A reusable piezoelectric immunosensor using antibody-adsorbed magnetic nanocomposite

Journal of Immunological Methods 332 (2008) 103 – 111 www.elsevier.com/locate/jim

Research paper

A reusable piezoelectric immunosensor using antibody-adsorbed magnetic nanocomposite Yun Zhang, Hua Wang ⁎, Bani Yan, Yuwei Zhang, Jishan Li, Guoli Shen ⁎, Ruqin Yu State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China Received 30 September 2007; received in revised form 21 November 2007; accepted 21 December 2007 Available online 28 January 2008

Abstract This paper reports a simple, sensitive, and reusable piezoelectric immunosensor using magnetic hydroxyapatite (HAP)/γ-Fe2O3/ Au nanocomposite. Use of porous HAP nanocrystals embedded with γ-Fe2O3 and colloidal gold nanoparticles resulted in a multifunctional HAP/γ-Fe2O3/Au nanocomposite. Under optimized conditions, the biocompatible nanocomposites were exploited for direct adsorption of large quantities of rabbit anti-human immunoglobulin G antibodies (anti-hIgG) with well-preserved immunoactivity. In a homogeneous bulk solution, the hIgG analytes were captured by the anti-hIgG-derivatized immunocomposites followed by magnetic separation/enrichment onto a bovine serum albumin (BSA)-sealed QCM probe before measuring. This QCM immunosensor can quantitatively determine concentrations of hIgG ranging from ~ 20 to 800 ng/ml, with a detection limit of ~ 15 ng/ ml. Moreover, regeneration of the immunosensor can be simply realized by canceling the controllable magnetic field. With the possibility of performing the analysis automatically and considering its ease of use, high sensitivity, and good reusability, this magnetic separation-assisted QCM immunosensor may have great potential to be further tailored as a general and promising alternative for a broad range of practical applications. © 2008 Elsevier B.V. All rights reserved. Keywords: Piezoelectric immunosensor; Biomolecule immobilization; Hydroxyapatite; Multifunctional composite; Magnetic separation

1. Introduction

Abbreviations: HAP, hydroxyapatite; hIgG, human immunoglobulin G; anti-hIgG, rabbit anti-human immunoglobulin G antibodies; pIgG, pig immunoglobulin G; cIgG, cavy immunoglobulin G; BSA, bovine serum albumin; PBS, phosphate-buffered saline; TMAOH, tetramethylammonium hydroxide; QCM, quartz crystal microbalance; EDS, energy-dispersive spectroscopy; TEM, transmission electron microscope; SD, standard deviation. ⁎ Corresponding authors. Tel./fax: +86 731 8821355. E-mail addresses: [email protected] (H. Wang), [email protected] (G. Shen). 0022-1759/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2007.12.019

Immunosensors, which exploit the coupling of highly specific recognition events between antibodies and antigens to appropriate transducers, have been used in many applications in recent decades as an alternative immunoassay technique (Luppa et al., 2001; Franek and Hruska, 2005). The primary challenge in developing such an analytical device is the choice of an appropriate immobilization method for biomolecules to fabricate a specific biorecognition layer with desirable properties (i.e., large loading, well-preserved bioactivity and good reversibility).

