Centre for Personalised Nanomedicine, Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, QLD 4072, Australia.
Nanoscale. 2017 Mar 9;9(10):3496-3503. doi: 10.1039/c6nr09928a.
Simple nucleic acid detection methods could facilitate the progress of disease diagnostics for clinical uses. An attractive strategy is label-free surface-enhanced Raman scattering (SERS) due to its capability of providing structural fingerprinting of analytes that are close to or on nanomaterial surfaces. However, current label-free SERS approaches for DNA/RNA biomarker detection are limited to short and synthetic nucleic acid targets and have not been fully realized in clinical samples due to two possible reasons: (i) low target copies in limited patient samples and (ii) poor capability in identifying specific biomarkers from complex samples. To resolve these limitations and enable label-free SERS for clinical applications, we herein present a novel strategy based on multiplex reverse transcription-recombinase polymerase amplification (RT-RPA) to enrich multiple RNA biomarkers, followed by label-free SERS with multivariate statistical analysis to directly detect, identify and distinguish between these long amplicons (∼200 bp). As a proof-of-concept clinical demonstration, we employed this strategy for non-invasive subtyping of prostate cancer (PCa). In a training cohort of 43 patient urinary samples, we achieved 93.0% specificity, 95.3% sensitivity, and 94.2% accuracy. We believe that our proposed assay could pave the way for simple and direct label-free SERS detection of multiple long nucleic acid sequences in patient samples, and thus facilitate rapid cancer molecular subtyping for personalized therapies.
简单的核酸检测方法可以促进临床疾病诊断的进展。一种有吸引力的策略是无标记的表面增强拉曼散射（SERS），因为它能够提供接近或在纳米材料表面的分析物的结构指纹。然而，目前用于 DNA/RNA 生物标志物检测的无标记 SERS 方法仅限于短的和合成的核酸靶标，并且由于两个可能的原因尚未在临床样本中完全实现：（i）有限的患者样本中靶标拷贝数低，（ii）从复杂样本中识别特定生物标志物的能力差。为了解决这些限制并实现临床应用的无标记 SERS，我们在此提出了一种基于多重逆转录-重组酶聚合酶扩增（RT-RPA）的新策略，用于富集多个 RNA 生物标志物，然后进行无标记 SERS 与多元统计分析，以直接检测、识别和区分这些长的扩增子（∼200 bp）。作为概念验证的临床演示，我们将该策略用于非侵入性前列腺癌（PCa）的亚型分类。在 43 例患者尿液样本的训练队列中，我们实现了 93.0%的特异性、95.3%的灵敏度和 94.2%的准确性。我们相信，我们提出的检测方法可以为患者样本中多种长核酸序列的简单、直接的无标记 SERS 检测铺平道路，从而促进快速的癌症分子亚型分类，以实现个体化治疗。
