@MIP) were obtained by in situ coating Cyt c imprinted materials to the surface of the carboxyl modified UCNPs through sol–gel technique. The structure and component of the prepared UCNPs@MIP was investigated by transmission electron microscopy (TEM), power X-ray diffraction (XRD), energy-dispersive X-ray analysis (EDXA) and X-ray photoelectron spectroscopic (XPS). The TEM showed the diameter of UCNPs was 40 nm, and thickness of MIP was 5–10 nm. The fluorescence intensity of UCNPs@MIP reduced gradually with the increase of Cyt c concentration. Under optimum conditions, the imprinting factor is 3.19, and the UCNPs@MIP showed selective recognition for Cyt c among other proteins such as bovine serum albumin (BSA) and Lysozyme (Lyz). Therefore, this new method for sensing protein is very promising for future applications. & 2015 Elsevier B.V. All rights reserved.
Keywords: Molecularly imprinted polymer Upconversion nanoparticles Optosensing material Cytochrome c
1. Introduction The increasing importance of nanotechnology in biological research has led to the development of high fluorescent nanomaterials (Bruchez et al., 1998; Chan and Nie, 1998; Wang and Liu, 2009; Sahu et al., 2012; Bigall et al., 2012; Wu and Chiu, 2013; Shen et al., 2012). Recently, upconversion nanoparticles (UCNPs) have been used as an important type of fluorescence probes for biomolecule detection (Cheng et al., 2013; Wang et al., 2014). Compared with traditional fluorescent materials such as fluorescence dyes and semiconductor quantum dots (QDs), UCNPs 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. Therefore, UCNPs have been widely applied in bioimaging, biological probe and optical amplifier (Wang and Liu, 2008; Wang et al., 2010; Yang et al., 2012; Zhou et al., 2010; Cheng et al., 2010). Cheng et al. (2011) prepared multifunctional nanoparticles based on UCNPs with combined optical and magnetic properties by a layer-by-layer self-assembly for multimodal imaging and dual-tareted photothermal therapy. Liu et al. (2011) synthesized n
Corresponding author. E-mail address: [email protected] (S. Wang). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bios.2015.06.058 0956-5663/& 2015 Elsevier B.V. All rights reserved.
sub-10 nm UCNPs with bright fluorescence through thermal decomposition in the presence only of oleylamine. The hydrophilic UCNPs were obtained from hydrophobic UCNPs through ligand exchange of oleylamine with citric acid, and acted as upconversion luminescence (UCL) probe for bioimaging in vivo. Zhou et al. (2011) reported a tri-modality probe with radioactivity, magnetic and upconversion fluorescence properties for multimodality positron emission tomography (PET), magnetic resonance imaging (MR) and laser scanning UCL imaging. However, the materials are difficult to image the cell or therapy target due to the lack of specificity. To solve the problem, the antibody was conjugated to the UCNPs for the detection, cell imaging, and diagnosis of disease (Wang et al., 2009; Mi et al., 2011; Kuningas et al., 2006). Recently, a dual-modality molecular tumor probe was prepared by covalently attaching antitumor antibody to polyethylene glycol (PEG) modified UCNPs. The results showed that both subcutaneously transplanted tumors and intraperitoneally transplanted tumors are successfully detected by MRI and UCL imaging (Liu et al., 2013a, 2013b). It is well known antibodies are widely used for sensing materials due to its high specificity (Funari et al., 2015). However, antibodies are costly and have low stability (McConnell et al., 2014). Molecular imprinting is a technique that creates complementary cavities for the specific target (Chen et al., 2011; Ellen et al., 2011). Compared with antibody, molecularly imprinted polymers (MIPs) possess excellent mechanical and chemical
T. Guo et al. / Biosensors and Bioelectronics 74 (2015) 498–503
stability, ease of preparation, low cost and are reusable. The recognition materials that combine the high selectivity of MIP with high sensitivity of QDs have been reported (Lee et al., 2010; Liu et al., 2013a, 2013b, 2014). Zhang et al. (2011) reported chemosensory materials which were prepared by coating MIP to the surface of the 3-mercaptopropionic acid (MPA) stabilized QDs for selective recognition of the Cytochrome c. The results of experiment showed that the composite could selectively recognize the template. They prepared a thermo-sensitive MIP coating on CdTe QDs though a surface imprinting process (Zhang et al., 2012). Tan et al. (2013) reported a new strategy which prepared a MIP layer on the surface of vinyl modified Mn-doped ZnS QDs. However, QDs are certain toxic and chemically instable. Furthermore, QDs are mainly excited by light in UV or visible spectral ranges, resulting in the significant auto-fluorescence from biological samples, limited depth of light penetration, and even the tissue photodamage. Thus, the sensitivity of the detection can be reduced. In our previous report, a novel core–shell UCNPs@MIP for detecting metolcarb was developed (Qian et al., 2013). In the synthesis process, the hexagonal nanorods UCNPs with diameters of about 110 nm and lengths of about 2 μm were simply embedded by MIP. In this research, the MIP coated hexagonal phase UCNPs with small size (40 nm) and stronger fluorescence compared with the nanorods UCNPs were obtained, which improved sensitivity and widen the further application. In a proof-of-concept experiment, we demonstrate a novel design, in which the UCNPs@MIP was prepared for the specific recognition of Cyt c. Cyt c which is a very basic redox heme-protein and used as a heme-protein model in the present investigation play an important role in the biological respiratory chain. The hydrophobic UCNPs were synthesized by solvothermal reaction and modified with polyacrylic acid (PAA) through ligand exchange. The carboxyl modified UCNPs were introduced as flurophores to provide fluorescent signal and support materials. The template protein was immobilized on the surface of the carboxyl modified hydrophilic UCNPs. The optosensing material was prepared in aqueous solutions by using 3-aminopropyltriethoxylsilane (APTES) as functional monomer and tetraethoxysilane (TEOS) as crosslinker. The characterizations of the sensing material were evaluated.
2. Materials and methods
499
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. Ultraviolet–visible (UV–vis) spectra over 200–500 nm were recorded on a Cary 50-Bio UV spectrometer (Victoria, Australia). Transmission electron microscopy (TEM) and energy-dispersive X-ray analysis (EDXA) were obtained by a 2010 FEF microscope (JEOL, Japan). X-ray photoelectron spectroscopic (XPS) measurements were performed on PHI-5000 Versaprobe (PHI, Japan). X-ray powder diffraction (XRD) patterns were performed on a D8 X-ray power diffractometer at a scanning rate of 1°/min in the 2θ range from 10° to 80° (Bruker, Germany). Fourier transform infrared (FT-IR) spectra (4000–400 cm 1) were recorded using KBr pellets in a Vector 22FT-IR spectrophotometer (Bruker, Germany). 2.3. Preparation of UCNPs 2.3.1. Preparation of hydrophobic UCNPs UCNPs were synthesized according to the previous method Li and Zhong (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. The mixture was heated to 160 °C, to form a transparent solution, and then cooled down to room temperature. 10 mL methanol solution containing NaOH (2.5 mmol) and NH4F (4 mmol) was added into the flask dropwise. The mixed solution was stirred for 30 min, which ensured that all NH4F has reacted completely. 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 under argon atmosphere. The resulted solution was cooled down naturally. UCNPs were collected, and washed with ethanol for three times. 2.3.2. Modification of hydrophobic UCNPs PAA-UCNPs were synthesized according to the previous literature with a modified procedure (Naccache et al., 2009). PAA (300 mg) was mixed with 30 mL DEG in a flask and heated to 110 °C, and resulted in 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 solution was heated to 240 °C and maintained for 1 h. The resulted solution was cooled down to room temperature, and the excess dilute hydrochloric aqueous solution was added. The PAA-UCNPs were obtained via centrifugation, and washed three times with water.
