Biosensors and Bioelectronics 66 (2015) 559–564

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Magnetic bead and gold nanoparticle probes based immunoassay for β-casein detection in bovine milk samples Y.S. Li a,1, X.Y. Meng a,1, Y. Zhou a,n, Y.Y. Zhang a, X.M. Meng b, L. Yang a, P. Hu a, S.Y. Lu a, H.L. Ren a, Z.S. Liu a, X.R. Wang a a b

Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun 130062, PR China Grain and Oil Food Processing Key Laboratory of Jilin Province, Jilin Business and Technology College, Changchun 130062, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 19 September 2014 Received in revised form 27 November 2014 Accepted 8 December 2014 Available online 9 December 2014

In this work, a double-probe based immunoassay was developed for rapid and sensitive determination of β-casein in bovine milk samples. In the method, magnetic beads (MBs), employed as supports for the immobilization of anti-β-casein polyclonal antibody (PAb), were used as the capture probe. Colloidal gold nanoparticles (AuNPs), employed as a bridge for loading anti-β-casein monoclonal antibody (McAb) and horseradish peroxidase (HRP), were used as the amplification probe. The presence of β-casein causes the sandwich structures of MBs-PAb–β-casein-McAb–AuNPs through the interaction between β-casein and the anti-β-casein antibodies. The HRP, used as an enzymatic-amplified tracer, can catalytically oxidize the substrate 3,3′,5,5′-tetramethylbenzidine (TMB), generating optical signals that are proportional to the quantity of β-casein. The linear range of the immunoassay was from 6.5 to 1520 ng mL  1. The limit of detection (LOD) was 4.8 ng mL  1 which was 700 times lower than that of MBs-antibody-HRP based immunoassay and 6–7 times lower than that from the microplate-antibody-HRP based assay. The recoveries of β-casein from bovine milk samples were from 95.0% to 104.3% that had a good correlation coefficient (R2 ¼0.9956) with those obtained by an official standard Kjeldahl method. For higher sensitivity, simple sample pretreatment and shorter time requirement of the antigen–antibody reaction, the developed immunoassay demonstrated the viability for detection of β-casein in bovine milk samples. & 2014 Published by Elsevier B.V.

Keywords: Probe Immunoassay β-casein Bovine milk

1. Introduction Protein content is one of particularly important milk quality parameters for characterizing its nutritional value (Kucheryavskiy et al., 2014). Casein (CN) is the main protein component of bovine milk which account for approximately 80% of the total milk protein content (Choi et al., 2011). There are four kinds of CNs in bovine milk and the concentration of each CN is consistent, namely αs1-, αs2-, β-, and κ-CNs in the ratios of 37%, 10%, 37% and 10%, respectively (Johansson et al., 2009). Therefore, each CN could be used as an index to evaluate the quality of bovine milk. Various analytical techniques have been proposed for determination of individual CN content, such as electrophoresis for κ-CN (Kaminaridesa and Koukiassa, 2002), liquid chromatography for β-CN (1–25) (Gaucheron et al., 1995), surface plasmon resonance (SPR) measurement for β-CN (Muller–Renaud et al., 2004) and αs1-CN (Muller-Renaud et al., 2005), reverse-phase highn

Corresponding author. E-mail address: [email protected] (Y. Zhou). 1 Both authors contributed equally to the preparation of this manuscript.

http://dx.doi.org/10.1016/j.bios.2014.12.025 0956-5663/& 2014 Published by Elsevier B.V.

performance liquid chromatography, hydrophobic interaction chromatography and ion-exchange chromatography for αs1-, αs2-, β-, and κ-CNs (Bonfatti et al., 2008; Bramanti et al., 2003; Holland et al., 2010). Although these techniques are reliable for determination of CNs, they require expensive instruments and specific technical skills for inconvenient sample pre-treatment processes. Immunoassays based on antibody are sensitive and high throughput procedure for quantitative detection and also successfully applied to measurement of milk protein content, such as enzyme-linked immunosorbent assay (ELISA) for αs1-CN (Black and Reynolds, 1998) and β-CN (Song et al., 2011; Zhou et al., 2013), immunomagnetic beads-based immunoassay for β-CN (Song et al., 2014), and nephelometric immunoassay for αs-CN and κ-CN (Collard-Bovy et al., 1991). Due to having high surface to volume ratio, nanoparticle-based sensing provides a comparably large surface area available for reaction within a small sample volume. On the other hand, the accelerated analyte species transport to the nanoparticle surface significantly reduce the time that is required to perform a measurement (Katelhon and Compton, 2014). Magnetic beads (MBs) can be easily separated from the reaction mixtures with a magnet and re-dispersed immediately following removal of the magnet

