系統性配分子指數增益演繹程序(systematic evolution of ligands by exponential enrichment, SELEX)是一種體外篩選技術，透過該技術能在任意序列核酸分子庫中，經由反覆結合、分離和進行聚合酶連鎖反應(Polymerase chain reaction, PCR)放大的方式，篩選出一段與標的分子具高特異性和高親和力之單股去氧核糖核酸(single stranded deoxyribonucleic acid, ssDNA)或核糖核酸(ribonucleic acid, RNA)片段，稱之為適合體(aptamer)。此類適合體利用其本身特殊三維結構，可應用於多種用途上，包括感測及診斷分析，甚至可作為新藥物設計等。然而，反覆結合、分離和放大的程序，費時費力，且需使用到數種大型儀器，更容易因人為操作產生誤差和汙染。近年，隨著微機電系統(micro-electro-mechanical-system, MEMS)製程和微流體(microfluidic)技術之成熟，藉由微機電各種創新之製程技術，可將數種大型生化分析儀器縮小並整合至一微小的微流體晶片中，以減少生物試劑耗量及成本，且具有高檢測效能、可拋棄式、可攜帶性等優點，因此在生物醫學中扮演了十分重要的角色。
本研究利用微機電製程和微流體技術之整合，提出兩個自動化SELEX晶片系統。此微型化晶片系統主要由兩大模組組成，分別為用於流體傳輸以及混合之微流體操控模組和一個用於產生溫度場以進行聚合酶連鎖反應之微型溫度控制模組。首先，利用和蛋白質具有高專一性鍵結之微型磁珠(magnetic microbeads)修飾上待測蛋白質，接著利用微流體操控模組，將磁珠和任意序列單股去氧核糖核酸均勻混合(incubation)，藉由流體造成之擾流，與標的蛋白質具有親合力之單股去氧核糖核酸能固定在磁珠表面，接著利用外加磁場將磁珠在樣品中進行分離及濃縮，藉由此步驟，檢體將被淨化且集中，最後再透過晶片上高升降溫速率之微型溫度控制模組，控制反應槽內溫度高低循環進行聚合酶連鎖反應，將磁珠上的單股去氧核糖核酸複製放大，如此便完成一次SELEX程序。利用此晶片系統，經由反覆的SELEX程序(5~6次)，可以快速在任意序列核酸分子庫中篩選出與C-反應蛋白(C-reactive protein, CRP)和甲型胎兒蛋白(alpha-fetoprotein, AFP)具高專一性和親合力之適合體，相較於傳統篩選方法，可以節省一半以上的檢測時間和試劑消耗。
即使經由SELEX程序篩選，仍只有部份的核酸最終被確認和標的分子具有足夠的親和力及專一性，因此，SELEX程序篩選過後的核酸還需利用競爭型測試(competitive assay)做進一步的確認，以節省之後定序、合成及親和力量測等費用。然而，傳統競爭試測除了要需要花費許多時間與人力外，繁覆的清洗步驟經常會有與待測物不具親和力之核酸殘留在試管之中造成汙染，不利最後的判斷；針對於這個問題，本研究提出競爭測試晶片，利用自動化的清洗裝置和流線型的管道設計避免殘留問題，達到精確篩選與標的分子具高親和力及高專一性的適合體。根據競爭試測實驗結果，C-反應蛋白和甲型胎兒蛋白適合體利用表面電漿共振(surface plasmon resonance, SPR)所測得之解離速率常數(dissociation constant)分別為3.51 nM 和 2.37 nM，與傳統抗體相較，適合體比抗體具有更高的親合力。
最後將所挑選出的C-反應蛋白和甲型胎兒蛋白之適合體實際應用於臨床檢測，由實驗結果可知，C-反應蛋白和甲型胎兒蛋白之適合體檢測極限約在0.0125 mg/L 和12.5 ng/mL，而C-反應蛋白在濃度為0.0125 mg/L到10 mg/L之間時，線性度0.9694 (R2 = 0.9694)，甲型胎兒蛋白在濃度為12.5 ng/mL到800 ng/mL之間時，線性度0.9747 (R2 = 0.9747)，皆符合臨床檢測上的應用，相信於不久的將來，本晶片系統所提供之自動化快速篩選平台，將對於利用適合體相關之分子生物研究有極大助益。

The Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is a screening technique that involves the progressive selection of highly specific ligands by repeated rounds of partition and amplification from a large combinatorial nucleic acid library. The products of this selection process are called target specific aptamers, which are short single stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) molecules, binding with a high affinity, attributed to their specific three-dimensional shapes, to a large variety of targets, ranging from small molecules to complex mixtures. The aptamers with the highest affinity binding that are isolated through the SELEX process can have great potential as biochemical detectors diagnostic indicators and are considered as potent therapeutic lead structures to be evaluated in preclinical disease models. However, SELEX is an iterative process requiring multiple rounds of selection and amplification that demand significant time and labor. In addition, several large-scale pieces of equipment are usually needed to perform the SELEX protocol and the bio-samples may also be wasted or be a source of contamination during manual operations.
