Improvement of Protein Immobilization and Bioactivity of Magnetic

Oct 30, 2015 - To achieve higher protein immobilization and bioactivity, as well as automatic manipulation, we prepared a new type of biocarrier based...
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Improvement of Protein Immobilization and Bioactivity of Magnetic Carriers Using a Brushed Beads-on-Beads Structure Peirui Wang, Ping Xu, Pingping Wang, Lingling Deng, Hongchen Gu, and Hong Xu* State Key Laboratory of Oncogenes and Related Genes, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200030, P. R. China S Supporting Information *

ABSTRACT: To achieve higher protein immobilization and bioactivity, as well as automatic manipulation, we prepared a new type of biocarrier based on the brushed beads-on-beads structure. Many poly(acrylic acid) (PAA) brushed nanoparticles were packed onto the surface of amino-functionalized magnetic particles through an efficient carbodiimide-assisted coupling reaction to attain a hierarchical structure, a unique three-dimensional (3D) space and automatic manipulation characteristics. The proposed biocarrier was evaluated in the recognition capability of the immunocomplex and showed a 6.7-fold increase compared with control beads with a hard surface. The results of this study suggest promising applications in targeted capture and highperformance biodetection processes. KEYWORDS: brushed beads-on-beads, fluidlike interface, higher protein immobilization, recognition capability, immunocomplex, automatic manipulation al. also reported that the fluidlike, spherical PAA brushes could not induce major conformational changes in proteins compared with a solid substrate with a “hard” surface, which had less negative influence on bioactivity.9 Although the nanoscale nature of SPBs allows high protein immobilization, it introduces new challenges for the separation and purification of the target biomolecules on SPBs. Combining SPBs with magnetic microspheres should exploit the high protein activity of brush conjugation and the rapid manipulation of magnetic carriers. Chen et al. synthesized a class of magnetic SPBs composed of a polystyrene core embedded with magnetite nanoparticles and a shell of PAA brushes by photoemulsion polymerization. However, the assynthesized magnetic PAA brushes do not typically form welldefined structures because their magnetic cores often lead to uncontrolled polymerization reactions, causing them to be unsuitable as biocarriers.10 In this study, we report a new type of biocarrier based on the brushed beads-on-beads structure. Fe3O4/polystyrene magnetic particles with amino groups (MPs-NH2) were used as host particles to facilitate separation and automation. PAA brushed silica nanoparticles (SiO2@PAA) were used as guest particles because of their advantageous and characteristic 3D structure and abundant carboxyl groups. As shown in Scheme 1, the brushed beads-on-beads particles (BBBs) were formed by

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urrently, magnetic microspheres with excellent superparamagnetic properties play an important role in bioseparation and biodetection platforms.1 As widely used biocarriers, they can significantly simplify the separation and detection operations when an external magnet is used.2 During these applications, magnetic microspheres will interact with the target receptor specifically through conjugated proteins on the outer surface. Hence, the effective immobilization and high bioactivity of the ligand proteins, such as antibodies, are the key issues for designing these carriers. Unfortunately, to date, a low loading capacity and a defective protein structure have limited protein immobilization to some extent when magnetic microspheres with hard surfaces are used as biocarriers.3 Among the substantial efforts to solve these problems, spherical polyelectrolyte brushes (SPBs) have been proposed as novel colloid particles for the immobilization of proteins in suspension systems.4 SPBs consist of a solid nanoparticle core onto which long linear polyelectrolyte chains, such as poly(acrylic acid) (PAA) or poly((2-methylpropenoyloxyethyl) trimethylammonium chloride) (PMPTAC), are grafted.5−7 Their nanoscale size endows these particles with a large surface area and excellent dispersity in suspensions. In addition, they exhibit a high binding capacity for proteins because of their three-dimensional (3D) structure, abundant functional groups, and ability to have a smart stimulation response.8 In our group’s preliminary study, we showed that PAA brushes with a molecular weight of 54 000 have a covalent binding capacity of ca. 2.6 mg of streptavidin (SA)/mg of brushes. This value is approximately 60-fold higher than that achieved by the conventional monolayer immobilization method.6 Ballauff et © XXXX American Chemical Society

