SENSORS AND ACTUATORS B-CHEMICAL
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2024
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IF 8
Susu Cui,Fan Wang,Weiwei Yang
Split aptamers have attracted extensive attention in sensing benefit of optimized concise structure and low-cost, but with lower binding affinity and stability. Here, we successfully engineer three pairs of split aptamers for PDGF-BB protein and develop a highly sensitive electrochemical sensor based on protein-templated split aptamer ligation reaction. The protein template directs the reassembly of split aptamers and accelerates the ligation reaction between azide and alkyne groups attached to the split fragments. The ligation yields of RS Split1 and RS Split2 with reserved binding sites are found to be 72.6 ± 1.8 % and 82.5 ± 1.3 %, respectively, while broken binding sites also obtain a ligation yield of 61.5 ± 2.1 % for BS Split3. We utilize the chemically modified split aptamers in place of the “capture” and “detection” antibodies used in ELISA, directly enabling the capture of electrochemically sensitive ligated aptamer to report target binding. This protein-templated ligation strategy improves the stability and sensitivity of the electrochemical sensor, with a low detection limit of 7 pM for PDGF-BB. The saturated electrochemical signal is approximately 3 times that of the non-ligated system. The proposed electrochemical sensor is also successfully applied to 50 % serum due to its excellent selectivity and bio-orthogonality. This strategy avoids the signal loss caused by traditional binding interruption during washing step via converting the ternary complex of aptamer/protein/aptamer to the binary complex of ligated aptamer/protein. This research offers a general sensing platform for other protein biomarkers and, more importantly, does not rely on expensive antibodies.
研究领域:
生物化学
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医学检测
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分子诊断
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DOI
高影响因子
TALANTA
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2025
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IF 6.1
Xuan Luo,Susu Cui,Weiwei Yang,Yongsheng Yu
Quinine is a natural antimalarial drug that also serves as a food additive; however, excessive consumption can result in harmful side effects. Here, we propose a simple and cost-effective electrochemical strategy for quinine detection based on the small molecule-templated split aptamer click ligation (SMT-SpA-CLR), where the integrity of the aptamer is restored through the template-promoted proximity effect. We first investigate the effects of two splitting sites on the affinity of quinine aptamer and find that the loop of the stem with more Watson-Crick base pairs is more suitable for splitting, which can maintain the affinity to a great extent. The affinity of the Split 1 design ( K d = 48 μM) is slightly reduced compared to the affinity of the parent aptamer. The stable quinine recognition and its sensitive detection are achieved by integrating the SMT-SpA-CLR with an electrochemical signal output. The linear response range for quinine detection is from 0.05 μM to 10 μM with a low detection limit of 25 nM (at S/N = 3). This strategy provides a novel ideal for the design of small molecule-templated split aptamer reaction and can potentially be expanded for detecting other small molecules and proteins.
研究领域:
生物化学
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分析化学
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PubMed
高影响因子
Analytical Methods
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2024
·Vol. 16
·期 8
·1281-1287
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IF 2.7
Keyu Li,Peng Xiao,Ningning Yuan,Shujuan Yan,Pei Zhao,Guowei Zuo
MiRNAs are critical regulators of target gene expression in many biological processes and are considered promising biomarkers for diseases. In this study, we developed a simple, specific, and sensitive miRNA detection method based on proximity ligation reaction, which is easy to operate. The method uses a pair of target-specific DNA probes immobilized on the same gold nanoparticles (AuNPs), which hybridize to the target miRNA. Hybridization brings the probes close together, allowing the formation of a continuous DNA sequence that can be amplified by Quantitative Real-time PCR (qPCR). This method eliminates the need for complex reverse transcription design and achieves high specificity for discriminating single base mismatches between miRNAs through a simple procedure. This method can sensitively measure three different miRNAs with a detection limit of 20 aM, providing high versatility and sensitivity, even distinguishing single-base variations among members of the miR-200 family with high selectivity. Due to its high selectivity and sensitivity, this method has important implications for the investigation of miRNA biological functions and related biomedical research.
研究领域:
分子生物学
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纳米生物技术
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医学检测
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PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2025
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IF 7.7
Huihong Yan,Xuan Luo,Chenglong Bao,Yongsheng Yu,Weiwei Yang
Quinine, a cinchona alkaloid, is utilized as an antimalarial agent or a preservative in aquatic products. Owing to its resistance to biodegradation, quinine readily persists in human body fluids and environmental water systems, posing risks of harmful side effects to human health. Here, we developed a highly sensitive and specific detection strategy for quinine based on a small molecule-templated split aptazyme click ligation reaction (SMT-SpAzyme-CLR). This strategy constructs a highly sensitive fluorescent biosensor by integrating a quinine-specific split aptamer with a Mg 2 + -dependent DNAzyme catalytic core, which undergoes quinine-induced click ligation and activates enzymatic cleavage activity. The recycling of the ligated SpAzyme facilitates a one-to-many signal amplification cascade, substantially enhancing the detection sensitivity. The proposed sensor exhibits a wide linear response range from 0.02 to 50 μM and achieves an impressively low detection limit of 5 nM, outperforming most existing aptasensors. It was successfully applied to the determination of quinine in tonic water samples, with recoveries ranging from 96.9 % to 104.9 %. Furthermore, a lateral flow immunoassay strip was implemented for visual semi-quantitative detection, demonstrating high specificity and strong anti-interference capability even in complex matrices. This work provides a sensing platform that combines the strengths of highly sensitive laboratory quantification and rapid on-site screening for quinine.