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(Wei et al., 2012) which allow for a nearly ‘in solution’ reaction (Kim et al., 2009), leading to shorter reaction times (Song et al., 2014). Various protocols based on MBs have been developed in a variety of research fields, such as environment monitoring (Schreier et al., 2012), clinical diagnosis (Eguilaz et al., 2010; Wei et al., 2012) and food safety (Xu et al., 2012). For having high surface areas, high chemical stability and unique size-dependent optical properties, colloidal gold nanoparticles (AuNPs) can be conjugated with more number of biomolecules for synthesis of probe to improve detection sensitivity (Jia et al., 2009), such as DNAs (Orza et al., 2010), antibodies (Omidfar et al., 2011) and enzymes (Zhou et al., 2012). In this study, MBs were immobilized with anti-β-CN polyclonal antibody (PAb) as the capture probe. In the probe, MBs were as carrier for PAb allowing easily manipulation of the antibody for improving the kinetic of the antibody–antigen immunointeraction. AuNPs were immobilized with anti-β-CN monoclonal antibody (McAb) and horseradish peroxidase (HRP) as the amplification probe to improve detection sensitivity with the assumption that the interference of sample matrix could be neglected by diluting the sample. As shown in Fig. 1, in the presence of β-CN, it was firstly captured by MBs probe. Followed by adding of AuNPs probe, the analyte was directly recognized by the McAb immobilized on AuNPs and detected by the amplified colorful products produced by the catalyzing oxidation of HRP and 3,3′,5,5′-tetramethylbenzidine (TMB). The absorbance value of the colorful products is proportional to the concentration of the target.

2. Materials and methods 2.1. Chemicals and apparatus

β-CN, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), horseradish peroxidase (HRP), 3,3′,5,5′-tetramethylbenzidine (TMB), and N-hydroxysuccinimide (NHS) were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Activated HRP (type B) kits and MBs (350 nm in diameter) were obtained from Tai Tianhe (Beijing, China) and Wa Wasaina (Wu Han, China), respectively. Anti-β-CN McAb and PcAb were produced in our previous study (Zhou et al., 2013). All reactions were carried out in 96-well polystyrene microtiter plates (Stripwell plate 2592, Costar, Changchun, China). The absorbance value was read with a MK3 microplate reader (Thermo, Shanghai, China). 2.2. Solutions and buffers Dilution buffer [phosphate buffered saline (PBS)], 10 mmol L  1 sodium phosphate buffer (pH 7.4) containing 140 mmol L  1 NaCl; PBST solution, PBS containing 0.05% (v/v) Tween 20; TMB solution, 50 mmol L  1 sodium citrate buffer (pH 5.0) containing 0.01% (w/v) TMB and 0.005% (v/v) H2O2. Distilled water was used throughout the experiments. 2.3. Preparation of MBs probe The MBs (400 μL, 10 mg mL  1) were firstly activated by using the mixture solution of EDC (300 μL, 50 mg mL  1) and NHS

Fig. 1. Synthesis illustration of MBs and AuNPs probes (A). Principle of the double-probe based immunoassay for rapid and sensitive detection of β-CN (B). The details are described in Section 2.5.

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(300 μL, 50 mg mL  1). After incubation for 30 min at room temperature, the mixture solution was washed thrice with PBST solution. Under slight stirring, 800 μg anti-β-CN PAb was added and incubated overnight at room temperature. Then the MBs were washed thrice with PBST to remove the unbonded PAb by magnetic separation process. The non-specific sites on MBs were blocked by using 2% BSA solution for 30 min at room temperature. The obtained MBs probes were stored at 4 °C for further use. 2.4. Preparation of AuNPs probe AuNPs (20 nm mean diameter) were prepared according to the method by the researchers themselves (Zhou et al., 2009).

