ANALYTICAL CHEMISTRY
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2022
·
IF 8.008
Lancheng Wang,Mingjie Xu,Huimin Zhou,Kun Yan,Shiqi Duan,Dandan Xue,Youmei Wang,Bin Di,Chi Hu
The target of typical PCR analysis is restricted to nucleic acids. To this end, we report here a novel strategy to simultaneously detect genetic and metabolic markers using commercial PCR kits with cucurbit[8]urils (CB[8]) implemented to manipulate the activity of Taq DNA polymerase. CB[8] binds with the nonionic surfactants and displaces them from the polymerase surface, resulting in decreased enzyme activity. Meanwhile, the inhibited enzyme can be reversibly activated when spermine, the downstream metabolite of ornithine decarboxylase (ODC), is present in the sample, which competitively binds to CB[8] and recovers polymerase activity. CB[8] was implemented in conventional PCR kits not only to reduce false-positive results but also to extend the detection range of PCR technology. With this novel method to detect ODC in cell lysates containing both the nucleotides and intracellular metabolites, positive results were only observed in highly active HEK 293T cells, whereas silent cells treated with ODC inhibitor showed negative readouts, therefore providing a simple but elegant dual-modality PCR method for precision diagnosis.
研究领域:
分子生物学
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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
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2018
·
IF 5.667
Jing Wang,Tingting Li,Haigang Li,Gongke Li,Shixin Wu,Liansheng Ling
AuNPs-DNA probes remain a challenge to recognize the double-stranded DNA (dsDNA) from routine PCR product despite its success in the recognition of the single-stranded (ssDNA) DNA of asymmetry-PCR product. Here, a universal colorimetric PCR biosensor is established with oligonucleotide modified AuNPs by recognizing the dsDNA of PCR product based on triplex DNA formation with the assistance of a triplex DNA intercalator Ru(phen) 2 dppx 2+ . The strategy was applied to detect different length dsDNA, including short dsDNA 1 (49 bp) and long dsDNA 2 (321 bp), both of which had the good linear relationship between the absorbance and the logarithm of the concentration of the targets. Moreover, the limit of detection (LOD) for short and long length dsDNA was 0.5 pM and 0.05 fg, lower than those of the previously reported methods, respectively. This strategy also displayed high selectivity against dsDNA 1 and dsDNA 2. These results demonstrate a potential strategy to detect all length of dsDNA, providing new opportunities in the life science, diagnosis, and medicine.
研究领域:
生物化学
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分子诊断
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纳米医学
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产品关联
DOI
高影响因子
TALANTA
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2023
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IF 6.1
Huizhen Li,Yunhui Li,Cenlin Gui,Daqi Chen,Lanfang Chen,Le Luo,Guobao Huang,Yang Yuan,Rong He,Fan Xia,Jiahai Wang
In this study, it is confirmed that without addition of organic solvent and embedding polymer hydrogel into glass nanopore, bare glass nanopore can faithfully measure various lengths of DNA duplexes from 200 to 3000 base pairs with 200 base pairs resolution, showing well-separated peak amplitudes of blockage currents. Furthermore, motivated by this readout capability of duplex DNA, amplicons from Polymerase Chain Reaction (PCR) amplification are straightforwardly discriminated by bare glassy nanopore without fluorescent labeling . Except simultaneous discrimination of up to 7 different segments of the same lambda genome, various pathogenic bacteria and viruses including SARS-CoV-2 and its mutants in clinical samples can be discriminated at high resolution. Moreover, quantitative measurement of PCR amplicons is obtained with detection range spanning from 0.75 aM to 7.5 pM and detection limit of 7.5 aM, which reveals that bare glass nanopore can faithfully disclose PCR results without any extra labeling.