Received: August 19, 2015 Accepted: October 30, 2015

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DOI: 10.1021/acsami.5b07733 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Scheme 1. Illustration of the Preparation of Brushed Beads-on-Beads Particles (BBBs) and Protein Immobilizationa

a

The BBBs were prepared in two steps: (1) carboxylated magnetic host particles were modified by branched poly(ether imide) (PEI), and (2) negatively charged guest particles (i.e., PAA brushes (SiO2@PAA)) were covalently coupled onto the surface of the host particles. As the model protein, SA was conjugated onto the BBBs in step (3) to obtain BBBs-SA complexes via a carbodiimide-assisted bioconjugation reaction.

conjugating a large number of guest PAA brushed nanoparticles (SiO2@PAA) onto the surface of amino-functionalized host particles (MPs-NH2) through a carbodiimide-assisted coupling reaction.11 The most prominent features of BBBs were the hierarchical structure, the unique 3D space and the soft polyelectrolyte chains from the guest PAA brushed particles. The long, linear poly(acrylic acid) chains of guest PAA brushed particles were used to form the host−guest structure and to create a 3D fabric with the dense brushed layer outstretched from the surface, thus facilitating the immobilization of targeted molecules. Additionally, the magnetism of BBBs would be beneficial for convenient bioseparation and automatic manipulation. In this study, SA was chosen as the model protein for immobilization on BBBs, and carboxyl-functionalized magnetic microspheres (MPs) with hard surfaces were chosen as control particles. Three sizes of biotinylated molecules were selected as target molecules to evaluate the bioactivity. Figures 1a, b show the transmission electron microscope (TEM) image of a typical type of guest SiO2@PAA brushed particles and the scanning electron microscope (SEM) image of host magnetic particles (MPs), respectively. Two types of PAA brushed particles, which are denoted as SiO2@PAA75 and SiO2@PAA300 (as shown in Figure S1), with 75 and 300 mean PAA chain polymerization degrees, were selected as guest particles. In this study, only SiO2@PAA75 particles were selected as an example for morphological analysis. The guest PAA brushed particles, for which polymer chains were not observed because of the low imaging contrast of the polymer, possessed a mean silica core size of 73 ± 6 nm (Figure S2a). The host particles were found to be spherical with a particle size of 596 ± 125 nm (Figure S2b). Both the host and guest particles exhibited monodisperse and uniform morphologies. Through a detailed investigation of the pH and salt concentration stimuli-responsive features of the PAA brushed particles (Figure S1), control over the density of the guest particles packed on the surface of the host particles was achieved (Table S1 and Figure S3). Figure 1c depicts the morphology of the conjugated host−guest spheres with the brushed beads-on-beads structure (BBBs). SiO2@PAA75 particles were found to be compactly packed on the surface of the host particles as a monolayer with a surface coverage of

Figure 1. (a) TEM image of nanoscale guest particles (SiO2@PAA75 particles were selected as an example). (b) SEM image of submicrometer-sized host particles. The corresponding DLS size distributions of particles are displayed in Figure S2. (c) SEM images of BBBs with a surface coverage of approximately 0.40. (d) Photos of the BBBs in aqueous suspension (left) and the directed movement under an external magnetic field (right).

approximately 0.40, as calculated by eqs 2 and 3 (see the Supporting Information). The BBBs exhibited a uniform morphology, and no aggregation of guest beads was observed, which is critical to achieve reproducible biomedical application results. In contrast to the conventional EDC/NHS chemicalconjugation method, the chemical-conjugation-after-electrostatic-adsorption strategy (paragraph 3, Supporting Information) would encourage the production of well-defined BBBs with a stable coupling density. Zeta potential provides another piece of evidence, as shown in Table S2, regarding the assembly of SiO2@PAA75 particles on the MPs. The zeta potential of the obtained BBBs changed from +44.5 mV to −33.9 mV, which indicated the presence of ample carboxyl groups on the BBBs B

