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Junhong Chen, et al.Aptamer-based cell-free detection system to detect target protein, Synthetic and Systems Biotechnology,Volume 6, Issue 3, September 2021, Pages 209-215

Abstract
Biomarkers of disease, especially protein, show great potential for diagnosis and prognosis. For detecting a certain protein, a binding assay implementing antibodies is commonly performed. However, antibodies are not thermally stable and may cause false-positive when the sample composition is complicated. In recent years, a functional nucleic acid named aptamer has been used in many biochemical analysis cases, which is commonly selected from random sequence libraries by using the systematic evolution of ligands by exponential enrichment (SELEX) techniques. Compared to antibodies, the aptamer is more thermal stable, easier to be modified, conjugated, and amplified. Herein, an Aptamer-Based Cell-free Detection (ABCD) system was proposed to detect target protein, using epithelial cell adhesion molecule (EpCAM) as an example. We combined the robustness of aptamer in binding specificity with the signal amplification ability of CRISPR-Cas12a′s trans-cleavage activity in the ABCD system. We also demonstrated that the ABCD system could work well to detect target protein in a relatively low limit of detection (50–100 nM), which lay a foundation for the development of portable detection devices. This work highlights the superiority of the ABCD system in detecting target protein with low abundance and offers new enlightenment for future design and development.

摘要
疾病的生物標(biāo)志物，尤其是蛋白質(zhì)，在診斷和預(yù)后方面顯示出巨大的潛力。為了檢測(cè)某種蛋白質(zhì)，通常會(huì)進(jìn)行抗體結(jié)合試驗(yàn)。然而，抗體不具有熱穩(wěn)定性，當(dāng)樣品成分復(fù)雜時(shí)，可能導(dǎo)致假陽(yáng)性。近年來(lái)，一種稱(chēng)為適體的功能性核酸已被用于許多生化分析案例中，該核酸通常是通過(guò)指數(shù)富集（SELEX）技術(shù)利用配體的系統(tǒng)進(jìn)化從隨機(jī)序列庫(kù)中選擇的。與抗體相比，適配子更熱穩(wěn)定，易于修飾、接合和擴(kuò)增。在此，以上皮細(xì)胞粘附分子（EpCAM）為例，提出了一種基于適體的無(wú)細(xì)胞檢測(cè)（ABCD）系統(tǒng)來(lái)檢測(cè)目標(biāo)蛋白。我們將適配子結(jié)合特異性的穩(wěn)健性與ABCD系統(tǒng)中CRISPR-Cas12a的反式切割活性的信號(hào)放大能力相結(jié)合。我們還證明了ABCD系統(tǒng)能夠很好地檢測(cè)目標(biāo)蛋白，其檢測(cè)范圍相對(duì)較低（50～100 nm），為便攜式檢測(cè)設(shè)備的研制奠定了基礎(chǔ)。這項(xiàng)工作突出了ABCD系統(tǒng)在檢測(cè)低豐度目標(biāo)蛋白方面的優(yōu)勢(shì)，并為未來(lái)的設(shè)計(jì)和開(kāi)發(fā)提供了新的啟示。

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