基於適體共軛奈米金粒子之D-二聚體比色分析法研究

Abstract

D-二聚體 (D-dimer) 是纖維蛋白的降解產物，在許多血栓患者中D-二聚體的含量都很高，D-二聚體是判定患者是否患有心血管疾病的重要因子，同時是檢驗抗凝血藥物在治療功效上的指標，因此如何精準地定量D-二聚體的含量成為了重要的議題，近年來，短片段的DNA適體 (Aptamer) 對特定蛋白質具有高度親合性與專一性，並且具有製造成本低、穩定性高以及容易修飾上官能基等優勢，逐漸成為替代抗體作為生物感測探針的工具，適體可以透過系統配位子指數增益演繹程序 (SELEX) 的方式篩選出來，此篩選出來的序列經過親和性與專一性的驗證後即可作為感測探針，並應用在基於比色法的感測器上來檢測D-二聚體，結合近年來蓬勃發展的奈米材料，適體-奈米金共軛比色感測法成為了一種新穎的感測方式，以奈米金、適體與其對應的蛋白質在鹽溶液中的交互作用達到感測效果，此方法的感測靈敏度高且偵測下限低，此外透過其溶液的顏色變化即可簡單的判讀蛋白質的含量，因此是非常有潛力作為快速感測D-二聚體的技術。 本研究旨在篩選出一條對於D-二聚體具有高親和性與專一性的適體序列，並應用此條適體作為感測元件應用於實際感測D-二聚體的含量，D-二聚體之適體使用基於玻璃微珠的SELEX從隨機序列庫中以定向演化的方式篩選出來，並經由定量聚合酶連鎖反應 (qPCR) 技術放大該序列於隨機序列庫的濃度，進一步定序後即可得到初步的適體候選序列，適體候選序列先經由表面電漿共振 (SPR) 確認其與D-二聚體之間的親和力與動力學特徵，從候選序列中找出表現最佳的序列作為後續感測用的適體，推算出此適體B5R10-4與D-二聚體的解離常數為18.3 nM，此B5R10-4適體透過高解析度熔解 (HRM) 分析及圓二色性 (CD) 光譜探討適體結構在不同溫度以及與D-二聚體結合作用下其構型的變化，最後，本研究應用自行篩選的B5R10-4適體結合到酶聯寡核苷酸分析法 (ELONA) 的感測平台去確認該適體於不同感測方法上的應用可行性，另一方面本研究試圖將B5R10-4適體推廣到新穎的適體-奈米金共軛比色分析法 (Apt-AuNP conjugates colorimetric assay)，並比較調整實驗參數和流程所取得的實驗表現，包含控制鹽類濃度、適體濃度、奈米金共軛適體機制以及奈米金粒徑大小等，綜合最佳的實驗條件顯示最佳的感測方法為使用粒徑小於約30 nm的奈米金粒子且利用結合式手法感測，其線性檢測範圍為0.125 – 10 nM，偵測下限低至0.25 nM，且靈敏度達到0.054，此適體感測裝置能簡易且強效的檢測D-二聚體，並有潛力發展為不須精密儀器即可判讀的快篩裝置或試劑，希望在不久的將來達成定點照護 (POC) 的感測目標。

D-dimer is a degradation product of fibrin. Elevated levels of D-dimer are commonly observed in patients with thrombosis. Thus, D-dimer is an important factor in determining whether a patient has cardiovascular disease. Meanwhile, it serves as an indicator of the efficacy of anticoagulant therapies. As a result, precise quantification of D-dimer levels has become a crucial issue. In recent years, short sequences of DNA aptamers have demonstrated high affinity and specificity for target proteins. Besides, they offer several advantages such as low production cost, high stability, and ease of functional modification. These properties have made aptamers a promising alternative to antibodies as biosensing probes. Aptamer can be selected through the systemic evolution of ligands by exponential enrichment (SELEX) process. After screening the affinity and specificity of the selected sequence, it can be used as a sensing probe in colorimetric sensors for detecting D-dimer. Coupled with the recent advancements in nanomaterials, aptamer-gold nanoparticle (Apt-AuNP) conjugates colorimetry has emerged as a novel sensing method. The sensing effect is based on the interactions between AuNPs, aptamers, and their corresponding proteins in saline solution. This method provides high sensitivity and low detection limits. Additionally, the color change in the solution allows for a simple visual readout of protein concentration. In light of this, this method holds great potential as a rapid detection technology for D-dimer. This study aims to select an aptamer sequence with high affinity and specificity for D-dimer and apply it as a sensing element for the quantification of D-dimer concentrations. The D-dimer aptamer was selected from a random sequence library using glass microbead-based SELEX. The sequence was then amplified through quantitative polymerase chain reaction (qPCR). After sequencing, preliminary aptamer candidates were obtained. The affinity and kinetic characteristics of these candidates were determined via surface plasmon resonance (SPR), and the best performing sequence was selected as the aptamer for sensing applications. The dissociation constant of this aptamer B5R10-4 and D-dimer was estimated to be 18.3 nM. The structural changes of the aptamer B5R10-4 at different temperature and in interaction with D-dimer were further investigated using high resolution melting (HRM) analysis and circular dichroism (CD) spectroscopy. Last but not least, the study applied the selected aptamer B5R10-4 to enzyme-linked oligonucleotide assay (ELONA) sensing platform to verify the applicability of the aptamer in different sensing platforms. On the other hand, this study aims to extend the application of the aptamer B5R10-4 to a novel Apt-AuNP conjugates colorimetric assay by adjusting experimental parameters and processes, including controlling salt concentration, aptamer concentration, Apt–AuNP conjugation mechanisms, and nanoparticle size. From the comprehensive experimental conditions, the colorimetric assay with the best performance involves using gold nanoparticles with a particle size smaller than approximately 30 nm, utilizing association-based approach. This method demonstrates a linear detection range of 0.125 – 10 nM, a detection limit as low as 0.25 nM, and achieves a sensitivity of 0.054. This aptamer-based sensor enables simple and robust detection of D-dimer. Furthermore, it has the potential to be developed into rapid diagnostic devices or kits that do not require sophisticated instruments to measure. The goal is to achieve a point-of-care (POC) sensing capability in the near future.