Key Laboratory of Luminescence Analysis and Molecular Sensing (Southwest University), Ministry of Education, Chongqing Engineering Laboratory of Nanomaterials & Sensor Technologies, School of Chemistry and Chemical Engineering, Southwest University, Chongqing, 400715, PR China.
Key Laboratory of Luminescence Analysis and Molecular Sensing (Southwest University), Ministry of Education, Chongqing Engineering Laboratory of Nanomaterials & Sensor Technologies, School of Chemistry and Chemical Engineering, Southwest University, Chongqing, 400715, PR China.
Anal Chim Acta. 2024 Mar 22;1295:342329. doi: 10.1016/j.aca.2024.342329. Epub 2024 Feb 2.
Modulating loop-mediated isothermal amplification (mLAMP) by short-stranded DNA segment trigger (T) to generate byproducts H ions (mLAMP/H) as signal transducer is intriguing for developing catalytic hairpin assembly (CHA)-cooperated amplifiable electrochemical biosensors. This would be a big challenge for traditional LAMP that is basically suitable for amplifying long-stranded oligonucleotides up to 200-300 nt. To address this inherent limitation of traditional LAMP, many researchers have put in efforts to explore improvements in this that would allow LAMP to be used for a wider range of target species amplification.
Here in this work, we are inspired to explore two-step loop-mediated amplification, firstly forming T-activated double-loop dumbbell structure (DLDS) intermediate by a recognition hairpin and a hairpin precursor, and next DLDS-guided mLAMP process with the aid of two primers to yield mLAMP/H during successive DNA incorporation via nucleophilic attacking interaction. To manipulate the mLAMP/H-directed transduction of input T, a pH-responsive triplex strand is designed with the ability of self-folding in Hoogsteen structure at slightly acidic conditions, resulting in the dehybridization of a fuel strand (FS) to participate in CHA between two hairpins on the modified electrode surface, in which FS is repetitively displaced and recycled to fuel the progressive CHA events. In the as-assembled dsDNA complexes, numerous electroactive ferrocene labels are immobilized in the electrode sensing interface, thereby generating significantly amplified electrochemical current signal that can sense the presented and varied T.
It is clear that we have creatively constructed a unique electrochemical biosensor for disease detection. Benefited from the rational combination of mLAMP and CHA, our electrochemical strategy is highly sensitive, specific and simplified, and would provide a new paradigm to construct various mLAMP/H-based biosensors for other short-stranded DNA or microRNAs markers.
通过短链 DNA 片段触发（T）来调制环介导等温扩增（mLAMP）以产生副产物 H 离子（mLAMP/H）作为信号转导物，这对于开发催化发夹组装（CHA）协同可扩增电化学生物传感器非常有趣。这对于基本适用于扩增长达 200-300nt 的长链寡核苷酸的传统 LAMP 来说将是一个巨大的挑战。为了解决传统 LAMP 的这种固有局限性，许多研究人员已经努力探索改进方法，以使 LAMP 能够用于更广泛的目标物种扩增。
在这项工作中，我们受到启发，探索两步环介导扩增，首先通过识别发夹和发夹前体形成 T 激活的双环哑铃结构（DLDS）中间体，然后在两个引物的辅助下进行 DLDS 引导的 mLAMP 过程，在通过亲核攻击相互作用连续 DNA 掺入过程中产生 mLAMP/H。为了操纵输入 T 引导的 mLAMP/H 转导，设计了一种 pH 响应的三链体，其在略酸性条件下具有在 Hoogsteen 结构中自折叠的能力，导致燃料链（FS）解杂交以参与在修饰电极表面上两个发夹之间的 CHA，其中 FS 被反复置换和回收以燃料渐进 CHA 事件。在组装的 dsDNA 复合物中，大量的电活性二茂铁标记物被固定在电极传感界面中，从而产生可检测存在和变化的 T 的显著放大的电化学电流信号。
很明显，我们创造性地构建了一种用于疾病检测的独特电化学生物传感器。受益于 mLAMP 和 CHA 的合理组合，我们的电化学生物传感器策略具有高度的灵敏性、特异性和简化性，并为构建其他基于短链 DNA 或 microRNAs 标记物的各种 mLAMP/H 基生物传感器提供了新范例。
