Bhalerao Ketki S, De Silva P I Thilini, Hiniduma Keshani, Grunbaum Ami, Rozza Nicholas, Kremer Richard, Rusling James F
Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, United States.
Department of Medicine, McGill University Health Centre, 1001 Decarie Blvd., Montreal, QC H3A 1A1, Canada.
ACS Omega. 2024 Jun 24;9(27):29320-29330. doi: 10.1021/acsomega.4c00735. eCollection 2024 Jul 9.
The "cytokine storm" often induced in COVID-19 patients contributes to the onset of "acute respiratory distress syndrome" (ARDS) accompanied by lung infection and damage, multiorgan failure, and even death. This large increase in pro-inflammatory cytokines in blood may be related to severity. Rapid, on-demand cytokine analyses can thus be critical to inform treatment plans and improve survival rates. Here, we report a sensitive, low-cost, semiautomated 3D-printed microfluidic immunoarray to detect 2 cytokines and CRP simultaneously in a single 10 μL serum sample in 25 min. Accuracy was validated by analyzing 80 COVID-19 patient serum samples, with results well correlated to a commercial Meso Scale protein immunoassay. Capture antibodies immobilized in detection microwells in a flat well plate-type flow chamber facilitate the immunoassay, with a programmable syringe pump automatically delivering reagents. Chemiluminescence signals were captured in a dark box with a CCD camera integrated for 30 s. This system was optimized to detect inflammation biomarkers IL-6, IFN-γ, and CRP simultaneously in blood serum. Ultralow limits of detection (LODs) of 0.79 fg/mL for IL-6, 4.2 fg/mL for CRP, and 2.7 fg/mL for IFN-γ with dynamic ranges of up to 100 pg/mL were achieved. ROC statistical analyses showed a relatively good diagnostic value related to the samples assigned WHO COVID-19 scores for disease severity, with the best results for IL-6 and CRP. Monitoring these biomarkers for coronavirus severity may allow prediction of disease severity as a basis for critical treatment decisions and better survival rates.
新冠病毒疾病（COVID-19）患者常引发的“细胞因子风暴”会导致“急性呼吸窘迫综合征”（ARDS）的发作，伴有肺部感染与损伤、多器官功能衰竭，甚至死亡。血液中促炎细胞因子的大幅增加可能与疾病严重程度相关。因此，快速、按需进行的细胞因子分析对于制定治疗方案和提高生存率至关重要。在此，我们报告一种灵敏、低成本、半自动的3D打印微流控免疫阵列，可在25分钟内于10微升血清样本中同时检测2种细胞因子和C反应蛋白（CRP）。通过分析80份COVID-19患者血清样本验证了准确性，结果与商业的中尺度蛋白质免疫测定法高度相关。固定在平板孔型流动室检测微孔中的捕获抗体便于进行免疫测定，可编程注射泵自动输送试剂。用集成了电荷耦合器件（CCD）相机的暗箱捕获30秒的化学发光信号。该系统经过优化，可同时检测血清中的炎症生物标志物白细胞介素-6（IL-6）、干扰素-γ（IFN-γ）和CRP。实现了IL-6的超低检测限（LOD）为0.79飞克/毫升、CRP为4.2飞克/毫升、IFN-γ为2.7飞克/毫升，动态范围高达100皮克/毫升。受试者工作特征（ROC）统计分析表明，与世界卫生组织（WHO）为疾病严重程度指定的COVID-19评分样本相关的诊断价值相对较好，IL-6和CRP的结果最佳。监测这些冠状病毒严重程度的生物标志物可能有助于预测疾病严重程度，作为关键治疗决策和提高生存率的依据。
