Clinical Biochemistry 47 (2014) 220–226
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Influence of the physical and chemical properties of magnetic nanoparticles on their performance in a chemiluminescence immunoassay Xiaoyan Dai a,b, Hong Xu b,⁎, Yongguang Li a, Hongchen Gu b, Meng Wei a,⁎⁎ a b
Division of Cardiology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, 200233, PR China Nano Biomedical Research Center, School of Biomedical Engineering, Med-X Research Institute, Shanghai Jiao Tong University, Shanghai 200030, PR China
a r t i c l e
i n f o
Article history: Received 13 October 2013 Received in revised form 2 December 2013 Accepted 7 December 2013 Available online 18 December 2013 Keywords: Magnetic nanoparticle Physicochemical property Chemiluminescence immunoassay Detection performance Myoglobin
a b s t r a c t Objectives: Magnetic nanoparticles (MNPs) are important carriers in immunoassays. In this study, we investigated the influence of the physical and chemical properties of MNPs on their performance in a detection process. Design and methods: A comparative study of the properties of two MNPs with sizes of 200 nm and 1 μm (MNP-200 nm and MNP-1 μm, respectively) was conducted using the following four aspects: nonspecific adsorption to proteins (IgG was used as a model protein), influence of magnetic nanoparticles on the chemiluminescence signal, response speed to an external magnetic field, and intensity of the detection signal. Results: MNP-1 μm exhibited lower nonspecific adsorption to IgG in serum, a weaker interference with the chemiluminescence signal, and a higher response speed to the external magnetic field than the same amount of MNP-200 nm. An automated chemiluminescence immunoassay system based on MNP-1 μm was also established. Conclusions: MNP-1 μm acts as an excellent carrier in an automated chemiluminescence immunoassay system for the analysis of serum samples from clinical patients. © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Magnetic nanoparticles (MNPs), due to their superparamagnetism, suspension stability, large surface area, and easy surface modification, have been widely applied in biomedical fields, including the separation and purification of biomolecules [1,2], immunoassays [3–6], targeted drug delivery [7,8], magnetic hyperthermia [9,10], and MRI contrast agents [11,12]. For in vitro immuno and molecular diagnostics, MNPs are usually used as solid carriers to realize the effective recognition, capture, and manipulation of the target molecules. By virtue of their efficient reaction kinetics and easy automation, MNPs have become an irreplaceable tool for immuno and molecular diagnostics in clinical applications. The current research interest in this field has focused on the development of new methods and detection agents for better detection performances [6,13–15]. However, the physical and chemical properties of MNPs, such as size, size distribution, surface structure, and density of functional groups, also have a direct impact on the detection performance
⁎ Correspondence to: H. Xu, Nano Biomedical Research Center, Med-X Research Institute, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai 200030, PR China. ⁎⁎ Correspondence to: M. Wei, Division of Cardiology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 600 Yishan Road, Shanghai 200233, PR China. E-mail addresses: [email protected] (H. Xu), [email protected] (M. Wei).
because these influence the state of the surface-capped biomolecules and the response speed of MNPs through the external magnetic field. However, few studies have been devoted to the investigation of the relationship between the physical and chemical properties of MNPs and their performance in immunoassays. Xie et al. reported that the detection signal obtained by a 100-nm MNP as the carrier is much higher than that obtained by a 1000- or 2800-nm MNP [16]. Harri et al. investigated the effect of the size and number of MNPs on the detection of PSA and concluded that the kinetics of a multiple microparticle assay (particle diameter = 4 μm, 500,000 particles per assay) was rapid due to a favorable surface-to-volume ratio [17]. Ding et al. studied the influence of the antibody coating density on MNPs on their immunoassay performance. These studies, while presenting some meaningful results concerning the relationship between the given properties of MNPs and their immunoassay performance, may not be universal considering the great variation in the surface structure of MNPs introduced by the different synthesis methods. Thus, a systematic study of this issue should be addressed. Herein, we present a systematic study of the relationship between the physical and chemical properties of MNPs and their performance in immunoassays. Two MNPs with different sizes (200 nm and 1 μm, denoted MNP-200 nm, MNP-1 μm, respectively) and similar functional group densities and surface structures were prepared through a hybrid miniemulsion method [18], and a comparative study of these two MNPs
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was conducted from the following four aspects: (1) nonspecific adsorption to IgG in serum, (2) effects of magnetic nanoparticles in the MNPs on the detection signal generated through chemiluminescence, (3) the intensity of the detection signal in myoglobin (model protein), and (4) the response speed to the external magnetic field. Based on the above-mentioned study, an automated chemiluminescence immunoassay system for myoglobin was established using MNP-1 μm as the carrier. In addition, we analyzed patients' serum samples using the proposed system and a commercial immunoassay system to investigate the correlation between the detection results obtained using the two systems.
Materials Apparatus A Nano ZS particle size analyzer was obtained from Malvern (Britain) to analyze MNPs hydrodynamic size, a 1420Multilabel Counter Victor3 instrument was purchased from PerkinElmer (Finland) to carry out nonspecific adsorption of MNPs to IgG and CLIA experiment, and a microplate shaker was obtained from Hengfeng (model WZ-2A, Jintan, China). Mini-table type vacuum pumps (GL-802) and a vortex mixer (QL-901) were purchased from QiLinBeiEr (Haimen, China). A magnetic separation rack and magnetic separation plate were obtained from Allrun (Shanghai, China), and a constant-temperature water bath oscillator was obtained from Bolaite (Taicang, China).
