A supramolecular approach for versatile biofunctionalization of magnetic nanoparticles

Changming Hu a, Jingxian Wu a, Ting Wei a, Wenjun Zhan a, Yangcui Qu a, Yue Pan *ab, Qian Yu *a and Hong Chen a
aState and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, 199 Ren’ai Road, Suzhou, 215123, P. R. China. E-mail: yuqian@suda.edu.cn; panyueps@gmail.com
bGuangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, 510120, P. R. China

Received 21st February 2018 , Accepted 19th March 2018

First published on 19th March 2018

A convenient and versatile approach for biofunctionalization of magnetic nanoparticles (MNPs) was developed based on supramolecular host–guest interaction. Adamantane groups were introduced on the surface of MNPs for further incorporation of specific biofunctional β-cyclodextrin derivatives, endowing MNPs with the desired bioactivity (e.g. biorecognition capability and biocidal activity).

In recent decades, magnetic iron oxide nanoparticles (MNPs) have attracted considerable attention for biomedical and biotechnology applications due to their unique properties, including superparamagnetism, high physical and chemical stability, good biocompatibility and low toxicity.1–3 In particular, MNPs supply a high surface area for post-modification, making them suitable as supports for the incorporation of bioactive molecules. These functionalized MNPs have been widely used in protein separation and enrichment, drug delivery, biocatalysis and biodetection.4–6 Traditionally, functionalization of MNPs relies on a chemical covalent bonding strategy. MNPs are pre-introduced with a thin organic layer with reactive functional groups (e.g., NH2, COOH, N3, etc.), and these groups serve as anchors for further conjugation of bioactive ligands or biomacromolecules through the formation of covalent bonds.7–9 Although effective, it should be noted that this covalent bonding strategy commonly involves a multi-step process, thus increasing the complexity of the entire process and the cost.10 Moreover, one covalent bonding method might only work for biomolecules with specific reactive groups. Therefore, a simple, facile and general method for the biofunctionalization of MNPs is highly desirable.

In contrast to chemical covalent bonding, supramolecular host–guest interaction offers an alternative method for incorporation of molecules via noncovalent molecular recognition.11–14 The recognition process usually can be performed in aqueous solution, supplying an environmentally friendly method without deterioration of the activities of the incorporated biomolecules.15 β-Cyclodextrin (β-CD) is one of the best investigated “host” molecules and has good capability for inclusion of various “guest” molecules (one typical guest molecule is adamantane (Ada)) to form complexes.16–19 Due to the specific and relatively strong binding interaction between β-CD and Ada, this pair has been used as a building block for the fabrication of complex structures and surface modification.20–22 Recently, several groups developed a series of β-CD modified MNPs for the incorporation of Ada derivatives to immobilize proteins and other biomolecules.23 For example, Díez et al. coated MNPs with β-CD and used them as supports for the immobilization of enzymes decorated with Ada groups via host–guest interaction. These enzyme-modified MNPs were further magnetically immobilized on an electrode surface for construction of electrochemical biosensors.24 In another report, Ravoo et al. fabricated β-CD-capped MNPs and further incorporated Ada–carbohydrate conjugates for selective capture and separation of lectins from a mixture of proteins in a magnetic field.25 In contrast, there are quite fewer reports on the Ada modified MNPs for incorporation of functional β-CD derivatives. One example is that Wang et al. introduced rhodamine-labelled β-CD on the surface of Ada functionalized MNPs to achieve a fluorogenic chemosensor for sensing Hg2+ efficiently in aqueous solution.26 It is noted that in all the systems mentioned above, one Ada group or β-CD was only conjugated with one bioactive ligand. In fact, β-CD has multiple binding sites for post-modification, making it suitable as a template for the conjugation of multiple ligands. Compared with monovalency, the multivalency ligands exhibit enhanced binding affinity and bioactivity.27 Using such multivalent β-CD derivatives, we have developed a series of supramolecular bioactive surfaces with multifunctionality or switchable bioactivity.28–35 However, to the best of our knowledge, few reports exist on the functionalization of MNPs using multivalent β-CD derivatives via host–guest interactions.

