MICROCHIMICA ACTA
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2020
·Vol. 187
·期 2
·1-8
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IF 6.232
Li Ruiying,Liu Qiang,Jin Yan,Li Baoxin
The authors have incidentally found that the three tandem repeats of a 13-mer G-rich oligomer (with sequence 5′-TGG GAA GGG AGG G-3′; referred to as G3) can directly fold into a stable G3 trimer. The G3 trimer/hemin DNAzyme exhibits an about 3-fold higher peroxidase-mimicking activity compared to the conventional G3/hemin DNAzyme. Combining this finding with rolling circle amplification (RCA), a colorimetric assay was developed for sensitive and specific determination of microRNA. In this method, each cycle of RCA generates three catalytic units. This leads to a significant signal amplification of the RCA. Using let-7a as a model analyte, the colorimetric method (best performed at 420 nm) exhibits high sensitivity toward microRNA-let-7a with a 37 fM detection limit and an analytical range that covers 3 orders of magnitude. The method was applied to the determination of let-7a in some cell lysates.
This G-triplex trimer-based rolling circle amplification (RCA) method can produce three catalytic units per RCA cycle, which can significantly improve the amplification efficiency of RCA.
研究领域:
生物化学
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分子生物学
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纳米技术
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分析化学
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产品关联
DOI
PubMed
高影响因子
BME Frontiers
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2025
·
IF 7.7
Yirui Zhang,Jieyu Yan,Wangheng Hou,Qian Gao,Tao Zhang,Yan Gao,Jingwen Li,Kun Han
Objective: Accurate and rapid detection of disease biomarkers is critical for early diagnosis, timely intervention, and effective disease management. Impact Statement: This strategy is exemplified through the development of 2 biosensors for detecting HIV-1 DNA and HIV-1 p24, biomarkers associated with acquired immunodeficiency syndrome (AIDS). Introduction: We propose a general biosensing strategy that leverages a treble signal amplification cascade, demonstrating its versatility and applicability across diverse biomolecular targets. Methods: The method integrates multiple amplification mechanisms to achieve unparalleled sensitivity. Initially, the hybridization of 2 aided probes with the target triggers isothermal amplification facilitated by polymerase and nicking enzyme, providing a robust preliminary signal enhancement. Repeated cycles of primer extension, nicking, and signal primer dissociation then generate multiple signal primers. These primers are further amplified via rolling circle amplification (RCA), resulting in an important secondary signal boost. Finally, the amplified products activate CRISPR-Cas12a-mediated trans-cleavage, achieving a tertiary level of signal enhancement. Results: This cascade amplification approach achieves remarkable sensitivity, with detection limits of 62 aM for nucleic acids and 8.48 pg/ml for proteins, positioning it as a broadly applicable framework. Clinical samples were assayed, which indicates its capability in clinical diagnosis. Conclusion: Beyond HIV detection, the modular design of this strategy allows adaptation for various biomarkers, showcasing its potential as a universal platform for molecular diagnostics in healthcare and research.
研究领域:
生物传感器
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分子诊断
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病毒学
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临床诊断
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生物医学工程
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核酸扩增
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蛋白质检测
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL BIOCHEMISTRY
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2022
·
IF 3.191
Ying Liang,Qionglin Wang,Xianwei Zhang,Mengxin Zhang,Bang Du,Weyland Cheng,Huanmin Wang,Lifeng Li,Guangjun Hou,Wancun Zhang
A dual isothermal amplification assay with dual fluorescence signal detection strategy, named dual isothermal amplification all-in-one approach, was developed for rapid, one-step, highly sensitive quantification of plasma circulating MYCN copy number of neuroblastoma (NB). The developed strategy consisted of rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) on a real-time PCR system using highly specific probe, molecular beacon (MB), as detection probe. The developed strategy possessing a broad linear dynamic range of 10 aM to 1 pM for both target gene ( MYCN ) and reference gene ( NAGK ) . The ratio of the MYCN copy number to NAGK copy number (M/N ratio) was detected by the developed approach in cell lines, NB tumor tissues, hepatoblastoma tumor tissues and Wilms’ tumor tissues, to which the M/N ratios were consistent with previous reports. In particular, the M/N ratio in NB clinical tissue specimens and NB plasma specimens detected with the developed approach were in keeping with the standard RT-PCR approach. More importantly, the M/N ratio in NB tissue samples and corresponding plasma samples of NB patients were consistent with each other with a correlation coefficient of 0.9690, indicating that plasma circulating MYCN is a promising indicator for the risk classification of NB.