研究领域:
生物物理学
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分子生物学
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医学诊断
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产品关联
DOI
PubMed
高影响因子
ACS Applied Bio Materials
·
2019
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IF 0
Zaizai Dong,Chuanhao Tang,Zhen Zhang,Wei Zhou,Rong Zhao,Lina Wang,Jiachao Xu,Yayun Wu,Jiang Wu,Xing Zhang,Li Xu,Libo Zhao,Xiaohong Fang
Exosomes, which are 30–150 nm extracellular vesicles containing a variety of biomolecules (i.e., proteins, RNA, DNA, and lipids), have emerged as important analytes in liquid biopsy. Although exosome detection offers a valuable chance to understand disease status in a multidimensional manner, comprehensive analysis of different exosomal biomolecules with limited specimens remains a challenge. Herein, we introduced an aptamer-based PCR (polymerase chain reaction) platform and realized highly sensitive detection of CD63 protein positive exosomes as low as 50 particles/μL. More importantly, simultaneous analysis of exosomal RNA and surface protein is available using this method. In a proof of concept experiment, simultaneous detection of two immunotherapy-related biomarkers, programmed death-ligand 1 (PD-L1) protein and indoleamine 2,3-dioxygenase 1 (IDO1) mRNA, was demonstrated with clinical samples. Our method provides a strategy for the comprehensive analysis of exosomes, with the advantages of high sensitivity and practicability.
研究领域:
生物技术
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分子生物学
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癌症研究
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医学诊断
✓ 已验证
DOI
PubMed
LAB ON A CHIP
·
2021
·Vol. 21
·期 20
·3933-3941
·
IF 6.799
Yu He,Zefan Lu,Hongliang Fan,Tao Zhang
Droplet microfluidics is a promising platform for various biological and biomedical applications. Among which, droplet-based digital PCR (ddPCR) is one of the most challenging examples, with practical issues involving possible fusion/fission of droplets during PCR thermocycling and difficulties of indexing them for real-time monitoring. While spatially trapped droplet arrays may be helpful, they currently are either of low trapping density or suffer from high droplet loss. In this paper, we, for the first time, report a photofabricated honeycomb micropillar array (PHMA) for high-density and loss-free droplet trapping. By rationally designing high-aspect-ratio micropillars into a honeycomb configuration, droplets can be captured at a density of 160–250 droplets per mm2 and, more interestingly, without any loss. The PHMA device can be fabricated from several photocurable materials, with one gasproof photopolymer being optimally selected herein to enable the simple design to avoid sample evaporation and tedious surface modification, thereby making the fabrication very convenient. Moreover, by using a photocurable oil as a continuous phase, the trapped droplets can be further immobilized, and thus, become more stable even in PCR thermocycling. With these features, the proposed PHMA has shown promising potential in ddPCR, and is expected to find a wide range of applications in various biological and biomedical research.
研究领域:
生物技术
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医学检测
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分子生物学
✓ 已验证
DOI
PubMed
高影响因子
JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE
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2025
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IF 3.5
Tianmi Liang,Wenjin Ma,Shijian Liu,Xiaoqi Tao
BACKGROUND As a traditional Chinese medicine, Colla Corii Asini is favored for its wide range of medicinal values. However, adulteration in the market is a serious problem, and effective detection methods are urgently needed. RESULTS The time-resolved fluorescence lateral flow assay coupled with PCR (TRF-LFA-PCR) method could detect a minimum content of 0.01% donkey hide in Colla Corii Asini , with recoveries ranging from 82.00% to 102.97% and a relative standard deviation between 1.10% to 12.98%. CONCLUSION The TRF-LFA-PCR technique is capable of quantitatively detecting the donkey hide content in Colla Corii Asini even in the presence of unknown components, thereby providing an innovative and effective solution for the detection of Colla Corii Asini adulteration and offering significant potential for further applications. © 2025 Society of Chemical Industry.
研究领域:
生物学
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医学
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药学
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分析化学
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DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2025
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IF 6.7
Ting Li,Tat San Lau,Junyou Li,Kaiqi Hu,Man Lung Lee,Pin You Chen,Chi Chiu Wang,Hung-Wing Li
Nonenzymatic autocatalytic DNA circuits, capable of exponential signal amplification, exhibit superior amplification efficiency compared to traditional single or cascade DNA amplification circuits, which offer only linear or quadratic signal amplification. However, autocatalytic DNA circuits are currently limited by complicated splitting designs, low autocatalytic efficiency, the need for additional probes, and a lack of universal design principles. Herein, we developed a PCR-like split-free autocatalytic amplification (SAA) DNA circuit with a simple design but extraordinary autocatalytic efficiency for effective biosensing. The SAA system could consecutively perform multiple cycles of a three-step process upon initiation, target recognition, replicating, and recycling, which is programmed to mimic the basic three reaction steps of PCR and constantly yield numerous new split-free target replicates to expedite the whole reaction, ultimately producing an exponentially amplified signal. The PCR-like sigmoidal kinetics, the ability to accurately execute the programmed instructions, and the high autocatalytic capability of the SAA system are demonstrated, which achieves the same target replication as the PCR but without the need for enzymes and precise control of temperature. The SAA circuit, characterized by exponential signal amplification, simple design, and minimal components, offers a promising approach for developing highly efficient universal DNA circuits. This enables the analysis of low-abundance biomarkers with minimal signal leakage, holding significant potential for biochemical research and clinical diagnosis.
