Kalarestaghi Alireza, Bayat Mansour, Hashemi Seyed Jamal, Razavilar Vadood
Department of Pathobiology, Faculty of Veterinary Specialized Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Food Microbiology Research center, Tehran University of Medical Sciences, Tehran, Iran.
Iran J Biotechnol. 2015 Sep;13(3):25-31. doi: 10.15171/ijb.1170.
Recently, some new nanobiosensors using different nanoparticles or microarray systems for detection of mycotoxins have been designed . However, rapid, sensitive and early detection of aflatoxicosis would be very helpful to distinguish high-risk persons.
We report a highly sensitive competitive immunoassay using magnetic/silica core shell as a signal intensifier for the determination of aflatoxin B1 using fluorescence resonance energy transfer (FRET) from Cd/Te quantum dots (antiaflatoxin B1 antibody immobilized on the surface of Cd/Te quantum dots) to Rhodamine 123 (Rho 123-labeled aflatoxin B1 bound to albumin). The specific immune-reaction between the anti-aflatoxin B1 antibody on the QDs and the labeledaflatoxin B1 brings the Rho 123 fluorophore (acting as the acceptor) and the QDs (acting as the donor) in close spatial proximity and causes FRET to occur upon photo-excitation of the QDs. Using magnetic/silica core shell to intensify the obtained signal is the novelty of this study.
Cd/Te QDs were synthesized by the simultaneous reduction of cadmium chloride and tellurium in the presence of sodium borohydride under nitrogen atmosphere. Magnetic nanoparticles were synthesized using FeSO and FeCl (1:2 molar ratio) and ammonia as an oxidizing agent under nitrogen atmosphere. The prepared magnetic nanoparticles shelled by silica using tetraethoxysilane in the presence of ammonia. Nanoparticles synthesis and monodispersity confirmed by TEM. Immobilization of Cd/Te QDs to antibodies and labeling of aflatoxin B1-albumin by Rho 123 were performed by EDC/NHS reaction in reaction mixture buffer, pH 6, at room temperature.
By using the magnetic/silica core shell sensitivity of the system changed from 2×10 in our previous study to 2×10 in this work. The feasibility of the method established by the detection of aflatoxin B1 in spiked human serum. There is a linear relationship between the decreased fluorescence intensity of Rho 123 with increasing concentration of aflatoxin B1 in spiked samples, over the range of 0.01-0.06 μmol.mL.
This homogeneous competitive detection scheme is simple, rapid and efficient, and does not require multiple separation steps and excessive washing.
最近，已经设计出了一些使用不同纳米颗粒或微阵列系统检测霉菌毒素的新型纳米生物传感器。然而，黄曲霉毒素中毒的快速、灵敏和早期检测对于区分高危人群非常有帮助。
我们报道了一种高灵敏度竞争免疫分析方法，该方法使用磁性/二氧化硅核壳作为信号增强剂，通过荧光共振能量转移（FRET）从Cd/Te量子点（抗黄曲霉毒素B1抗体固定在Cd/Te量子点表面）到罗丹明123（Rho 123标记的与白蛋白结合的黄曲霉毒素B1）来测定黄曲霉毒素B1。量子点上的抗黄曲霉毒素B1抗体与标记的黄曲霉毒素B1之间的特异性免疫反应使Rho 123荧光团（作为受体）和量子点（作为供体）在空间上紧密靠近，并在量子点光激发时导致FRET发生。使用磁性/二氧化硅核壳增强获得的信号是本研究的新颖之处。
在氮气气氛下，在硼氢化钠存在下通过同时还原氯化镉和碲合成Cd/Te量子点。在氮气气氛下，使用硫酸亚铁和氯化铁（摩尔比1:2）以及氨作为氧化剂合成磁性纳米颗粒。在氨存在下，使用四乙氧基硅烷将制备的磁性纳米颗粒包覆二氧化硅。通过透射电子显微镜（TEM）确认纳米颗粒的合成和单分散性。在室温下，在pH 6的反应混合物缓冲液中，通过EDC/NHS反应将Cd/Te量子点固定到抗体上，并将Rho 123标记到黄曲霉毒素B1-白蛋白上。
通过使用磁性/二氧化硅核壳，系统的灵敏度从我们之前研究中的2×10变为本研究中的2×10。通过检测加标人血清中的黄曲霉毒素B1证实了该方法的可行性。在加标样品中，随着黄曲霉毒素B1浓度的增加，Rho 123荧光强度的降低在0.01 - 0.06 μmol·mL范围内呈线性关系。
这种均相竞争检测方案简单、快速且高效，不需要多个分离步骤和过度洗涤。
