Analytical Methods
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2023
·Vol. 16
·期 1
·51-61
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IF 3.1
Jing Qi,Qianyi Qi,Zhou Zhou,Yixuan Wu,Aiting Cai,Jinran Wu,Bairong Chen,Qingxiang Wang,Lin Chen,Feng Wang
The detection of epidermal growth factor receptor (EGFR) mutation L858R in circulating tumor DNA (ctDNA) is beneficial for the clinical diagnosis and personalized therapy of non-small cell lung cancer (NSCLC). Herein, for the first time, the combination of the primer exchange reaction (PER) and clustered regularly interspaced short palindromic repeats (CRISPR) and its associated nucleases (Cas) 14a was used in electrochemical biosensor construction for the detection of ctDNA EGFR L858R. EGFR L858R, as the target, induced the isothermal amplification of the PER reaction, and then the CRISPR/Cas14a system was activated; subsequently, the substrate ssDNA-MB was cleaved and the electron on the surface of the gold electrode transferred, resulting in the fluctuation of the electrochemical redox signal on the electrode surface, whereas the electrochemical signal will be stable when EGFR L858R is absent. Therefore, the concentration of EGFR L858R can be quantified by electrochemical signal analysis. The low detection limit is 0.34 fM and the dynamic detection range is from 1 fM to 1 μM in this work. The PER-CRISPR/Cas14a electrochemical biosensor greatly improved the analytical sensitivity. In addition, this platform also exhibited excellent specificity, reproducibility, stability and good recovery. This study provides an efficient and novel strategy for the detection of ctDNA EGFR L858R, which has great potential for application in the diagnosis and treatment of NSCLC.
研究领域:
分子生物学
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临床诊断
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癌症研究
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生物传感技术
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产品关联
DOI
PubMed
高影响因子
iScience
·
2021
·
IF 5.458
Xidong Yang,Meiyan Liao,Hanfei Zhang,JinBo Gong,Fan Yang,Mengying Xu,Pier-Luc Tremblay,Tian Zhang
Summary A liquid biopsy is a noninvasive approach for detecting double-stranded circulating tumor DNA (ctDNA) of 90–320 nucleotides in blood plasma from patients with cancer. Most techniques employed for ctDNA detection are time consuming and require expensive DNA purification kits. Electrochemiluminescence resonance energy transfer (ECL-RET) biosensors exhibit high sensitivity, a wide response range, and are promising for straightforward sensing applications. Until now, ECL-RET biosensors have been designed for sensing short single-stranded oligonucleotides of less than 45 nucleotides. In this work, an ECL-RET biosensor comprising graphitic carbon nitride quantum dots was assessed for the amplification-free detection in the blood plasma of DNA molecules coding for the EGFR L858R mutation, which is associated with non-small-cell lung cancer. Following a low-cost pre-treatment, the highly specific ECL-RET biosensor quantified double-stranded EGFR L858R DNA of 159 nucleotides diluted into the blood within a linear range of 0.01 fM to 1 pM, demonstrating its potential for noninvasive biopsies.
研究领域:
医学诊断
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生物传感技术
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癌症研究
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DOI
PubMed
高影响因子
ACS Sensors
·
2021
·
IF 7.711
Lei Cao,Xiaojin Guo,Ping Mao,Yulin Ren,Zedong Li,Minli You,Jie Hu,Miao Tian,Chunyan Yao,Fei Li,Feng Xu
Digital polymerase chain reaction (dPCR) has found widespread applications in molecular diagnosis of various diseases owing to its sensitive single-molecule detection capability. However, the existing dPCR platforms rely on the auxiliary procedure to disperse DNA samples, which needs complicated operation, expensive apparatus, and consumables. Besides, the complex and costly dPCR readers also impede the applications of dPCR for point-of-care testing (POCT). Herein, we developed a portable digital loop-mediated isothermal amplification (dLAMP) platform, integrating a microscale hydrogel (microgel) array chip for sample partition, a miniaturized heater for DNA amplification, and a hand-held reader for digital readout. In the platform, the chip with thousands of isolated microgels holds the capability of self-absorption and partition of DNA samples, thus avoiding auxiliary equipment and professional personnel operations. Using the integrated dLAMP platform, λDNA templates have been quantified with a good linear detection range of 2–1000 copies/μL and a detection limit of 1 copy/μL. As a demonstration, the epidermal growth factor receptor L858R gene mutation, a crucial factor for the susceptibility of the tyrosine kinase inhibitor in non-small-cell lung cancer treatment, has been accurately identified by the dLAMP platform with a spiked plasma sample. This work shows that the developed dLAMP platform provides a low-cost, facile, and user-friendly solution for the absolute quantification of DNA, showing great potential for the POCT of nucleic acids.