DOI: 10.1021/acsami.5b07733 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces and the feasibility of using BBBs in further biomedical applications. Figure 1d shows that the as-synthesized BBBs could be easily captured and manipulated using an external magnet within 1 min; the related magnetization curves are displayed in Figure S4. The saturation magnetization value of BBBs was 36.5 emu/g at 300 K, and the value of MPs was 47.2 emu/g. The reversible hysteresis behavior with ultralow coercivity (Hc) and remanence effect (Mr) demonstrated the superparamagnetic nature of the two prepared particles, thus indicating their potential application as biocarriers for bioseparation and automated bioassays. We have demonstrated that two factors were critical for the determination of the assembly density when constructing the BBBs structure: (i) the length of the polymer chains of the guest PAA brushed particles, and (ii) a suitable ionic strength for the assembly process. Regarding the former factor, the PAA chain collapses at pH values near its pKa and exhibits a fully stretched formation under slightly basic conditions. The reason for the strong stretching behavior observed may derive from the fact that the PAA chains are gradually ionized and become more negatively charged as the pH is increased.8 In addition, the brush thickness decreases when the ionic strength is increased by adding large amounts of salt. This phenomenon is significant in SiO2@PAA300, for which the particle diameter rapidly varies from 295 to 257 nm as the NaCl concentration is increased to 25 mM. Subsequently, as the concentration of NaCl is increased from 25 to 100 mM, the particle diameter changes slowly to 245 nm. This behavior can be explained by the fact that high ionic strength will diminish the electrostatic repulsion interaction among the functional groups of the brushes.8 However, these characteristics are not apparent in SiO2@PAA75 because the polymer chains are relatively short, making the stimulation response relatively small. Regarding the latter factor, a suitable ionic strength will effectively shield the electrostatic repulsion between the guest PAA brushed particles, which leads to the smallest possible space between the PAA brushed particles and results in a high assembly density.12 In this study, the optimum assembly condition of SiO2@PAA75 guest particles deposited onto the surface of the host particles was found to occur with 25 mM MES-T (pH 4.0). The variation of the coverage in both the same and different batches of the as-synthesized BBBs was less than 12.5%, indicating good controllability and reproducibility of the assembly process. For subsequent biocarrier evaluation experiments, we selected the as-synthesized 3D structured BBBs (here, MPs@ SiO2@ PAA75 particles were used with an assembly density of 0.40) and carboxyl-modified host MPs with a conventional 2D hard surface for comparison. The key structural parameters of the two particles investigated are listed in Table 1. The results showed that the effective surface area of the BBBs improved by up to 1.8-fold compared with that of the MPs, which agreed with the fact that a binary topology can increase the surface area of these materials.13 The amount of carboxyl groups in the assynthesized BBBs per unit mass was 2.6 times larger than that of control MPs, thus providing abundant binding sites for protein covalent immobilization (Table 1). This value could be converted to 19.5 × 107 carboxyl groups per BBB compared with 5.9 × 107 carboxyl groups per MP, corresponding to a 3.3fold improvement. SA was selected as the model protein for conjugation onto both particles using EDC/NHS coupling chemistry. The binding capacity of differently scaled, biotinylated molecules was further determined based on the

Table 1. Comparison of Structural Parameters between BBBs and MPsa entry

BBBs

diameter (nm)b

host MPs 596

amount of carboxyl (μmol/g)c mass of each particle ( × 10−13g)d effective surface area of each particle ( × 106nm2)e particle quantity ( × 109/mg)f

guest SiO2@ PAA75 73 1380 2.34 2.06 4.27

MPs 596

530 1.85 1.12 5.40

a

MPs@SiO2@PAA75 with high surface coverage of 0.4 was selected as an example. bDiameters were determined based on TEM results and calculated by eq 1 (see the Supporting Information). cThe amount of carboxyl was measured by conductometric titration. dThe mass of each particle was estimated by the diameter, density, and surface coverage. e The effective surface area of BBB depended on the sum of the spherical areas of all the guest particles on a particular host particle. f Particle quantity was determined from the mass of each particle.

