Assaying Carcinoembryonic Antigens by Normalized Saturation Magnetization

Huang et al. Nanoscale Research Letters (2015):7 DOI 10.1186/s11671-015-0964-6 NANO EXPRESS Open Access Assaying Carcinoembryonic Antigens by Normalized Saturation Magnetization Kai-Wen Huang2,3, Jen-Jie Chieh1*, Jin-Cheng Shi1 and Ming-Hsien Chiang4 Abstract Biofunctionalized magnetic nanoparticles (BMNs) that provide unique advantages have been extensively used to develop immunoassay methods. However, these developed magnetic methods have been used only for specific immunoassays and not in studies of magnetic characteristics of materials. In this study, a common vibration sample magnetometer (VSM) was used for the measurement of the hysteresis loop for different carcinoembryonic antigens (CEA) concentrations (ΦCEA) based on the synthesized BMNs with anti-CEA coating. Additionally, magnetic parameters such as magnetization (M), remanent magnetization (MR), saturation magnetization (MS), and normalized parameters (ΔMR/MR and ΔMS/MS) were studied. Here, ΔMR and ΔMs were defined as the difference between any ΦCEA and zero ΦCEA. The parameters M, ΔMR, and ΔMS increased with ΦCEA, and ΔMS showed the largest increase. Magnetic clusters produced by the conjugation of the BMNs to CEAs showed a ΔMS greater than that of BMNs. Furthermore, the relationship between ΔMS/MS and ΦCEA could be described by a characteristic logistic function, which was appropriate for assaying the amount of CEAs. This analytic ΔMS/MS and the BMNs used in general magnetic immunoassays can be used for upgrading the functions of the VSM and for studying the magnetic characteristics of materials. Keywords: Magnetic immunoassays; Saturation magnetization; Magnetic clusters; Carcinoembryonic antigen; Biofunctionalized magnetic nanoparticles Background Magnetic nanoparticles interest researchers because of their potential applications in biomedicine, such as protein purification [1], magnetofection [2], tomographic imaging [3], magnetic resonance imaging [4–6], magnetic immunoassays [7, 8], tumor diagnosis [9], and hyperthermia therapy [10]. In magnetic immunoassays, magnetic nanoparticles are first biofunctionalized with antibodies to obtain biofunctionalized magnetic nanoparticles (BMNs), which are then dissolved in solutions to form magnetic reagents. To assay a biotarget, a magnetic reagent is mixed with a sample solution containing the biotarget. The conjugation of BMNs with the biotarget produces magnetic clusters because of molecular interaction (Fig. 1), and the magnetic properties of the reagent changes. Biological samples, unconjugated BMNs, and magnetic clusters of conjugated biotargets show a negligible magnetic background individually and differ in their * Correspondence: 1 Institute of Electro-Optical Science and Technology, National Taiwan Normal University, 116 Taipei, Taiwan Full list of author information is available at the end of the article magnetic characteristics. Hence, it is possible to develop magnetic immunoassays on the basis of several parameters and phenomena such as magnetic relaxation [11, 12], remanent magnetization (MR) [13, 14], saturation magnetization (MS) [15], magnetic resonance [16, 17], and alternating current (ac) susceptibility (χac) [8, 18–21]. In addition, because signal changes associated with the magnetic characteristics of BMNs are always small, a high-sensitivity high-critical-temperature superconducting quantum interference device (SQUID) sensor is usually used to enhance the signal-to-noise ratio and mu-metal shielding is provided to reduce environmental noise. A cryogenic biodetection system involving SQUIDs is difficult to construct. Washing processes are sometimes required to separate magnetic clusters from reagents for measuring magnetic characteristics; however, they are time-consuming. Therefore, developing a biodetection system featuring an alternative detection mechanism and high detection sensitivity is crucial. A wash-free immunomagnetic reduction (IMR) method based on ac magnetic susceptibility reduction has been proposed [19], and various studies have © 2015 Huang et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Huang et al. Nanoscale Research Letters (2015):7 Page 2 of 7 Fig. 1 A scheme of CEAs, Fe3O4-anti-CEA, and Fe3O4-anti-CEA-CEA. Some Fe3O4-anti-CEAs become as magnetic cluster, Fe3O4-anti-CEA-CEA, after binding to CEA antigen demonstrated the sensitive detection of biomolecules, such as nucleic acids [20], biomarkers (for diagnosing Alzheimer’s disease) [6], alpha-fetoprotein (for detecting liver tumors) [7], and human C-reactive protein (for diagnosing inflammation) [15]. In this study, we proposed a magnetic immunoassay method based on the BMNs used in magnetic immunoassay methods, like IMR; the proposed method does not require a SQUID sensor or washing process. The method involves the use of a vibration sample magnetometer (VSM) for measuring the hysteresis loop, from which the major magnetic characteristics can be inferred, and does not require a specific magnetic instrument for magnetic immunoassays. The magnetic parameters of the hysteresis loop were studied to determine the analytic method of magnetic immunoassay. When the method is applied to magnetic immunoassays, the magnetic parameters of the analytics are determined from the hysteresis loop. Methods Figure 1 shows a schematic of the clustering process involving BMNs and dextran-coated Fe3O4 nanoparticles. The procedures used for synthesizing BMNs consisting of anticarcinoembryonic antigens (anti-CEAs) coated on dextran-coated Fe3O4 nanoparticles (MF-DEX-0060, MagQu Corp., Taiwan) were similar to those used in a previous study for synthesizing dextran-coated Fe3O4 nanoparticles coated with anti-goat C-reactive protein [22]. Dextran-coated Fe3O4 nanoparticles was oxidized using NaIO4 to create aldehyde groups (−CHO), and dextran reacted with the antibodies of anti-CEAs (10CCR2014M5, Fitzgerald, MA, USA) through −CH = N- to covalently conjugate the antibodies of anti-CEAs. After magnetic separation, the unbound antibodies were separated from conjugated BMNs consisting of dextran-coated Fe3O4 nanoparticles coated with anticarcinoembryonic antigens (Fe3O4-anti-CEAs). Subsequently, a reagent was synthesized by dissolving the BMNs in phosphatebuffered saline. The biotargets were carcinoembryonic antigens (CEAs; 30-AC30, Fitzgerald, MA, USA). These antigens are typically used as a tumor marker for colorectal cancers, which are caused by uncontrolled cell growth in the colon or rectum [23] and are the second leading cause of cancer death in adults worldwide [24]. The mean value of the hydrodynamic diameter of the BMNs was 40.8 nm, as detected through dynamic laser scattering (Nanotrac 150, Microtrac, PA, USA). The conjugation capability of BM (...truncated)