2.1. Materials and chemicals 2.4. Synthesis of the MIP-coated UCNPs (UCNPs@MIP) All reagents used in this study were of at least analytical grade. Oleic acid (OA, 90%), 1-octadecene (ODE, 90%) were obtained from Alfa Aesar Co. Ltd. (Massachusetts, USA). Y (CH3COO)3 4H2O (99.9%), Yb (CH3COO)3 4H2O (99.9%), Er (CH3COO)3 xH2O (99.9%), PAA (M ¼1800) and diethylene glycol (DEG) were purchased from Sigma Aldrich Co. Ltd. (St Louis, USA). TEOS, APTES were purchased from Hubei Wuhan University Silicone New Material Co. Ltd. (Wuhan, China). Bovine serum albumin (BSA, molecular weight (MW) 67 kDa, isoelectric point (pI) 4.9), bovine hemoglobin (BHb, MW 66 kDa, pI 6.7), Cyt c (MW 12.4 kDa, pI 10.2), Lysozyme (Lyz, MW 14.4 kDa, pI 11), Ovalbumin (OVA, MW 45 kDa, pI 4.7) were obtained from Sangon Biotech Co. Ltd. (Shanghai, China). Horseradish Peroxidase (HRP) was purchased from Roche (Mannheim, Germany). 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 Fluorescence measurements were performed on an F-2500
10 mg of template protein, 10 mg of UCNPs and 10 mL Tris (pH 7.2 0.01 M) were added in a 25 mL flask and stirred for 30 min. Then 150 μL of APTES was added to the mixture and stirred for 1 h at room temperature. Subsequently, 330 μL of TEOS and 0.2 mL of the NH3 H2O ((w/v) 25%) was added, and then reacted for 24 h. The UCNPs@MIP was centrifuged 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. 2.5. Characterization of the UCNPs@MIP In the experiments, fluorescence measurements were performed on an F-2500 fluorescence spectrometer attached with an external 980 nm laser instead of internal excitation source. The external diode laser was set at 1 W. For equilibrium binding experiments, 2.5 mg of UCNPs@MIP or UCNPs@NIP and 2 mL of protein solution with a given concentration were added into 4 mL
500
T. Guo et al. / Biosensors and Bioelectronics 74 (2015) 498–503
Eppendorf tube. The mixture was shaken at room temperature for a period of time and measured quickly. For rebinding kinetics experiments, 2.5 mg of UCNPs@MIP and 2 mL of Cyt c solution with concentration of 8 μM were shaken for different times (0.5, 1, 2, 4, 6, 8, 10, 12, 14 and 16 h) at room temperature and measured quickly. The specificity experiment was studied by choosing Lyz and BSA as competitive protein. 2.5 mg of UCNPs@MIP and 2 mL of protein solution containing Cyt c and Lyz or Cyt c and BSA were mixed and shaken at room temperature for a period of time and determined quickly.
3. Results and discussion 3.1. Response of UCNPs for different proteins It is well-known that NaYF4 has been the most efficient host material for UCNPs (Liu et al., 2011; Boyer et al., 2010). In the research, NaYF4 nanocrystals doped with Yb3 þ , Er3 þ were synthesized by a solvothermal method in OA and ODE. Owing to the organic ligand on the surface of the nanocrystals, UCNPs were well dispersed in nonpolar solvents. FT-IR spectrum of OA-UCNPs and PAA-UCNPs were shown in Fig. S1a and Fig. S1b, respectively. In Fig. S1a, the strong peaks at 2926 cm 1 and 2853 cm 1 were observed, which were associated with the asymmetric and symmetric stretching vibrations respectively of methylene groups (–CH2 ) of OA in the long alkyl chain. In addition, the two strong bands at respectively 1421 cm 1 and 1560 cm 1, which were attributed to the asymmetric and symmetric stretching vibrations of carboxylate anions (COO ) on the surface of UCNPs. The OAUCNPs were converted into hydrophilic UCNPs by ligand exchange of OA with PAA, which was convenient for further application. In Fig. S1b, the band at 1736 cm 1 was attributed to the stretching vibrations of the carbonyl group (C ¼O), which suggested the existence of carboxylic group (–COOH) on the surface of UCNPs. Meanwhile, the peak intensities at 2926 cm 1 and 2854 cm 1 became weak. The results demonstrated that OA molecules were exchanged with PAA on the surface of UCNPs. Fig. S2 showed the fluorescence spectra of OA-UCNPs and PAA-UCNPs excited with a 980 nm laser with 1 W output. The results indicated that the difference of fluorescence intensity between OA-UCNPs and PAAUCNPs was not obvious. It can be seen from the insert photograph that the green fluorescence intensity of UCNPs is strong. For the UCNPs, two green emissions at 529.5 nm and 543.5 nm were assigned to the 2H11/2-4I15/2 and 4S3/2-4I15/2 transitions; a red emission at 660.5 nm was assigned to the 4F9/2-4I15/2 transition. To investigate the interactions between UCNPs and protein through the changes of fluorescence intensity, different protein solutions at same concentration (8 μM) were prepared. The results in Fig. 1 showed that the all of the six proteins could quench the fluorescence of the UCNPs, however, the decrement of fluorescence intensity was different between six proteins, which was due to different distribution of functional groups on the surface of protein. It can be found that the fluorescence quench by Cyt c was the biggest, which was possible due to a photo-induced electron transfer process. Herein, the Cyt c was selected as the template for further studies. 3.2. Fluorescence response of UCNPs to Cyt c A series of Cyt c solutions at different concentrations were prepared to investigate the interaction between UCNPs and Cyt c. Fig. 2 is the fluorescence spectra of UCNPs with the concentration of Cyt c from 0 to 24 μM. It can be seen that as the concentration of Cyt c increased, the fluorescence intensity reduced gradually.
Fig. 1. Fluorescence intensity changes of the UCNPs with different proteins.
Fig. 2. Fluorescence emission spectra of UCNPs with Cyt c of different concentrations. Inset A is the Stern–Volmer curve, and inset B is the relationship between F/F0 and concentration of Cyt c. F and F0 are the fluorescence intensity of UCNPs in the presence and absence of Cyt c, respectively.