principle of the specific recognition between SA and biotin or biotinylated biomolecules, which have been used in many biotechnology applications.14 Figure 2 shows the biological performance of BBBs and MPs according to the values of the SA immobilization and binding capacity. Specifically, the saturated SA immobilization value of BBBs was determined to be 125.4 μg/mg, which is approximately 1.4 times larger than that of MPs (e.g., 89.3 μg/mg; Figure S5). This value could be equivalent to 2.7 × 105 SA per BBB compared with the 1.5 × 105 SA per MP, which is 1.8 times’ normalization result shown in the first histogram in Figure 2. This difference could be attributed to the many binding sites on the surface of the BBBs resulting from the 3D architecture of the outstretched PAA brushes. SA was immobilized onto the polymer chains’ ends or in the outer region of the 3D structure because of the unique “Donnan effect” of polyelectrolyte brushes.15 It should be noted that SA could be stably immobilized onto the particles primarily through covalent chemical conjugation, while nonspecifically adsorbed SA would be removed through a subsequent washing procedure (Figure S6). In addition, the larger surface areas of the raspberrylike morphology obtained using SiO2@PAA in the outer shell of the BBBs could account for the enhanced protein binding efficiency. To fully exploit the high affinity interaction between biotin and SA, we tested the biotin binding capacity of the surfacebound SA using the enzyme competitive inhibition method.16 First, approximately 5.7 × 105 biotin molecules were assumed to be accessible on the surface of each BBB, whereas only approximately 2.7 × 105 biotin molecules were accessible per MP. This corresponded to a 2.1-fold difference relative to the value shown in the second histogram in Figure 2. The mean numbers of biotin binding sites in the BBBs-SA and MPs-SA complexes were 2.1 per SA and 1.8 per SA, respectively, indicating a 1.2-fold increase in the activity shown in the fourth histogram in Figure 2. The binding capacity between SA and biotin decreased significantly from 4 per SA in the free solution to ca. 2 per SA on the particles because of the reduced accessibility and mobility of SA when SA was immobilized onto the solid phase, which is in agreement with the findings of a previous study.17 These results indicate that the biological activity of SA immobilized on both particles persists after the conjugation process. The marginal superiority of BBBs over MPs may be ascribed to the fewer and less important C

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Figure 2. (Left) Normalization results of the immobilization of SA on BBBs and the binding capacity of BBBs-SA complexes compared with MPs. (a) The three histograms are representative of the immobilization of SA on each particle and the binding capacity of each particle-SA complex with biotin and HRP-biotin, respectively. (b) The two histograms are obtained from the results in panel a through mathematical calculations. (Right) Related schematic diagram. The upside describes the interaction between BBBs-SA complexes and biotin or HRP-biotin, and the downside describes the MPs-SA complexes.

Figure 3. Plots of intensity induced by the immune complex immobilized on BBBs (red points) and MPs (blue points) as a function of the added amount of HBsAg and fitted curves. Two sets of comparisons between BBBs and control particles are shown. The SA immobilization values of BBBs and MPs were 57.2 and 59.5 μg/mg, respectively (hollow pattern, ○ and □). (a) Data obtained in a PBST buffer system, and (b) data collected in a 30% v/v serum system. The details of the immune assay are shown at the bottom of the figure. The left column describes the MPs, and the right column describes the BBBs.

conformational changes of the protein on the fluidlike BBBs interface, which has long PAA chains.9 To investigate the binding activity to different sized targets, we selected biotinylated horseradish peroxidase (HRP-biotin) as a medium-molecular weight compound to visually represent the binding capacity of SA on the above two types of particles. The binding capacity per BBB was found to increase by 9.5-fold compared with the MPs according to the signal intensity obtained by capturing the HRP-biotin, as shown in the third

histogram in Figure 2. These results correspond to a 5.3-fold overmatch per SA, as shown in the fifth histogram in Figure 2. Compared to the simple 2D morphology of MPs, the BBBs exhibited the characteristics of a special stereoscopic structure and flexible polymer chains. An average distance between the attached PAA chains was calculated as 2 nm based on the reported grafting density.6 The hydrodynamic diameter of the SA in solution was ca. 5 nm;18 therefore, SA on the BBBs likely bonded to the PAA chains’ ends or to the outer region of the D

DOI: 10.1021/acsami.5b07733 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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have potential applications as biological carriers in biodetection systems. In summary, we proposed a simple and reproducible method for the preparation of BBBs with spherical PAA brushes as outer guest particles, providing a hierarchical structure and an abundant number of carboxyl groups. BBBs were shown to be easily manipulated by an external magnet when a magnetic core was used as a host component. Higher SA immobilization was observed on the BBBs compared to the MPs because of their abundant carboxyl contents and 3D structures. When selecting the raspberrylike BBBs as carriers to recognize and capture different sizes of biotinylated molecules, the binding capacity was shown to be superior to that of MPs, which may be attributed to the fewer conformational changes induced by the flexible BBB surface. Thus, BBBs could be used as novel carriers for the recognition and manipulation of biomolecules and in high-performance biodetection systems.

