在過去的幾十年中，微珠式三明治免疫法已經引起了科學界的興趣，這是因為它們在檢測各種生物標誌物方面具有快速和複數檢測的能力。然而，由於濃縮的方法有限，應用在檢測低濃度分析物方面仍然是一個挑戰。為此，我們研究提出了一種通過光電動平台進行微珠濃縮的方法，隨後以此技術檢測了雙重的生物標記物，用於篩檢稱為糖尿病性視網膜病（DR）的眼睛疾病。在這項研究中，在目標抗原存在下，微珠的表面形成了夾心的免疫複合物，免疫結構包含抓取式抗體-抗原（生物標誌物）-探測性抗體。然後，通過光電動平台將各個微珠集中，以增強其信號。信號可在10 秒內迅速升高，並且偵測極限可達到100 pg / mL。我們以前的研究顯示，快速光電動圖紋法中微珠的行為與所提供的電場頻率和微珠的大小有關。以前的研究已成功分離三個不同大小（1、3和5 μm）的微珠。在這項研究中，相同粒徑的微珠分別修飾上螢光微珠和螢光二抗，並且將微珠通過不同的表面性質分開。為了進行實際的DR檢測，我們使用了兩種生物標誌物脂質運載蛋白1（LCN1）和血管內皮生長因子（VEGF）以增加準確性與特異性。從檢測患者收集到約20 μL的眼淚樣品。此兩種生物標誌物的濃度均顯示具有出隨糖尿病視網膜病變的嚴重程度而升高的趨勢。根據檢測到的曲線，從24個臨床樣本中確認了LCN1和VEGF的兩個生物標誌物在增生型糖尿病視網膜病變中的濃度閾值在此光電平台上分別為250 µg/ml與10 ng/ml 。最後為了驗證兩種閾值的有效性，我們對五個隨機受試者的臨床淚液樣本進行了單盲測試。該驗證的最終結果顯示檢測正確的準確度至少為 80％以上。這種非侵入性檢測為DR的早期診斷提供了一種新的方法，並且希望將來可以增加對高危險糖尿病患者的篩檢率。

Bead-based immunosensors have been intriguing the scientific community over the past decades due to their rapid and multiplexed capabilities in the detection of various biological targets. Nevertheless, their use in the detection of low-abundance analytes remains a continual challenge because of the limited number of active enrichment approaches. To this end, our research presents a delicate microbead enrichment by an optoelectrokinetic platform, followed by the detection of dual biomarkers for the sensitive screening of an eye disease termed diabetic retinopathy (DR). In this study, microbeads turned fluorescent as their surfaces formed sandwiched immunocomplexes in the presence of target antigens. Tiny fluorescent dots were then concentrated by the optoelectrokinetic platform for the enhancement of their signals. The signal rapidly escalated in 10 s, and the optimal limit of detection was nearly 100 pg/mL. Our previous studies have shown that the behavior of microbeads in REP is related to the frequency of the electric field provided and the size of the bead. It has been successfully separated and mixed with three beads of different sizes (1, 3 and 5 μm). In this study, the microbeads of the same particle size are used to modify the fluorescent microbeads and the fluorescent secondary antibody respectively, and the microbeads are separated by different surface properties. For practical DR screening, two biomarkers, lipocalin 1 (LCN1) and vascular endothelial growth factor (VEGF), were used. Approximately 20 μL of analytes was collected from the tear samples of the tested patients. The concentrations of both biomarkers showed escalating trends with the severity of DR. Two concentration thresholds of LCN1 250 µg/ml and VEGF 10 ng/ml that indicate proliferative DR were determined out of 24 clinical samples based on the receiver operating characteristic curves. For verification, a single-blinded test was conducted with additional clinical tear samples from five random subjects. The final outcome of this evaluation showed an accuracy of >80%. This noninvasive screening provides a potential means for the early diagnosis of DR and may increase the screening rate among the high-risk diabetic population in the future.

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