The fluorescence quenching in this system followed the Stern– Volmer equation
F0/F = 1 + KSV [Q ]
(1)
F and F0 are the fluorescence intensity of UCNPs in the presence and absence of Cyt c, respectively, Ksv is the quenching constant of Cyt c, and [Q] represents the concentration of Cyt c. Inset A in Fig. 2 is the Stern–Volmer curve, and inset B is the relationship between F/F0 and concentration of Cyt c, respectively. An upward sloping curve in Inset A indicated that the quenching intensity of Cyt c to UCNPs increased with increasing concentration of Cyt c. Inset B showed that a good linear relationship was obtained between the relative fluorescence intensity F/F0 and concentration of Cyt c. 3.3. Preparation of UCNPs@MIP We first proposed carboxyl modified UCNPs as core for preparing protein imprinted optosensing material. Fig. 3 presented the scheme to illustrate the synthesis process and the mechanism of protein recognition. In the first step, the template protein was immobilized on the surface of the carboxyl modified UCNPs which were introduced as support materials and as fluorophores to provide fluorescent signal by hydrogen bond. Then, the complex was interacted with functional monomer (APTES) by non-covalent interaction. Under TEOS as crosslinker, the composites were
T. Guo et al. / Biosensors and Bioelectronics 74 (2015) 498–503
501
Fig. 3. Preparative procedures for the fluorescent UCNPs@MIP.
prepared through the hydrolysis and condensation reaction of APTES. After removal of the template protein, the recognition sites were formed. The fluorescence intensity of sensing material is quenched when the template protein is rebound to the sites of material. When the template protein is extracted from the sensing material, the fluorescence intensity of the sensing material recovers. The functional monomer and crosslinker have a great influence on response of sensing materials, because the stability cavity cannot be formed when the quantity of crosslinker was not enough. In contrast, too much crosslinker resisted to remove template from material. It can be observed from Table S1, as the amount of APTES increasing, the quenching of UCNPs@MIP and UCNPs@NIP in fluorescence intensity increased initially, then decreased. And TEOS followed the similar trend in fluorescence intensity as the amount of it increased. The results showed the sensing material with the best imprinting effect was prepared by using 150 μL APTES and 330 μL TEOS. 3.4. Characterization of sensing material 3.4.1. TEM of sensing material The TEM images of the UCNPs and UCNPs@MIP are shown in Fig. 4a and b, respectively. Fig. 4 showed the size of UCNPs was 40 nm, and thickness of MIP was 5–10 nm. The elements of UCNPs were determined by EDXA, as shown in Fig. S3, and the results showed that UCNPs were mainly composed of F, Na, Y and Yb. Er element could not be determined due to the fact that its content
was only 2 mol% in the UCNPs. 3.4.2. XRD of UCNPs and UCNPs@MIP The structure of UCNPs and UCNPs@MIP were measured by XRD (Fig. S4). The XRD pattern of the UNCPs showed the good agreement with the identified JCPDS card 16-0334, indicating that the structure of the UCNPs was hexagonal phase. Meanwhile, the XRD pattern of UCNPs@MIP was similar to that of the UCNPs, which illustrated the existence of hexagonal phase in UCNPs@MIP. It was noteworthy that the pattern of UCNPs@MIP was lower response due to the effect of the MIP shell, which was attributed to the successful coating procedure. 3.4.3. XPS of UCNPs and UCNPs@MIP To investigate the surface the component of the UCNPs@MIP, XPS was performed. The Fig. S5a showed that signals of F1s at 685 eV, Na1s at 1072 eV, Y3d at 160 eV and Yb4d at 187 eV, which illustrated again the UCNPs were composed of F, Na, Y and Yb. The Fig. S5b showed that signals of C1s at 285 eV, N1s at 400 eV and O1s at 532 eV, which indicated that the material was prepared by hydrolysis and condensation of APTES and TEOS, and successfully coated on the surface of the UCNPs. 3.5. Fluorescent optosensing of the template protein 3.5.1. Effect of pH The pH value had a great influence on the fluorescence intensity of the UCNPs@MIP, because the pH value affected not only
Fig. 4. TEM images of UCNPs (a) and UCNPs@MIP (b).
502
T. Guo et al. / Biosensors and Bioelectronics 74 (2015) 498–503
Fig. 5. Fluorescence emission spectra of UCNPs@MIP (a) and UCNPs@NIP (b) with addition of the indicated concentration of protein Cyt c in PBS (0.01 M, pH 7.4) solution. RSD is 1.0–4.9%.
the surface environment of the material but also the charge of the protein. Fig. S6 showed the influence of pH on recognition. The best imprinting effect was observed at pH 7.4, so pH of 7.4 was chosen for further experiment. 3.5.2. Equilibrium binding To demonstrate the rebind ability of the UCNPs@MIP versus that of the UCNPs@NIP, the recognition of Cyt c was performed at different concentrations ranging from 0 to 24 μM, as shown in Fig. 5. It can be seen from Fig. 5a that the fluorescence intensity of the UCNPs@MIP was quenched gradually with the increasing concentration of Cyt c. In this research, the fluorescence was quenched mainly due to the specific interaction between the UCNPs with fluorescent signal and the template protein when the imprinted sites close to the template protein in rebinding process. From inset A and B of Fig. 5, it was clearly seen that the decrement of the UCNPs@MIP in fluorescence intensity was much larger than that of UCNPs@NIP under the same template concentration. The imprinting factor (IF), which was the ratio of the slopes of inset A and B in Fig. 5, used to evaluate the specificity of the composite materials. The result showed that the IF (KMIP/KNIP) was 3.19, which demonstrated that the sensing material with imprinted sites can greatly recognize the template protein. In this text, to analysis the rebinding kinetics performance of the UCNPs@MIP, the experiment was carried out by fixing the concentration of Cyt c (8 μM). According to Fig. S7, it can be seen that 67.4% binding was completed within 4 h and UCNP@MIP reached the adsorption equilibrium within 10 h.
3.6. Specificity study To further investigate the performance of the UCNPs@MIP, the specificity experiments were carried out by preparing a series of protein solution of Cyt-Lyz or Cyt-BSA and fixing the concentration of Cyt c and increasing the concentration of Lyz or BSA, respectively (Fig. S8). It can be seen from Fig. 6 that the intensity of fluorescence was small affected with the increase of the ratio of CLyz/CCyt or CBSA/CCyt. As competitive protein, BSA is different from Cyt c in PI and the molecular volume of BSA is larger than that of Cyt c. BSA could not access the recognition sites due to the steric hindrance of the material, so there was almost no change in fluorescence of the material. Although the competitive protein, Lyz, is similar to the Cyt c in pI and molecular weight, the recognition ability of the Lyz was much weaker than that of the Cyt c. Thus, the recognition mechanism of the UCNPs@MIP was based on the interactions of the size, shape and functional group of the template protein. The template protein was strongly bound to the UCNPs@MIP and caused a significant reduction of fluorescence intensity. However, the recognition sites were not complementary to the competitive proteins, so the fluorescence intensity was not obviously reduced by the competitive proteins. 3.7. Detection range and limit In order to further demonstrate the performance of UCNPs@MIP, the detection range and limit were investigated. The UCNPs@MIP exhibited a linear in the range of 1–24 μM (Table S3) with a correlation of 0.997 for Cyt c. The detection limit, which
Fig. 6. Influence of the competitive protein BSA (a) or Lyz (b) in the recognition of Cyt c on the UCNPs@MIP. The error bars were from three parallel experiments.
