Technical Note pubs.acs.org/ac
Surface-Imprinted Nanoparticles Prepared with a His-Tag-Anchored Epitope as the Template Senwu Li,†,‡ Kaiguang Yang,† Jianxi Liu,†,‡ Bo Jiang,† Lihua Zhang,*,† and Yukui Zhang† †
National Chromatographic R&A Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, People’s Republic of China ‡ University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China S Supporting Information *
ABSTRACT: The specific recognition of biomolecules by artificial antibodies has inspired fascination among chemists and biologists. Herein, we propose a new method to prepare epitope-oriented surface-imprinted nanoparticles with high template utilization efficiency. Using a His-tag as the anchor to facilitate the epitope immobilization/removal and the self-polymerization of dopamine to control the imprinted shell thickness, the prepared epitope-imprinted nanoparticles show specific recognition of the target protein. Moreover, with improved hydrophilicity of the His-tag-anchored epitope, this method opens up a universal route for imprinting epitopes with various polarities.
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polymerization. Later, Gajovic-Eichelmann achieved Cytochrome c epitope immobilization on a gold electrode surface through the Au−S bond and removal by electrochemical stripping.20 Although present epitope imprinted polymers show good selectivity to target proteins, such strategies are limited to the preparation of molecule imprinted polymers (MIPs) in the form of a film. With the advantages of good mechanical stability, high adsorption capacity, and easy operation, nanoparticles have been widely applied in the field of molecular imprinting. Therefore, it is imperative to develop novel epitope surface-imprinting methods to prepare such MIPs. The His-tag, which is an amino acid motif composed of at least six histidine residues, has been widely used in the purification of recombinant protein, because histidine has a strong interaction with Ni2+, which can be easily destroyed by a competitive reagent to release the purified protein.21,22 However, to the best of our knowledge, the His-tag has never been used to facilitate the immobilization and removal of templates to prepare biomolecule surface-imprinted nanoparticles. Herein, a His-tag-anchored epitope imprinting approach was developed to achieve specific recognition of a target protein.
he specific recognition of biomolecules is of great significance in separation, diagnostics, and biology.1,2 Molecular imprinting is a tailor-made synthesis technique to achieve specific recognition of target molecules, with advantages of good stability, resistance to harsh environments, and selective recognition, even for molecules without antibodies.3−6 Although the imprinting of small molecules has been achieved through various approaches, its extension to proteins has, thus far, posed a great challenge, because of the huge size, structural complexity, and conformational flexibility of the templates.7−9 Inspired by the recognition between antigen and antibody, epitope imprinting has recently been proposed.10 Compared to whole protein imprinting, the template is not only of rigid configuration, but is also easy to acquire and beneficial to the preparation of imprinted materials to achieve the recognition of the protein of interest.11,12 Until now, for reported epitope imprinting techniques, the template has been either blended with the functional monomers or immobilized on the substrate surface to generate imprinted sites.12−20 In the former case, the template is submerged in a polymer network, making template removal difficult and target protein accessibility limited. In the latter case, the recognition sites are formed on the surface of the imprinted materials, which is beneficial to solving the abovementioned problems. Therefore, epitope surface imprinting is more attractive to achieve target protein recognition. Shea’s group carried out milestone research on epitope surface imprinting in 2006.12 In their work, the epitope was immobilized on the surface of glass through an amidation reaction and later removed by striking out the substrate after © XXXX American Chemical Society
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EXPERIMENTAL SECTION The entire procedure used to prepare surface-imprinted magnetic nanoparticles is shown in Scheme 1. In brief, a silica Received: December 19, 2014 Accepted: April 17, 2015
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DOI: 10.1021/ac5047246 Anal. Chem. XXXX, XXX, XXX−XXX
Technical Note
Analytical Chemistry
than that of Fe3O4@SiO2@IDA. Under the optimal preparation condition, the highest imprinting factors (IF) for the epitope and HSA were 4.26 and 2.12, respectively. The equilibrium adsorption of our prepared MIPs for HSA was studied. As shown in Figure 2, at each concentration, the
Scheme 1. Preparation of His-Tag-Anchored Epitope Surface-Imprinted Magnetic Nanoparticles
shell was coated on the surface of Fe3O4 nanoparticles via the sol−gel method. The IDA group was modified onto the silica shell through reaction with GLYMO-IDA, followed by Ni2+ modification to fabricate Fe3O4@SiO2@IDA@Ni2+. After that, the His-tag anchored epitope dissolved in buffer containing 0.5 M NaCl, 20 mM imidazole, and 50 mM PB (pH 7.4) was introduced. Finally, the imprinted shell was fabricated through the self-polymerization of dopamine under weakly alkaline conditions, followed by the removal of the His-tag-anchored epitope by washing with 200 mM EDTA-2Na.