研究领域:
分子生物学
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生物化学
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诊断学
✓ 已验证
DOI
PubMed
高影响因子
FOOD CONTROL
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2022
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IF 6.652
Zhi Li,Chuqin Yang,Wenying Lu,Zhaohui Chu,Jingwen Zhang,Ming Li,Qingjiang Wang
It was desired to develop the ultrasensitive and efficient strategy for detecting ultratrace aflatoxin B 1 (AFB 1 ) and protecting consumers' health. In this study, a magnetic immuno-PCR method (MIPCR) based on the barcode DNA was described and evaluated for detecting AFB 1 in agricultural by-product samples. Under the optimal MIPCR , the limit of detection (LOD) was 0.01 pg mL −1 , the half inhibitive concentration (IC 50 ) was 1.72 pg mL −1 , and the detection range was between 0.01 and 100 pg mL −1 . The LOD and IC 50 showed 480-fold and 21-fold lower than that of ELISA , respectively. The sensitivity of MIPCR had reached an ultratrace level and considerably improved. The reasonable specificity, precision and accuracy had been demonstrated. Furthermore, the high reliability and applicability of the MIPCR had been further confirmed by the good correlation of LC-MS/MS for the authentic samples. The proposed MIPCR had significantly shortened the analytical procedure, testing time and workload through the powerful probes and magnetic separation, which indicated the high-efficiency performance. This study could provide an alternative approach to detect the AFB 1 contamination, and a critical reference for the ultrasensitive and efficient detection of other hazardous chemicals.
研究领域:
食品安全
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分子生物学
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分析化学
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产品关联
DOI
高影响因子
ADVANCED FUNCTIONAL MATERIALS
·
2022
·Vol. 32
·期 48
·2207879
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IF 19.924
Lexiang Zhang,Wenzhao Li,Rokshana Parvin,XiaoChen Wang,Qihui Fan,Fangfu Ye
Exosomes are cell-derived extracellular vesicles which enclose genetic substances from their origin cells, offering a good source of biomarkers. Many efforts have been made to develop non-invasive exosome-based clinical diagnosis, which are urgently needed for prostate cancer, as traditional methods are criticized due to the inaccuracy and discomfort. In this study, a simple, universal process for genotyping of exosome-derived microRNAs using nanoparticles to capture exosomes and the novel photothermal droplet digital polymerase chain reaction (ddPCR) assay to detect target molecules is presented. To detect the target sensitively and simply, the smart ddPCR thermocycling of the agarose microcarriers can be controllably realized via embedding photothermal MXene and magnetic nanoparticles composite, and then irradiating with a homemade automated near-infrared control module. Duplex photothermal-driven ddPCR cooperating with dual TaqMan probes enabled simultaneously and quantitatively detecting copy numbers of prostate oncogenes microRNA-21-5p, microRNA-375-3p, and metastasis microRNA-574-3p along with the control microRNA-30a-5p within the extracted exosomes. The results show that the expression levels of these biomarkers in prostate cancer cell lines PC-3 and DU145 are 7–210 folds higher than those in healthy control cells HaCaT and HUVEC. Thus, it is believed that these versatile exosomal microRNA genotyping approaches are potentially valuable for diverse clinical applications.