In this work, by leveraging the advantages of the multivalent effect of β-CD derivatives and the flexibility of supramolecular host–guest interactions between Ada and β-CD, we developed a simple and convenient functionalization method to endow MNPs with the desired bioactivity. Ada-functionalized core–shell structured magnetic composite nanoparticles (known as MNP@SiO2–Ada) were prepared, and the Ada groups on the surface served as binding sites for further incorporation of biofunctional β-CD derivatives (referred to as CD–X, where X is the bioactive ligand conjugated on the narrower rim of β-CD). The functionalization process was conducted under mild conditions in aqueous solution at room temperature. Three different CD–X molecules (where X = biotin, mannose, or quaternary ammonium salt (QAS)) were used in this work, and the results of the corresponding bioassays indicated that the functionalized MNPs exhibited unique bioactivity (capture of a specific protein or killing of bacteria) according to the ligands on CD–X.

The synthesis route for the MNP@SiO2–Ada composite particles is illustrated in Scheme 1. First, magnetic porous Fe3O4 particles stabilized by citrate groups were synthesized via a one-pot hydrothermal method, as reported previously.36 As observed using transmission electron microscopy (TEM), the pristine MNPs are monodisperse, uniform, and nearly spherical in shape with an average diameter of 200 nm (Fig. 1a). Moreover, the scanning electron microscopy (SEM) image showed that the surface of the MNPs is coarse (Fig. 1d), indicating that the porous particles are composed of small irregularly shaped primary nanoparticles, in accordance with previous reports.37 A thin layer of dense silica was subsequently coated on the surface of MNPs using a sol–gel process to form core–shell structured MNP@SiO2 particles. A clear core–shell structure with a dark Fe3O4 core and a gray SiO2 layer was observed using TEM (Fig. 1b). The MNP@SiO2 particles displayed a smooth surface and an increase in diameter of 50 nm, indicating that a silica layer of 25 nm was formed. Finally, Ada-terminated organic silanes were introduced on the surface of the MNP@SiO2 particles via a silane coupling method to obtain MNP@SiO2–Ada composite particles. No remarkable changes in the size or shape of the particles before and after the introduction of Ada groups were observed using TEM and SEM. The formation of the SiO2 layer and the introduction of Ada groups were further confirmed via zeta potential measurements and Fourier transform infrared spectroscopy (FTIR). The zeta potential of pristine MNPs was −42.4 mV due to the existence of citrate groups, but after coating of SiO2 and introduction of Ada groups, the zeta potential increased to −38.2 mV and −28.8 mV, respectively (Fig. 2a). Moreover, as shown in Fig. 2b, compared with the pristine MNPs, the MNP@SiO2 particles showed a new absorbance peak at 1092 cm−1, indicating the stretching vibration of Si–O–Si and thus confirming the existence of the SiO2 layer. The new peaks at 2922 cm−1 and 2956 cm−1 in the spectrum of the MNP@SiO2–Ada particles were attributed to the methylene asymmetric C–H stretching and methylene symmetric C–H stretching, respectively, indicating the successful introduction of Ada groups. The MNP@SiO2–Ada particles formed a stable dark dispersion in water at room temperature and could be quickly attracted to the sidewall, which was positioned close to a regular magnet (Fig. S1, ESI), being beneficial for further magnetic-related applications.

image file: c8tb00490k-s1.tif
Scheme 1 Preparation procedure for MNP@SiO2–Ada and incorporation with CD–X.

image file: c8tb00490k-f1.tif
Fig. 1 Representative TEM images (a–c) and SEM images (d–f) of MNP (a and d), MNP@SiO2 (b and e) and MNP@SiO2–Ada (c and f).

image file: c8tb00490k-f2.tif
Fig. 2 (a) Zeta potential of MNP, MNP@SiO2 and MNP@SiO2–Ada in water and (b) FTIR spectra of MNP, MNP@SiO2 and MNP@SiO2–Ada in water.