3D structure, while SA on MPs may have directly contacted with the solid surface. Greater conformational changes in protein structure upon adsorption have been observed on larger particles, along with a higher loss fraction of activity.3 A higher surface curvature of the fluidlike PAA nanoscaled brushes would help to protect the native conformation of proteins.19 Thus, it follows that proteins maintain more activity on BBBs compared to the solid surfaces of MPs. In addition, this could be partially explained by a lower stereohindrance effect between SA and HRP-biotin caused by the outstretched flexible PAA chains. On the basis of the above results regarding the binding capacity of biotin and HRP-biotin, it can be concluded that the obtained raspberrylike, monodispersed BBBs exhibited excellent SA immobilization and high binding bioactivity compared to MPs, as shown by the 1.2-fold improvement in the small-molecular biotin binding capacity and the 5.3-fold improvement in the medium-molecular weight HRP-biotin binding. The different improvements observed can be primarily attributed to the varied size of the biological molecules investigated. Compared with biotin, the medium-sized molecules of HRP-biotin are typically more difficult to bind by SA immobilized on smooth MPs having a 2D design because of the steric hindrance between the biomolecules; however, the effect of the steric hindrance was relatively small in the fluidlike polymer chains. For further proof of the high bioactivity of BBBs, the macromolecule hepatitis B surface antigen (HBsAg) immunocomplex was shown to interact with the surface of SAimmobilized particles, and a chemiluminescence immunoassay was used to evaluate the binding capacity of the HBsAg immunocomplex. Figure 3 shows that the luminescence intensity increased linearly as the HBsAg concentration was increased from 0 to 500 ng/mL. The slope of the fitted curve for the detection was utilized as an indicator of the binding capacity between the particles and the macromolecules. In this study, we investigated the dissimilarity of the recognition capability of SA on the particles when an equivalent amount of immobilized SA was used, which revealed the structure’s influence on the recognition capability. The amounts of SA immobilization achieved by the BBBs and MPs were determined to be 57.2 and 59.5 μg/mg, respectively. The slope of the fitted curve showing the binding capacity of BBBs was approximately 2.3 times larger per SA in the PBST buffer system (Figure 3a) and 6.7 times larger per SA in the 30% v/v serum system (Figure 3b) compared with that of the MPs. Since an approximately equal amount of SA was immobilized on the particles (i.e., 1.2 × 105 SA per BBB and 1.0 × 105 SA per MP), the advantage shown by the BBBs could only be explained by the additional active sites on the SA, which may be ascribed to the fewer conformational changes of the protein induced by the fluid-like polymer chains. In addition, the superiority of BBBs over MPs is shown to be approximately 1.4 times larger per SA in the PBST buffer system and 4.1 times larger per SA in the 30% v/v serum system when the SA immobilization reaches saturation (Figure S7).The prominent advantage of BBBs over MPs in the serum system shown in Figure 3b revealed the protective effect of PAA chains against the interference of impurities in the serum system, in which thousands of nontarget proteins or other complex components exist and an ultralow nonspecific adsorption property is present. This indicates that the novel BBBs can provide a more sensitive detection and a wide linearity range, which suggests that BBBs

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b07733. Detailed properties of the guest particles, as well as the preparation of BBBs and their SEM images. Related protein immobilization and the recognition capability of the immunocomplex are given (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

The manuscript was written through contributions from all authors. All authors have approved the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the 863 High Tech Program (2012AA020103), STCSM(12DZ1941500), SJTU funding (YG2013MS29), and the Shmec Project (14ZZ023).

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REFERENCES

(1) Laurent, S.; Forge, D.; Port, M.; Roch, A.; Robic, C.; Vander Elst, L.; Muller, R. N. Magnetic Iron Oxide Nanoparticles: Synthesis, Stabilization, Vectorization, Physicochemical Characterizations, and Biological Applications. Chem. Rev. 2008, 108, 2064−2110. (2) Colombo, M.; Carregal-Romero, S.; Casula, M. F.; Gutiérrez, L.; Morales, M. P.; Böhm, I. B.; Heverhagen, J. T.; Prosperi, D.; Parak, W. J. Biological Applications of Magnetic Nanoparticles. Chem. Soc. Rev. 2012, 41, 4306−4334. (3) Roach, P.; Farrar, D.; Perry, C. C. Surface Tailoring for Controlled Protein Adsorption: Effect of Topography at the Nanometer Scale and Chemistry. J. Am. Chem. Soc. 2006, 128, 3939−3945. (4) Wang, S.; Chen, K.; Li, L.; Guo, X. Binding between Proteins and Cationic Spherical Polyelectrolyte Brushes: Effect of pH, Ionic Strength, and Stoichiometry. Biomacromolecules 2013, 14, 818−827. (5) Guo, X.; Weiss, A.; Ballauff, M. Synthesis of Spherical Polyelectrolyte Brushes by Photoemulsion Polymerization. Macromolecules 1999, 32, 6043−6046. (6) Qu, Z.; Hu, F.; Chen, K.; Duan, Z.; Gu, H.; Xu, H. A Facile Route to the Synthesis of Spherical Poly (Acrylic Acid) Brushes via RAFT E