Reagents Tween-20 was provided by BBI (Canada). MES (2-[N-morpholino] ethane sulfonic acid) was purchased from Alfa Aesar (USA), and potassium dihydrogen phosphate (KH2PO4), sodium chloride (NaCl), potassium persulfate (KPS), hydroxylamine hydrochloride, and 1,10phenanthroline monohydrate were obtained from Sinopharm Chemical Reagents (Shanghai, China). Potassium chloride (KCl) was provided by SCRC (Shanghai, China), glycine was obtained from Sigma (USA), and bovine serum albumin (BSA) was obtained from Genview (USA). N-Hydroxysuccinimide (NHS), O-phenylenediamine (OPD), hydrogen peroxide (H2O2), the BCA protein assay kit, and the chemiluminescent substrates were purchased from Pierce (USA), and 1-ethyl-3-[3dimethylaminopropyl]carbodiimide hydrochloride (EDC) was obtained from Medpe (Shanghai, China). The quality control serum was purchased from Randox (UK), and the myoglobin antigen and two antihuman myoglobin monoclonal antibodies (mAb1-4E2 and mAb2-7C3), one of which (mAb2-7C3) was tagged with horseradish peroxidase (HRP), were purchased from Hytest (Turku, Finland). HRP-labeled goat anti-human IgG was obtained from Bioss (Beijing, China). The test human serum was collected from Shanghai Sixth People's Hospital. This study was approved by the Medical Ethics Committee of Shanghai Sixth People's Hospital Affiliated with Shanghai Jiaotong University, and all of the patients provided signed informed consent.
Methods
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Nonspecific adsorption of MNPs to IgG in serum In this study, the relative adsorption quantity of MNPs to IgG in serum was measured to evaluate the antifouling property of the MNPs. MNP-200 nm and MNP-1 μm (30, 50, 100, 200, and 300 μg) were introduced into a 96-well microplate and washed twice with PBST (0.05 M, pH 7.5). One hundred microliters of the quality control serum were added to each well, and the plate was incubated at 37 °C for 30 min. After incubation, the MNPs were collected by an external magnetic field and washed three times with PBST and deionized water. One hundred microliters of a diluted solution of HRP-labeled goat-anti-human IgG were then added to the MNPs, and the mixtures were incubated for 30 min at 37 °C. After magnetic separation and discarding the supernatant, 150 μL of the substrate solution was added (OPD + H2O2) to each well, and the plate was incubated for 15 min at 37 °C to afford the color reaction. After magnetic separation, 100 μL of the supernatant was transferred to another clean 96-well microplate, and 25 μL of the stopping solution (2 M H2SO4) was added to each well. The absorption of the solution was measured at the wavelength of 490 nm, and the intensity was used to evaluate the relative quantity of nonspecific adsorption of the MNPs to IgG. Response speed of MNPs to an external magnetic field The response speed of MNPs to a magnetic field was evaluated by monitoring the concentration of Fe at different time points after an external magnetic field was applied to an MNP suspension. First, 1 mg of MNPs (MNP-200 nm and MNP-1 μm) suspended in 800 μL of deionized water was added to a 1.5-mL centrifuge tube, which was then placed in a magnetic separator (a total of 12 samples, six for each MNP). Then, 700 μL of the supernatant were removed 0, 20, 40, 60, 90, and 120 s after the external magnetic field was applied. Four hundred microliters of HCl (37 wt.%) were added to each sample, and the samples were incubated for 48 h to dissolve the MNPs. The concentration of Fe in the supernatant at the different time points was determined through a colorimetric method: 1 mL of 100 g/L hydroxylamine hydrochloride solution was added to the supernatant, and the mixture was shaken for 5 min. Then, 1 mL of 4 g/L 1,10-phenanthroline monohydrate solution and 5 mL of HAc–NaAc buffer were successively added. Deionized water was then added to obtain a final volume of 50 mL. The absorbance of the solution was measured at the wavelength of 510 nm. Influence of MNPs on the signal of a chemiluminescence immunoassay (CLIA) First, 0–100 μg of the MNPs (MNP-200 nm and MNP-1 μm) were added to a 96-well microplate and washed twice with PBST. To each well, 100 μL of a mixed solution of enzyme-labeled antibody (1 mg/mL, 10,000-fold dilution) and chemiluminescence agent (1:9 v/v) was added and mixed with the MNPs. Then, 90 μL of the mixture was transferred to a white microplate and incubated in the dark for 5 min before measuring the chemiluminescence intensity. To evaluate the effect of the content of the magnetite nanoparticles on the CLIA signal, the same quantity of three MNPs with different contents of magnetic nanoparticles (25 wt.%, 48 wt.%, and 74 wt.%) was used. The experimental procedure was the same as that described above.
Synthesis of MNPs Establishment of the CLIA system Two carboxylated MNPs with sizes of approximately 200 nm and 1 μm (MNP-200 nm and MNP-1 μm, respectively) were synthesized through hybrid miniemulsion polymerization as described in our previous work [18]. The MNPs were composed of Fe3O4 nanoparticles, and the content of magnetite nanoparticles (25 wt.%, 48 wt.%, and 74 wt.%) was adjusted by changing the concentration of magnetic aggregates during the synthesis.
Preparation of immuno-MNPs First, 2 mg of MNPs (MNP-200 nm and MNP-1 μm) were added to a 2-mL centrifuge tube and re-dispersed in 200 μL of MEST (25 mM, pH 6) by washing twice with MEST. Then, 2 mg of EDC and NHS were added to the tube, and the reaction was allowed to proceed at ambient temperature for 15 min. After the reaction, the excess reactants were
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removed by washing three times with MEST. Then, 300 μg of myoglobin monoclonal antibody was added, and the reaction was allowed to proceed at ambient temperature for 2 h. The MNP-myoglobin complexes were blocked with 200 μL of blocking solution (0.05 M PBST containing 0.1% BSA and 0.3% Gly) overnight at 4 °C. The complexes were then redispersed in 200 μL of storing solution (0.05 M PBST containing 2% BSA) at 4 °C. The binding capacity of the antibody was determined using a BCA protein quantification kit with the depletion method. A control tube that underwent the same procedure without the addition of antibody was used to eliminate the interference. Chemiluminescence detection of myoglobin The obtained immuno-MNP was applied for the chemiluminescence detection of myoglobin using the following procedure: To a microplate, 30 μg of immuno-MNP (30 μL), 60 μL of antigen solution, and 100 μL of HRP-labeled antibody solution (1 mg/mL, 2000-fold dilution) were added and incubated at 37 °C for 30 min to obtain MNP–immune complexes. After the reaction, the obtained MNP–immune complexes were washed five times with Tris–HCl (0.02 M, pH 7.4), and the supernatant was discarded with the help of an external magnetic field. Then, 100 μL of the chemiluminescence agent was added and mixed with the MNP– immune complexes. The mixture was placed in the dark for 5 min, and the chemiluminescence signal was then measured. Establishment of automated MYO CLIA system The myoglobin chemiluminescence enzyme immunoassay system (Horseradish peroxidase as label enzyme, Luminol as enzyme substrate) was established according to the above-mentioned procedure using MNP-1 μm as the carrier. The detection standard curve, sensitivity, precision, and dynamic range of the system were determined. Twenty-six split serum samples were collected from 26 patients hospitalized in Shanghai Sixth People's Hospital (two samples from each patient). The concentration of myoglobin in one sample from a patient was determined using the system proposed in this paper, and the concentration of myoglobin in the other sample from the same patient was determined using the Beckman chemiluminescence immunoassay kit and instrument. The results obtained by the two systems were analyzed through a correlation analysis.