After confirmation of the successful preparation of the MNP@SiO2–Ada particles, we used these particles for the incorporation of functional CD–X molecules to provide specific bioactivity. First, a β-CD derivative conjugated with seven biotin units (CD–B) was chosen as a model CD–X molecule because biotin is a typical ligand for biotechnology due to its high affinity to avidin.28,33 The MNP@SiO2–Ada particles were incubated in CD–B solution (1 mg mL−1 in water) for 12 h to incorporate CD–B via host–guest interaction, and the resulting particles were referred to as MNP@SiO2–B. To investigate the recognition and binding ability of MNP@SiO2–B to avidin, these particles and three control particles (MNP, MNP@SiO2, MNP@SiO2–Ada) were incubated in a fluorescein isothiocyanate-labelled avidin (FITC-avidin) solution (0.1 mg mL−1 in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH = 7.4) for 20 min, and the particles were attracted to the side by applying an external magnetic field. The changes in fluorescence intensity of the supernatant were determined using a fluorescence spectrophotometer. As shown in Fig. 3a, after treatment of the MNP@SiO2–B particles, a remarkable decrease in fluorescence intensity was observed, and the extent of decrease was associated with the concentration of the particles, suggesting good binding ability of these particles to avidin. The binding capabilities of different particles were quantified as described in the ESI, and the results are summarized in Fig. 3b. It was found that the MNP@SiO2–B particles exhibited much higher binding ability to avidin compared with those of the three control particles, indicating that most avidin molecules were bound through specific recognition to the biotin units on the particle surface and that non-specific physical adsorption was limited. Each CD–B molecule has seven biotin units and thus exhibits enhanced affinity to avidin due to the multivalent nature.27,38 Moreover, the MNP@SiO2–Ada particles offer many Ada groups for the incorporation of CD–B on the surface. The combination of the “multivalent effect” from the CD–B molecule and abundant binding sites from the MNP@SiO2–Ada particles together increases both the local density and total amount of biotin units and thus enhances the binding affinity to avidin.

image file: c8tb00490k-f3.tif
Fig. 3 (a) Fluorescence emission spectra of the supernatants from FITC–avidin solution after incubation with different concentrations of MNP@SiO2–B and separation via a magnetic field; (b) adsorption of 0.1 mg mL−1 FITC–avidin on MNP, MNP@SiO2, MNP@SiO2–Ada and MNP@SiO2–B; (c) adsorption kinetics of avidin and BSA on MNP@SiO2–B; and (d) adsorption of FITC–avidin on MNP@SiO2-B from 0.1 mg mL−1 FITC–avidin solution in HEPES, 0.1 mg mL−1 BSA in HEPES, and 10% plasma diluted in HEPES. Error bars represent the standard deviation of the mean (n = 3).

To further investigate the specificity of MNP@SiO2–B particles, the adsorption kinetics and binding capabilities of these particles to the target protein avidin and a model non-specific protein, bovine serum albumin (BSA), were compared. As shown in Fig. 3c, the binding of avidin increased rapidly until 15 min and subsequently plateaued to reach equilibrium, suggesting that these particles show a rapid enrichment behavior within the first 15 min. In contrast, no obvious change in adsorption kinetics was observed for the non-specific protein BSA. The final binding capability of BSA was 14.6 mg g−1, which is much lower than that of avidin (151.5 mg g−1). The difference in binding capabilities of these particles to BSA and avidin was also confirmed via dynamic light scattering (DLS) measurements (Fig. S2, ESI). Furthermore, the selectivity of the MNP@SiO2–B particles was tested in complex media containing “interference” proteins and other biomolecules. These particles were incubated in three different media with the same FITC–avidin concentration (0.1 mg mL−1), namely, HEPES, HEPES containing 0.1 mg mL−1 BSA, and 10% human plasma diluted in HEPES. The binding capabilities were compared, and no significant changes were found among the three media (Fig. 3d). These results together indicated that the MNP@SiO2–B particles can bind the target protein specifically and selectively and can also resist non-specific protein adsorption to avoid interference from a complex biological environment, which is beneficial for applications such as protein enrichment and separation.

