Optical-fiber sensors for dissociation constant measurement of aptamer and rapid detection of ampicillin in river water and milk

IF 6.1 2区化学 Q1 CHEMISTRY, ANALYTICAL

Analytica Chimica Acta Pub Date : 2026-04-15 Epub Date: 2026-02-09 DOI:10.1016/j.aca.2026.345221

Guangxin Zhang , Haixu Zhao , Xiaoqi Li , Yi Zhang, Haijing Jia, Qiaoqiao Zhang, Tianyu Dai, Xinhui Lou

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引用次数: 0

Abstract

Background

The conflicted affinity evaluation results of small molecule-binding aptamers (SM-Apts) are increasing, raising the concerns on if a reported aptamer is a real aptamer. AMP17 is the first published controversial aptamer, which has been applied in at least 37 reported aptasensors for the detection of ampicillin, but was disproved by several homogeneous affinity methods including isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, and mass spectrometry. Only the original paper reported its dissociation constant (K_D = 13.4 nM) by the affinity column-based fluorescence method. If is AMP17 a real aptamer? What are the possible reasons that result in the conflicted results?

Results

We carried out four most popularly used homogeneous assays (FRET-based strand displacement assays, circular dichroism, EvaGreen and thioflavin T–based label-free fluorescence assays, and DNase I assay). None of them proved the binding of AMP17 to ampicillin. We then successfully measured the K_D using a fiber optic evanescent wave sensor (FOEW), in which AMP17 was covalent immobilized on the SiO₂ fiber without (K_D = 2.6 ± 0.2 nM) or with BSA coating (K_D = 5.1 ± 0.5 nM). The high specificity of AMP17 was proved by testing other molecules and replacing AMP17 by a non-binding sequence. We finally constructed a FOEW for the detection of ampicillin via one-step competitive or two step-noncompetitive detection modes, respectively. The latter showed 25.9-fold lower limit of detection (LOD = 2.8 fM, S/N = 3) than the former. Both detection modes possessed the good anti-matrix interference capability and were capable for the detection of ampicillin spiked in river water and milk without the need of sample purification.

Significance

Our results support that AMP17 is a real aptamer with high affinity and specificity. The surface immobilization favors the binding of AMP17 with ampicillin. Our study highlights the limited application scope of homogeneous assays for affinity evaluation of small molecule-binding aptamers. FOEW is a facile platform for both K_D measurements and rapid detection.

Abstract Image

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光纤传感器解离常数测量及河水和牛奶中氨苄西林的快速检测

摘要小分子结合适体（SM-Apts）亲和性评价结果的冲突越来越多，引起了人们对报道的适体是否为真正的适体的关注。AMP17是第一个发表的有争议的适配体，它已被应用于至少37个报道的用于氨苄西林检测的适配体传感器中，但被几种均匀亲和力方法（包括等温滴定量热法、核磁共振波谱法和质谱法）所否定。仅用亲和柱荧光法报道了其解离常数（KD=13.4 nM）。如果AMP17是一个真正的适配体？导致结果冲突的可能原因是什么？结果我们进行了四种最常用的均相分析（基于fret的链位移分析，圆二色性，evgreen和基于硫黄素的无标记荧光分析，以及dna酶I分析）。它们都没有证明AMP17与氨苄西林的结合。然后，我们使用光纤倏变波传感器（FOEW）成功地测量了KD，其中AMP17被共价固定在没有（KD= 2.6±0.2 nM）或有BSA涂层（KD=5.1±0.5 nM）的SiO2光纤上。通过检测其他分子并用非结合序列替代AMP17，证实了AMP17的高特异性。最后，我们分别通过一步竞争和两步非竞争检测模式构建了检测AMP17的FOEW。后者的检出下限（LOD=2.8 fM, S/N=3）是前者的25.9倍。两种检测方式均具有良好的抗基质干扰能力，无需对样品进行纯化即可检测河水和牛奶中添加的AMP。我们的结果支持AMP17是一个真正的适体，具有高亲和力和特异性。表面固定有利于AMP17与氨苄西林的结合。我们的研究突出了小分子结合适体亲和性评价的均相分析的有限应用范围。FOEW是一个简便的KD测量和快速检测平台。

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来源期刊

Analytica Chimica Acta 化学-分析化学

CiteScore

10.40

自引率

6.50%

发文量

1081

审稿时长

38 days

期刊介绍： Analytica Chimica Acta has an open access mirror journal Analytica Chimica Acta: X, sharing the same aims and scope, editorial team, submission system and rigorous peer review. Analytica Chimica Acta provides a forum for the rapid publication of original research, and critical, comprehensive reviews dealing with all aspects of fundamental and applied modern analytical chemistry. The journal welcomes the submission of research papers which report studies concerning the development of new and significant analytical methodologies. In determining the suitability of submitted articles for publication, particular scrutiny will be placed on the degree of novelty and impact of the research and the extent to which it adds to the existing body of knowledge in analytical chemistry.