T. Guo et al. / Biosensors and Bioelectronics 74 (2015) 498–503
was calculated as the concentration of Cyt c that quenched three times the standard deviation of the blank signal, divided by the slope of the standard curve, was 0.73 μM (Table S3). The precision for three replicate measurements of 8 μM Cyt c was 1.80% (relative standard deviation).
4. Conclusions In summary, we have reported a novel method that combined the high selectivity of molecular imprinting with the strong fluorescence of upconversion nanoparticles (UCNPs) for sensing of Cytochrome c (Cyt c). The recognition characterizations of the UCNPs@MIP were evaluated, and the results showed that the recognition of the UCNPs@MIP was better than that of the UCNPs@NIP and the imprinting factor was 3.19. The proposed sensing materials combine the high selectivity of the molecular imprinting with strong fluorescence of the UCNPs, which are regarded as a promising optosensing material for future applications.
Acknowledgments The authors are grateful for the financial support provided by the Ministry of Science and Technology of the people’s republic of China (Project no. 2013AA102202) and National Natural Science Foundation of China (Project nos. 21375094 and 31225021).
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.2015.06.058.
References Bruchez, M., Moronne, M., Gin, P., Weiss, S., Alivisatos, A.P., 1998. Science 281, 2013–2016. Bigall, N.C., Parak, W.J., Dorfs, D., 2012. Nano Today 7, 282–296. Boyer, J.C., Manseau, M.P., Murray, J.I., Van Veggel, F.C.J.M., 2010. Langmuir 26,
503
1157–1164. Chan, W.C.W., Nie, S.M., 1998. Science 281, 2016–2018. Cheng, L., Wang, C., Liu, Z., 2013. Nanoscale 5, 23–37. Cheng, L., Yang, K., Zhang, S., Shao, M.W., Lee, S., Liu, Z., 2010. Nano Res. 3, 722–732. Cheng, L., Yang, K., Li, Y.G., Chen, J.H., Wang, C., Shao, M.W., 2011. Angew. Chem. Int. Ed. 50, 7385–7390. Chen, L.X., Xu, S.F., Li, J.H., 2011. Chem. Soc. Rev. 40, 2922–2942. Ellen, V., Joris, P.S., Martin, V.W., Marie-Astrid, D., Wim, E.H., Cornelus, F.V.N., 2011. Biomaterials 32, 3008–3020. Funari, R., Della, V.B., Carrieri, R., Morra, L., Lahoz, E., Gesuele, F., Altucci, C., Velotta, R., 2015. Biosens. Bioelectron. 67, 224–229. Kuningas, K., Ukonaho, T., Pakkila, H., Rantanen, T., Rosenberg, J., Lovgren, T., Soukka, T., 2006. Anal. Chem. 78, 4690–4696. Liu, Q., Sun, Y., Yang, T.S., Feng, W., Li, C.G., Li, F.Y., 2011. J. Am. Chem. Soc. 33, 17122–17125. Liu, C.Y., Gao, Z.Y., Zeng, J.F., Hou, Y., Fang, F., Li, Y.L., Qiao, R.R., Shen, L., Lei, H., Yang, W.S., Gao, M.Y., 2013a. ACS Nano 7, 7227–7240. Lee, M.H., Chen, Y.C., Ho, M.H., Lin, H.Y., 2010. Anal. Bioanal. Chem. 397, 1457–1466. Li, Z.Q., Zhong, Y., 2008. Nanotechnology 19, 345606–345611. Liu, H.L., Fang, G.Z., Zhu, H.D., Li, C.M., Liu, C.C., Wang, S., 2013b. Biosens. Bioelectron. 47, 127–132. Liu, H.L., Fang, G.Z., Wang, S., 2014. Biosens. Bioelectron. 55, 127–132. Mi, C.C., Tian, Z.H., Cao, C., Wang, Z.J., Mao, C.B., Xu, S.K., 2011. Langmuir 27, 14632–14637. McConnell, A.D., Zhang, X., Macomber, J.L., Chau, B., Sheffer, J.C., Rahmnian, S., Hare, E., Spasojevic, V., Horlick, R.A., King, D.J., Bowers, P.M., 2014. mAbs 6, 1274–1282. Naccache, R., Vetrone, F., Mahalingam, V., Cuccia, L.A., Capobianco, L.A., 2009. Chem. Mater. 21, 717–723. Qian, K., Fang, G.Z., Wang, S., 2013. RSC Adv. 3, 3825–3828. Sahu, S., Behera, B., Maiti, T.K., Mohapatra, S., 2012. Chem. Commun. 48, 8835–8837. Wu, C.F., Chiu, D.T., 2013. Angew. Chem. Int. Ed. 52, 3086–3109. Shen, Y.Y., Li, L.L., Lu, Q., Ji, J., Fei, R., Zhang, J.R., Abdel-Halim, E.S., Zhu, J.J., 2012. Chem. Commun. 48, 2222–2224. Tan, L., Rang, C.C., Xu, S.Y., Tang, Y.W., 2013. Biosens. Bioelectron. 48, 216–223. Wang, F., Liu, X.G., 2008. J. Am. Chem. Soc. 130, 5642–5643. Wang, F., Liu, X.G., 2009. Chem. Soc. Rev. 38, 976–989. Wang, F., Han, Y., Lim, C.S., Lu, Y.H., Wang, J., Xu, J., Chen, H.Y., Zhang, C., Hong, M.H., Liu, X.G., 2010. Nature 463, 1061–1065. Wang, M., Mi, C.C., Wang, W.X., Liu, C.H., Wu, Y.F., Xu, Z.R., Mao, C.B., Xu, S.K., 2009. ACS Nano 3, 1580–1586. Wang, X., Yang, C.X., Chen, J.T., Yan, X.P., 2014. Anal. Chem. 86 (7), 3263–3267. Yang, T.S., Sun, Y., Liu, Q., Feng, W., Yang, P.Y., Li, F.Y., 2012. Biomaterials 33, 3733–3742. Zhou, J., Sun, Y., Du, X.X., Xiong, L.Q., Hu, H., Li, F.Y., 2010. Biomaterials 31, 3287–3295. Zhou, J., Yu, M.X., Sun, Y., Zhang, X.Z., Zhu, X.J., Wu, Z.H., Wu, D.M., Li, F.Y., 2011. Biomaterials 32, 1148–1156. Zhang, W., He, X.W., Chen, Y., Li, W.Y., Zhang, Y.K., 2011. Biosens. Bioelectron. 26, 2553–2558. Zhang, W., He, X.W., Li, W.Y., Zhang, Y.K., 2012. Chem. Commun. 48, 1757–1759.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Molecularly imprinted upconversion nanoparticles for highly selective and sensitive sensing of Cytochrome c Ting Guo 1, Qiliang Deng 1, Guozhen Fang, Cuicui Liu, Xuan Huang, Shuo Wang n 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
art ic l e i nf o
a b s t r a c t
Article history: Received 6 March 2015 Received in revised form 18 June 2015 Accepted 25 June 2015 Available online 30 June 2015
A novel method combined the high selectivity of molecular imprinting technology with the strong fluorescence property of upconversion nanoparticles (UCNPs) for sensing of Cytochrome c (Cyt c) was proposed. The molecularly imprinted material-coated upconversion nanoparticles (UCNPs@MIP) were obtained by in situ coating Cyt c imprinted materials to the surface of the carboxyl modified UCNPs through sol–gel technique. The structure and component of the prepared UCNPs@MIP was investigated by transmission electron microscopy (TEM), power X-ray diffraction (XRD), energy-dispersive X-ray analysis (EDXA) and X-ray photoelectron spectroscopic (XPS). The TEM showed the diameter of UCNPs was 40 nm, and thickness of MIP was 5–10 nm. The fluorescence intensity of UCNPs@MIP reduced gradually with the increase of Cyt c concentration. Under optimum conditions, the imprinting factor is 3.19, and the UCNPs@MIP showed selective recognition for Cyt c among other proteins such as bovine serum albumin (BSA) and Lysozyme (Lyz). Therefore, this new method for sensing protein is very promising for future applications. & 2015 Elsevier B.V. All rights reserved.
Keywords: Molecularly imprinted polymer Upconversion nanoparticles Optosensing material Cytochrome c
1. Introduction The increasing importance of nanotechnology in biological research has led to the development of high fluorescent nanomaterials (Bruchez et al., 1998; Chan and Nie, 1998; Wang and Liu, 2009; Sahu et al., 2012; Bigall et al., 2012; Wu and Chiu, 2013; Shen et al., 2012). Recently, upconversion nanoparticles (UCNPs) have been used as an important type of fluorescence probes for biomolecule detection (Cheng et al., 2013; Wang et al., 2014). Compared with traditional fluorescent materials such as fluorescence dyes and semiconductor quantum dots (QDs), UCNPs 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. Therefore, UCNPs have been widely applied in bioimaging, biological probe and optical amplifier (Wang and Liu, 2008; Wang et al., 2010; Yang et al., 2012; Zhou et al., 2010; Cheng et al., 2010). Cheng et al. (2011) prepared multifunctional nanoparticles based on UCNPs with combined optical and magnetic properties by a layer-by-layer self-assembly for multimodal imaging and dual-tareted photothermal therapy. Liu et al. (2011) synthesized n
Corresponding author. E-mail address: [email protected] (S. Wang). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bios.2015.06.058 0956-5663/& 2015 Elsevier B.V. All rights reserved.
sub-10 nm UCNPs with bright fluorescence through thermal decomposition in the presence only of oleylamine. The hydrophilic UCNPs were obtained from hydrophobic UCNPs through ligand exchange of oleylamine with citric acid, and acted as upconversion luminescence (UCL) probe for bioimaging in vivo. Zhou et al. (2011) reported a tri-modality probe with radioactivity, magnetic and upconversion fluorescence properties for multimodality positron emission tomography (PET), magnetic resonance imaging (MR) and laser scanning UCL imaging. However, the materials are difficult to image the cell or therapy target due to the lack of specificity. To solve the problem, the antibody was conjugated to the UCNPs for the detection, cell imaging, and diagnosis of disease (Wang et al., 2009; Mi et al., 2011; Kuningas et al., 2006). Recently, a dual-modality molecular tumor probe was prepared by covalently attaching antitumor antibody to polyethylene glycol (PEG) modified UCNPs. The results showed that both subcutaneously transplanted tumors and intraperitoneally transplanted tumors are successfully detected by MRI and UCL imaging (Liu et al., 2013a, 2013b). It is well known antibodies are widely used for sensing materials due to its high specificity (Funari et al., 2015). However, antibodies are costly and have low stability (McConnell et al., 2014). Molecular imprinting is a technique that creates complementary cavities for the specific target (Chen et al., 2011; Ellen et al., 2011). Compared with antibody, molecularly imprinted polymers (MIPs) possess excellent mechanical and chemical
T. Guo et al. / Biosensors and Bioelectronics 74 (2015) 498–503
stability, ease of preparation, low cost and are reusable. The recognition materials that combine the high selectivity of MIP with high sensitivity of QDs have been reported (Lee et al., 2010; Liu et al., 2013a, 2013b, 2014). Zhang et al. (2011) reported chemosensory materials which were prepared by coating MIP to the surface of the 3-mercaptopropionic acid (MPA) stabilized QDs for selective recognition of the Cytochrome c. The results of experiment showed that the composite could selectively recognize the template. They prepared a thermo-sensitive MIP coating on CdTe QDs though a surface imprinting process (Zhang et al., 2012). Tan et al. (2013) reported a new strategy which prepared a MIP layer on the surface of vinyl modified Mn-doped ZnS QDs. However, QDs are certain toxic and chemically instable. Furthermore, QDs are mainly excited by light in UV or visible spectral ranges, resulting in the significant auto-fluorescence from biological samples, limited depth of light penetration, and even the tissue photodamage. Thus, the sensitivity of the detection can be reduced. In our previous report, a novel core–shell UCNPs@MIP for detecting metolcarb was developed (Qian et al., 2013). In the synthesis process, the hexagonal nanorods UCNPs with diameters of about 110 nm and lengths of about 2 μm were simply embedded by MIP. In this research, the MIP coated hexagonal phase UCNPs with small size (40 nm) and stronger fluorescence compared with the nanorods UCNPs were obtained, which improved sensitivity and widen the further application. In a proof-of-concept experiment, we demonstrate a novel design, in which the UCNPs@MIP was prepared for the specific recognition of Cyt c. Cyt c which is a very basic redox heme-protein and used as a heme-protein model in the present investigation play an important role in the biological respiratory chain. The hydrophobic UCNPs were synthesized by solvothermal reaction and modified with polyacrylic acid (PAA) through ligand exchange. The carboxyl modified UCNPs were introduced as flurophores to provide fluorescent signal and support materials. The template protein was immobilized on the surface of the carboxyl modified hydrophilic UCNPs. The optosensing material was prepared in aqueous solutions by using 3-aminopropyltriethoxylsilane (APTES) as functional monomer and tetraethoxysilane (TEOS) as crosslinker. The characterizations of the sensing material were evaluated.
2. Materials and methods
499
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. Ultraviolet–visible (UV–vis) spectra over 200–500 nm were recorded on a Cary 50-Bio UV spectrometer (Victoria, Australia). Transmission electron microscopy (TEM) and energy-dispersive X-ray analysis (EDXA) were obtained by a 2010 FEF microscope (JEOL, Japan). X-ray photoelectron spectroscopic (XPS) measurements were performed on PHI-5000 Versaprobe (PHI, Japan). X-ray powder diffraction (XRD) patterns were performed on a D8 X-ray power diffractometer at a scanning rate of 1°/min in the 2θ range from 10° to 80° (Bruker, Germany). Fourier transform infrared (FT-IR) spectra (4000–400 cm 1) were recorded using KBr pellets in a Vector 22FT-IR spectrophotometer (Bruker, Germany). 2.3. Preparation of UCNPs 2.3.1. Preparation of hydrophobic UCNPs UCNPs were synthesized according to the previous method Li and Zhong (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. The mixture was heated to 160 °C, to form a transparent solution, and then cooled down to room temperature. 10 mL methanol solution containing NaOH (2.5 mmol) and NH4F (4 mmol) was added into the flask dropwise. The mixed solution was stirred for 30 min, which ensured that all NH4F has reacted completely. 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 under argon atmosphere. The resulted solution was cooled down naturally. UCNPs were collected, and washed with ethanol for three times. 2.3.2. Modification of hydrophobic UCNPs PAA-UCNPs were synthesized according to the previous literature with a modified procedure (Naccache et al., 2009). PAA (300 mg) was mixed with 30 mL DEG in a flask and heated to 110 °C, and resulted in 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 solution was heated to 240 °C and maintained for 1 h. The resulted solution was cooled down to room temperature, and the excess dilute hydrochloric aqueous solution was added. The PAA-UCNPs were obtained via centrifugation, and washed three times with water.
2.1. Materials and chemicals 2.4. Synthesis of the MIP-coated UCNPs (UCNPs@MIP) All reagents used in this study were of at least analytical grade. Oleic acid (OA, 90%), 1-octadecene (ODE, 90%) were obtained from Alfa Aesar Co. Ltd. (Massachusetts, USA). Y (CH3COO)3 4H2O (99.9%), Yb (CH3COO)3 4H2O (99.9%), Er (CH3COO)3 xH2O (99.9%), PAA (M ¼1800) and diethylene glycol (DEG) were purchased from Sigma Aldrich Co. Ltd. (St Louis, USA). TEOS, APTES were purchased from Hubei Wuhan University Silicone New Material Co. Ltd. (Wuhan, China). Bovine serum albumin (BSA, molecular weight (MW) 67 kDa, isoelectric point (pI) 4.9), bovine hemoglobin (BHb, MW 66 kDa, pI 6.7), Cyt c (MW 12.4 kDa, pI 10.2), Lysozyme (Lyz, MW 14.4 kDa, pI 11), Ovalbumin (OVA, MW 45 kDa, pI 4.7) were obtained from Sangon Biotech Co. Ltd. (Shanghai, China). Horseradish Peroxidase (HRP) was purchased from Roche (Mannheim, Germany). 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 Fluorescence measurements were performed on an F-2500
10 mg of template protein, 10 mg of UCNPs and 10 mL Tris (pH 7.2 0.01 M) were added in a 25 mL flask and stirred for 30 min. Then 150 μL of APTES was added to the mixture and stirred for 1 h at room temperature. Subsequently, 330 μL of TEOS and 0.2 mL of the NH3 H2O ((w/v) 25%) was added, and then reacted for 24 h. The UCNPs@MIP was centrifuged 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. 2.5. Characterization of the UCNPs@MIP In the experiments, fluorescence measurements were performed on an F-2500 fluorescence spectrometer attached with an external 980 nm laser instead of internal excitation source. The external diode laser was set at 1 W. For equilibrium binding experiments, 2.5 mg of UCNPs@MIP or UCNPs@NIP and 2 mL of protein solution with a given concentration were added into 4 mL
500
T. Guo et al. / Biosensors and Bioelectronics 74 (2015) 498–503
Eppendorf tube. The mixture was shaken at room temperature for a period of time and measured quickly. For rebinding kinetics experiments, 2.5 mg of UCNPs@MIP and 2 mL of Cyt c solution with concentration of 8 μM were shaken for different times (0.5, 1, 2, 4, 6, 8, 10, 12, 14 and 16 h) at room temperature and measured quickly. The specificity experiment was studied by choosing Lyz and BSA as competitive protein. 2.5 mg of UCNPs@MIP and 2 mL of protein solution containing Cyt c and Lyz or Cyt c and BSA were mixed and shaken at room temperature for a period of time and determined quickly.
3. Results and discussion 3.1. Response of UCNPs for different proteins It is well-known that NaYF4 has been the most efficient host material for UCNPs (Liu et al., 2011; Boyer et al., 2010). In the research, NaYF4 nanocrystals doped with Yb3 þ , Er3 þ were synthesized by a solvothermal method in OA and ODE. Owing to the organic ligand on the surface of the nanocrystals, UCNPs were well dispersed in nonpolar solvents. FT-IR spectrum of OA-UCNPs and PAA-UCNPs were shown in Fig. S1a and Fig. S1b, respectively. In Fig. S1a, the strong peaks at 2926 cm 1 and 2853 cm 1 were observed, which were associated with the asymmetric and symmetric stretching vibrations respectively of methylene groups (–CH2 ) of OA in the long alkyl chain. In addition, the two strong bands at respectively 1421 cm 1 and 1560 cm 1, which were attributed to the asymmetric and symmetric stretching vibrations of carboxylate anions (COO ) on the surface of UCNPs. The OAUCNPs were converted into hydrophilic UCNPs by ligand exchange of OA with PAA, which was convenient for further application. In Fig. S1b, the band at 1736 cm 1 was attributed to the stretching vibrations of the carbonyl group (C ¼O), which suggested the existence of carboxylic group (–COOH) on the surface of UCNPs. Meanwhile, the peak intensities at 2926 cm 1 and 2854 cm 1 became weak. The results demonstrated that OA molecules were exchanged with PAA on the surface of UCNPs. Fig. S2 showed the fluorescence spectra of OA-UCNPs and PAA-UCNPs excited with a 980 nm laser with 1 W output. The results indicated that the difference of fluorescence intensity between OA-UCNPs and PAAUCNPs was not obvious. It can be seen from the insert photograph that the green fluorescence intensity of UCNPs is strong. For the UCNPs, two green emissions at 529.5 nm and 543.5 nm were assigned to the 2H11/2-4I15/2 and 4S3/2-4I15/2 transitions; a red emission at 660.5 nm was assigned to the 4F9/2-4I15/2 transition. To investigate the interactions between UCNPs and protein through the changes of fluorescence intensity, different protein solutions at same concentration (8 μM) were prepared. The results in Fig. 1 showed that the all of the six proteins could quench the fluorescence of the UCNPs, however, the decrement of fluorescence intensity was different between six proteins, which was due to different distribution of functional groups on the surface of protein. It can be found that the fluorescence quench by Cyt c was the biggest, which was possible due to a photo-induced electron transfer process. Herein, the Cyt c was selected as the template for further studies. 3.2. Fluorescence response of UCNPs to Cyt c A series of Cyt c solutions at different concentrations were prepared to investigate the interaction between UCNPs and Cyt c. Fig. 2 is the fluorescence spectra of UCNPs with the concentration of Cyt c from 0 to 24 μM. It can be seen that as the concentration of Cyt c increased, the fluorescence intensity reduced gradually.
Fig. 1. Fluorescence intensity changes of the UCNPs with different proteins.
Fig. 2. Fluorescence emission spectra of UCNPs with Cyt c of different concentrations. Inset A is the Stern–Volmer curve, and inset B is the relationship between F/F0 and concentration of Cyt c. F and F0 are the fluorescence intensity of UCNPs in the presence and absence of Cyt c, respectively.
