ANALYTICAL CHEMISTRY
·
2017
·
IF 6.32
Shanglin Li,Yin Gu,Yi Lyu,Yan Jiang,Peng Liu
An integrated graphene oxide purification-lateral flow test strip (iGOP-LFTS) was developed for on-strip purifying and visually detecting polymerase chain reaction (PCR) products with an improved sensitivity as well as a more stringent specificity. PCR products amplified with a pair of biotin- and digoxin-labeled primers were directly pipetted onto GO pads, on which graphene oxide selectively adsorbed residual primers and primer-dimers with the aid of a running buffer containing MgCl2 and Tween 20. By stacking up three GO pads to increase the surface area for adsorption, 83.4% of double-stranded DNA with a length of 30 bp and 98.6% of 20-nt primers could be removed from a 10-μL DNA mixture. Since no primers interfered with detection, the increase of the sample loading volume from 5 to 20 μL could improve the signal-to-noise ratio of the test line 1.6 fold using the iGOP-LFTS while no changes were observed using the conventional LFTS. The limit of detection of the iGOP-LFTS was determined to be 30 copies of bacteriophage λ-DNA with naked eyes and this limit could be further decreased to 3 copies by loading 20 μL of the sample, which corresponded to a 1000-fold improvement compared to that of the LFTS detected by naked eyes. When the ImageJ analysis was employed, a 100-fold decrease of the detection limit can be obtained. In addition, due to the removal of the primer-dimers, the dim test line observed in the negative control of the LFTS was eliminated using the iGOP-LFTS. A mock clinical specimen spiked with defective HIV-1 (human immunodeficiency virus) viruses was successfully analyzed using a two-step reverse transcription-PCR with 30 amplification cycles followed by the iGOP-LFTS detection. These significant improvements were achieved without introducing any additional hands-on operations and instrumentations.
研究领域:
分子生物学
·
分析化学
·
生物医学工程
·
病毒学
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产品关联
DOI
PubMed
高影响因子
Plant Stress
·
2024
·
IF 6.8
Haiyang Chi,Shasha Bai,Chenxiao Chen,Yuying Bao,Xiaoxin Qu,Shitao Sun,Jiangpeng Pan,Xiushi Yang,Chunsheng Hou,Yanchun Deng
Squalene plays a crucial role in plant growth, development, and stress tolerance. To select the best reference genes (RGs) for expression profile analysis of genes involved in squalene biosynthesis in olives under stress tolerance, the expression stability of 22 candidate RGs across four tissues (root, stem, leaf, and fruit) and five representative cultivars under drought stress was assessed using six methods. Our study showed that, ubiquitin-conjugating enzyme1 ( UBC1 ) and 60S ribosomal protein L18-2-like ( 60S ) were the most stable RGs across five different cultivars and various tissues. Additionally, elongation factor 1-alpha-like ( EF1-α2) and UBC1 were the most stable RG in olives under drought stress. UBC1 was the appropriate RG for further study, qPCR analysis showed that five certain genes involved in the squalene biosynthesis exhibited significant differential expression under varying conditions. Specially, the expression level of squalene synthase (SQS) in the leaves was highest, and HPLC analysis showed that squalene content was the highest in leaves. Likewise, the expression levels of SQS and farnesyl diphosphate synthase ( FPPS ) in the leaves of the cultivar Arbosana and Chelmsford Lal were significantly higher than those of the other cultivars, respectively, and HPLC analysis also showed that squalene content was highest in the two cultivars. Interestingly, the amount of squalene increased dramatically in olive under drought stress, as lupeol synthase ( LUPS ) was significantly up-regulated. To date, this study firstly provided the comprehensive analysis of RGs related to squalene biosynthesis in various cultivars and tissues of olives under drought stress.