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高影响因子
BIOSENSORS & BIOELECTRONICS
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2014
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IF 6.451
Lu Lu,Jing Cao Si,Zhong Feng Gao,Yu Zhang,Jing Lei Lei,Hong Qun Luo,Nian Bing Li
A dual strategy that combines the adenosine triphosphate (ATP)-dependent enzymatic ligation reaction with self-cleaving DNAzyme-amplified electrochemical detection is employed to construct the biosensor. In this design, the methylene blue-labeled hairpin-structured DNA was self-assembled onto a gold electrode surface to prepare the modified electrode through the interaction of Au–S bond. In the procedure of ATP-dependent ligation reaction, when the specific cofactor ATP was added, the two split oligonucleotide fragments of 8-17 DNAzyme were linked by T4 DNA ligase and then released to hybridize with the labeled hairpin-structured DNA substrate. The linked 8-17 DNAzyme catalyzes the cleavage of the hairpin-structured substrate by the addition of Zn 2+ , causing the methylene blue which contains high electrochemical activity to leave the surface of the gold electrode, therefore generating a dramatic decrease of electrochemical signal. The decrease of peak current was readily measured by square wave voltammetry and a relatively low detection limit (0.05 nM) was obtained with a linear response range from 0.1 to 1000 nM. By taking advantage of the highly specific cofactor dependence of the DNA ligation reaction, the proposed ligation-induced DNAzyme cascades demonstrate ultrahigh selectivity toward the target cofactor ATP. A catalytic and molecular beacons strategy is further adopted to amplify the electrochemical signal detection achieved by cycling and regenerating the 8-17 DNAzyme to realize enzymatic multiple turnover, thus one DNAzyme can catalyze the cleavage of several hairpin-structured substrates, which improves the sensitivity of the newly designed electrochemical sensing system.
研究领域:
生物化学
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电化学
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生物传感器
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纳米技术
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PubMed
高影响因子
ANALYTICA CHIMICA ACTA
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2025
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IF 6
Jingpu Zhang,Fahui Dai,Yejing Shi,Peng Zou,Xiao Zhi,Mei Liu,Nannan Wu,Tie Fu Liu
Background DNA and RNA virus detections commonly rely on qPCR (quantitative polymerase chain reaction) -based methods that need dyes or Taqman probes as signaling probes and expertized instruments. Isothermal amplification such as linear rolling-circle amplification (LRCA) has attracted large interest in its role to act as a supplement to the traditional qPCR. However, the current LRCA-based signaling probes suffered from long time-consumption and complex procedures for nucleic acid detections. Novel systems are urgently needed to integrate the simplicity of LRCA with rapid signal amplification of label-free probes to spread the advantages of isothermal amplification for virus detections. Results A primer-independent LRCA system utilizing AgNCs/DNA as signaling probes was developed for rapid virus detection. LRCA was optimized into a one-step reaction by integrating target-triggered ligation reaction of padlock probes (PlPs) with DNA amplification. After an easy H 2 O 2 treatment to minimize dithiothreitol's interference, LRCA product with tandem repeats of G-rich sequence was hybridized with AgNCs/DNA, promptly amplifying fluorescence signals through the G-proximity enhancement effect. The system precisely discriminated single-base mismatch at ligation junctions of PlPs. The formation of 3′-hairpin structure in PlPs was found to be prioritized over 5′-exposed linear sequences to maximize the fluorescence signals. It requires only 1 μL sample and costs 40 min at 25–30 °C, facilitating a sensitive and broad linear range of detection for E (10 -9 -10 μM) gene in RNA virus (SARS-CoV-2) and VP2/3 gene (10 −9 - 1 μM) in DNA virus (BK human polyomavirus, BKV), and successful application in urine samples. Significance AgNCs/DNA lighting up LRCA products with rapid signal amplifications alongside the extension of single strand DNA enables the direct use of LRCA for nucleic acids detections. The modular design of PlPs, universal signal probes, near-room temperature reaction conditions, small sample requirements and rapid reaction make it adapted for various DNA detections, and give it great promise for future point-of-care tests.
研究领域:
分子诊断
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病毒学
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纳米生物技术
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核酸扩增
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生物传感器
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DOI
PubMed
高影响因子
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