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The general methods mainly involve covalent binding of biomolecules to transducer surfaces pre-activated with functional films by using linkage reagents. Of the functional films available, self-assembled monolayers (SAMs) of sulfur-containing molecules have been widely explored, as they allow for controlled and stable biorecognition layers on metal substrates (e.g., platinum, silver and principally gold) (Ulman, 1996; Fung and Wong, 2001; Frederix et al., 2003). However, SAM-based covalent methods may inherently suffer from the main disadvantage associated with limited loading and low bioactivity, of biomolecules immobilized on planar transducers. In recent years, various nanomaterials with unique physical and chemical properties have been widely applied to achieve improved immobilization of biomolecules. Some research endeavors have led to development of immobilization methods combining nanomaterials with covalent binding procedures (Tang et al., 1992; Li et al., 2003; Wang et al., 2004a,b, 2005b; Okunoa et al., 2007). Although these techniques enable relatively large loading, they are still inconvenient and complicated due to the requirement for pre-activation of the nanomaterial surfaces. Moreover, covalent coupling can cause loss of bioactivity which in turn can decrease the binding efficiency of the analytes. Alternatively, efforts have been made to adsorb bioelements to biocompatible materials such as colloidal gold. A number of immobilization procedures using gold nanoparticles have proved to be capable of retaining high bioactivity of the adsorbed biomolecules (Li et al., 2005; Wu et al., 2005; Zhang et al., 2007). Nevertheless, time-consuming pre-treatments were required to assemble the gold nanoparticles onto the transducer surfaces. In addition, the irreversible chemisorption of the biorecognition layer might make the regeneration of the sensor practically impossible. Multifunctional composite material may offer a promising alternative. This composite material has not only the inherited advantages from the component materials, but also improved properties due to “synergic action” (Cai et al., 2006). Encouraged by the recent success of hydroxyapatite-colloidal gold (HAP/Au) nanocomposites for efficient immobilization of biomolecules (Ding et al., 2007), we tried to prepare paramagnetic HAP/γFe2O3/Au nanocomposites using both colloidal gold and γ-Fe2O3 nanoparticles. HAP, a porous biomaterial with good biocompatibility and particular multi-adsorbing sites, has been successfully applied in protein chromatography and biomedical implants (Narasaraju and Phebe, 1996; Luo and Joseph, 1998; Wang et al., 2004a,b). Compared with pure HAP, the HAP/Au nanocomposite shows greatly improved dispersity and higher surface area, enabling more efficient immobilization of biomole-

cules and may find broader bioapplications (Ding et al., 2007). The magnetic separation technique has been widely used for purification of cells (Shimazaki et al., 1988), proteins (Haukanes and Kvam, 1993) and nucleic acids (Xu et al., 2005), and design of the regenerative biosensing interface (Li et al., 2003; Wang et al., 2005b). In this paper, a simple, sensitive, and reusable immunosensor was developed by combining the magnetic separation technique with quartz crystal microbalance (QCM) measurement. Human immunoglobulin G (hIgG) was chosen as a model analyte. First, γ-Fe2O3 and gold nanoparticles were assembled onto the porous HAP surface to fabricate HAP/γ-Fe2O3/Au nanocomposites for direct adsorption of rabbit-anti-hIgG antibodies (anti-hIgG), followed by capture of hIgG in a homogeneous bulk solution. Subsequently, the immunocomplexes formed were introduced into a laboratory-made detection vessel for magnetic separation-based QCM measurement, where the regeneration of the immunosensor could be accomplished by canceling the magnetic field (schematically illustrated in Fig. 1). The newly prepared nanomaterials were characterized using transmission electron microscopy (TEM) and energy-dispersive spectroscopy (EDS). The experimental variables, including pH, ionic strength, and adsorption time, were optimized for adsorption immobilization of anti-hIgG. The real-time frequency response and detection characteristics of the QCM immunosensor for hIgG were further studied in detail. 2. Materials and methods 2.1. Reagents and materials Human immunoglobulin G (hIgG), rabbit-anti-hIgG antibody (anti-hIgG), pig immunoglobulin G (pIgG), cavy immunoglobulin G (cIgG) and bovine serum albumin (BSA) were obtained from Beijing Dingguo Biotech. Co. Ltd. (Beijing, China). Phosphate-buffered saline (PBS) solutions at various pH values were prepared using 0.01 M Na2HPO4 and 0.01 M KH2PO4. Hot “Piranha” solution is a 7/3 mixture of concentrated H2SO4 and 30% H2O2 (caution: piranha reacts violently with organic compounds!). All other reagents were of analytical grade. Ultrapure water (electric resistance N 18.3 MW) was used throughout the experiments. 2.2. Apparatus Shape characterization and energy-dispersive spectroscopic (EDS) analysis were performed on a high-resolution transmission electron microscope (TEM, JEM 3010) supplied by Japan Electron Optics Laboratory Co. Ltd. (Tokyo,