研究领域:
植物科学
·
分子生物学
·
胁迫生理学
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DOI
高影响因子
FOOD CONTROL
·
2021
·
IF 5.548
Shan Shan,Yanmei Huang,Zhaohong Huang,Zhonger Long,Chengwei Liu,Xuelong Zhao,Keyu Xing,Xiaoyue Xiao,Jintao Liu,Yunhong Huang,Weihua Lai,Daofeng Liu
Shiga toxin-producing Escherichia coli (STEC) is known as a kind of foodborne pathogens that can produce Shiga toxin type1, type 2, or both, encoded by stx1 and stx2 genes, respectively. Rapid and accurate detection of STEC and stx1 / stx2 subtyping assay in food and environment is essential for foodborne outbreak investigation. In this study, a method for the accurate and rapid subtyping of STEC single colony using asymmetric polymerase chain reaction (asPCR) combined with lateral flow immunoassay (LFIA) was established. AsPCR was conducted using biotinylated stx1 and digoxin-labeled stx2 primers to obtain several single stranded DNA amplicons labeled with biotin or/and digoxin. Subsequently, these amplicons were tagged with specific probes labeled with carboxylate-modified red polystyrene microspheres using sequential hybridization. Finally, STEC with different Shiga toxin gene types could be distinguished depending on the appearance of different test lines on an LFIA strip. Thus, the STEC single colony could be identified and subtyped accurately and conveniently using this method. This method showed high specificity. Twenty-four strains of STEC in milk were detected and subtyped using our method, which combines asPCR with LFIA.
研究领域:
微生物学
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分子生物学
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食品安全
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病原体检测
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产品关联
DOI
高影响因子
ANALYTICA CHIMICA ACTA
·
2019
·
IF 5.256
Hai-Bo Wang,Li-Hong Ma,Ting Zhang,Kai-Chen Huang,Yuan-Di Zhao,Tian-Cai Liu
Single nucleotide polymorphism (SNP) was associated with many human diseases, therefore, SNP detection was important for early diagnosis and clinical prognosis. Herein, a simple and accurate method for visual detection SNP sites (A/A, G/G, A/G) in CYP1A1 gene related to cancers based on colloidal gold nucleic acid strip biosensor and primer-specific polymerase chain reaction (PCR) was established. This method could directly distinguish SNP sites on strip biosensor by introducing twice PCR amplifications. The second PCR (primer-specific PCR) was performed using specific product of the first PCR as template, thus this twice PCR could reduce non-specific amplification greatly and obtain target product. In addition, single-strand or double-strand DNA (ssDNA or dsDNA) was accurately produced by introducing mismatched base at the 3’ end of forward primers in primer-specific PCR. The designed strip biosensor could only combine with the ssDNA, thus visual detection of SNP could be achieved within 10 min by color difference of a pair of strips. 61 human blood samples by this method were identical with those of pyrosequencing. This method had the advantages of rapid, visual and low-cost and was expected to be applied in medical diagnosis.
研究领域:
分子生物学
·
医学诊断
·
遗传学
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DOI
PubMed
高影响因子
LAB ON A CHIP
·
2023
·
IF 6.1
Bo Yang,Ping Wang,Zhenqing Li,Qingxiang You,Shinichi Sekine,Junshan Ma,Songlin Zhuang,Dawei Zhang,Yoshinori Yamaguchi
Based on time to place conversion, continuous flow polymerase chain reaction (CF-PCR) can realize a rapid amplification of DNA by running the PCR reagent in a serpentine microchannel but a larger space is required for each sample, which greatly reduces the efficiency of the CF-PCR. Herein, we propose a multiplex circular array shaped CF-PCR microfluidic chip for on-site detection of bacteria. There were 12 serpentine microchannels which were distributed on the disc in an annular form, and each microchannel consisted of an inlet for sample injection, and an outlet for the detection of the PCR products based on fluorescence. Samples could be simultaneously driven into each inlet by a one-to-twelve diverter through a syringe. Moreover, the method of adding fluorescent dyes at the end of the microchannel can solve the inhibition effect of excessive fluorescent dyes on the PCR reaction. The process finished with simultaneous amplification of 12 different target genes from Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Escherichia coli, and on-site detection of their corresponding positives within 23 min. The fastest detectable PCR reaction time was 5.38 ± 0.2 min at a flow rate of 1 mL h−1. For E. coli, the minimum detectable concentration was 2.5 × 10−3 ng μL−1 in this microfluidic system. Such a system can increase the throughput of CF-PCR for point-of-care testing of pathogens.