Table 1 Characterization of magnetic nano-particles. MNP
Hydrodynamic diameter (nm)
Fe3O4 content (wt.%)
Carboxyl group density (mmol/g)
MNP-1 μm MNP-200 nm
1380 204
~50 ~60
~0.5 0.57
electron microscope (TEM) image of the MNPs. Both MNPs are composed of 10-nm Fe3O4 nanoparticles with a narrow size distribution. In addition, their contents of magnetite nanoparticles and their functional group densities are similar (Table 1). Nonspecific adsorption to IgG In a clinical immunoassay, the recognition and capture of the target molecule by MNPs is usually performed in serum and plasma. Therefore, the nonspecific adsorption to IgG in serum or plasma will influence the specificity and accuracy of the assay. To evaluate the nonspecific adsorption of MNPs in the detection of serum samples, a comparative study of the nonspecific adsorption properties of MNP-200 nm and MNP-1 μm to IgG in serum was performed. As shown in Fig. 2a, for both MNPs, the optical density (OD) increases with an increase in the amount of the MNPs (30 μg–300 μg). When the amounts of the two MNPs were the same, the OD of MNP200 nm was always higher than that of MNP-1 μm, indicating a higher nonspecific adsorption. This difference can be ascribed to the larger specific surface area of MNP-200 nm due to its smaller size, which provides a larger area for protein adsorption. The results were converted into OD/nm2 to show the nonspecific adsorption capacity of the MNPs per unit area. As shown in Fig. 2b, for both MNPs, the nonspecific adsorption per unit area decreased with an increase in the MNP amount. In addition, the nonspecific adsorption of MNP-200 nm was always lower than that of MNP-1 μm. This result suggests that the interaction between MNP-200 nm and the protein is weaker, which possibly results from the larger curvature of MNP200 nm [19,20]. In contrast, although MNP-200 nm has a lower nonspecific adsorption per unit area, it possesses a higher nonspecific adsorption per unit mass due to its larger specific surface area. Therefore, a higher background was obtained for MNP-200 nm in the immunoassay.
Results and discussion Response speed to the external magnetic field Synthesis and characterization of MNPs Carboxylated MNPs with sizes of approximately 200 nm and 1 μm (MNP-200 nm, MNP-1 μm, respectively) were synthesized through hybrid miniemulsion polymerization. Fig. 1 shows the transmission
MNPs act as carriers for the separation of biomolecules in CLIA. Their response speed to the external magnetic field not only influences the detection time but also plays a decisive role in the realization of the automated detection procedure. Hereby, we evaluated the responses
Fig. 1. TEM of the superparamagnetic beads with diameters of 200 nm (a) and 1 μm (b) synthesized through hybrid miniemulsion polymerization.
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Fig. 2. Non-specific adsorption of MNP-200 nm and MNP-1 μm as a function of the mass of the MNPs. (a) Total adsorption. (b) Adsorption per unit surface area. ** indicates p b 0.01.
of MNP-200 nm and MNP-1 μm by detecting the residual Fe concentration in the supernatant as a function of the capture time after applying the external magnetic field. As shown in Fig. 3, for both MNPs, the Fe concentration in the supernatant decreases with the prolongation of the magnetic capture time. The Fe concentration in the supernatant of MNP-1 μm decreased more rapidly and almost reached zero at 40 s. In contrast, MNP-200 nm is not fully captured even at 120 s because the motion velocity of MNP in a magnetic field is proportional to the square of its radius as well as the magnetic substances [21]. If the contents of magnetite nanoparticles of MNP-200 nm and MNP-1 μm are similar (approximately 60 wt.% and 50 wt.%, respectively), the larger MNP1 μm exhibits a rapid response speed to the external magnetic field. In practical applications of CLIA, the slow response speed of MNP-
200 nm will greatly increase the detection time and cannot meet the demands of automation. Therefore, MNP-1 μm is more suitable for automated CLIA based on its detection time and the demands of automation.
Fig. 3. Magnetic response speed of MNP-200 nm and MNP-1 μm to the external magnetic field. This response was evaluated by the decrease in the residual Fe concentration in the supernatant as a function of time after applying the external magnetic field.
Fig. 4. Quenching effect of MNP-200 nm and MNP-1 μm on the chemiluminescence signal. The MNPs were directly added to a mixture of the enzyme-labeled antibody and the chemiluminescence agent.
Quenching of the chemiluminescence signal by MNPs The immunoassay system established in this study is a luminol chemiluminescence system based on horseradish peroxidase (HRP)labeled IgG. The MNPs were suspended in the chemiluminescence substrate throughout the detection process and thus may quench the chemiluminescence signal. In this work, we studied the influence of
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The influences of MNPs with different contents of magnetite nanoparticles on the chemiluminescence signal were also investigated. Three MNPs with a similar size and different contents of magnetite nanoparticles (25 wt.%, 48 wt.%, and 74 wt.%) were used. As shown in Fig. 5, the quenching effect increases with an increase in the MNP amount for all three MNPs, and the quenching effect is stronger for MNPs with a higher content of magnetite nanoparticles. The Fe3O4 nanoparticles inside the MNPs are most likely responsible for the quenching of the chemiluminescence signal due to their strong nonspecific adsorption in the ultraviolet–visible spectrum. As a result, MNPs with a higher content of magnetite nanoparticles exhibit a stronger quenching effect. Hence, there exists a balance between the content of magnetic substances and its quenching effect. The selection of the proper MNP as the carrier in an immunoassay should reach an optimal point between these two factors in order to achieve an effective separation and a low quenching of the detection signal. Fig. 5. Quenching effect of MNPs with different contents of magnetic substances. The experimental procedure was the same as that used to obtain the results shown in Fig. 4.