研究领域:
癌症生物学
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分子诊断
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纳米技术
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生物医学工程
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产品关联
DOI
高影响因子
Separations
·
2023
·Vol. 10
·期 4
·271
·
IF 2.6
Bo Yang,Jiaxin Huang,Chunxian Tao,Zhenqing Li,Dawei Zhang,Yoshinori Yamaguchi
Continuous-flow PCR (CF-PCR) can realize rapid DNA amplification because of the high temperature variation rate. However, off-line detection methods for PCR may induce cross contamination. To overcome this problem, we herein fabricated an integrated CF-PCR and electrophoresis microfluidic chip. The optimal voltage applied in the electrophoresis part of the microfluidic chip was achieved by simulation in COMSOL. Coating the inside wall of the microchannel can inhibit electroosmotic flow and improve the resolution for DNA fragments. The temperature distribution of the serpentine part can meet the PCR and has no obvious suppressive effect on sample separation. Finally, we have performed the amplification of target genes forPorphyromonas gingivalis,Tannerella forsythia, andTreponema denticolaand detected the corresponding PCR products in the microfluidic chip within 11 min. Such work provides a new method for the rapid detection of bacteria.Keywords:capillary electrophoresis;periodontal pathogens;continuous flow PCR;microfluidic chip;fluorescence detection
研究领域:
生物医学工程
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分子生物学
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牙科科学
✓ 已验证
产品关联
DOI
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2025
·
IF 8.5
Hang Yuan,Samaila Boyi Ajeje,Yunzhe Wen,Chonlong Chio,Yun Hu,Shaohua Dou,Wensheng Qin,Ezhen Zhang,Qun Wu,Fubao Sun
Endo-1,4-β-glucanase plays a crucial role in converting cellulose from lignocellulosic biomass into fermentable sugars for biofuel production. However, its commercial utility is hindered by poor catalytic performance under extreme conditions. This study enhanced the catalytic activity of the endo-1,4-β-glucanase ( Tn CelB) from Thermotoga neapolitana through error-prone PCR directed evolution. After screening >4000 colonies, three mutants with enhanced activity were obtained. Mutants Tn CelB Y88F , Tn CelB A233T , and Tn CelB W219R displayed 52.14 U/mg, 44.90 U/mg, and 34.70 U/mg of specific activity on CMC, respectively, which is 1.9, 1.7, and 1.3 times higher than that of the wild-type (26.74 U/mg), correspondingly. Likewise, their enzyme activity on barley β-D-glucan increased by 3.5, 2.2, and 1.8 times, respectively. Notably, Tn CelB Y88F maintained over 90 % activity after 60 mins at high temperatures (80–100 °C), indicating an exceptional thermostability. Protein docking revealed that Tn CelB Y88F had higher binding affinity, aligned with kinetic studies. Tn CelB was capble of released more non-oxidized sugars from the hydrolysis of regenerated amorphous cellulose (RAC) by synergy with auxiliary action family 10 ( AA 10), which is potential in development of efficient lignocellulosic saccharification. This study can provide useful insights for the future engineering of other endoglucanases in the glycoside hydrolases family 12.
研究领域:
生物化学
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分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Applied Sciences-Basel
·
2022
·Vol. 12
·期 13
·6415
·
IF 2.838
Fangbin Xiao,Xuekun Bai,Keyu Wang,Yifan Sun,Hengyi Xu
Featured ApplicationRapid magnetic enrichment strategy can significantly improve the sensitivity of multiplex PCR analysis and shorten the pre-concentration time. The simultaneous and sensitive detection of pathogenicListeriaspp. was achieved.AbstractListeria monocytogenesandListeria ivanoviiare important pathogenicListeriaspp. that cause infections in humans and animals. Establishing a rapid and sensitive method for the simultaneous screening of pathogenicListeriaspp. is of great significance for ensuring food safety. Multiplex polymerase chain reaction (mPCR) has been extensively reported to simultaneously detect several pathogens in food with high sensitivity, but a time-consuming pre-enrichment process is necessary. In this study, we report the usage of surface-modified polyethyleneimine-coated positively charged magnetic nanoparticles (PEI-MNPs) for rapid enrichment of pathogenicListeriaspp. through electrostatic interactions. The enrichment process takes only 10 min with high capture efficiency (more than 70%) at a wide pH range and ionic strength. Combined with mPCR analysis, the PEI-MNPs-mPCR strategy can simultaneously, rapidly, and sensitively detect pathogenicListeriaspp. without a time-consuming pre-concentration process. Under the optimal conditions, the detection limits ofL. monocytogenesandL. ivanoviiin lettuce were both as low as 101CFU/mL, which was a hundred times lower than that without magnetic enrichment. In conclusion, the magnetic enrichment strategy based on charge interaction combined with mPCR analysis has great application potential in shortening the pre-concentration time of foodborne pathogens and improving the detection sensitivity.Keywords:magnetic enrichment;multiplex PCR;electrostatic interaction;bacteria detection;method sensitivity;food safety
研究领域:
食品科学
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微生物学
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分子生物学
✓ 已验证
产品关联
DOI
高影响因子
危险品化学品经营许可证（带存储）