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Fig. 1. Schematic representation of the magnetic separation-based QCM measurement, including (a) the introduction of the immunocomplex-coated HAP/γ-Fe2O3/Au nanocomposites, (b) the magnetic separation/enrichment of the magnetic matrix onto the BSA-modified crystal probe, and (c) the regeneration of the contaminated detection system for the next run.

Japan). The peristaltic power pump was purchased from Haitian Electron Instruments Ltd. (Zhejiang, China). The ultrapure water system was provided by Barnstead/ Thermolyne Co. (Dubuque, USA). The quartz crystal analyzer (QCA, 922) used for monitoring sensor responses was obtained from Princeton Applied Research (Oak Ridge, USA). The AT-cut quartz crystal microbalance (QCM, 9 MHz, gold electrodes), each side of which was further sealed with a silicone O-ring, were the products of Chenxing Radio Equipment (Beijing, China). A laboratory-made detection vessel with a test chamber measuring 1 cm3 was designed as reported elsewhere (Wang et al., 2005b), in which the prepared QCM probe modified with bovine serum albumin (BSA) was hermetically held in place. 2.3. Preparation of nanomaterials 2.3.1. Preparation of HAP nanocrystal HAP nanocrystals were synthesized according to the method reported by Wang et al. (2004a,b) with some minor modifications. Briefly, 0.5 M Ca(NO3)2·4H2O (pH 10, 150 ml) and 0.5 M (NH4)2·HPO4 (pH 10, 20 ml) prepared in 25% ethanol solution and ultrapure water, respectively, were mixed under vigorous stirring. Then, 70 ml of 0.5 M (NH4)2·HPO4 (pH 10) was added dropwise into this mixture over 30 min, and the total molar ratio between Ca and P was adjusted to 1.67. During the addition of (NH4)2·HPO4, the pH of the reaction system was controlled at about 10 by using concentrated ammonia. After another 2 h, the resultant

milky suspension was aged for 24 h without stirring. The precipitate was isolated by centrifuge (6000 rpm), washed with ultrapure water and ethanol, and then dried at 90 °C. 2.3.2. Preparation of γ-Fe2O3 nanoparticle Paramagnetic γ-Fe2O3 nanoparticles were prepared according to the method reported by Lyon et al. (2004). Briefly, a mixture of 0.4 M FeCl2·4H2O and 0.8 M FeCl3·6H2O prepared in 10 mM HCl (25 ml) was added dropwise to 1.5 M NaOH solution (250 ml) with vigorous mechanical stirring. The black precipitate that formed immediately was washed gradually with 0.1 M HNO3 (250 ml) and 0.01 M HNO3 (250 ml), each for 30 min. The suspension obtained was heated at 90 °C for 30 min until the color changed to brown–red, and then cooled to room temperature. The precipitate was isolated using a permanent magnet, washed with ultrapure water, and suspended in 0.1 M tetramethylammonium hydroxide (TMAOH, 250 ml). 2.3.3. Preparation of colloidal gold Colloidal gold was prepared according to the method reported by Turkevick et al. (1951). In a 250-ml roundbottom flask, 0.01% HAuCl4·4H2O solution (95 ml) was brought to boil with mechanical stirring. To this solution 1% sodium citrate solution (5 ml) was rapidly added. The mixture turned deep blue and subsequently salmon pink within 1 min. Boiling was continued for an additional 10 min, then the colloid was cooled to room temperature with continued stirring and stored at 4 °C.