研究领域:
肿瘤学
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分子生物学
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临床诊断
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2021
·
IF 6.558
Yutong Wang,Zhengzheng Wang,Zhongxu Zhan,Ju Liu,Tingting Deng,Hengyi Xu
Staphylococcus aureus is a common foodborne pathogen that can cause a suppurative infection after eating contaminated food. Detection of S. aureus plays an important role in the food industry. In this study, a strategy for the detection of S. aureus using magnetic separation (MS) technology combined with rolling circle amplification (MS-RCA) was proposed. The strategy used antibiotics to capture bacteria and employed RCA products as signal output probes. Vancomycin (Van), as a commonly used antibiotic, can recognize peptidoglycan on the cell wall of Gram-positive bacteria and can effectively identify target bacteria. Therefore, we prepared BSAylated-Van functionalized magnetic beads (Van-MBs) for the pre-enrichment of S. aureus . To ensure the selectivity of this method, we used biotin-pig IgG to bind S. aureus . In addition, to amplify the output signal of the MS-RCA strategy, we introduced streptavidin (SA) and successfully obtained the [email protected] S. aureus @biotin-pig [email protected] @biotin-RCA probe complex and used the biotin-avidin-system (BAS) by combining magnetic separation technology and RCA technology to realize the enrichment and specific detection of S. aureus . Furthermore, by optimizing the experimental conditions such as the magnetic separation time and the amount of Van-MBs, the detection performance of this method was improved. Under the optimal conditions, the detection limit of this method for S. aureus was 3.3 × 10 2 CFU/mL in fruit juice, and it was less affected by other bacteria.
研究领域:
食品科学
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微生物学
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生物技术
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产品关联
DOI
PubMed
高影响因子
FOOD CONTROL
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2019
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IF 4.248
Zhongxu Zhan,Hui Li,Ju Liu,Guoyang Xie,Fangbing Xiao,Xin Wu,Zoraida P. Aguilar,Hengyi Xu
Listeria monocytogenes ( L. monocytogenes ) is a foodborne pathogen which can cause significant threats to human health, thus, a highly specific and accurate method for detection in food is urgently needed. In this study, an enzyme-linked aptasensor with rolling circle amplification (ELARCA) assay was developed for the sensitive and specific detection of L. monocytogenes. The assay was based on the competition between the aptamer (that was specific for L. monocytogenes ) that was bound to the biotin probe 1 (BP1~A) and the RCA probe that was complementary to the BP1. Addition of the bacteria released the BP1 making it available for the RCA probe which initiated the RCA process. In the presence of the RCA buffer, multiple copies of the DNA were formed which attached the biotin probe 3 (BP3). The biotin in BP3 interacted with streptavidin labeled with horse radish peroxidase (SA-HRP) to complete the assay complex. In the presence of the enzyme substrate, HRP produced a chromophore that led to the colorimetric detection. Under the optimal conditions, the ELARCA assay for L. monocytogenes showed a limit of detection (LOD) of 4.6 × 10 2 CFU/mL in pure culture, which was three orders of magnitude higher than without RCA. The ELARCA assay in spiked fresh lettuce showed an LOD of 6.1 × 10 3 CFU/g. The method developed was fast, low-cost, sensitive, and highly specific for L. monocytogenes . By changing the specificity of the aptamer, the proposed ELARCA method provided a potential platform for the detection of other pathogenic bacteria.
