糖尿病視網膜病變是糖尿病常見的併發症。根據衛生福利部疾病管制署統計，此疾病在美國和台灣等多國已成為成年人失明的主因。目前臨床診斷此疾病的檢測方法有眼部斷層掃描和螢光血管照影，但前者無論是儀器或是檢測費用都較昂貴，後者則為侵入式的檢測方式，必須在靜脈施打螢光染劑，且這兩種檢測方法皆無法量化。為了增加民眾的檢測意願，本研究希望藉由發展熱擴散量測技術搭配微珠式免疫接合法，以人體淚液中的腫瘤壞死因子(TNF-α)含量來快速篩檢疾病。
研究原理主要利用在微觀尺度下，粒子產生的布朗運動進行擴散度分析。根據斯托克斯-愛因斯坦方程式，當粒徑增加時，粒子運動的擴散度會隨之下降，造成布朗運動變慢。實驗過程中，我們將螢光奈米粒子表面修飾有腫瘤壞死因子(TNF-α)之抗體、抗原和奈米金進行三明治免疫分析，在螢光顯微鏡下觀測其目標物之螢光，藉以量測粒子布朗運動之變化。
本研究利用兩種免疫檢測法進行實驗，分別為以三明治免疫法建立標準檢測曲線和以競爭免疫法開發二分法檢測。為了最佳化實驗系統，我們分別進行了不同粒徑的螢光粒子、螢光粒子數量和增加粒徑改變量的不同奈米金粒子測試。此外，我們也藉由初步的實驗，驗證此系統可以藉由施打綠光雷射對奈米金粒子產生的表面電漿共振，來提升高濃度與低濃度組別間的擴散度變化。
由本研究所開發的檢測平台，可以使用微量非侵入式檢體快速檢測指標性蛋白(TNF-α)。由此研究系統所建立的標準檢測曲線之檢測敏感度為100 pg/mL，而二分法之檢測敏感度可檢測至與控制組別差異五倍之濃度樣品，且檢測敏感度可下降至10 pg/mL。為了可以讓操作者簡易的執行檢測分析免去繁複的操作步驟，目前此檢測平台已開發成使用者圖形介面。此外，本研究所開發之檢測平台，可以藉由改變不同的指標性蛋白進行不同疾病的檢測分析。

Diabetic retinopathy (DR) is a common complication of diabetes mellitus. According to the statistical results from Centers for Disease Control and Prevention (CDC), DR has become the leading cause of blindness among adults in the US and Taiwan. The current methods for diagnosis of DR are Optical Coherent Tomography (OCT) and Fluorescein Angiography (FAG). However, the diagnostic cost of OCT is expensive and FAG requires the injection of a special dye into an arm vein. In addition, both methods are not quantitative. To encourage the patients’ willingness of diagnosis, we wanted to develop a rapid screening technique based on diffusometry with functionalized beads to detect the concentration of TNF-α in human tear fluid.
The concentration of the analyte was determined by measuring the Brownian diffusion of the functionalized nanobeads. With the presence of target analytes, particle diameter increased due to sandwiched binding of antibodies, target analytes and gold nanoparticles to the particle. According to Stoke Einstein equation, increased particle diameter results in decreased diffusivity. Therefore, Brownian motion can be used in analytes screening.
Two methods based on different types of immunoassays were developed. Sandwiched immunoassay was used to establish a standard curve while competitive immunoassay was used in dichotomy screening. We investigated different parameters such as particle diameter, number of particles and the size and shape of gold nanoparticles to optimize the experimental system. In addition, we learned from our preliminary experiments that the diffusivity of different concentrations of analytes could be enhanced by applying a laser beam.
In conclusion, the screening technique combining bead-based immunoassays with evaluations of diffusometry is able to detect different concentrations of biomarker (TNF-α) noninvasively and rapidly while using low-volume samples. We established an optimal standard curve using conjugation of 80 nm spherical gold nanoparticles to 200 nm particles and the limit of detection (LOD) is 100 pg/mL. The dichotomy screening method can detect a 5-fold difference in concentration and the LOD is 10 pg/mL. Furthermore, this technique was successfully developed into analytes screening platform with an included graphical user interface. Lastly, our platform can be applied to detect biomarkers in other diseases by altering the capture antibodies on the particles to perform rapid screening.

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