Dugan Maria B, Anderson Jared L
Department of Chemistry, Iowa State University, 2415 Osborn Drive, Ames, IA, 50011-1021, USA.
Department of Chemistry, Iowa State University, 2415 Osborn Drive, Ames, IA, 50011-1021, USA.
Anal Chim Acta. 2025 Nov 8;1374:344534. doi: 10.1016/j.aca.2025.344534. Epub 2025 Aug 13.
Advancements in nucleic acid (NA) analysis rely on the development of efficient extraction and amplification techniques that deliver high sensitivity, reliability, and streamlined design. Traditional organic solvents used in NA extraction often present challenges related to their toxicity, volatility, and limited biocompatibility. Deep eutectic solvents (DESs) have emerged as a promising class of solvents due to their customizable properties and potential for biomolecular applications. This study addresses a fundamental challenge in molecular diagnostics by evaluating DESs as both extraction media for NAs and direct additives within quantitative polymerase chain reaction (qPCR) assays, thereby eliminating the need for intermediate purification.
In this study, fifty-three DESs from seven distinct classes were evaluated for their compatibility with qPCR and utility as extraction solvents for NA analysis. Incorporating DESs directly into qPCR revealed key benefits like enhanced DNA amplification efficiency and the ability to tailor conditions for diverse extraction requirements. Two DNA sequences, a 98 base pair (bp) BRAF and 121 bp uidA fragment, were analyzed to assess their amplification in the presence of DESs with varied chemical composition. A subset of thirty thermally stable DESs proved promising under simulated PCR conditions. However, when the DESs were added directly to qPCR, six permitted successful amplification, while the others interfered with amplicon generation. Three of these DESs yielded qPCR efficiencies of 97.22 ± 5.41 % for the 121 bp uidA fragment. Findings revealed that the nature of hydrogen bond acceptor and donor play a crucial role in maintaining qPCR enzymatic activity and preserving NA integrity.
This work is the first to systematically evaluate a broad range of DESs for integrated NA extraction and direct amplification with qPCR. The findings offer valuable insight into the DES structure-function relationships that govern assay compatibility. By eliminating traditional purification steps, DESs can simplify workflows and increase throughput for molecular diagnostics. This platform opens new opportunities for the development of automated, field-deployable NA testing systems using customizable solvent systems with enhanced analytical performance.
核酸（NA）分析的进展依赖于高效提取和扩增技术的发展，这些技术需具备高灵敏度、可靠性和简化的设计。NA提取中使用的传统有机溶剂常常因其毒性、挥发性和有限的生物相容性而带来挑战。由于其可定制的性质以及在生物分子应用中的潜力，深共熔溶剂（DESs）已成为一类有前景的溶剂。本研究通过评估DESs作为NA的提取介质以及定量聚合酶链反应（qPCR）测定中的直接添加剂，解决了分子诊断中的一个基本挑战，从而无需中间纯化步骤。
在本研究中，对来自七个不同类别的53种DESs进行了评估，以确定它们与qPCR的兼容性以及作为NA分析提取溶剂的效用。将DESs直接纳入qPCR显示出关键优势，如提高DNA扩增效率以及能够针对不同提取要求调整条件。分析了两个DNA序列，一个98碱基对（bp）的BRAF和121 bp的uidA片段，以评估它们在具有不同化学组成的DESs存在下的扩增情况。在模拟PCR条件下，30种热稳定的DESs子集显示出前景。然而，当将DESs直接添加到qPCR中时，六种允许成功扩增，而其他的则干扰扩增子生成。其中三种DESs对121 bp的uidA片段产生的qPCR效率为97.22±5.41%。研究结果表明，氢键受体和供体的性质在维持qPCR酶活性和保持NA完整性方面起着关键作用。
这项工作首次系统地评估了广泛的DESs用于NA的综合提取和qPCR直接扩增。这些发现为支配测定兼容性的DES结构 - 功能关系提供了有价值的见解。通过消除传统纯化步骤，DESs可以简化工作流程并提高分子诊断的通量。该平台为使用具有增强分析性能的可定制溶剂系统开发自动化、可现场部署的NA检测系统开辟了新机会。