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The dispersity and diameters of AuNPs were checked by a transmission electron microscope (TEM, H-7650). The pH of AuNPs solution for anti-β-CN McAb and HRP immobilization was adjusted to 8.3 with 0.1 mol L  1 K2CO3 and then filtered through a 0.22 mm cellulose nitrate filter to remove any floating aggregates. Under gentle stirring, anti-β-CN McAb (20 mL, 1 mg mL  1) and HRP (80 mL, 1 mg mL  1) were mixed and then added into 10 mL of the AuNPs solution. After incubation for 20 min, the mixture solution was centrifuged at 10,100g for 30 min to remove unconjugated protein molecules. The obtained probes were resuspended in 10 mL of PBS solution (containing 1% BSA) and stored at 4 °C for use.

Fig. 2. TEM images of the bare MBs (A) and the synthesized MBs probe (B). (C) The relationship between the absorbance value and the amount of PAb immobilized on the surface of MBs. TEM images of the bare AuNPs (D) and AuNPs probe (E). (F) DLS analysis of the bare AuNPs before (left) and after (right) the modification process. (G) The relationship between the absorbance value and the ratio of HRP to McAb. (H) Calibration curve for the determination of β-CN (a) and detection curve obtained from calibration curve (b). Each point represents the mean 7 standard deviation from three determinations (n¼3). (I) Selectivity of the assay. Absorbance value (a) and color (b) changes of the result upon addition of 0.5 mg of β-CN and other proteins, respectively. (1) β-CN; (2) αs1-CN; (3) αs2-CN; (4) BSA; (5) OVA; (6) α-lactalbumin; and (7) κ-CNs. The absorbance value was obtained by using a MK3 microplatereader. Data represent mean 7 SD (n¼3). (J) The stability of the probes. No loss of activity was observed within 6 weeks preserved at 4 °C. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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2.5. Procedures of the assay The immunoassay was based on MBs probe and AuNPs probe (Fig. 1A). The procedures of the assay are as follows (Fig. 1B): Aliquots of 50 μL MBs probe (1 mg mL  1) were added into microtiter plate well and mixed with 100 μL of sample solution. After 10 min incubation at 37 °C, the plate was positioned on the magnet for 5 s and the supernatant was discarded. Subsequently, the conjugations of MBs probe and β-CN were washed with 200 μL of washing buffer for 3 times to remove unbound β-CN and other substances. Then 100 μL of AuNPs probe was added into the well and incubated for 10 min at 37 °C. After another washing step, 100 μL TMB solution was added and incubated for 5 min at 37 °C. The reaction was terminated by 50 μL of 2 M H2SO4 and the absorbance was measured at 450 nm wavelength with a MK3 microplate reader. The calibration curve was obtained by plotting values of positive/negative (P/N) against the logarithm of β-CN concentration. 2.6. Application study To investigate the applicability of the assay, three brand bovine milk samples were purchased from local supermarket and fortified with β-CN at the concentration of 10, 30 and 50 mg mL  1. After being diluted to 1  105 times with bidistilled water, the concentration of β-CN was detected by the developed assay and the results were standardized by the officially recognized standard Kjejdahl method (Kamizake et al., 2003).

3. Results and discussion 3.1. Synthesis confirmation of MBs probe The MBs probe, synthesized by modification of MBs with anti-

