Sensitive and specific detection of ctDNA using Copper-Free click chemistry and magnetic bead Technology

IF 3.6 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods Pub Date : 2026-02-01 Epub Date: 2025-11-30 DOI:10.1016/j.ymeth.2025.11.010

Reza Didarian , Dilek Kanarya , Sonya Sahin , Canan Özyurt , Serap Evran , Dilek Odaci , Nimet Yildirim-Tirgil

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引用次数: 0

Abstract

Liquid biopsy, particularly the analysis of circulating tumor DNA (ctDNA), offers immense potential for non-invasive cancer diagnosis and monitoring. It provides a less invasive alternative to traditional tissue biopsies, enabling earlier detection and real-time assessment of disease progression. However, a significant hurdle in its widespread adoption is the extremely low concentration of ctDNA in biological samples, especially during the early stages of cancer, making sensitive and specific detection challenging. This work addresses the critical problem of developing a highly sensitive and specific method for low abundance ctDNA detection.

We developed a novel, highly sensitive, and specific method for ctDNA analysis, employing copper-free click chemistry (strain-promoted azide-alkyne cycloaddition, SPAAC) for enzyme-free amplification, coupled with magnetic bead-assisted fluorometric detection. This enzyme-free approach significantly enhanced specificity and reduced background noise. We meticulously optimized parameters, including primer length and annealing temperature, finding that 30-base primers and a 50 °C annealing temperature yielded optimal amplification efficiency. Our method successfully detected ctDNA at concentrations as low as 10 pM (15 bp primer). Agarose gel electrophoresis confirmed highly specific amplification with minimal non-specific products, and the assay demonstrated excellent allelic discrimination, accurately distinguishing single-nucleotide mutations. Importantly, the method proved robust in complex human serum samples, demonstrating its practical applicability.

This innovative, cost-effective, and enzyme-free platform overcomes many limitations of current ctDNA detection technologies. By enabling highly sensitive and specific detection of low abundance ctDNA, this methodology represents a significant leap forward for non-invasive cancer diagnostics, paving the way for earlier disease detection, improved treatment monitoring, and the broader implementation of personalized medicine.

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利用无铜点击化学和磁珠技术对ctDNA进行灵敏和特异的检测。

液体活检，特别是循环肿瘤DNA （ctDNA）的分析，为非侵入性癌症诊断和监测提供了巨大的潜力。它为传统的组织活检提供了一种侵入性较小的替代方法，能够更早地发现和实时评估疾病进展。然而，其广泛采用的一个重大障碍是生物样品中ctDNA的浓度极低，特别是在癌症的早期阶段，使得敏感和特异性检测具有挑战性。这项工作解决了开发低丰度ctDNA检测的高灵敏度和特异性方法的关键问题。我们开发了一种新的、高灵敏度和特异性的ctDNA分析方法，采用无铜点击化学（菌株促进叠氮化物-炔环加成，SPAAC）进行无酶扩增，结合磁珠辅助荧光检测。这种无酶的方法显著提高了特异性并降低了背景噪声。我们精心优化了引物长度和退火温度等参数，发现30碱基的引物和50 °C的退火温度产生了最佳的扩增效率。我们的方法成功地检测到浓度低至10 pM（15 bp引物）的ctDNA。琼脂糖凝胶电泳证实了高特异性扩增和最小的非特异性产物，并且该试验显示了出色的等位基因区分，准确区分单核苷酸突变。重要的是，该方法在复杂的人血清样本中证明了鲁棒性，证明了其实用性。这种创新、经济、无酶的平台克服了当前ctDNA检测技术的许多局限性。通过实现低丰度ctDNA的高灵敏度和特异性检测，该方法代表了非侵入性癌症诊断的重大飞跃，为早期疾病检测、改进治疗监测和更广泛地实施个性化医疗铺平了道路。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Methods 生物-生化研究方法

CiteScore

9.80

自引率

2.10%

发文量

222

审稿时长

11.3 weeks

期刊介绍： Methods focuses on rapidly developing techniques in the experimental biological and medical sciences. Each topical issue, organized by a guest editor who is an expert in the area covered, consists solely of invited quality articles by specialist authors, many of them reviews. Issues are devoted to specific technical approaches with emphasis on clear detailed descriptions of protocols that allow them to be reproduced easily. The background information provided enables researchers to understand the principles underlying the methods; other helpful sections include comparisons of alternative methods giving the advantages and disadvantages of particular methods, guidance on avoiding potential pitfalls, and suggestions for troubleshooting.