Anal Chem. 2019 Sep 17;91(18):11848-11855. doi: 10.1021/acs.analchem.9b02572. Epub 2019 Aug 26.
The extraction of bioanalytes is the first step in many diagnostic and analytical assays. However, most bioanalyte extraction methods require extensive dilution-based washing processes that are not only time-consuming and laborious but can also result in significant sample loss, limiting their applications in rare sample analyses. Here, we present a method that enables the efficient extraction of multiple different bioanalytes from rare samples (down to 10 cells) without washing-centrifugation-assisted immiscible fluid filtration (CIFF). CIFF utilizes centrifugal force to drive the movement of analyte-bound glass microbeads from an aqueous sample into an immiscible hydrophobic solution to perform an efficient, simple, and nondilutive extraction. The method can be performed using conventional polymerase chain reaction (PCR) tubes with no requirement of specialized devices, columns, or instruments, making it broadly accessible and cost-effective. The CIFF process can effectively remove approximately 99.5% of the aqueous sample in one extraction with only 0.5% residual carryover, whereas a traditional "spin-down and aspirate" operation results in a higher 3.6% carryover. Another unique aspect of CIFF is its ability to perform two different solid-phase bioanalytes extractions simultaneously within a single vessel without fractionating the sample or performing serial extractions. Here we demonstrate efficient mRNA and DNA extraction from low-input samples (down to 10 cells) with slightly higher to comparable recovery compared to a traditional column-based extraction technique and the simultaneous extraction of two different proteins in the same tube using CIFF.
生物分析物的提取是许多诊断和分析测定的第一步。然而，大多数生物分析物提取方法需要广泛的基于稀释的洗涤过程，不仅耗时费力，而且还会导致显著的样品损失，限制了它们在稀有样品分析中的应用。在这里，我们提出了一种方法，无需洗涤-离心辅助不混溶流体过滤（CIFF）即可从稀有样品（低至 10 个细胞）中高效提取多种不同的生物分析物。CIFF 利用离心力将结合分析物的玻璃微珠从水性样品驱动到不混溶的疏水性溶液中，从而进行高效、简单和非稀释的提取。该方法可以使用常规聚合酶链反应（PCR）管进行，不需要特殊的设备、柱子或仪器，因此具有广泛的适用性和成本效益。CIFF 过程可以在一次提取中有效去除约 99.5%的水性样品，而传统的“离心和吸液”操作会导致更高的 3.6%残留。CIFF 的另一个独特方面是它能够在单个容器内同时进行两种不同的固相生物分析物提取，而无需对样品进行分馏或进行串行提取。在这里，我们证明了从低输入样品（低至 10 个细胞）中高效提取 mRNA 和 DNA，与传统的基于柱的提取技术相比，回收率略高或相当，并且可以同时使用 CIFF 在同一管中提取两种不同的蛋白质。