β-CN PAb, was first confirmed by the transmission electron mi-

croscopy (Fig. 2A, and B). After modification with PAb, the average diameter of the composite microspheres slightly increases from 350 nm to 365 nm. The results indicate that the immobilized MBs were uniform and well-distributed. In the developed assay, the MBs are as solid support for anti-β-CN PAb allowing easily manipulation of the antibody for improving the kinetic of the immunoreactions (Tudorache et al., 2008), subsequently leading to a shorter reaction time of the assay (Song et al., 2014). Therefore, the amount of PAb immobilized on the MBs should be optimized. As shown in Fig. 2C, the absorbance value increased with the higher amount of PAb immobilized on the surface of MBs. The highest absorbance value was observed when the amount of PAb was 400 μg and higher amount could not increase the absorbance value. Therefore, 400 μg was selected as the optimal amount. The amount of MBs probe in the detection system was optimized by using 10, 30, and 50 μL MBs probe (1 mg mL  1). After 5 min substrate incubation, 50 μL of MBs probe provided appropriate absorbance values (about 1.0). Higher amounts of the MBs probe were not tested because they would have implied the use of higher immunoreaction substance concentrations and consequently higher limit of detections (Reverte et al., 2013). 3.2. Synthesis and characterization of AuNPs probe The AuNPs probe was synthesized by immobilization of anti-βCN McAb and HRP based on the electrostatic and hydrophobic interactions of the proteins and AuNPs (Cheung-Lau et al., 2014). The immobilization was confirmed by TEM images and dynamic light scattering (DLS) analysis. The existence of the protein halo around the AuNPs confirmed the successful synthesis of AuNPs

probe (Fig. 2E). DLS images showed that the hydrodynamic diameter of the AuNPs before and after the modification process increased from 20 nm to 55 nm (Fig. 2F), indicating successful immobilization of McAb and HRP molecules. The narrow width and symmetry of the graphs also indicated that the synthesized AuNPs probe was monodispersed. The success modification of AuNPs with HRP was further confirmed by adding TMB solution to detect the absorbance at 450 nm. As shown in Fig. S1 (provided in Supplementary materials), only AuNPs with HRP-binding showed the activity of HRP. The absorbance of AuNP probes reached to 2.6 while bare AuNPs and AuNP-IgG showed no HRP activity after addition of TMB solution. The results indicated success modification of AuNPs with HRP. The ratio of HRP to McAb can affect the sensitivity of the assay which was optimized. As shown in Fig. 2G, the absorbance value increased with the increasing ratios of 1:1, 2:1 and 3:1 and decreased with the increasing ratios more than 3:1, because higher amount of HRP on the AuNPs hinder the interaction between McAb and β-CN. Therefore, the ratio of 3:1 was selected as the optimal ratio of HRP to McAb for further experiments. 3.3. Calibration curve As shown in Fig. 2H, a calibration curve was obtained using the relationship between the logarithm of β-CN concentration and the P/N values (Fig. 2H (a)). The linear range of the assay for the detection of β-CN was 6.5–1520 ng mL  1. The linear regression equation was: y¼3.5878x  1.45 with a coefficient correlation R2 ¼0.9967 (Fig. 2H (b)). The limit of detection (LOD), calculated as the mean value of 10 blank samples plus 3 times standard deviations of the mean (Peng et al., 2008), was 4.8 ng mL  1, which was more than 700 times lower than that obtained from MBs-antibody-HRP based immunoassay (Song et al., 2014), 6–7 times lower than those of microplate-antibody-HRP based assay (Zhou et al., 2013) and AuNPs aggregation-based assay (Li et al., 2014). While the procedure of the double-probe based immunoassay is as simple as that of these methods. 3.4. Reaction dynamics of each antigen–antibody reaction step Antibody immobilized MBs can be dispersed in solution to allow for pseudohomogeneous reaction with antigens which improve the efficiency of antigen–antibody reaction. It also can be easily separated from the unreactive substances by applying a magnet and re-dispersed immediately following removal of the magnet (Wei et al., 2012). Each individual antigen–antibody reaction step was studied by monitoring the P/N value using MBsPAb as capture probe and AuNPs-McAb as amplification probe. The concentration of β-CN was 2.0 mg mL  1. As shown in Fig. S2 (provided in Supplementary materials), the P/N value has significantly affected by the incubation time of MBs-PAb and AuNPsMcAb with β-CN. The P/N value display saturation within 5 min incubation of MBs-PAb and 6 min incubation of AuNPs-McAb, which indicates that the detection after 5 and 6 min incubation of MBs-PAb and AuNPs-McAb can produce a stable P/N value. The LOD, detection range and antigen–antibody reaction time of the developed method along with reported papers for the detection of β-CN have been listed in the Table S1 (provided in Supplementary materials). The results indicated that the developed method has the advantages of higher sensitivity and shorter antigen–antibody reaction time. 3.5. Selectivity of the assay To evaluate the selectivity of the developed immunoassay, αs1-, αs2-, β-, κ-CNs, α-lactalbumin, BSA and OVA were detected by