DOI: 10.1021/acsami.5b07733 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Polymerization for High-capacity Protein Immobilization. J. Colloid Interface Sci. 2013, 398, 82−87. (7) Mei, Y.; Lu, Y.; Polzer, F.; Ballauff, M.; Drechsler, M. Catalytic Activity of Palladium Nanoparticles Encapsulated in Spherical Polyelectrolyte Brushes and Core-Shell Microgels. Chem. Mater. 2007, 19, 1062−1069. (8) Ballauff, M. Spherical Polyelectrolyte Brushes. Prog. Polym. Sci. 2007, 32, 1135−1151. (9) Wittemann, A.; Ballauff, M. Secondary Structure Analysis of Proteins Embedded in Spherical Polyelectrolyte Brushes by FT-IR Spectroscopy. Anal. Chem. 2004, 76, 2813−2819. (10) Chen, K.; Zhu, Y.; Zhang, Y.; Li, L.; Lu, Y.; Guo, X. Synthesis of Magnetic Spherical Polyelectrolyte Brushes. Macromolecules 2011, 44, 632−639. (11) Zhao, B.; Collinson, M. M. Well-Defined Hierarchical Templates for Multimodal Porous Material Fabrication. Chem. Mater. 2010, 22, 4312−4319. (12) Hu, Y.; Guo, T.; Ye, X.; Li, Q.; Guo, M.; Liu, H.; Wu, Z. Dye Adsorption by Resins: Effect of Ionic Strength on Hydrophobic and Electrostatic Interactions. Chem. Eng. J. 2013, 228, 392−397. (13) Matsunaga, T.; Maeda, Y.; Yoshino, T.; Takeyama, H.; Takahashi, M.; Ginya, H.; Aasahina, J.; Tajima, H. Fully Automated Immunoassay for Detection of Prostate-Specific Antigen Using Nanomagnetic Beads and Micro-polystyrene Bead Composites, ‘Beads on Beads’. Anal. Chim. Acta 2007, 597, 331−339. (14) Li, J.; Zhu, J. J. Quantum Dots for Fluorescent Biosensing and Bio-imaging Applications. Analyst 2013, 138, 2506−2515. (15) Henzler, K.; Haupt, B.; Lauterbach, K.; Wittemann, A.; Borisov, O.; Ballauff, M. Adsorption of β-Lactoglobulin on Spherical Polyelectrolyte Brushes: Direct Proof of Counterion Release by Isothermal Titration Calorimetry. J. Am. Chem. Soc. 2010, 132, 3159− 3163. (16) Gan, Z.; Marquardt, R. R. Colorimetric Competitive Inhibition Method for the Quantitation of Avidin, Streptavidin and Biotin. J. Biochem. Biophys. Methods 1999, 39, 1−6. (17) Dai, X.; Xu, H.; Zhang, X.; Zhu, W.; Gu, H.; Wei, M. Determination of the Affinity Constant of Streptavidin-coupled Magnetic Particles and a Biotinylated Antibody for High Performance of Magnetic Solid Carrier in Immunoassays. Mater. Sci. Eng., C 2014, 34, 422−428. (18) Weisenhorn, A. L.; Schmitt, F. J.; Knoll, W.; Hansma, P. K. Streptavidin Binding Observed with an Atomic Force Microscope. Ultramicroscopy 1992, 42, 1125−1132. (19) Qu, Z.; Xu, H.; Xu, P.; Chen, K.; Mu, R.; Fu, J.; Gu, H. Ultrasensitive ELISA Using Enzyme-loaded Nanospherical Brushes as Labels. Anal. Chem. 2014, 86, 9367−9371.

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DOI: 10.1021/acsami.5b07733 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX