Development of a Stable Dual Functional Coating with Low Non

ARTICLE pubs.acs.org/Langmuir

Development of a Stable Dual Functional Coating with Low Non-specific Protein Adsorption and High Sensitivity for New Superparamagnetic Nanospheres Xian’an Zhang,† Weifeng Lin,† Shengfu Chen,*,† Hong Xu,*,‡ and Hongchen Gu‡ †

State Key Laboratory of Chemical Engineering, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, People’s Republic of China ‡ Nano Biomedical Research Center, Med-X Research Institute, Shanghai Jiaotong University, Shanghai 200030, People’s Republic of China ABSTRACT: To overcome major challenges of non-specific protein adsorption on nanoparticles for nanosensing and nanodiagnosis, an efficient method for robust chemical modification was developed to achieve excellent specific biorecognition and long-term stability in complex biomedia. This method is demonstrated by a highly specific and sensitive immunoassay (IA), using superparamagnetic nanospheres (NSs) with high magnetite content. The non-specific protein adsorption on the NSs was suppressed dramatically when modified with dual functional poly(carboxybetaine methacrylate) (polyCBMA) via surface-initiated atom transfer radical polymerization (SI-ATRP) and chemically grafted with antibodies of the β subunit of human chorionic gonadotrop (anti-β-hCG). The response to hCG of IA NSs with polyCBMA coatings was highly consistent in either phosphate-buffered saline (PBS) or 50% fetal bovine serum (FBS), which is far less variable than the response of the IA NSs without polyCBMA coatings. After all, a very robust platform for IA NSs with excellent specific biorecognition was obtained. It is expected that this method for nanoparticle modification could be widely used in ultrasensitive nanosensing and nanodiagnosis in the future.

1. INTRODUCTION With the development of nanotechnologies, nanoparticle (NP)-based bioassays show great advantages over conventional methods because of their ultrasensitivity, quick response, high efficiency, and cost effectiveness.1
3 NPs are also promising candidates for cell and tissue imaging in vivo.4
6 However, to realize these advantages of NPs, three major hindrances of NPs in nanosensing and nanodiagnosis, non-specific protein adsorption, poor long-term stability, and easy aggregation, should be overcome. The non-specific protein adsorption on NPs becomes a big challenge to achieve ultrasensitive nanosensing in vitro when NPs exposed to complex media,7 such as whole blood and highconcentration serum or plasma. Moreover, this is also the key reason for reducing the performance of nanodiagnosis in vivo, which causes blood clot and quick clearance of NPs by the reticuloendothelial system, such as liver, spleen, and monocytes in blood.8 Up to date, nearly all surfaces are modified by oligo(ethylene glycol) (OEG) to improve surface resistance to nonspecific protein adsorption and specific biorecognition.9
11 However, there is a lack of versatility for many NP-based biomedical applications through OEG-based coatings,7,12 which always face a compromise between maximizing the numbers of functional groups for immobilization of biorecognition molecules and maintaining resistance to non-specific adsorption. Conventionally, OEG with reactive end groups,7,12 such as COOH and NH2, are always partially mixed in OEG-based r 2011 American Chemical Society

coatings for chemical immobilization of biorecognition molecules. The unreacted end groups after immobilization become surface defects for non-specific protein adsorption.12 On the other hand, the possible interaction between OEG/or OEG-like tetraglyme and complement control protein C3 in serum was observed because of its strong activation.13 Thus, OEG coatings become unfavorable for future application in nanosensing and nanodiagnosis in vivo. With the systematic investigation in nonfouling materials, Chen and his collaborators have developed a straightforward method to design and prepare various nonfouling materials,14,15 such as bioinspired poly(carboxybetaine) (polyCB), possessing its bifunctionality in excellent resistance to protein adsorption,16 and chemically immobilized biomolecules.17 Moreover, polyCBs show capabilities to stabilize NPs in detection media through strong electrostatic hydration and provide plenty of functional carboxyl groups for immobilization. Furthermore, those unreacted N-hydroxysuccinimide (NHS)-activated carboxyl groups after the immobilization step could be hydrolyzed back to the original carboxybetaine (CB) format to maintain resistance to protein adsorption. Thus, polycarboxybetaine methacrylate (polyCBMA) has been proven as a novel polyzwitterionic material with excellent resistance to non-specific protein adsorption and Received: July 7, 2011 Revised: October 4, 2011 Published: October 04, 2011 13669