Figure 2. Equilibrium adsorption of HSA. Amount of MIPs and NIPs, 0.4 mg; volume, 0.3 mL; binding media, 1 × PBS (pH 7.4); incubation time, 3 h. The data represent the mean values of three parallel incubations.
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equilibrium adsorption capacity of MIPs toward HSA is much higher than that of nonimprinted polymers (NIPs), demonstrating the high affinity to the target protein. Furthermore, the adsorbed HSA can be released by 10% SDS solution with efficiency of 89% (see Figure S-1 in the Supporting Information). To evaluate the recognition selectivity, the prepared MIPs were incubated with HSA (pI 5.92), BSA (pI 5.82), and Cyc (pI 9.59), and the obtained IFs were 2.12, 1.45, and 1.11, respectively, demonstrating the highest binding affinity toward HSA. Besides, to investigate the possible interference caused by the discarded His-tag imprinted sites, a comparative peptides with His-tag, AYLKKATNEHHHHHH, was incubated with MIPs. The resulting IF of 0.72 indicated the upper recognition sites for the target epitope could block the nontarget protein with histidine residues to reach the recognition sites for the His-tag. Therefore, peptides or proteins contain His-tag do not affect the adsorption selectivity. In addition, because the grand average of hydropathicity (GRAVY) value of the His-tag is −3.2, the hydrophilicity of the His-tag-anchored epitope could be clearly improved, evidenced by our work showing that the GRAVY value of the HSA epitope was decreased from 1.122 to −0.607 after attaching the His-tag on the C-terminus of HSA. Therefore, dopamine selfpolymerization, usually performed in aqueous media, could be applied to fabricate the imprinting shell, with the epitope’s natural configuration being well reserved. It could be anticipated that our proposed His-tag-anchored epitope surface imprinting method is applicable to imprinting both polar and nonpolar epitopes in an aqueous polymerization solution, which is beneficial for achieving further target protein recognition in physiological environments. To study the imprinting process fundamentally, a coefficient, template utilization efficiency (ETU), was herein applied to illustrate the percentage of initial feeding template used to generate recognition sites for the specific binding of targets, which could be calculated according the following equation:
RESULTS AND DISCUSSION The selection of an appropriate epitope is important to achieve the specific target protein recognition.23 Although both the Nand C-terminal nonapeptides of target proteins have been successfully used as epitopes for protein imprinting,16−20 the Nterminal is always accompanied by various post-translation modifications.24,25 Therefore, in this work, the His-tag modified C-terminal nonapeptide of human serum albumin (HSA), AASQAALGL-His-tag, was selected as the template to prepare epitope surface-imprinted nanoparticles. Compared with whole protein imprinting, the recognition selectivity of epitope imprinted materials is more sensitive to the imprinted shell thickness. Because dopamine can form a thickness-controllable surface adherent film,26 dopamine was applied as the monomer to fabricate the imprinted shell, in the optimized amount of 15 mg. Furthermore, the effect of AASQAALGL-His-tag concentration on the recognition specificity of HSA was studied, and 0.5 mg/mL was selected as the best value. As shown in Figure 1A, the peak at 1628 cm−1, corresponding to the stretching vibration of the phenyl groups, appeared in the FT-IR spectrum of MIPs, indicating the formation of the dopamine shell. Furthermore, the total shell thickness of the MIPs was ca. 51 nm (Figure 1B), 6 nm larger