研究领域:
微生物学
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食品安全
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生物技术
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分析化学
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产品关联
DOI
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2023
·
IF 12.6
Siqi Zhao,Yaxue Jia,Anping Wang,Jinlan Yang,Li Yang
We present a novel dual-mode DNA-walker based optical fiber biosensor (DMDW-Opt biosensor) for sensitive assay of micro-RNAs in bio-samples. In the sensor system , we develop a new strategy for the cascade amplification, DNA-walker/rolling cycle amplification (RCA), by the use of the residue track of the walker. The strategy can significantly improve the response of the sensor and avoid any tedious operation procedure. Dual-mode readouts, i.e., fluorescence and chemiluminescence , are measured independently without interfering with each other to achieve reliable and accurate analysis. Optical fibers with the surface modified by gold nanoparticles are utilized as the support for fabrication of the sensor, which would be benefit for developing miniaturized and portable sensing devices. The performance of the proposed method is evaluated by using micro-RNAs (MiR-155 and MiR-21) as the analytical target. The method is successfully applied for accurate determination of micoRNAs in human serum and MCF-7 cells. Our method can perform sensitive assays of MiR-155 with limit-of-detection as low as 97.72 fM and 11.22 fM, MiR-21 with limit-of-detection as low as 107.15 fM and 8.32 fM for the fluorescence- and the chemiluminescence-readout respectively, and the biosensor exhibits excellent specificity, reproducibility and storage stability, indicating its valuable potential applications for sensing trace-amount targets in complicated real samples.
研究领域:
生物传感
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纳米技术
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分子生物学
·
分析化学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2019
·
IF 9.518
Lilin Ge,Bin Li,Houxi Xu,Wenyuan Pu,Hang Fai Kwok
A simple and feasible electrochemical immunosensing protocol with glucometer readout was designed for the detection of low-abundance disease-related biomarker (alpha-fetoprotein; AFP) on the basis of backfilling rolling cycle amplification (RCA) with invertase-DNA 2 conjugates on the detection antibody. The assay consisted of the immunoreaction, RCA reaction, DNA 2 -invertase hybridization and glucose measurement. Initially, a sandwiched immunoreaction was carried out between anti-AFP capture antibody-coated microplate between nanogold-labeled pAb 2 detection antibody conjugated with DNA 1 primer (DNA 1 -AuNP-pAb 2 ) in the presence of target ATP . Thereafter, the carried primers triggered the RCA reaction in the presence of circular DNA template, polymerase and dNTP, to produce numerous repeated oligonucleotide sequences for hybridization with many invertase-DNA 2 conjugates. The carried invertase molecules accompanying the hybridization reaction hydrolyzed sucrose into glucose, thereby resulting in the amplification of the detectable signal on a handheld personal glucometer (PGM). Under optimum conditions, the developed immunoassay exhibited high sensitivity for the quantitative screening of AFP within a dynamic range of 0.1–100 ng mL −1 at a low detection limit of 0.087 ng mL −1 . Other biomarkers and proteins did not interfere the signals of this system. In addition, this method was utilized to determine human serum samples containing target AFP, and received well-matched results with the referenced enzyme-linked immunosorbent assay (ELISA) method.
研究领域:
生物技术
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医学诊断
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电化学
·
分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2013
·
IF 5.437
Sai Bi,Yangyang Cui,Ying Dong,Ningbo Zhang
A biosensing system is established for the multi-amplified detection of DNA or specific substrates of aptamers under isothermal conditions , which combines nicked rolling circle amplification (N-RCA) and beacon assisted amplification (BAA) with sensitive colorimetric technique by using DNAzymes as reporter units. According to the configuration, the analysis of DNA is accomplished by recognizing the target to capture nucleic acid-functionalized magnetic particles, followed by the self-assembly of the other two nucleic acids into multicomponent DNA supramolecular structure on magnetic particles. After magnetic separation, the circularization with ligase and the fragmentation with polymerase activate N-RCA and BAA in the presence of polymerase, dNTPs , and the nicking endonuclease , successively producing horseradish peroxidase (HRP)-mimicking DNAzymes that act as colorimetric reporter to catalyze the oxidation of ABTS 2− by H 2 O 2 in the presence of hemin . Under the optimized conditions, we obtain a wide dynamic range for DNA analysis over 6 orders of magnitude from 1.0×10 −14 to 1.0×10 −9 M with a low limit of detection of 6.8×10 −15 M. In the absence of a target, neither self-assembly of nucleic acids nor amplification process can be initiated, indicating an excellent selectivity of the proposed strategy. Similarly, an analogous system is activated by cancer cells or lysozyme through cooperative self-assembly of nucleic acids on magnetic particles in the presence of respective substrates of aptamers to synthesize HRP-mimicking DNAzymes that give the readout signal for the recognition events, achieving LODs of 81 Ramos cells and 7.2×10 −15 M lysozyme, respectively.
研究领域:
生物传感
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纳米技术
·
分子生物学
·
分析化学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
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