MNP-200 nm and MNP-1 μm on the chemiluminescence signal as a function of the added amount of MNPs. As shown in Fig. 4, the chemiluminescence signal decreases with an increase in the MNPs amount, and this decreasing trend is more prominent at smaller MNP amounts. The decrease in the chemiluminescence signal obtained with MNP200 nm is faster than that obtained with MNP-1 μm. This difference is because the number of MNP-200 nm particles required to obtain a specific mass of MNPs is approximately two orders of magnitude higher than the corresponding number of MNP-1 μm particles. An increase in the number of MNPs leads to a stronger scattering, which in turn exerts a stronger influence on the chemiluminescence signal.
Effect of particle size of MNPs on the CLIA signal A comparative study of the intensities of the detection signals of MNP-200 nm and MNP-1 μm was conducted using myoglobin as the model analyte. First, immuno-MNPs coated with myoglobin monoclonal antibody were prepared by conjugating anti-myoglobin monoclonal antibody to the MNPs via EDC/NHS coupling chemistry. The binding capacities of MNP-200 nm were determined to be 29 μg/mg and 25 μg/mg, respectively. The myoglobin antigen at concentrations of 10 ng/mL, 500 ng/mL, and 1000 ng/mL was detected using the chemiluminescence immunoassay method. As shown in Fig. 6a, the detection signal obtained by MNP-200 nm is much higher than MNP-1 μm at the three concentration levels tested, even though the coupling amounts of antibody per unit mass of MNPs are almost the same. A possible reason is that the small surface curvature of the larger MNP-1 μm leads to a
Fig. 6. Detection signal obtained with MNP-200 nm and MNP-1 μm in a chemiluminescence immunoassay. (a) Relative light unit (RLU) obtained with the same mass (30 μg) of MNPs. (b) RLU obtained with the same surface area (1 × 10−4 m2) of MNPs. The insets show the amplified image of the detection signal for 10 ng/mL MYO. ** indicates p b 0.01.
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stronger interaction with the antibody, which further induces an alteration in the protein conformation and thereby reduces its activity [22,23]. As a result, the antibody on MNP-1 μm may have a lower affinity to the myoglobin antigen. Converting the results in Fig. 6a into the detection signal obtained with the same MNP surface area, we obtained Fig. 6b. As shown, when the surface area of the two MNPs are the same (1 × 10−4 m2), the detection signal obtained with MNP-200 was higher than that obtained with MNP-1 μm in the presence of 10 ng/mL and 500 ng/mL antigen, whereas the opposite results were obtained with an antigen concentration of 1000 ng/mL. A possible reason is that the MNP surface antibody at the antigen concentrations of 10 ng/mL and 500 ng/mL is found in excess compared with the immune-complex, and the signal is determined by the reactivity of the surface antibody. As a result, a higher signal was obtained for MNP-200 due to the higher reactivity of the antibody on its surface. When the antigen concentration was increased to 1000 ng/mL, the number of antigen molecules becomes larger than the number of surface antibody molecules, and the density of the surface antibody becomes a decisive factor. Because the antibody coating amounts per mass of MNP were similar for the two MNPs, MNP-1 μm possesses a higher density of surface antibody due to its lower specific surface area. Consequently, a higher signal was obtained with MNP-1 μm in the presence of an antigen concentration of 1000 ng/mL. The described studies evaluated the properties of MNP-200 nm and MNP-1 μm in CLIA from several aspects. Although the detection signal obtained with MNP-1 μm is lower than that obtained with MNP200 nm, MNP-1 μm can generally meet the demands of the immunoassay and exhibits a lower background interference. The most important aspect is that MNP-1 μm can be rapidly captured by the external magnetic field to fulfill automated detection. Therefore, MNP-1 μm is more suitable to be used as the carrier in an automated CLIA. Comparison between the present chemiluminescence immunoassay system and a commercial system The automated chemiluminescence immunoassay system for the detection of myoglobin antibody was established using MNP-1 μm as the carrier. The system was applied for the detection of the myoglobin concentration in serum samples from clinical patients using an automated chemiluminescence spectrometer, and the results were compared with those obtained using a commercial system produced by Beckman. As shown in Fig. 7, a good interrelation was obtained between the two systems (R = 0.935). This result indicates that MNP-1 μm is an
Fig. 7. Comparison and correlation analysis of the two systems for myoglobin detection. SHJD: the detection system established in this study; Beckman: the detection system produced by Beckman, including the reagents and the apparatus.
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excellent carrier for an automated chemiluminescence immunoassay and that the immunoassay system established in this study generally meets the demands associated with clinical detection.
Conclusion MNPs are widely used as carriers in immunoassays. The physical and chemical properties of MNPs, including their size and contents of magnetic nanoparticles, have a direct influence on their performance in an immunoassay. In this study, we conducted a comparative study of the properties of two MNPs with micron and sub-micron sizes and discovered that MNP-1 μm presents a lower nonspecific adsorption to protein, a weaker interference with the chemiluminescence signal, and a more rapid response speed to the external magnetic field compared with MNP-200 nm. These properties make MNP-1 μm more suitable to be used as the carrier in automated CLIAs. Thus, the study of the relationship between the physical and chemical properties of MNPs and their performance in an immunoassay can provide both theoretical and experimental guidance for the research and development of MNPs to be used in immunoassays. In addition, a CLIA system for the detection of myoglobin antigen was established in this study using MNP-1 μm as the carrier. The system exhibits excellent performance and successfully meets the demands of a clinical immunoassay.