One of the remarkable advantages of our MNP@SiO2–Ada particles is that they supply a general platform with the ability to incorporate diverse bioactive ligands in a simple and mild process. To explore the versatility, we further used these particles for the incorporation of another two CD–X molecules with different activities (a β-CD derivative conjugated with seven mannose units (CD–M) and a β-CD derivative conjugated with seven QAS units (CD–Q)). Mannose is a well-known ligand with specific affinity to lectins such as the concanavalin A (ConA) and FimH proteins presented on type I fimbria of Gram-negative bacteria (e.g., Escherichia coli) via carbohydrate–protein interactions.39,40 QAS is a typical biocidal group with a permanent positive charge that can disrupt the negatively charged outer membrane of bacteria, leading to the death of bacteria.41,42 To test whether the functionalized particles exhibited the corresponding activity, pristine MNPs and MNP@SiO2–M particles (1 mg mL−1) were incubated in FITC–ConA solution (0.1 mg mL−1 in HEPES) for 20 min, and the binding capacities were compared (Fig. 4a). Similar to the MNP@SiO2–B particles, the MNP@SiO2–M particles exhibited high binding capacity to the target protein ConA, at a rate 8.3 times higher than that of pristine MNPs. Moreover, these particles also showed resistance to adsorption of the non-specific protein BSA (Fig. S3–S5, ESI). In addition, pristine MNPs and MNP@SiO2–M particles (2 mg mL−1) were incubated in Escherichia coli (E. coli) suspensions (1 × 105 CFU mL−1 in phosphate buffered saline (PBS), pH = 7.4) for 30 min for comparison of the bacteria capture ability. As shown in Fig. 4b, less than 20% of bacteria were captured by the pristine MNPs, whereas the introduction of CD–M led to a dramatic increase (∼5.2 times higher) in bacterial capture ability. The bacterial capture showed a trend similar to that of the ConA adsorption, suggesting that bacterial adhesion was specific to the mannose moieties on CD–M via the well-established mannose–FimH interaction.39,40 In addition, the increased local density of mannose ligands of CD–M is suggested to enhance their affinity to bacteria.29,31

image file: c8tb00490k-f4.tif
Fig. 4 (a) Adsorption of 0.1 mg mL−1 FITC–ConA on MNP and MNP@SiO2–M and (b) bacterial capture efficiency of MNP and MNP@SiO2–M for E. coli. Error bars represent the standard deviation of the mean (n = 3).

In another example, to test the bactericidal ability, pristine MNPs and MNP@SiO2–Q particles (2 mg mL−1 in PBS) were incubated in a model pathogenic bacterium Staphylococcus aureus (S. aureus) suspension (105 CFU mL−1 in PBS) for 3 h, and the viability of the bacteria was quantified using a conventional colony-counting assay. As shown in Fig. 5, no significant difference was observed in the colonies formed on the plate from untreated bacteria and bacteria treated with MNPs, indicating that the MNPs themselves could not kill bacteria. In contrast, after incorporation of CD–Q, the functionalized MNPs exhibited a strong biocidal activity that killed more than 99% bacteria due to the existence of abundant QAS groups (Fig. S6, ESI), because QAS is a typical positive charged biocide that can kill bacteria via disruption of the negatively charged cell membranes and destabilization of the intracellular matrix of a bacterium through a contact mechanism.41,42 Taken together, the results of these two examples demonstrated that it is easy to realize a modular design to endow the MNPs with the desired functionality according to the requirements of various applications.

image file: c8tb00490k-f5.tif
Fig. 5 Typical photographs of S. aureus colonies formed on agar plates at a density of 1 × 105 CFU mL−1 (a) and after being treated with MNP (b) and MNP@SiO2-Q (c) for 3 h, respectively.

In summary, we developed a simple and versatile approach for the functionalization of MNPs using host–guest interactions. Core–shell MNP@SiO2–Ada composite particles were prepared as a versatile platform for the further functionalization of β-CD-based multivalent ligands. The process can be conducted under mild conditions in an aqueous medium at room temperature. The final bioactivity of MNPs can be modularly designed and easily tailored according to the diverse requirements of applications. The general applicability of this approach was demonstrated by the incorporation of three different CD–X molecules on MNP@SiO2–Ada particles and could be extended for the incorporation of other CD–X molecules to endow MNPs with the desired functionality.

Conflicts of interest

There are no conflicts to declare.


This work was supported by the National Natural Science Foundation of China (21774086, 21334004, 21474071, and 51402203), the National Key Research and Development Program of China (2016YFC1100402), the Science and Technology Program of Suzhou (SYG201736), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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Electronic supplementary information (ESI) available. See DOI: 10.1039/c8tb00490k

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