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2.3.4. Preparation of HAP/γ-Fe2O3/Au nanocomposite Some HAP powder (5 mg) was dispersed in 50 μg/ml γ-Fe2O3 suspension (pH 7, 10 ml) and sonicated for 30 min. After isolation with a permanent magnet, the precipitate was washed with ultrapure water, and dispersed in a colloidal gold suspension (pH 7, 10 ml). This mixture was then sonicated for 30 min. After isolation, HAP/γ-Fe2O3/Au nanocomponents with satisfactory dispersity were obtained. The nanocomponents were washed with ultrapure water and dispersed in PBS solution containing 40 mM NaCl (pH 7.7, 10 ml). 2.4. Preparation of anti-hIgG-coated HAP/γ-Fe2O3/Au nanocomposite The anti-hIgG-coated HAP/γ-Fe2O3/Au nanocomposites were prepared by adding 0.2 mg/ml anti-hIgG (1 ml) into 0.5 mg/ml nanocomposite suspension containing 40 mM NaCl (pH 7.7, 10 ml) at room temperature. After 2.5 h, the precipitate was isolated and suspended in 10 mg/ml BSA solution containing 40 mM NaCl (pH 7.7, 1 ml) for another 2.5 h, followed by several rinses with PBS solution. Finally, the immunocomposites were suspended in PBS solution containing 40 mM NaCl (pH 7.7, 1 ml) and stored at 4 °C. 2.5. Magnetic separation-assisted QCM measurement Immunoassays for hIgG were carried out using a laboratory-made QCM detection vessel; a schematic representation of the detection principle is shown in Fig. 1. The detection vessel with a BSA-modified QCM probe was first installed. Then, a test PBS solution containing 40 mM NaCl (pH 7) was added and driven by a peristaltic power pump resulting in a circular flow. Meantime, the anti-hIgG-coated HAP/γ-Fe2O3/Au nanocomposite suspension was pre-reacted with each of the hIgG samples at different concentrations at 37 °C for 30 min. After frequency stabilization of the probe, the immunocomplex suspension formed was introduced into the flow system and circulated with the test solution to be magnetically separated (enriched) onto the probe surface with a permanent magnet. After each run, the permanent magnet was removed and the used test solution was discharged. Subsequently, a fresh PBS solution was introduced to regenerate the entire contaminated system until the permissible frequency recovery of the QCM probe. The control test using only anti-hIgG-coated nanocomposites was conducted in the same way. The frequency produced was further subtracted from that of the test solution to define the real immunosensing signal.

2.6. Content analysis According to Sauerbrey's pioneering equation (Sauerbrey, 1959) and the equation derived by Wang et al. (2005a), for a 9-MHz AT-cut QCM with a constant frequency of 167 kHz, density of quartz of 2.65 g/cm3 and a surface area of 0.28 cm2, the biomolecule content of carriers, which is assumed to be the molecular number per gamma (c, n/μg), can be calculated: c ¼ 9:21  1014

DF Mm

ð1Þ

where ΔF is the maximal frequency change, M is the molar mass of the biomolecule (the molecular weight of hIgG was assumed to be 150 kDa), and m is the mass of the carriers (the mass of anti-hIgG-coated HAP/γ-Fe2O3/Au nanocomposites was assumed to be 5 μg). 3. Results and discussion 3.1. Preparation and characterization of the HAP/γFe2O3/Au nanocomposites As mentioned above, multifunctional composite material has more advantages than its component materials due to “synergic action”. Porous HAP, a well-known biomaterial with great biocompatibility and adsorbability, has found wide practical application (Narasaraju and Phebe, 1996; Luo and Joseph, 1998; Wang et al., 2004a,b). But the bioapplication of pure HAP is limited due to its unsatisfactory dispersity in aqueous solution. In this work, HAP as a support was assembled with γ-Fe2O3 and gold nanoparticles to fabricate multifunctional HAP/γ-Fe2O3/ Au nanocomposites with improved properties. Typical TEM images of the prepared nanomaterials are shown in Fig. 2. Most of the pure HAP crystals showed unique uniform rod-like nanostructures (Fig. 2a). EDS analysis further indicated the formation of HAP; the molar ratio of Ca and P was calculated to be 1.66 (Fig. 2a, insert). This value was approximately equal to the natural ratio of 1.67 in human and animal bone and teeth. As shown in Fig. 2b, the HAP matrix has been doped with γ-Fe2O3 and gold nanoparticles with estimated diameter of ~10 nm and ~20 nm, respectively. Compared with pure HAP, the assembled gold nanoparticles conferred improved dispersity, higher surface area and greater adsorption capacity on the HAP/γ-Fe2O3/Au nanocomposites (Ding et al., 2007). Thus the nanocomposites were able to adsorb biomolecules in large loading with wellpreserved bioactivity. Paramagnetic γ-Fe2O3 nanoparticles could further allow the nanocomposites to be used in

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Fig. 2. TEM images of (a) the HAP nanocrystals and (b) the magnetic HAP/γ-Fe2O3/Au nanocomposites. Insert shows the EDS data for (a). Scale bar, 50 nm.

immunomagnetic separation/enrichment analysis and in the design of a regenerative biosensing interface.

3.3. Dosage of the anti-hIgG-coated HAP/γ-Fe2O3/Au nanocomposites

3.2. Optimization of the anti-hIgG immobilization

Anti-hIgG-coated HAP/γ-Fe2O3/Au nanocomposite suspensions at various concentrations were used to investigate the effects of immunocomposite dosage on the proposed QCM immunosensor (Fig. 6). The frequency changes in the hIgG immunoassays peaked at a nanocomposite concentration of 0.5 mg/ml, denoting the recommended dosage for the experiments. It was observed

HAP surfaces are abundant in positive as well as negative charge ions (i.e., Ca2+, PO43− , and OH−), which may influence biomolecule adsorption (Luo and Joseph, 1998). We tried to optimize the experimental conditions for anti-hIgG immobilization on HAP/γ-Fe2O3/Au nanocomposite, including pH values, ionic strength and adsorption time. Fig. 3 shows the effects of pH values of the adsorption solution on anti-hIgG immobilization. pH has an important effect on anti-hIgG immobilization. The largest frequency change was obtained with the PBS solution at pH 7.7, suggesting this is the optimum value. The optimum ionic strength for anti-hIgG immobilization was also investigated (Fig. 4). The frequency shifted from 345 to 540 Hz when the NaCl concentration increased from 10 to 40 mM. Thus, the NaCl concentration of 40 mM was chosen for the experiments. The results indicate that the appropriate pH and ionic strength in the PBS medium might lead to the stable surface charge characteristics of the HAP/γ-Fe2O3/Au nanocomposite necessary for reliable anti-hIgG immobilization. In addition, the effects of adsorption time were studied (Fig. 5). The optimum adsorption time for saturated antihIgG adsorption was found to be 2.5 h, when a plateau appears in the frequency change. This indicates that all the active adsorbing sites on the nanocomposite surface might have been completely held by anti-hIgG. Thus, the adsorption time of 2.5 h was chosen for anti-hIgG immobilization.

Fig. 3. The pH-dependence of adsorption of anti-hIgG onto the magnetic HAP/γ-Fe2O3/Au nanocomposites; each of the 0.5 mg/ml nanocomposite suspensions at various pH values containing 40 mM NaCl was exposed in 0.2 mg/ml anti-hIgG solution for 2.5 h. The resultant suspension was reacted with the 500 ng/ml hIgG sample at 37 °C for 30 min, followed by the magnetic separation-assisted QCM measurement as described in Fig. 1.