The fluorescence quenching in this system followed the Stern– Volmer equation
F0/F = 1 + KSV [Q ]
(1)
F and F0 are the fluorescence intensity of UCNPs in the presence and absence of Cyt c, respectively, Ksv is the quenching constant of Cyt c, and [Q] represents the concentration of Cyt c. Inset A in Fig. 2 is the Stern–Volmer curve, and inset B is the relationship between F/F0 and concentration of Cyt c, respectively. An upward sloping curve in Inset A indicated that the quenching intensity of Cyt c to UCNPs increased with increasing concentration of Cyt c. Inset B showed that a good linear relationship was obtained between the relative fluorescence intensity F/F0 and concentration of Cyt c. 3.3. Preparation of UCNPs@MIP We first proposed carboxyl modified UCNPs as core for preparing protein imprinted optosensing material. Fig. 3 presented the scheme to illustrate the synthesis process and the mechanism of protein recognition. In the first step, the template protein was immobilized on the surface of the carboxyl modified UCNPs which were introduced as support materials and as fluorophores to provide fluorescent signal by hydrogen bond. Then, the complex was interacted with functional monomer (APTES) by non-covalent interaction. Under TEOS as crosslinker, the composites were
T. Guo et al. / Biosensors and Bioelectronics 74 (2015) 498–503
501
Fig. 3. Preparative procedures for the fluorescent UCNPs@MIP.
prepared through the hydrolysis and condensation reaction of APTES. After removal of the template protein, the recognition sites were formed. The fluorescence intensity of sensing material is quenched when the template protein is rebound to the sites of material. When the template protein is extracted from the sensing material, the fluorescence intensity of the sensing material recovers. The functional monomer and crosslinker have a great influence on response of sensing materials, because the stability cavity cannot be formed when the quantity of crosslinker was not enough. In contrast, too much crosslinker resisted to remove template from material. It can be observed from Table S1, as the amount of APTES increasing, the quenching of UCNPs@MIP and UCNPs@NIP in fluorescence intensity increased initially, then decreased. And TEOS followed the similar trend in fluorescence intensity as the amount of it increased. The results showed the sensing material with the best imprinting effect was prepared by using 150 μL APTES and 330 μL TEOS. 3.4. Characterization of sensing material 3.4.1. TEM of sensing material The TEM images of the UCNPs and UCNPs@MIP are shown in Fig. 4a and b, respectively. Fig. 4 showed the size of UCNPs was 40 nm, and thickness of MIP was 5–10 nm. The elements of UCNPs were determined by EDXA, as shown in Fig. S3, and the results showed that UCNPs were mainly composed of F, Na, Y and Yb. Er element could not be determined due to the fact that its content
was only 2 mol% in the UCNPs. 3.4.2. XRD of UCNPs and UCNPs@MIP The structure of UCNPs and UCNPs@MIP were measured by XRD (Fig. S4). The XRD pattern of the UNCPs showed the good agreement with the identified JCPDS card 16-0334, indicating that the structure of the UCNPs was hexagonal phase. Meanwhile, the XRD pattern of UCNPs@MIP was similar to that of the UCNPs, which illustrated the existence of hexagonal phase in UCNPs@MIP. It was noteworthy that the pattern of UCNPs@MIP was lower response due to the effect of the MIP shell, which was attributed to the successful coating procedure. 3.4.3. XPS of UCNPs and UCNPs@MIP To investigate the surface the component of the UCNPs@MIP, XPS was performed. The Fig. S5a showed that signals of F1s at 685 eV, Na1s at 1072 eV, Y3d at 160 eV and Yb4d at 187 eV, which illustrated again the UCNPs were composed of F, Na, Y and Yb. The Fig. S5b showed that signals of C1s at 285 eV, N1s at 400 eV and O1s at 532 eV, which indicated that the material was prepared by hydrolysis and condensation of APTES and TEOS, and successfully coated on the surface of the UCNPs. 3.5. Fluorescent optosensing of the template protein 3.5.1. Effect of pH The pH value had a great influence on the fluorescence intensity of the UCNPs@MIP, because the pH value affected not only
Fig. 4. TEM images of UCNPs (a) and UCNPs@MIP (b).
502
T. Guo et al. / Biosensors and Bioelectronics 74 (2015) 498–503
Fig. 5. Fluorescence emission spectra of UCNPs@MIP (a) and UCNPs@NIP (b) with addition of the indicated concentration of protein Cyt c in PBS (0.01 M, pH 7.4) solution. RSD is 1.0–4.9%.
the surface environment of the material but also the charge of the protein. Fig. S6 showed the influence of pH on recognition. The best imprinting effect was observed at pH 7.4, so pH of 7.4 was chosen for further experiment. 3.5.2. Equilibrium binding To demonstrate the rebind ability of the UCNPs@MIP versus that of the UCNPs@NIP, the recognition of Cyt c was performed at different concentrations ranging from 0 to 24 μM, as shown in Fig. 5. It can be seen from Fig. 5a that the fluorescence intensity of the UCNPs@MIP was quenched gradually with the increasing concentration of Cyt c. In this research, the fluorescence was quenched mainly due to the specific interaction between the UCNPs with fluorescent signal and the template protein when the imprinted sites close to the template protein in rebinding process. From inset A and B of Fig. 5, it was clearly seen that the decrement of the UCNPs@MIP in fluorescence intensity was much larger than that of UCNPs@NIP under the same template concentration. The imprinting factor (IF), which was the ratio of the slopes of inset A and B in Fig. 5, used to evaluate the specificity of the composite materials. The result showed that the IF (KMIP/KNIP) was 3.19, which demonstrated that the sensing material with imprinted sites can greatly recognize the template protein. In this text, to analysis the rebinding kinetics performance of the UCNPs@MIP, the experiment was carried out by fixing the concentration of Cyt c (8 μM). According to Fig. S7, it can be seen that 67.4% binding was completed within 4 h and UCNP@MIP reached the adsorption equilibrium within 10 h.
3.6. Specificity study To further investigate the performance of the UCNPs@MIP, the specificity experiments were carried out by preparing a series of protein solution of Cyt-Lyz or Cyt-BSA and fixing the concentration of Cyt c and increasing the concentration of Lyz or BSA, respectively (Fig. S8). It can be seen from Fig. 6 that the intensity of fluorescence was small affected with the increase of the ratio of CLyz/CCyt or CBSA/CCyt. As competitive protein, BSA is different from Cyt c in PI and the molecular volume of BSA is larger than that of Cyt c. BSA could not access the recognition sites due to the steric hindrance of the material, so there was almost no change in fluorescence of the material. Although the competitive protein, Lyz, is similar to the Cyt c in pI and molecular weight, the recognition ability of the Lyz was much weaker than that of the Cyt c. Thus, the recognition mechanism of the UCNPs@MIP was based on the interactions of the size, shape and functional group of the template protein. The template protein was strongly bound to the UCNPs@MIP and caused a significant reduction of fluorescence intensity. However, the recognition sites were not complementary to the competitive proteins, so the fluorescence intensity was not obviously reduced by the competitive proteins. 3.7. Detection range and limit In order to further demonstrate the performance of UCNPs@MIP, the detection range and limit were investigated. The UCNPs@MIP exhibited a linear in the range of 1–24 μM (Table S3) with a correlation of 0.997 for Cyt c. The detection limit, which
Fig. 6. Influence of the competitive protein BSA (a) or Lyz (b) in the recognition of Cyt c on the UCNPs@MIP. The error bars were from three parallel experiments.