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Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Influence of the physical and chemical properties of magnetic nanoparticles on their performance in a chemiluminescence immunoassay Xiaoyan Dai a,b, Hong Xu b,⁎, Yongguang Li a, Hongchen Gu b, Meng Wei a,⁎⁎ a b
Division of Cardiology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, 200233, PR China Nano Biomedical Research Center, School of Biomedical Engineering, Med-X Research Institute, Shanghai Jiao Tong University, Shanghai 200030, PR China
a r t i c l e
i n f o
Article history: Received 13 October 2013 Received in revised form 2 December 2013 Accepted 7 December 2013 Available online 18 December 2013 Keywords: Magnetic nanoparticle Physicochemical property Chemiluminescence immunoassay Detection performance Myoglobin
a b s t r a c t Objectives: Magnetic nanoparticles (MNPs) are important carriers in immunoassays. In this study, we investigated the influence of the physical and chemical properties of MNPs on their performance in a detection process. Design and methods: A comparative study of the properties of two MNPs with sizes of 200 nm and 1 μm (MNP-200 nm and MNP-1 μm, respectively) was conducted using the following four aspects: nonspecific adsorption to proteins (IgG was used as a model protein), influence of magnetic nanoparticles on the chemiluminescence signal, response speed to an external magnetic field, and intensity of the detection signal. Results: MNP-1 μm exhibited lower nonspecific adsorption to IgG in serum, a weaker interference with the chemiluminescence signal, and a higher response speed to the external magnetic field than the same amount of MNP-200 nm. An automated chemiluminescence immunoassay system based on MNP-1 μm was also established. Conclusions: MNP-1 μm acts as an excellent carrier in an automated chemiluminescence immunoassay system for the analysis of serum samples from clinical patients. © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Magnetic nanoparticles (MNPs), due to their superparamagnetism, suspension stability, large surface area, and easy surface modification, have been widely applied in biomedical fields, including the separation and purification of biomolecules [1,2], immunoassays [3–6], targeted drug delivery [7,8], magnetic hyperthermia [9,10], and MRI contrast agents [11,12]. For in vitro immuno and molecular diagnostics, MNPs are usually used as solid carriers to realize the effective recognition, capture, and manipulation of the target molecules. By virtue of their efficient reaction kinetics and easy automation, MNPs have become an irreplaceable tool for immuno and molecular diagnostics in clinical applications. The current research interest in this field has focused on the development of new methods and detection agents for better detection performances [6,13–15]. However, the physical and chemical properties of MNPs, such as size, size distribution, surface structure, and density of functional groups, also have a direct impact on the detection performance
⁎ Correspondence to: H. Xu, Nano Biomedical Research Center, Med-X Research Institute, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai 200030, PR China. ⁎⁎ Correspondence to: M. Wei, Division of Cardiology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 600 Yishan Road, Shanghai 200233, PR China. E-mail addresses: [email protected] (H. Xu), [email protected] (M. Wei).
because these influence the state of the surface-capped biomolecules and the response speed of MNPs through the external magnetic field. However, few studies have been devoted to the investigation of the relationship between the physical and chemical properties of MNPs and their performance in immunoassays. Xie et al. reported that the detection signal obtained by a 100-nm MNP as the carrier is much higher than that obtained by a 1000- or 2800-nm MNP [16]. Harri et al. investigated the effect of the size and number of MNPs on the detection of PSA and concluded that the kinetics of a multiple microparticle assay (particle diameter = 4 μm, 500,000 particles per assay) was rapid due to a favorable surface-to-volume ratio [17]. Ding et al. studied the influence of the antibody coating density on MNPs on their immunoassay performance. These studies, while presenting some meaningful results concerning the relationship between the given properties of MNPs and their immunoassay performance, may not be universal considering the great variation in the surface structure of MNPs introduced by the different synthesis methods. Thus, a systematic study of this issue should be addressed. Herein, we present a systematic study of the relationship between the physical and chemical properties of MNPs and their performance in immunoassays. Two MNPs with different sizes (200 nm and 1 μm, denoted MNP-200 nm, MNP-1 μm, respectively) and similar functional group densities and surface structures were prepared through a hybrid miniemulsion method [18], and a comparative study of these two MNPs
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was conducted from the following four aspects: (1) nonspecific adsorption to IgG in serum, (2) effects of magnetic nanoparticles in the MNPs on the detection signal generated through chemiluminescence, (3) the intensity of the detection signal in myoglobin (model protein), and (4) the response speed to the external magnetic field. Based on the above-mentioned study, an automated chemiluminescence immunoassay system for myoglobin was established using MNP-1 μm as the carrier. In addition, we analyzed patients' serum samples using the proposed system and a commercial immunoassay system to investigate the correlation between the detection results obtained using the two systems.
Materials Apparatus A Nano ZS particle size analyzer was obtained from Malvern (Britain) to analyze MNPs hydrodynamic size, a 1420Multilabel Counter Victor3 instrument was purchased from PerkinElmer (Finland) to carry out nonspecific adsorption of MNPs to IgG and CLIA experiment, and a microplate shaker was obtained from Hengfeng (model WZ-2A, Jintan, China). Mini-table type vacuum pumps (GL-802) and a vortex mixer (QL-901) were purchased from QiLinBeiEr (Haimen, China). A magnetic separation rack and magnetic separation plate were obtained from Allrun (Shanghai, China), and a constant-temperature water bath oscillator was obtained from Bolaite (Taicang, China).