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Fig. 4. The ion strength-dependence of adsorption of anti-hIgG onto the magnetic HAP/γ-Fe2O3/Au nanocomposites under the experimental conditions described in Fig. 3, except for various NaCl concentrations (CNaCl) in 0.5 mg/ml nanocomposite suspension (pH 7.7).

that if the nanocomposite concentration was too high, either very little or very large change in frequency occurred (data not shown). There might be two reasons for the abnormal frequency responses of the immunosensor. First, the total amount of magnetic immunocomplexcoated nanocomposites collected on the QCM probe might be too large to deal with the mass loading capacity

Fig. 5. The adsorption time-dependence of adsorption of anti-hIgG onto the magnetic HAP/γ-Fe2O3/Au nanocomposites under the experimental conditions described in Fig. 3, except for various adsorption times in 0.2 mg/ml anti-hIgG solution (pH 7.7).

Fig. 6. The dosage-dependence of the magnetic separation-assisted QCM immunoassay under the experimental conditions described in Fig. 3, except for various anti-hIgG loaded HAP/γ-Fe2O3/Au nanocomposite concentrations in PBS solution (pH 7.7).

of the crystal. Second, excessive increase in the surface loading on the QCM probe could inherently influence the characteristics of the interface (e.g., surface free energy and viscoelasticity). 3.4. Response characteristics of the magnetic separationassisted QCM immunosensor The incorporation of the QCM measurement and selective immunomagnetic separation resulted in a reusable immunosensor with some highlighted properties (Wang et al., 2005b). The real-time frequency response characteristics of the magnetic separation-assisted QCM immunosensor in one run were investigated under optimized conditions (Fig. 7). Each addition into the system produces a decrease in frequency response change, then the curve flattens and no further decrease is expected. The biggest decrease in frequency response occurred within 3 min after the addition of the immunocomplex-coated HAP/γ-Fe2O3/Au nanocomposites. This was attributed to a mass of magnetic matrix that had been rapidly and magnetically collected onto the BSA-modified QCM probe surface under the magnetic field. When the simple regeneration procedure was carried out, recovery of the original frequency was achieved as shown at the end of the curve. The sensing system can then be used for the next run. The total analytical time for one run was estimated to be ~35 min. A short incubation time of 30 min at 37 °C for mixing anti-hIgG-adsorbed nanocomposites with hIgG sample,

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spectively. In addition, 0.5 mg/ml BSA, pIgG and cIgG were used to evaluate the effect of non-specific binding. The average frequency changes introduced were 35 Hz, 22 Hz and 27 Hz, respectively. This indicated that the frequency change of the QCM immunosensor resulted from the specific antibody–antigen reactions, and nonspecific binding was negligible. These results clearly show the excellent performance of the QCM immunosensor, i.e., high analytical sensitivity, high selectivity and good reproducibility. 3.6. Reusability of the immunosensor

Fig. 7. Typical characteristics of real-time frequency responses of the circular magnetic separation-assisted QCM immunosensor to (a) the addition of the test PBS solution, (b) the addition of the immunocomplex-loaded magnetic HAP/γ-Fe2O3/Au nanocomposite suspension, (c) the removal of the permanent magnet and the outpour of the used PBS solution, (d) addition of the regenerating PBS solution, and (e) addition of the fresh test PBS solution.

would lead to the completion of the antibody–antigen immunoreaction. This may be in agreement with the fact that the exposed structures of the nanocomposites could provide the adsorbed anti-hIgG with a good degree of flexibility and accessibility to hIgG. Suspension of the anti-hIgG-adsorbed nanocomposites in homogeneous solution might also offer the advantage of a rapid diffusion rate, favoring rapid antibody–antigen reaction.