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Table 1 Recoveries of β-CN from spiked bovine milk samples. Milk samplea

Huishan

Mengniu

Wandashan

Spiked concentration (mg mL  1)

Detected concentration (mg mL  1)b

Recovery (%)

Kjeldahl methodc

Developed assay

Kjeldahl method

Developed assay

0 10 30 50

8.17 0.4 18.4 7 0.7 38.9 7 0.9 60.3 7 1.3

8.3 7 0.6 18.7 7 0.9 37.8 7 1.2 57.3 7 1.6

102.8 102.6 104.3

103.7 98.5 97.9

0 10 30 50

8.8 7 0.6 18.6 7 0.8 40.3 7 1.1 56.3 7 1.5

8.7 7 0.3 18.4 7 0.5 38.2 7 0.9 58.6 7 1.3

97.9 104.9 95.0

96.7 98.4 99.8

0 10 30 50

8.3 7 0.8 18.4 7 0.7 39.6 7 1.2 56.9 7 1.4

8.6 7 0.7 18.3 7 0.8 38.6 7 1.1 57.3 7 1.6

100.7 104.3 97.1

97.2 100.1 97.4

a

Each spiked sample was diluted to 1  105 times with bidistilled water, then detected by using the two methods. Data are the means of triplicates. c The pH of the sample was firstly adjusted to 4.6 with trichloroacetic acid (150 mg/mL). Then the precipitate was collected and determined by the Kjeldahl method (Tang et al., 2009). b

adding 100 mL (5.0 mg mL  1) of each protein respectively into microplate well. As shown in Fig. 2I, no cross reactivity was observed with other proteins which indicated that the developed immunoassay could be used for selective detection of β-CN.

higher and the antigen–antibody reaction time is shorter than those of reported microplate-antibody-HRP based assays.

Acknowledgment 3.6. Applicability of the assay Three brand bovine milk samples were spiked with β-CN at the concentrations of 10, 30 and 50 mg mL  1 and then diluted to 1  105 times with bidistilled water. The applications of the developed assay were evaluated to determine the concentration of βCN in the milk samples and the results were validated with the officially recognized standard Kjejdahl method (Kamizake et al., 2003). As shown in Table 1, the concentration of β-CN in Huishan, Mengniu and Wandasahn milk samples was 8.1, 8.8 and 8.3 mg mL  1, respectively. The recoveries of β-CN from three brand samples were 100.0%, 98.3% and 98.2%, respectively. The correlation coefficient (R2) of the results obtained from the two methods was 0.9956. The results indicated that the developed method could be used for β-CN detection in bovine milk samples. In order to avert the drawback of the 96-well-microplate based assay which requires more than 30 min for each antigen–antibody reaction step (Zhou et al., 2013), we used MBs as a solid support that only 10 min was needed for each antigen–antibody reaction step. We also used AuNPs as a bridge for loading more amounts of HRP molecules which increased the sensitivity of the assay. 3.7. Stability of the probes The stability was examined by running the procedure described in Section 2.5 using 2.5 μg mL  1 of β-CN solution instead of sample solution. Fig. 2J showed the response of the probes. The absorbance value did not changed in 6 weeks, which indicated that the probes can be used for repeated measurements after 6 weeks storage at 4 °C.

4. Conclusions In the current study, we described the development of a double-probe based immunoassay for sensitive detection of β-CN in bovine milk. For application of the assay, bovine milk sample only need to be diluted to 1  105 times with distilled water without trivial pretreatment. The sensitivity of the developed method is

The authors are thankful to the financial support of the National Nature Science Foundation of China (NSFC, Nos. 61171022, 60971011 and 30771657) and the financial support of the Science and Technology Department of Jilin Province (20140101012JC).

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.12.025.

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Magnetic bead and gold nanoparticle probes based immunoassay for β-casein detection in bovine milk samples.

In this work, a double-probe based immunoassay was developed for rapid and sensitive determination of β-casein in bovine milk samples. In the method, ...
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