Another issue involved in the aptamer screening process is that it still requires a lengthy process of competitive assay to verify the selectivity of aptamers, before the selected ssDNA can be subsequently sequenced and synthesized. This is because only a few of the selected ssDNA aptamers are useful for further testing. Therefore, a competitive assay which screens the selected ssDNAs after the SELEX process is in great need. However, the traditional method for performing a competitive assay still requires repeated, time-consuming selection rounds and delicate manual processes. For example, it has proven challenging to control in a reproducible manner the washing conditions to avoid dead volumes in test tubes, which has a direct impact on the resulting aptamers that are extracted.
In order to address these problems, this study presents a magnetic bead-based microfluidic system which performs ssDNA incubation, extraction and nucleic acid amplification processes facilitating the entire SELEX process. More importantly, the selected ssDNA are further examined for their affinity and specificity to target molecules in a new competitive assay chip, which demonstrates exceptional separation efficiency in removing any weakly bound or unbound ssDNA to rapidly identify target-specific aptamers. With this approach, a C-reactive protein (CRP)-specific aptamer and an alpha-fetoprotein (AFP)-specific aptamer, which are biomarkers for cardiovascular diseases and liver cancers, respectively, have been successfully purified and enriched from a whole combinatorial ssDNA library. The resulting dissociation constant of the CRP-specific aptamer and the AFP-specific aptamer was 3.51 nM and 2.37 nM, respectively, which is comparable to the affinities for the CRP antibody and AFP antibody reported from different clones (10-7~10-9 M).
When compared to the traditional equipment that is used for SELEX processing, the microfluidic system is more compact in size and consumes fewer samples and reagents. It only takes approximately 60 min for the microfluidic SELEX chip to perform a single round of the SELEX process, which is much faster than that of a traditional SELEX process (at least 150 min). Moreover, the total sample volume which is consumed in each operation is only 40 μL, which is significantly less than that required in a large system (100 μL). Furthermore, the selected ssDNA has been further examined for its affinity and the specificity with the target in a new competitive assay chip. This process is much faster than the traditional competitive assay method and can significantly reduce the cost for ssDNA sequencing, synthesis and SPR detection.
In order to demonstrate the capabilities of the developed chips, the detection of CRP and AFP by using the SELEX selected CRP-specific aptamer and the AFP-specific aptamer have further been demonstrated. Experimental results show that the detection limit for CRP and AFP are 0.0125 mg/L and 12.5 ng/mL, respectively. Linear ranges (R2 = 0.9694 and R2 = 0.9747) have been obtained over concentrations ranging from 0.0125 mg/L to 10 mg/L and 12.5 ng/mL to 800 ng/mL, respectively, which makes it suitable for clinical applications. These developed microsystems are capable of faster screening of aptamers and can be used as a powerful tool to select analyte-specific aptamers for future biomedical applications.

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