The reusability of an immunosensor is a significant criterion for the development of an immunoassay technique with practical possibilities. In this work, the regeneration of the QCM immunosensor could be easily accomplished as follows. After each run, the permanent magnet was first removed from the top of the detection vessel. The immunocomplex-coated HAP/γ-Fe2O3/Au nanocomposites deposited on the QCM probe surface were then discharged along with the used test solution flowing out of the detection system. Finally, fresh PBS solution was added to wash the system until recovery of the probe's original frequency was achieved. The QCM immunosensor regenerated in such a way is then ready for the next run. The desquamation of the magnetic matrix from the probe surface could be accelerated by placing the

3.5. Quantitative analysis The quantitative analysis capabilities of the QCM immunosensor were investigated under the optimized conditions using a series of hIgG samples with various analyte concentrations. Fig. 8 shows the calibration curve describing the relationship between the frequency change (ΔF) of immunoreactions and hIgG concentration. It was found that the linear detection range of hIgG concentrations was ~20–800 ng/ml. The detection limit was estimated to be ~15 ng/ml, according to the 3 standard deviation (SD) rule. The hIgG content of the anti-hIgGcoated HAP/γ-Fe2O3/Au nanocomposites, defined as the molecular number per gamma (c, n/μg), was also evaluated. From Fig. 8, the maximal ΔF value was found to be ~810 Hz. Thus, the hIgG content of the nanocomposites was estimated to be ~1.0 × 1012/μg according to Eq. (1). The reproducibility of the immunosensor was investigated and the average relative standard deviations (RSD) of intra-assay, inter-assay and day-to-day assay were 10.5% (n = 5), 11.9% (n = 4) and 12.1% (n = 4), re-

Fig. 8. The calibration curve showing the relationship between the frequency shifts (ΔF) of the hIgG-anti-hIgG immunoreactions and the varying hIgG concentrations under the optimized experimental conditions. Each data point represents the average frequency response of triplicate measurements.

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magnet under the vessel for a reverse magnetic field. Therefore, the regeneration performance could be improved (data not shown). Otherwise, the contaminated probe was uninstalled from the analytical vessel, treated with hot “Piranha” solution and washed with ultrapure water. A “complete” regeneration of the QCM immunosensor was thus realized and leaving it ready for new test runs after addition of BSA. 4. Conclusions A simple, sensitive, and reusable piezoelectric immunosensor has been successfully fabricated, based on biomolecules directly adsorbed on paramagnetic HAP/ γ-Fe2O3/Au nanocomposites. This new single-step immobilization strategy offers several distinct advantages over the common methods. First, the porous HAP/γFe2O3/Au nanocomposites provide a large loading capacity of biomolecules with well-preserved bioactivity. Second, the recognition biomolecules immobilized on the nanocomposites with exposed structure possess a good degree of flexibility and accessibility to the analyte. The suspension of the immunocomposites in bulk solution also offers an advantage associated with a fast diffusion rate favoring rapid antigen–antibody interaction. Third, the direct adsorption procedure is simple and rapid, thus avoiding the need for chemical linkers for pre-functionalization of the transducer surface. Finally, the paramagnetic properties of the biomoleculecoated nanocomposites enables a regenerative biosensing interface via a controllable magnetic field. The magnetic separation-assisted QCM immunosensor shows excellent analytical performance, e.g. high sensitivity, good selectivity, and good reproducibility and reusability. It may have further potential to be tailored as a general and promising alternative for many applications. Further research work is now underway in our group. Acknowledgments This work was supported by the NNSF of China (Nos. 20775020, 20435010), and “973” National Basic Research Program of China (No. 2007CB310500). References Cai, W.Y., Xu, Q., Zhao, X.N., Zhu, J.J., Chen, H.Y., 2006. Porous gold-nanoparticle-CaCO3 hybrid material: preparation, characterization, and application for horseradish peroxidase assembly and direct electrochemistry. Chem. Mater. 18, 279. Ding, Y.J., Liu, J., Wang, H., Shen, G.L., Yu, R.Q., 2007. A piezoelectric immunosensor for the detection of a-fetoprotein using an interface of gold/hydroxyapatite hybrid nanomaterial. Biomaterials 28, 2147.

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