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Scheme 1. Synthesis Route of the Initiator for ATRP

capability for antibody immobilization in the macroscale, such as on surfaces and hydrogels.17 In fact, recent reports on zwitterionic NP coatings showed promising results in biocompatibility.18 NPs with zwitterionic coatings are very stable over a broad pH range and even in saturated NaCl solution and show minimal non-specific protein adsorptions. However, the long-term stability of NPs in solution is also a major problem. Up to date, most surface modification on NPs still depends upon thiol or silane chemistry.7 The long-term stability in solution, especially in complex biomedia, must be improved because the bond for immobilization can be oxidized or hydrolyzed in its buffer during long-term storage. NP encapsulated in polystyrene
poly(acrylic acid) (PS
PAA) copolymer was proven to be a stable and convenient protection method.19 However, the non-specific protein adsorption is still relatively high after protein immobilization and treatment by conventional methods, such as with physical adsorption of bovine serum albumin (BSA), to achieve inert properties. This difficulty is believed to be caused by the very high curvature of NPs compared to flat surfaces. Thus, a nonfouling layer with stable chemical bonds on NPs is desired for nanosensing and nanodiagnosis. To achieve high sensitivity from magnetic-related signals and easy capture in a fully automatic immunoassay (IA) system, superparamagnetic nanospheres (NSs) with very high and uniform magnetite content (Fe3O4) were developed by mini-emulsion/ emulsion polymerization by authors and other groups.19
21 In comparison to a conventional magnetite microsphere, these superparamagnetic NSs showed great advantages for bioassay and diagnosis in vitro or in vivo because of their nano effect and high magnetite content.1
3,5,22 However, the high density and heterogeneous defect surface caused by loading a large amount of heavy magnetite content in the NSs will lead to low hydrodynamic stability and more non-specific protein adsorption, which require higher hydration and a larger coverage layer than those on normal NPs. Thus, polyCBMA is chosen as a protection material for the NSs because of its strong surface hydration via electrostatic interactions and flexibility for surface modification. In this work, a convenient and reliable method through forming multilayer core
shell NSs with NPs encapsulated in PS
PAA co-polymer, chemically immobilized polyCBMA, and antibody on shell (Scheme 2) was developed. The high sensitivity of superparamagnetic IA NSs with excellent specific biorecognition and long-term stability in complex biomedia was achieved.

2. MATERIALS AND METHODS 2.1. Materials. Superparamagnetic NSs were synthesized by Med-X Institute of Shanghai Jiao Tong University.14 Human chorionic gonadotrophin (hCG, 100 mIU/mL), anti-α-hCG antibody (6.4 mg/mL), and anti-β-hCG antibody (10 mg/mL) were purchased from Hangzhou Clongene Biotech Co. Ltd. Horseradish peroxidase (HRP)-conjugated goat anti-human immunoglobulin G (IgG) was purchased from Beijing

Biosynthesis Biotechnology Co. Ltd. Fetal bovine serum (FBS) was purchased from Hangzhou Sijiqing Biological Engineering Materials Co. Ltd. 2-(N,N0 -dimethylamino)ethylmethacrylate (DMAEM, 99%), 1-ethyl3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDC, 98.5%), hydroxyl-2,5-dioxopyrolidine-3-sulfonic acid sodium salt (sulfo-NHS, 98%), HRP (>300 mIU/mL), ethylenediamine anhydrous (EDA, 99%), triethylamine (Et3N, 99.5%), copper(I) bromide (CuBr, 99.999%), bromoisobutyryl bromide (BIBB, 98%), 2,20 -bipyridine (BPY, 99%), Tween20 (CP), and o-phenylenediamine (OPD, 98%) were purchased from Aladdin Reagent Co. β-Propiolactone (98%) was purchased from J&K Scientific Ltd. Di-tert-butyl dicarbonate (98%) was purchased from Shanghai Medpep Co. Ltd. Tetrahydrofuran (THF) and acetone were purchased from Sinopharm Chemical Reagent Beijing Co. Ltd.; these solvents were distilled overnight and dried prior to use. 2.2. Synthesis of the CBMA Monomer.23 2-Carboxy-N,N-dimethyl-N-(20 -(methacryloyloxy)ethyl)ethanaminium inner salt (carboxybetaine methacrylate, CBMA) was synthesized by the reaction of DMAEM and β-propiolactone. β-Propiolactone (10.32 g, 143.1 mmol) in 40 mL of dried acetone was added dropwise to a solution of DMAEM (21.67 g, 137.8 mmol) dissolved in 80 mL of anhydrous acetone. The reaction mixture was stirred under argon protection at room temperature for 5 h. The white precipitate was washed with anhydrous acetone 3 times and dried under reduced pressure to obtain the final CBMA monomer product (22.3 g; yield, 70.7%). 1H NMR (D2O): 1.84 (CH3
C
), 2.63 (CH2
COO), 3.09 [(CH3)
N], 3.59 (CH2
CH2
COO), 3.70 (N
CH2
CH2
O), 4.54 (O
CH2
CH2
N), 5.67 (CHHdC), 6.05 (CHHdC). 2.3. Synthesis of the Initiator.24 With argon protection, a solution of di-tert-butyl dicarbonate (37.98 g, 0.174 mol) in 80 mL of THF was slowly added to a stirred solution of EDA (107.8 g, 1.793 mol) in 340 mL of THF over a period of 2 h at 0 °C. The temperature of the reaction was then changed to room temperature. After 24 h, the precipitate was filtered off and both THF and excess EDA were removed under vacuum. Water (400 mL) was added to the residue, and bis(N,N0 tert-butyloxycarbonyl)-1,2-diaminoethane was removed by filtration. The aqueous solution was saturated with sodium chloride and extracted 3 times with dichloromethane (250 mL). The organic phase was dried over sodium sulfate, and dichloromethane was evaporated under vacuum to obtain a colorless oil (1) (15.62 g; yield, 56.1%). 1H NMR (CDCl3): 1.39 [(CH3)3C
], 2.74 (
CH2NH2), 3.11 (
CH2NH
) (Scheme 1). In the second step, a solution of BIBB (19.72 g, 85.8 mmol) in 40 mL of THF was slowly added at 0 °C to a solution of compound 1 (9.16 g, 57.2 mmol) in THF (150 mL) in the presence of Et3N (11.58 g, 114.4 mmol). After 2 h, the reaction was left for 26 h with continuous stirring at room temperature. Triethylammonium bromide was formed as a white precipitate and filtered off. After removal of the solvent under vacuum, a yellow solid was dissolved in ethyl acetate and extracted twice with the aqueous solution saturated with Na2CO3. The organic phase was dried over sodium sulfate, and ethyl acetate was evaporated under vacuum to obtain a pale yellow solid. The solid was washed with hexane 3 times and dried under reduced pressure to obtain a white solid (2) (6.17 g; yield, 34.9%). 1H NMR (CDCl3): 1.45 [(CH3)3C
], 1.96 [(CH3)2C
], 3.34 (
CH2
NH
COO
), 3.38 (
CH2
NH
CO
). In the third step, hydrogen chloride gas was synthesized by the reaction of concentrated H2SO4 and concentrated HCl. The HCl gas was introduced into a solution of compound 2 (3.09 g, 10.0 mmol) in 13670

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Scheme 2. Route of the IA Superparamagnetic NS Preparation

50 mL of ethyl acetate. When the solution turned to slightly cloudy, ethyl acetate was evaporated under vacuum to obtain a white solid. The solid was dissolved in water, and the white precipitate was filtered off. After freeze drying, the aqueous solution afforded a white solid (3) (2.18 g; yield, 88.5%). 1H NMR (D2O): 1.93 [(CH3)2C
], 3.16 (
CH2
NH2), 3.54 (
CH2
NH
CO
).

2.4. Preparation of Anti-α-hCG Antibody
HRP (Anti-α-hCG
HRP) Conjugates.25 A fresh aqueous solution of 0.1 M sodium

periodate (60 μL) was added to a solution of HRP (10 mg) in 1 mL of water, shielded from light, and shaken for 15 min at 25 °C. A total of 60 μL of 0.4 M Na2SO3 was added to quench the oxidation reaction for 2 min. Then, anti-α-hCG antibody diluted by carbonate buffer (pH 9.5) to 2.2 mg/mL was added. The reaction solution was then shielded from light and shaken for 2 h at 25 °C; a fresh aqueous solution of 5 M NaHB4 (26 μL) was added; and the mixture was set at 4 °C for 2 h. The final solution was dialyzed overnight with dialysis bags at 4 °C to obtain anti-α-hCG
HRP.

2.5. Modification on the Surface of Superparamagnetic NSs.7,26,27 A total of about 1.71 mg of superparamagnetic NSs was set into each of four tubes, washed twice with 0.01 M 4-morpholineethanesulfonic buffer (MES, pH 5.0, containing 0.05% Tween-20), and finally, dispersed in 800 μL of MES buffer. Different weights of EDC and initiator (3) were added into the four tubes. After all of the tubes of superparamagnetic NSs were shaken at 25 °C for 2 h, they were washed with phosphate-buffered saline (PSB) containing 0.05% Tween-20 (PBST, pH 7.4) 6 times to obtain NS 5, as show in the Scheme 2. After superparamagnetic NSs with ATRP initiator self-assembled monolayers (SAMs) were prepared, polyCBMA films were synthesized via surface-initiated atom transfer radical polymerization (SI-ATRP). An amount of 30.3 mg (0.21 mmol) of CuBr and 62.4 mg (0.40 mmol) of BPY were dissolved in 3 mL of methanol, and the solution was degassed for 30 min. Meanwhile, a solution of CBMA (0.84 g, 3.65 mmol) in methanol/water (1 mL/0.5 mL) was degassed for 30 min. A volume of 200 μL of methanol containing CuBr and BPY was added to the sealed tubes with superparamagnetic NSs. Another 250 μL of solution containing CBMA was transferred in the same way under argon protection. After 2 h, the superparamagnetic NSs were washed with PBS 6 times to obtain NS 6.

2.6. Functionalization on the Surface of Superparamagnetic NSs. A sample of ∼0.134 g of NS 6 coated with polyCBMA was removed from PBS solution and washed with MES buffer twice. A general procedure of functionalization on the surfaces of NPs was followed according to these steps: the carboxylate groups of the polyCBMA surface were activated by injection of a freshly prepared solution of 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDC) (0.4 M) and hydroxyl-2,5-dioxopyrolidine-3-sulfonic acid sodium salt (sulfo-NHS) (0.1 M) in the MES buffer for 15 min at 25 °C. After the MES buffer was removed, a solution of anti-β-hCG antibody (2.5 mg/mL) in 200 μL of borate buffer (pH 8.5, containing

Figure 1. Relative values of non-specific adsorption of HRP-conjugated anti-β-hCG and the corresponding film thickness of the IA superparamagnetic NSs. 0.05% Tween-20) was added. After reaction for 2 h at 25 °C, the sample of NSs was washed with PBS 6 times to obtain IA superparamagnetic NS 7. In the same way, IA superparamagnetic NS 8 were synthesized from the bare IA superparamagnetic NS 4, as show in Scheme 2.

2.7. Measurements of Non-specific Protein Adsorption and Specific Adsorption of Antigen.28 To measure the nonspecific protein adsorption on the surface of superparamagnetic NSs, both compounds 4 and 6 with different modifications were treated by the following steps: The amount of ∼11.3 μg of superparamagnetic NSs was incubated with HRP-conjugated goat anti-human IgG (∼1 μg/mL) for 90 min at 25 °C and washed with PBS 6 times. A total of 1/5 of the sample was taken out and put into a new tube. A solution of o-phenylenediamine (OPD) in citrate
phosphate buffer (800 μL, pH 5.0, 1 mg/mL) containing 0.03% hydrogen peroxide was added. Enzyme activity was stopped by adding an equal volume of H2SO4 (2 N) after 15 min. The tangerine color is measured at 492 nm. The total non-specific protein adsorption on the superparamagnetic NSs modified with polyCBMA was shown as Figure 1, when the non-specific protein adsorption of bare superparamagnetic NSs was set as 100%. The response of the antibody grafted on IA superparamagnetic NSs to antigen was measured by a standard sandwich method. Both NSs 7 and 8 (∼11.3 μg) were divided into six tubes. After two washes with PBS, a solution of hCG (0, 2.5, 5, 15, 25, and 50 mIU/mL) in PBS was added into six tubes, respectively. After shaking for 1 h at 25 °C, all of the samples of NSs were washed with PBS once and incubated with a solution of anti-α-hCG
HRP (∼10 μg/mL) in PBS for 1 h at 25 °C, followed by six washes with PBS. Applying the same method as before 13671

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Table 1. ζ Potentials and the Diameters of NSs before and after Modification serial number

0

EDC (mg/mL) ζ potential (mV, modified with initiator)

2

3

4

20

50

100

200


21.8 ( 0.5


18 ( 1


17 ( 1


16.7 ( 0.7


14.6 ( 0.6


11.1 ( 0.2


11 ( 1


11.5 ( 0.8


11.1 ( 0.8

231 ( 3

360 ( 20

340 ( 10

310 ( 10

281 ( 5

ζ potential (mV, coated with polyCBMA) diameter (nm, coated with polyCBMA)

1

Figure 2. TEM images of NSs: (4) bare NSs, (5) NSs modified with initiator, and (6) NSs coated with polyCBMA. Dark scale bars represent 0.2 μm. (measurement of the non-specific protein adsorption on the surface of superparamagnetic NSs), 1/5 of the 12 samples was taken out and put into a new tube to measure the absorbency. In the same way, the response of anti-β-hCG antibody grafted on NSs to hCG in 50% FBS (1:1 FBS/PBS) was also measured.

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of IA Superparamagnetic NSs. The superparamagnetic NSs were activated by

EDC and modified by N-(2-aminoethyl)-2-bromo-2-metylpropanamide (compound 3) in 0.01 M MES buffer (pH 5.0) with 0.05% Tween-20. With the protection of 0.05% Tween-20, the hydrophobic
hydrophobic interaction between NSs after modification of compound 3 was minimized. Thus, the size of NSs did not show any obvious increase by dynamic light scattering (DLS). The ζ potential of modified NSs slightly increased from ∼
22 to
14 mV with the increase of the EDC concentration (Table 1). This indicated that the density of COOH on original NSs decreased because of more immobilization of neutral compound 3 by the increase of the EDC concentration. PolyCBMA brushes on the modified NSs were formed by following 2 h SI-ATRP and measured 25
64 nm by DLS. This is the optimal range of thickness for the lowest non-specific protein adsorption.27 The results show that the thickness decreased with the increase of the density of the initiator on NSs and all ζ potentials of NSs coated with polyCBMA are close to
11 mV. The comparison of resistance to non-specific protein adsorption shows that almost all NSs with a polyCBMA protection layer could reduce protein adsorption to about 5% of the control, original NSs, as show in Figure 1. However, with the increase of the initiator on NSs, the non-specific protein adsorptions decrease even lower to 3.2 ( 0.2%, which indicates that the density of polyCBMA on NSs is more effective in preventing non-specific protein adsorption rather than the thickness of polyCBMA. This result agrees with our previous results of zwitterionic materials because they are so effective even when they are in a monolayer.28 In comparison to the resistance to non-specific protein adsorption on surface plasmon resonance (SPR) surfaces, this result indicates that both complex three-dimensional structures and surface distribution of chemical groups of NSs made it more difficult to reduce

non-specific protein adsorption. It is believed that the defects caused by a small amount of uncovered surfaces by carboxyl groups might be the major reason to cause such a protein adsorption. On the other hand, the modified NSs were very stable in both nonfouling and hydrodynamic properties, of which diameters showed no obvious change after being stored in pH 7.4 PBST buffer over 3 months at 4 °C. After all, NSs with covalently bonded polyCBMA are in very good conditions to prepare IA NSs. To reduce the cross-link through anti-hCG between IA NSs, the surface modification of NSs by anti-hCG is prepared by a twostep method. The polyCBMA-modified NSs were first activated by sulfo-NHS with EDC in acidic buffer and reacted with anti-hCG in mild basic buffer after removing activation solution. The optimization of both reaction conditions are performed (data not shown). The highest surface coverage of anti-hCG could be achieved by activating NSs under 0.1 M sulfo-NHS/0.4 M EDC pH 5.0 MEST buffer for 15 min at 25 °C, followed by PBST washes 6 times, and conjugating with 2.5 mg/mL anti-β-hCG in pH 8.5 PBST. The highest surface coverage on the polyCBMA-modified NSs reaches ∼20% on the original NSs, which was measured through immobilized labeled protein (HRP-conjugated goat anti-human IgG) on both surfaces. This surface coverage is lower than those on SPR surfaces.29 This might be caused by the different surface structures between a flat surface and NSs. However, the non-specific protein adsorption after being modified by anti-β-hCG still keeps the same level (less than 5%), which indicated no obvious interference on nonfouling properties before and after surface modification. More structural details of polyCBMA-modified NSs were also investigated by transmission electron microscopy (TEM), as shown in Figure 2. The TEM result showed that the increase in the diameter of dry polyCBMA-modified NSs is negligible compared to the diameter measured by DLS. Such a difference should come from the collapsed dry polyCBMA layers of NSs in TEM. A clear difference is the heterogeneous distribution of Fe3O4 NPs in NSs after the polyCBMA layer formed. This might be caused by the partial dissolving of Fe3O4 NPs in the SI-ATRP reaction when Fe3O4 NPs were not well-protected by the hydrophobic PS layer. Thus, porous structures on polyCBMA-modified NSs might exist, which could be a possible reason for higher non-specific protein adsorption on NSs rather than flat SPR surfaces. 13672

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polyCBMA platform. The surface modification method of NSs is rather reliable and straightforward through routine chemistry, such as the formation of polyCBMA by SI-ATRP and conjugated antibody by EDC/NHS chemistry. Meanwhile, highly sensitive IA superparamagnetic NSs are obtained through applying both advantages of bifunctional polyCBMA and uniform magnetite content. The bifunctional polyCBMA on IA NSs successfully surpassed the nonfouling protein adsorption and also covalently immobilized anti-hCG. The key properties of IA in specificity, sensitivity, and reliability are superior to those of IA NSs without polyCBMA and conventional ELISA. Their specific responses to antigen in either PBS or 50% FBS are very consistent, and the detection limit is 1 order of magnitude lower than the detection limit of conventional ELISA. In conclusion, the polyCBMA platform is more efficient and reliable than conventional PEG for biodetection and diagnosis. Figure 3. Relative responses to different hCG concentrations of IA superparamagnetic NSs with and without polyCBMA layers in PBS and 50% FBS.