Reagents Tween-20 was provided by BBI (Canada). MES (2-[N-morpholino] ethane sulfonic acid) was purchased from Alfa Aesar (USA), and potassium dihydrogen phosphate (KH2PO4), sodium chloride (NaCl), potassium persulfate (KPS), hydroxylamine hydrochloride, and 1,10phenanthroline monohydrate were obtained from Sinopharm Chemical Reagents (Shanghai, China). Potassium chloride (KCl) was provided by SCRC (Shanghai, China), glycine was obtained from Sigma (USA), and bovine serum albumin (BSA) was obtained from Genview (USA). N-Hydroxysuccinimide (NHS), O-phenylenediamine (OPD), hydrogen peroxide (H2O2), the BCA protein assay kit, and the chemiluminescent substrates were purchased from Pierce (USA), and 1-ethyl-3-[3dimethylaminopropyl]carbodiimide hydrochloride (EDC) was obtained from Medpe (Shanghai, China). The quality control serum was purchased from Randox (UK), and the myoglobin antigen and two antihuman myoglobin monoclonal antibodies (mAb1-4E2 and mAb2-7C3), one of which (mAb2-7C3) was tagged with horseradish peroxidase (HRP), were purchased from Hytest (Turku, Finland). HRP-labeled goat anti-human IgG was obtained from Bioss (Beijing, China). The test human serum was collected from Shanghai Sixth People's Hospital. This study was approved by the Medical Ethics Committee of Shanghai Sixth People's Hospital Affiliated with Shanghai Jiaotong University, and all of the patients provided signed informed consent.
Methods
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Nonspecific adsorption of MNPs to IgG in serum In this study, the relative adsorption quantity of MNPs to IgG in serum was measured to evaluate the antifouling property of the MNPs. MNP-200 nm and MNP-1 μm (30, 50, 100, 200, and 300 μg) were introduced into a 96-well microplate and washed twice with PBST (0.05 M, pH 7.5). One hundred microliters of the quality control serum were added to each well, and the plate was incubated at 37 °C for 30 min. After incubation, the MNPs were collected by an external magnetic field and washed three times with PBST and deionized water. One hundred microliters of a diluted solution of HRP-labeled goat-anti-human IgG were then added to the MNPs, and the mixtures were incubated for 30 min at 37 °C. After magnetic separation and discarding the supernatant, 150 μL of the substrate solution was added (OPD + H2O2) to each well, and the plate was incubated for 15 min at 37 °C to afford the color reaction. After magnetic separation, 100 μL of the supernatant was transferred to another clean 96-well microplate, and 25 μL of the stopping solution (2 M H2SO4) was added to each well. The absorption of the solution was measured at the wavelength of 490 nm, and the intensity was used to evaluate the relative quantity of nonspecific adsorption of the MNPs to IgG. Response speed of MNPs to an external magnetic field The response speed of MNPs to a magnetic field was evaluated by monitoring the concentration of Fe at different time points after an external magnetic field was applied to an MNP suspension. First, 1 mg of MNPs (MNP-200 nm and MNP-1 μm) suspended in 800 μL of deionized water was added to a 1.5-mL centrifuge tube, which was then placed in a magnetic separator (a total of 12 samples, six for each MNP). Then, 700 μL of the supernatant were removed 0, 20, 40, 60, 90, and 120 s after the external magnetic field was applied. Four hundred microliters of HCl (37 wt.%) were added to each sample, and the samples were incubated for 48 h to dissolve the MNPs. The concentration of Fe in the supernatant at the different time points was determined through a colorimetric method: 1 mL of 100 g/L hydroxylamine hydrochloride solution was added to the supernatant, and the mixture was shaken for 5 min. Then, 1 mL of 4 g/L 1,10-phenanthroline monohydrate solution and 5 mL of HAc–NaAc buffer were successively added. Deionized water was then added to obtain a final volume of 50 mL. The absorbance of the solution was measured at the wavelength of 510 nm. Influence of MNPs on the signal of a chemiluminescence immunoassay (CLIA) First, 0–100 μg of the MNPs (MNP-200 nm and MNP-1 μm) were added to a 96-well microplate and washed twice with PBST. To each well, 100 μL of a mixed solution of enzyme-labeled antibody (1 mg/mL, 10,000-fold dilution) and chemiluminescence agent (1:9 v/v) was added and mixed with the MNPs. Then, 90 μL of the mixture was transferred to a white microplate and incubated in the dark for 5 min before measuring the chemiluminescence intensity. To evaluate the effect of the content of the magnetite nanoparticles on the CLIA signal, the same quantity of three MNPs with different contents of magnetic nanoparticles (25 wt.%, 48 wt.%, and 74 wt.%) was used. The experimental procedure was the same as that described above.
Synthesis of MNPs Establishment of the CLIA system Two carboxylated MNPs with sizes of approximately 200 nm and 1 μm (MNP-200 nm and MNP-1 μm, respectively) were synthesized through hybrid miniemulsion polymerization as described in our previous work [18]. The MNPs were composed of Fe3O4 nanoparticles, and the content of magnetite nanoparticles (25 wt.%, 48 wt.%, and 74 wt.%) was adjusted by changing the concentration of magnetic aggregates during the synthesis.
Preparation of immuno-MNPs First, 2 mg of MNPs (MNP-200 nm and MNP-1 μm) were added to a 2-mL centrifuge tube and re-dispersed in 200 μL of MEST (25 mM, pH 6) by washing twice with MEST. Then, 2 mg of EDC and NHS were added to the tube, and the reaction was allowed to proceed at ambient temperature for 15 min. After the reaction, the excess reactants were
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removed by washing three times with MEST. Then, 300 μg of myoglobin monoclonal antibody was added, and the reaction was allowed to proceed at ambient temperature for 2 h. The MNP-myoglobin complexes were blocked with 200 μL of blocking solution (0.05 M PBST containing 0.1% BSA and 0.3% Gly) overnight at 4 °C. The complexes were then redispersed in 200 μL of storing solution (0.05 M PBST containing 2% BSA) at 4 °C. The binding capacity of the antibody was determined using a BCA protein quantification kit with the depletion method. A control tube that underwent the same procedure without the addition of antibody was used to eliminate the interference. Chemiluminescence detection of myoglobin The obtained immuno-MNP was applied for the chemiluminescence detection of myoglobin using the following procedure: To a microplate, 30 μg of immuno-MNP (30 μL), 60 μL of antigen solution, and 100 μL of HRP-labeled antibody solution (1 mg/mL, 2000-fold dilution) were added and incubated at 37 °C for 30 min to obtain MNP–immune complexes. After the reaction, the obtained MNP–immune complexes were washed five times with Tris–HCl (0.02 M, pH 7.4), and the supernatant was discarded with the help of an external magnetic field. Then, 100 μL of the chemiluminescence agent was added and mixed with the MNP– immune complexes. The mixture was placed in the dark for 5 min, and the chemiluminescence signal was then measured. Establishment of automated MYO CLIA system The myoglobin chemiluminescence enzyme immunoassay system (Horseradish peroxidase as label enzyme, Luminol as enzyme substrate) was established according to the above-mentioned procedure using MNP-1 μm as the carrier. The detection standard curve, sensitivity, precision, and dynamic range of the system were determined. Twenty-six split serum samples were collected from 26 patients hospitalized in Shanghai Sixth People's Hospital (two samples from each patient). The concentration of myoglobin in one sample from a patient was determined using the system proposed in this paper, and the concentration of myoglobin in the other sample from the same patient was determined using the Beckman chemiluminescence immunoassay kit and instrument. The results obtained by the two systems were analyzed through a correlation analysis.
