Celiac disease is an autoimmune disorder represented by the ingestion of the gluten protein usually found in wheat, barley and rye. To date, ELISA has been the most accurate method for determining the presence of anti-gliadin, which is cumbersome, expensive (compared to a suspension microarray technique), and requires extensive sample preparation. In this study, in order to establish a more accurate assay to identify gliadin at lower concentrations, optical nano biosensors using an indirect immunoassay method for gliadin detection was designed and fabricated. For this, polycaprolactone (PCL) nano- to micro-beads were fabricated as a platform for the gliadin antigen which were optimized and nano functionalized with amine groups for such purposes. The gliadin antibody, which is selective to gliadin, was then added to the beads. Static light scattering tests were conducted to determine PCL particle size distribution and sizes were found from 0.1 to 30 μm, which is suitable for flowcytometry detection devices. Anti-gliadin detection was performed using an anti IgG mouse antibody conjugated with FITC in a flow cytometry device to detect the smallest particle. Fluorescence intensity was investigated at different concentrations of anti-gliadin and a standard curve used to determine gluten concentration based on fluorescence intensity. Results showed that the fluorescence intensity increased with greater concentrations of anti-gliadin providing a very effective method of detection due to selectivity at a 5 ppm detection limit. This represents a new highly sensitive and fast method for anti-gliadin detection. Further, the disuse of a cross linker and the use of a dedicated antibody at a very low level (1 μl) made this new method very economical to identify anti-gliadin concentrations at the nano level. In summary, this study provides a new, more accurate and sensitive, as well as less expensive system to detect anti-gliadin for the improved diagnosis of celiac disease.

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基于悬浮微阵列的乳糜泻抗麦醇溶蛋白检测纳米生物传感器的开发

乳糜泻是一种自身免疫性疾病，以摄入小麦、大麦和黑麦中常见的面筋蛋白为代表。迄今为止，ELISA 一直是确定抗麦醇溶蛋白存在的最准确方法，这种方法既麻烦又昂贵（与悬浮微阵列技术相比），并且需要大量的样品制备。在这项研究中，为了建立更准确的检测方法来识别较低浓度的麦醇溶蛋白，设计和制造了使用间接免疫测定法检测麦醇溶蛋白的光学纳米生物传感器。为此，制造了聚己内酯 (PCL) 纳米到微珠作为麦醇溶蛋白抗原的平台，这些抗原经过优化并用胺基进行了纳米功能化。然后将对麦醇溶蛋白具有选择性的麦醇溶蛋白抗体添加到珠子中。进行静态光散射测试以确定PCL粒径分布，发现粒径为0.1至30μm，适用于流式细胞仪检测装置。使用与 FITC 偶联的抗 IgG 小鼠抗体在流式细胞仪中进行抗麦醇溶蛋白检测，以检测最小的颗粒。在不同浓度的抗醇溶蛋白和用于基于荧光强度确定面筋浓度的标准曲线下研究荧光强度。结果表明，荧光强度随着抗醇溶蛋白浓度的增加而增加，由于在 5 ppm 检测限下的选择性，提供了一种非常有效的检测方法。这代表了一种新的高灵敏度和快速的抗麦醇溶蛋白检测方法。更远，交联剂的废弃和使用非常低水平（1 μl）的专用抗体使得这种新方法在确定纳米级抗麦醇溶蛋白浓度时非常经济。总之，本研究提供了一种新的、更准确、更灵敏、更便宜的检测抗醇溶蛋白的系统，以改进乳糜泻的诊断。