Figure 1. Characterization of prepared nanoparticles: (A) FT-IR spectra of MIPs (spectrum a) and Fe 3 O 4 @SiO 2 @IDA@Ni 2+ (spectrum b); (B) TEM image of MIPs. B
DOI: 10.1021/ac5047246 Anal. Chem. XXXX, XXX, XXX−XXX
Technical Note
Analytical Chemistry Table 1. Comparison of ETU Obtained by Our Method with Others in Reported References
a
substrate
template
QMIPs − QNIPs
ETU (%)
source
Fe3O4@SiO2@IDA, 50 mg PES, 200 mg CNTs@SiO2, 1 g SiO2@-MPS, 40 mg MWNTs, 60 mg Fe3O4-TTCA, 65.6 mg MWNTs, 200 mg Fe3O4@SiO2, 40 mg
epitope, 0.5 mg epitope, 31.8 μmol triclosan, 1 g estriol, 0.26 mmol brucine, 0.1 mmol ofloxacin, 0.2 mmol emodin, 0.4 mmol tadalafil, 0.26 mmol
2.38 mg/g 12.8 μmol/g 2.3 mg/g 206.8 μmol/g 42.5 μmol/g 72.3 μmol/g 28.8 μmol/g 112.7 μmol/g
48.25a 10.98a 0.23 3.18 2.55 2.37 1.44 1.73
our method ref 19 ref 27 ref 28 ref 29 ref 30 ref 31 ref 32
ETU‑epitope.
E TU =
Notes
(Q MIPs − Q NIPs)/M
The authors declare no competing financial interest.
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QT / M T
ACKNOWLEDGMENTS We gratefully acknowledge funding from the National Basic Research Program of China (No. 2012CB910601), the National Nature Science Foundation (Nos. 21375128 and 21190043), the Creative Research Group Project of the NSFC (No. 21321064), and the National High Technology Research and Development Program of China (No. 2012AA020202).
where QMIPs (mg/g) and QNIPs (mg/g) are the adsorption capacities of MIPs and NIPs toward the target, i.e., epitopes or proteins. M is the molecular weight of the target. QT (mg/g) and MT are, respectively, the initial amount and the molecular weight of the template. For the adsorption of the epitope, AASQAALGL, QMIPs and QNIPs were measured as 3.11 mg/g and 0.73 mg/g, respectively. ETU for AASQAALGL (ETU‑epitope) was calculated to be 48.25%, which was noticeably improved, compared with previously reported results, as shown in Table 1. Moreover, for the adsorption of the target protein, the ETU for protein recognition, was calculated to be 5.11%, which is much higher than that obtained by in-bulk epitope imprinting without template orientation (0.98%). Such high ETU values for both the epitope and the protein should be attributed to the following reasons: (1) the template could be effectively immobilized by the interaction between Ni2+ and the His-tag anchored to the C-terminus of the epitope, with the efficiency calculated up to 100% according to eq S-1 in the Supporting Information; (2) the immobilized template could be easily removed by EDTA-2Na, with an efficiency of 44.2%, according to eq S-2 in the Supporting Information; and (3) oriented imprinted sites and the well-tuned imprinted shell thickness guaranteed that most recognition sites were accessible to the target.
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CONCLUSION In summary, we developed a novel oriented surface imprinting method using a His-tag-anchored epitope as the template. With high template utilization efficiency and well-controlled imprinted shell thickness, the molecule imprinted polymers (MIPs) exhibit specific recognition capacity toward the epitope and the protein. Because of the good hydrophilicity of the anchored His-tag, such a strategy could be applied to the imprinting of epitopes with various polarities.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Address: 457 Zhongshan Road, Dalian 116023, China. Tel./ Fax: +86-411-84379720. E-mail:
[email protected]. C
DOI: 10.1021/ac5047246 Anal. Chem. XXXX, XXX, XXX−XXX
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DOI: 10.1021/ac5047246 Anal. Chem. XXXX, XXX, XXX−XXX
