Kaladari Fatema, El-Maghrabey Mahmoud, Kishikawa Naoya, El-Shaheny Rania, Kuroda Naotaka
Department of Analytical Chemistry for Pharmaceuticals, Course of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan.
Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt.
Biosensors (Basel). 2025 Apr 16;15(4):256. doi: 10.3390/bios15040256.
This study seeks to develop and implement a non-enzymatic fluorescent labeling for immunoassay and immunochromatographic assay (ICAs) targeting SARS-CoV-2, to meet the extensive interest and need for effective COVID-19 diagnosis. In this manuscript, we delineate the development, synthesis, and evaluation of a novel quinone polymer zinc hybrid nanoarchitecture, referred to as polymerized alizarin red-inorganic hybrid nanoarchitecture (PARIHN), which integrates an antibody for direct use in fluorescent immunoassays, offering enhanced sensitivity, reduced costs, and improved environmental sustainability. The designed nanoarchitecture can enhance the sensitivity of the immunoassay and enable rapid results without the complexities associated with enzymes, such as their low stability and high cost. At first, a chitosan-alizarin polymer was synthesized utilizing quinone-chitosan conjugation chemistry (QCCC). Then, the chitosan-alizarin polymer was embedded with the detection antibody using zinc ion, forming PARIHN, which was proven to be a stable label with the ability to enhance the assay stability and sensitivity of the immunoassay. PARIHN can react with phenylboronic acid (PBA) or boric acid through its alizarin content to produce fluorescence signals with an LOD of 15.9 and 2.6 pm for PBA and boric acid, respectively, which is the first use of a boric acid derivative in signal generation in the immunoassay. Furthermore, PARIHN demonstrated high practicality in detecting SARS-CoV-2 nucleoprotein in fluorescence (PBA and boric acid) systems with an LOD of 0.76 and 10.85 pm, respectively. Furthermore, owing to the high brightness of our PARIHN fluorogenic reaction, our labeling approach was extended to immunochromatographic assays for SARS-CoV-2 with high sensitivity down to 9.45 pg/mL.
本研究旨在开发并实施一种针对严重急性呼吸综合征冠状病毒2(SARS-CoV-2)的非酶促荧光标记,用于免疫测定和免疫层析测定(ICA),以满足对有效的2019冠状病毒病(COVID-19)诊断的广泛关注和需求。在本论文中,我们描述了一种新型醌聚合物锌杂化纳米结构的开发、合成和评估,该结构被称为聚合茜素红-无机杂化纳米结构(PARIHN),它整合了一种抗体,可直接用于荧光免疫测定,具有更高的灵敏度、更低的成本和更好的环境可持续性。所设计的纳米结构可以提高免疫测定的灵敏度,并能快速得出结果,而无需面对与酶相关的复杂性,如酶的稳定性低和成本高。首先,利用醌-壳聚糖共轭化学(QCCC)合成了壳聚糖-茜素聚合物。然后,使用锌离子将壳聚糖-茜素聚合物与检测抗体包埋,形成PARIHN,事实证明它是一种稳定的标记物,能够提高免疫测定的稳定性和灵敏度。PARIHN可以通过其茜素成分与苯硼酸(PBA)或硼酸反应,分别产生检测限为15.9和2.6皮摩尔的荧光信号,这是硼酸衍生物首次用于免疫测定中的信号产生。此外,PARIHN在荧光(PBA和硼酸)系统中检测SARS-CoV-2核蛋白时表现出很高的实用性,检测限分别为0.76和10.85皮摩尔。此外,由于我们的PARIHN荧光反应具有高亮度,我们的标记方法被扩展到用于SARS-CoV-2的免疫层析测定,灵敏度高达9.45 pg/mL。
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