T. Guo et al. / Biosensors and Bioelectronics 74 (2015) 498–503
was calculated as the concentration of Cyt c that quenched three times the standard deviation of the blank signal, divided by the slope of the standard curve, was 0.73 μM (Table S3). The precision for three replicate measurements of 8 μM Cyt c was 1.80% (relative standard deviation).
4. Conclusions In summary, we have reported a novel method that combined the high selectivity of molecular imprinting with the strong fluorescence of upconversion nanoparticles (UCNPs) for sensing of Cytochrome c (Cyt c). The recognition characterizations of the UCNPs@MIP were evaluated, and the results showed that the recognition of the UCNPs@MIP was better than that of the UCNPs@NIP and the imprinting factor was 3.19. The proposed sensing materials combine the high selectivity of the molecular imprinting with strong fluorescence of the UCNPs, which are regarded as a promising optosensing material for future applications.
Acknowledgments The authors are grateful for the financial support provided by the Ministry of Science and Technology of the people’s republic of China (Project no. 2013AA102202) and National Natural Science Foundation of China (Project nos. 21375094 and 31225021).
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.2015.06.058.
References Bruchez, M., Moronne, M., Gin, P., Weiss, S., Alivisatos, A.P., 1998. Science 281, 2013–2016. Bigall, N.C., Parak, W.J., Dorfs, D., 2012. Nano Today 7, 282–296. Boyer, J.C., Manseau, M.P., Murray, J.I., Van Veggel, F.C.J.M., 2010. Langmuir 26,
503
1157–1164. Chan, W.C.W., Nie, S.M., 1998. Science 281, 2016–2018. Cheng, L., Wang, C., Liu, Z., 2013. Nanoscale 5, 23–37. Cheng, L., Yang, K., Zhang, S., Shao, M.W., Lee, S., Liu, Z., 2010. Nano Res. 3, 722–732. Cheng, L., Yang, K., Li, Y.G., Chen, J.H., Wang, C., Shao, M.W., 2011. Angew. Chem. Int. Ed. 50, 7385–7390. Chen, L.X., Xu, S.F., Li, J.H., 2011. Chem. Soc. Rev. 40, 2922–2942. Ellen, V., Joris, P.S., Martin, V.W., Marie-Astrid, D., Wim, E.H., Cornelus, F.V.N., 2011. Biomaterials 32, 3008–3020. Funari, R., Della, V.B., Carrieri, R., Morra, L., Lahoz, E., Gesuele, F., Altucci, C., Velotta, R., 2015. Biosens. Bioelectron. 67, 224–229. Kuningas, K., Ukonaho, T., Pakkila, H., Rantanen, T., Rosenberg, J., Lovgren, T., Soukka, T., 2006. Anal. Chem. 78, 4690–4696. Liu, Q., Sun, Y., Yang, T.S., Feng, W., Li, C.G., Li, F.Y., 2011. J. Am. Chem. Soc. 33, 17122–17125. Liu, C.Y., Gao, Z.Y., Zeng, J.F., Hou, Y., Fang, F., Li, Y.L., Qiao, R.R., Shen, L., Lei, H., Yang, W.S., Gao, M.Y., 2013a. ACS Nano 7, 7227–7240. Lee, M.H., Chen, Y.C., Ho, M.H., Lin, H.Y., 2010. Anal. Bioanal. Chem. 397, 1457–1466. Li, Z.Q., Zhong, Y., 2008. Nanotechnology 19, 345606–345611. Liu, H.L., Fang, G.Z., Zhu, H.D., Li, C.M., Liu, C.C., Wang, S., 2013b. Biosens. Bioelectron. 47, 127–132. Liu, H.L., Fang, G.Z., Wang, S., 2014. Biosens. Bioelectron. 55, 127–132. Mi, C.C., Tian, Z.H., Cao, C., Wang, Z.J., Mao, C.B., Xu, S.K., 2011. Langmuir 27, 14632–14637. McConnell, A.D., Zhang, X., Macomber, J.L., Chau, B., Sheffer, J.C., Rahmnian, S., Hare, E., Spasojevic, V., Horlick, R.A., King, D.J., Bowers, P.M., 2014. mAbs 6, 1274–1282. Naccache, R., Vetrone, F., Mahalingam, V., Cuccia, L.A., Capobianco, L.A., 2009. Chem. Mater. 21, 717–723. Qian, K., Fang, G.Z., Wang, S., 2013. RSC Adv. 3, 3825–3828. Sahu, S., Behera, B., Maiti, T.K., Mohapatra, S., 2012. Chem. Commun. 48, 8835–8837. Wu, C.F., Chiu, D.T., 2013. Angew. Chem. Int. Ed. 52, 3086–3109. Shen, Y.Y., Li, L.L., Lu, Q., Ji, J., Fei, R., Zhang, J.R., Abdel-Halim, E.S., Zhu, J.J., 2012. Chem. Commun. 48, 2222–2224. Tan, L., Rang, C.C., Xu, S.Y., Tang, Y.W., 2013. Biosens. Bioelectron. 48, 216–223. Wang, F., Liu, X.G., 2008. J. Am. Chem. Soc. 130, 5642–5643. Wang, F., Liu, X.G., 2009. Chem. Soc. Rev. 38, 976–989. Wang, F., Han, Y., Lim, C.S., Lu, Y.H., Wang, J., Xu, J., Chen, H.Y., Zhang, C., Hong, M.H., Liu, X.G., 2010. Nature 463, 1061–1065. Wang, M., Mi, C.C., Wang, W.X., Liu, C.H., Wu, Y.F., Xu, Z.R., Mao, C.B., Xu, S.K., 2009. ACS Nano 3, 1580–1586. Wang, X., Yang, C.X., Chen, J.T., Yan, X.P., 2014. Anal. Chem. 86 (7), 3263–3267. Yang, T.S., Sun, Y., Liu, Q., Feng, W., Yang, P.Y., Li, F.Y., 2012. Biomaterials 33, 3733–3742. Zhou, J., Sun, Y., Du, X.X., Xiong, L.Q., Hu, H., Li, F.Y., 2010. Biomaterials 31, 3287–3295. Zhou, J., Yu, M.X., Sun, Y., Zhang, X.Z., Zhu, X.J., Wu, Z.H., Wu, D.M., Li, F.Y., 2011. Biomaterials 32, 1148–1156. Zhang, W., He, X.W., Chen, Y., Li, W.Y., Zhang, Y.K., 2011. Biosens. Bioelectron. 26, 2553–2558. Zhang, W., He, X.W., Li, W.Y., Zhang, Y.K., 2012. Chem. Commun. 48, 1757–1759.