Table 1 Characterization of magnetic nano-particles. MNP
Hydrodynamic diameter (nm)
Fe3O4 content (wt.%)
Carboxyl group density (mmol/g)
MNP-1 μm MNP-200 nm
1380 204
~50 ~60
~0.5 0.57
electron microscope (TEM) image of the MNPs. Both MNPs are composed of 10-nm Fe3O4 nanoparticles with a narrow size distribution. In addition, their contents of magnetite nanoparticles and their functional group densities are similar (Table 1). Nonspecific adsorption to IgG In a clinical immunoassay, the recognition and capture of the target molecule by MNPs is usually performed in serum and plasma. Therefore, the nonspecific adsorption to IgG in serum or plasma will influence the specificity and accuracy of the assay. To evaluate the nonspecific adsorption of MNPs in the detection of serum samples, a comparative study of the nonspecific adsorption properties of MNP-200 nm and MNP-1 μm to IgG in serum was performed. As shown in Fig. 2a, for both MNPs, the optical density (OD) increases with an increase in the amount of the MNPs (30 μg–300 μg). When the amounts of the two MNPs were the same, the OD of MNP200 nm was always higher than that of MNP-1 μm, indicating a higher nonspecific adsorption. This difference can be ascribed to the larger specific surface area of MNP-200 nm due to its smaller size, which provides a larger area for protein adsorption. The results were converted into OD/nm2 to show the nonspecific adsorption capacity of the MNPs per unit area. As shown in Fig. 2b, for both MNPs, the nonspecific adsorption per unit area decreased with an increase in the MNP amount. In addition, the nonspecific adsorption of MNP-200 nm was always lower than that of MNP-1 μm. This result suggests that the interaction between MNP-200 nm and the protein is weaker, which possibly results from the larger curvature of MNP200 nm [19,20]. In contrast, although MNP-200 nm has a lower nonspecific adsorption per unit area, it possesses a higher nonspecific adsorption per unit mass due to its larger specific surface area. Therefore, a higher background was obtained for MNP-200 nm in the immunoassay.
Results and discussion Response speed to the external magnetic field Synthesis and characterization of MNPs Carboxylated MNPs with sizes of approximately 200 nm and 1 μm (MNP-200 nm, MNP-1 μm, respectively) were synthesized through hybrid miniemulsion polymerization. Fig. 1 shows the transmission
MNPs act as carriers for the separation of biomolecules in CLIA. Their response speed to the external magnetic field not only influences the detection time but also plays a decisive role in the realization of the automated detection procedure. Hereby, we evaluated the responses
Fig. 1. TEM of the superparamagnetic beads with diameters of 200 nm (a) and 1 μm (b) synthesized through hybrid miniemulsion polymerization.
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Fig. 2. Non-specific adsorption of MNP-200 nm and MNP-1 μm as a function of the mass of the MNPs. (a) Total adsorption. (b) Adsorption per unit surface area. ** indicates p b 0.01.
of MNP-200 nm and MNP-1 μm by detecting the residual Fe concentration in the supernatant as a function of the capture time after applying the external magnetic field. As shown in Fig. 3, for both MNPs, the Fe concentration in the supernatant decreases with the prolongation of the magnetic capture time. The Fe concentration in the supernatant of MNP-1 μm decreased more rapidly and almost reached zero at 40 s. In contrast, MNP-200 nm is not fully captured even at 120 s because the motion velocity of MNP in a magnetic field is proportional to the square of its radius as well as the magnetic substances [21]. If the contents of magnetite nanoparticles of MNP-200 nm and MNP-1 μm are similar (approximately 60 wt.% and 50 wt.%, respectively), the larger MNP1 μm exhibits a rapid response speed to the external magnetic field. In practical applications of CLIA, the slow response speed of MNP-
200 nm will greatly increase the detection time and cannot meet the demands of automation. Therefore, MNP-1 μm is more suitable for automated CLIA based on its detection time and the demands of automation.
Fig. 3. Magnetic response speed of MNP-200 nm and MNP-1 μm to the external magnetic field. This response was evaluated by the decrease in the residual Fe concentration in the supernatant as a function of time after applying the external magnetic field.
Fig. 4. Quenching effect of MNP-200 nm and MNP-1 μm on the chemiluminescence signal. The MNPs were directly added to a mixture of the enzyme-labeled antibody and the chemiluminescence agent.
Quenching of the chemiluminescence signal by MNPs The immunoassay system established in this study is a luminol chemiluminescence system based on horseradish peroxidase (HRP)labeled IgG. The MNPs were suspended in the chemiluminescence substrate throughout the detection process and thus may quench the chemiluminescence signal. In this work, we studied the influence of
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The influences of MNPs with different contents of magnetite nanoparticles on the chemiluminescence signal were also investigated. Three MNPs with a similar size and different contents of magnetite nanoparticles (25 wt.%, 48 wt.%, and 74 wt.%) were used. As shown in Fig. 5, the quenching effect increases with an increase in the MNP amount for all three MNPs, and the quenching effect is stronger for MNPs with a higher content of magnetite nanoparticles. The Fe3O4 nanoparticles inside the MNPs are most likely responsible for the quenching of the chemiluminescence signal due to their strong nonspecific adsorption in the ultraviolet–visible spectrum. As a result, MNPs with a higher content of magnetite nanoparticles exhibit a stronger quenching effect. Hence, there exists a balance between the content of magnetic substances and its quenching effect. The selection of the proper MNP as the carrier in an immunoassay should reach an optimal point between these two factors in order to achieve an effective separation and a low quenching of the detection signal. Fig. 5. Quenching effect of MNPs with different contents of magnetic substances. The experimental procedure was the same as that used to obtain the results shown in Fig. 4.