3.2. Performance of IA Superparamagnetic NSs. The

responses to different hCG concentrations of IA NSs were measured by a standard sandwich method (Figure 3). The results show that the responses of IA NSs with polyCBMA in PBST solution increase linearly with the hCG concentration in a low hCG concentration range (e15 mIU/mL), which is the range to judge the early pregnancy. In contrast, the responses of NSs directly conjugated with anti-β-hCG exhibited a huge variation at a low hCG concentration range. The difference of the detection response in the range of 0
15 mIU/mL cannot be told. Furthermore, the responses in 50% FBS of IA NSs with polyCBMA showed almost identical values as those in PBST. This clearly indicates the consistency of the response of IA NSs with polyCBMA to the concentration of hCG even in very challenging biomedia (50% FBS). However, the response of IA NSs without polyCBMA is interfered by the condition change of detection. Such a reliable detection is obtained by covalent conjugation of the anti-β-hCG antibody on the nonfouling polyCBMA background because no other protein in FBS could change the density and orientation of anti-β-hCG on NSs. The detection limit of IA NSs with polyCBMA was also investigated in detail, which is about 15 ng/mL (data not shown) and 1 order of magnitude lower than the detection limit measured by the conventional enzyme-linked immunosorbent assay (ELISA) using 96-well plates in PBS when the same pair of antibodies and antigens were used. This clearly shows that highly sensitive and reliable IA NSs are obtained through surpassing the non-specific protein adsorption by the polyCBMA layer. The high value of the detection limit might be caused by the relative low quality of antibody and antigen pairs because the apparent association constant (Ka) of anti-α-hCG
HRP and hCG is on the microgram level. A much lower detection limit could be achieved if a high-quality antibody
antigen pair with Ka values on the nanogram level had been used. On the other hand, the immobilized amount of anti-β-hCG on NSs should be improved, which could also make IA NSs more sensitive.

4. CONCLUSION Three major hindrances of nanoparticles in nanosensing and nanodiagnosis, non-specific protein adsorption, low long-term stability, and easy aggregation, can be overcome by the novel

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected] (S.C.); [email protected] (H.X.).

’ ACKNOWLEDGMENT This work is supported by the National Nature Science Foundation of China (20974095, 20936005, and 21075082) and Qianjiang Talent Program (2009R10014). ’ REFERENCES (1) 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 nano-superparamagnetic beads and micro-polystyrene bead composites, ‘beads on beads’. Anal. Chim. Acta 2007, 597 (2), 331–339. (2) Hsing, M.; Xu, Y.; Zhao, W. Micro- and nano-superparamagnetic particles for applications in biosensing. Electroanalysis 2007, 19, 755–768. (3) Zhang, R.; Hirakawa, K.; Seto, D.; Soh, N.; Nakano, K.; Masadome, T.; Nagata, K.; Sakamoto, K.; Imato, T. Sequential injection chemiluminescence immunoassay for nonionic surfactants by using superparamagnetic microbeads. Anal. Chim. Acta 2007, 600, 105–113. (4) Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Quantum dot bioconjugates for imaging, labelling and sensing. Nat. Mater. 2005, 4 (6), 435–446. (5) Shi, D. L.; Cho, H. S.; Chen, Y.; Xu, H.; Gu, H. C.; Lian, J.; Wang, W.; Liu, G. K.; Huth, C.; Wang, L. M.; Ewing, R. C.; Budko, S.; Pauletti, G. M.; Dong, Z. Y. Fluorescent polystyrene
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