研究领域:
分子诊断
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生物医学工程
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癌症研究
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产品关联
DOI
PubMed
高影响因子
ANALYST
·
2019
·Vol. 144
·期 16
·4757-4771
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IF 4.019
Xiurui Zhu,Shisheng Su,Mingzhu Fu,Zhiyong Peng,Dong Wang,Xiao Rui,Fang Wang,Xiaobin Liu,Baoxia Liu,Lingxiang Zhu,Wenjun Yang,Na Gao,Guoliang Huang,Gaoshan Jing,Yong Guo
Droplet digital PCR (ddPCR) is a single-molecule amplification technology with broad applications in precision medicine and clinical diagnosis. Dual-fluorescence and four-cluster ddPCR (two/four-ddPCR) assay is an effective way to quantify copy numbers. Currently, two/four-ddPCR data are usually classified with manual thresholds. For clinical applications, automatic and accurate methods are required to avoid subjectivity in diagnosis. Although there are some automatic classification algorithms, their accuracy and robustness still need to be improved to meet the needs of clinical diagnosis. Therefore, a new method is in high demand to automatically classify two/four-ddPCR data in an accurate and robust way. Here, a novel density-watershed algorithm (DWA) method was developed for the accurate, automatic and unsupervised classification of two/four-ddPCR data. First, data gridding was applied to a scatter plot of the fluorescence signal intensity to calculate data densities. Based on the data densities, the watershed algorithm was used to divide the gridded scatter plot into isolated regions automatically. Next, an optimal cluster pattern was determined based on these isolated regions, and excess regions were merged. Finally, the two/four-ddPCR data were classified based on the merged regions, and DNA template copy numbers were calculated accordingly. Using the DWA method for the quantification of both wild types and mutants of epidermal growth factor receptor (EGFR) L858R and T790M, the classification results were highly consistent with expectations, and significantly better than commonly-used automatic algorithms for now. The computed template copy numbers scaled proportionally to the relative concentration of input templates (r2 > 0.998) in four orders of magnitude with a good reproducibility, and achieved a limit of detection over 40 times lower than the commonly-used automatic algorithms. Furthermore, the DWA method was validated on 254 clinical DNA samples derived from frozen tissues, formalin-fixed paraffin-embedded tissues and peripheral blood. In most cases, the DWA method derived accurate and valid classification results. This highly effective DWA method may be widely used for automatically classifying two/four-ddPCR data, and it will greatly promote the application of ddPCR in clinical diagnosis.
研究领域:
生物信息学
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分子生物学
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临床诊断
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精准医疗
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产品关联
DOI
PubMed
高影响因子
LAB ON A CHIP
·
2023
·
IF 6.1
Yulin Ren,Jingcheng Ji,Haoqing Zhang,Lei Cao,Jie Hu,Feng Xu,Zedong Li
Droplet digital polymerase chain reaction (ddPCR) technology has found widespread applications in the ultrasensitive analysis of nucleic acids, where integrated ddPCR platforms with the capability of sample dispersion, followed by in situ amplification and data analysis, are highly expected. However, current integrated ddPCR platforms are usually limited by either difficultly mass-produced materials or lack of integrated control instruments, restricting their practical application. This paper proposes an integrated three-in-one ddPCR platform with high user-friendliness and practicability, which is composed of an easy-to-use chip and a matching control instrument. The chip was made of thermally resistant and easily mass-produced polycarbonate (PC) material, and the benchtop control instrument was designed to perform droplet generation, in situ amplification, and fluorescence reading. The droplet generation and in situ heating on the chip were well characterized. Finally, the performance of the platform was validated through the analysis of the EGFR L858R mutation in lung cancer. The proposed three-in-one ddPCR platform shows great practicability in ultrasensitive nucleic acid testing. By virtue of its sensitivity, practicability, and cost-effectiveness, the ddPCR can serve as a universal detection platform for monitoring nucleic acid in the fields of tumor diagnosis, pathogen detection, and prenatal diagnosis.
研究领域:
生物技术
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分子生物学
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医学诊断
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基因工程
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产品关联
DOI
PubMed
高影响因子
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