MNP-200 nm and MNP-1 μm on the chemiluminescence signal as a function of the added amount of MNPs. As shown in Fig. 4, the chemiluminescence signal decreases with an increase in the MNPs amount, and this decreasing trend is more prominent at smaller MNP amounts. The decrease in the chemiluminescence signal obtained with MNP200 nm is faster than that obtained with MNP-1 μm. This difference is because the number of MNP-200 nm particles required to obtain a specific mass of MNPs is approximately two orders of magnitude higher than the corresponding number of MNP-1 μm particles. An increase in the number of MNPs leads to a stronger scattering, which in turn exerts a stronger influence on the chemiluminescence signal.
Effect of particle size of MNPs on the CLIA signal A comparative study of the intensities of the detection signals of MNP-200 nm and MNP-1 μm was conducted using myoglobin as the model analyte. First, immuno-MNPs coated with myoglobin monoclonal antibody were prepared by conjugating anti-myoglobin monoclonal antibody to the MNPs via EDC/NHS coupling chemistry. The binding capacities of MNP-200 nm were determined to be 29 μg/mg and 25 μg/mg, respectively. The myoglobin antigen at concentrations of 10 ng/mL, 500 ng/mL, and 1000 ng/mL was detected using the chemiluminescence immunoassay method. As shown in Fig. 6a, the detection signal obtained by MNP-200 nm is much higher than MNP-1 μm at the three concentration levels tested, even though the coupling amounts of antibody per unit mass of MNPs are almost the same. A possible reason is that the small surface curvature of the larger MNP-1 μm leads to a
Fig. 6. Detection signal obtained with MNP-200 nm and MNP-1 μm in a chemiluminescence immunoassay. (a) Relative light unit (RLU) obtained with the same mass (30 μg) of MNPs. (b) RLU obtained with the same surface area (1 × 10−4 m2) of MNPs. The insets show the amplified image of the detection signal for 10 ng/mL MYO. ** indicates p b 0.01.
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stronger interaction with the antibody, which further induces an alteration in the protein conformation and thereby reduces its activity [22,23]. As a result, the antibody on MNP-1 μm may have a lower affinity to the myoglobin antigen. Converting the results in Fig. 6a into the detection signal obtained with the same MNP surface area, we obtained Fig. 6b. As shown, when the surface area of the two MNPs are the same (1 × 10−4 m2), the detection signal obtained with MNP-200 was higher than that obtained with MNP-1 μm in the presence of 10 ng/mL and 500 ng/mL antigen, whereas the opposite results were obtained with an antigen concentration of 1000 ng/mL. A possible reason is that the MNP surface antibody at the antigen concentrations of 10 ng/mL and 500 ng/mL is found in excess compared with the immune-complex, and the signal is determined by the reactivity of the surface antibody. As a result, a higher signal was obtained for MNP-200 due to the higher reactivity of the antibody on its surface. When the antigen concentration was increased to 1000 ng/mL, the number of antigen molecules becomes larger than the number of surface antibody molecules, and the density of the surface antibody becomes a decisive factor. Because the antibody coating amounts per mass of MNP were similar for the two MNPs, MNP-1 μm possesses a higher density of surface antibody due to its lower specific surface area. Consequently, a higher signal was obtained with MNP-1 μm in the presence of an antigen concentration of 1000 ng/mL. The described studies evaluated the properties of MNP-200 nm and MNP-1 μm in CLIA from several aspects. Although the detection signal obtained with MNP-1 μm is lower than that obtained with MNP200 nm, MNP-1 μm can generally meet the demands of the immunoassay and exhibits a lower background interference. The most important aspect is that MNP-1 μm can be rapidly captured by the external magnetic field to fulfill automated detection. Therefore, MNP-1 μm is more suitable to be used as the carrier in an automated CLIA. Comparison between the present chemiluminescence immunoassay system and a commercial system The automated chemiluminescence immunoassay system for the detection of myoglobin antibody was established using MNP-1 μm as the carrier. The system was applied for the detection of the myoglobin concentration in serum samples from clinical patients using an automated chemiluminescence spectrometer, and the results were compared with those obtained using a commercial system produced by Beckman. As shown in Fig. 7, a good interrelation was obtained between the two systems (R = 0.935). This result indicates that MNP-1 μm is an
Fig. 7. Comparison and correlation analysis of the two systems for myoglobin detection. SHJD: the detection system established in this study; Beckman: the detection system produced by Beckman, including the reagents and the apparatus.
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excellent carrier for an automated chemiluminescence immunoassay and that the immunoassay system established in this study generally meets the demands associated with clinical detection.
Conclusion MNPs are widely used as carriers in immunoassays. The physical and chemical properties of MNPs, including their size and contents of magnetic nanoparticles, have a direct influence on their performance in an immunoassay. In this study, we conducted a comparative study of the properties of two MNPs with micron and sub-micron sizes and discovered that MNP-1 μm presents a lower nonspecific adsorption to protein, a weaker interference with the chemiluminescence signal, and a more rapid response speed to the external magnetic field compared with MNP-200 nm. These properties make MNP-1 μm more suitable to be used as the carrier in automated CLIAs. Thus, the study of the relationship between the physical and chemical properties of MNPs and their performance in an immunoassay can provide both theoretical and experimental guidance for the research and development of MNPs to be used in immunoassays. In addition, a CLIA system for the detection of myoglobin antigen was established in this study using MNP-1 μm as the carrier. The system exhibits excellent performance and successfully meets the demands of a clinical immunoassay.
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