DataSheet1_Development of ZnCdSe/ZnS quantum dot-based fluorescence immunochromatographic assay for the rapid visual and quantitative detection 25⁃hydroxyvitamins D in human serum.docx

Vitamin D deficiency is associated with various diseases such as obesity, digestive problems, osteoporosis, depression, and infections, and has therefore emerged as a topic of great interest in public healthcare. The quantitative assessment of 25-hydroxyvitamin D (25-OH VD) in human serum may accurately reflect the nutritional status of vitamin D in the human body, which is significant for the prevention and treatment of vitamin D-deficient patients. In this study, we developed an assay for quantitative detection of 25-OH VD based on the 25-OH VD monoclonal antibody (mAb), and identified the optimal process parameters. The following process settings were found to be suitable for the test strips: pH of 7.6, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) ratio of 1:2000, and the anti-25-OH VD mAb ratio was 1:8. The equilibration time of the immune dynamic assay was 15 min. Under optimal conditions, the quantum dot nanoparticle-based fluorescent immunochromatographic assay (QDs-FICA) exhibited dynamic linear detection of 25-OH VD in PBS, from 5 ng/mL to 100 ng/mL, and the strip quantitative curve could be represented by the following regression equation: y = −0.02088 logx)+1.444 (R2 = 0.9050). The IC50 of the QDs-FICA was 39.6 ± 1.33 ng/mL. The specificity of the QDs-FICA was evaluated by running several structurally related analogues, including 25-OH VD2, 25-OH VD3, 1,25-OH2VD3, 1,25-OH2VD2, VD2, and VD3. The coefficients of variation were all below 10%. The shelf life of the test strips in this study was about 160 days at room temperature. Briefly, this study is the first to perform QDs-FICA for the rapid visual and quantitative detection of 25-OH VD, with great potential significance for clinical diagnosis of vitamin D-associated diseases.

维生素D缺乏与肥胖、消化系统疾病、骨质疏松症、抑郁症和感染等多种疾病相关联，因此已成为公共卫生领域备受关注的议题。对人类血清中25-羟基维生素D（25-OH VD）的定量评估能够准确反映人体内维生素D的营养状况，这对于预防及治疗维生素D缺乏症患者具有重要意义。在本研究中，我们基于25-OH VD单克隆抗体（mAb）开发了一种定量检测25-OH VD的检测方法，并确定了最佳工艺参数。研究发现，pH值为7.6，1-乙基-3-(3-二甲基氨基丙基)碳二亚胺（EDC）比例为1:2000，以及抗25-OH VD单克隆抗体比例为1:8的工艺设置适用于试纸测试。免疫动态测定的平衡时间为15分钟。在最佳条件下，基于量子点纳米颗粒的荧光免疫层析测定（QDs-FICA）在磷酸盐缓冲溶液（PBS）中对25-OH VD的检测呈现动态线性，检测范围从5 ng/mL至100 ng/mL，试纸定量曲线可用以下回归方程表示：y = −0.02088 logx)+1.444（R2 = 0.9050）。QDs-FICA的半抑制浓度（IC50）为39.6 ± 1.33 ng/mL。通过运行包括25-OH VD2、25-OH VD3、1,25-OH2VD3、1,25-OH2VD2、VD2和VD3在内的多个结构相关类似物，评估了QDs-FICA的特异性，其变异系数均低于10%。本研究中，试纸在室温下的保质期约为160天。简而言之，本研究首次采用QDs-FICA进行25-OH VD的快速视觉和定量检测，对于维生素D相关疾病的临床诊断具有重大的潜在意义。