研究领域:
微流控技术
·
分子生物学
·
生物技术
·
即时诊断
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产品关联
DOI
PubMed
高影响因子
ANALYST
·
2022
·Vol. 147
·期 21
·4876-4887
·
IF 5.227
Zengming Zhang,Shuhao Zhao,Lei Jiang,Junjun Wu,Wenhan Zhao,Xiaoniu Guo,Niancai Peng,Fei Hu
As the gold standard for nucleic acid detection, full-process polymerase chain reaction (PCR) analysis often falls into the dilemma of complex workflow, time-consuming, and high equipment costs. Therefore, we designed and optimized a DNA quantification microfluidic system by strategically integrating sample pretreatment and a smartphone-readable gradient plasmonic photothermal (GPPT) continuous-flow PCR (CF-PCR). Through preloading and sequential injection of immiscible extraction reagents, combined with magnetic bead (MB) manipulation, the microfluidic chip successfully purified and concentrated 100 μL of HBV-DNA spiked plasma into a 20-μL purified sample within 14 minutes. With a digital PCR platform, the optimized experiments showed that the DNA extraction efficiency can reach 69% at an immiscible reagent configuration ratio of 10 : 10 : 1 : 12 : 2 (sample : lysis/binding buffer : MB : silicone oil : eluent) and a flow rate of 25 μL min−1. For the first time, we used gold nanorod (AuNR)-doped PDMS to prepare a CF-PCR submodule for the amplification of a 40 μL PCR mixture. Due to the plasmonic photothermal effect of AuNRs and the gradient intensity of an expanded laser spot, the PCR thermal gradient was formed on a coin-sized area. The compact annular thermal-microfluidic layout, optimized DNA dye concentration, and chip transmittance synergistically enable a rarely reported smartphone-based fluorescence CF-PCR, greatly simplifying thermal control and detection setup. Prototype construction and validation experiments show that the microsystem can complete the sample-to-answer quantification of HBV-DNA with a dynamic linear range from 1.2 × 101 to 1.2 × 106 copies per μL in ∼37 minutes. This novel microfluidic solution effectively bridges the technical gap between the CF-PCR, sample pretreatment and result characterization, making the workflow standardized and rapid and requiring <15% of the commercial instrument cost. The simplicity, rapidity and low cost of this work make it promising for applications in decentralized laboratories and low-resource settings.
研究领域:
生物医学工程
·
分子诊断
·
微流控技术
·
纳米技术
·
即时检验
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DOI
PubMed
高影响因子
ANALYST
·
2019
·Vol. 144
·期 16
·4757-4771
·
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
·
临床诊断
·
精准医疗
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产品关联
DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2022
·
IF 6.911
Yuan Su,Xuetong Li,Longjiao Zhu,Huashuo Chu,Yangzi Zhang,Jingjing Tian,Shuxia Lyu,Wentao Xu
To develop an on-site, thermostatic and rapid sensor for the detection of Vibrio parahaemolyticus (V. parahaemolyticus) , A single cross-priming fluorescence (SCPF) sensor was designed using a 3D nano-nucleic acid hybrid material that termed mesoporous silica nanoparticle/nucleic acid-doped nanoflower (MSN/NA-doped nanoflower). In addition, a portable polymerase chain reaction (PCR) tube fluorescence reader was built. Further analysis of the MSN/NA-doped nanoflower morphology and the enhancement mechanism indicated that the MSNs aggregated into larger nanoclusters by adsorbing single cross-priming amplification (sCPA) components, forming MSNs/NAs-doped nanoflowers, and increasing the local concentrations to enhance sCPA efficiency. Cyclic amplification relied mainly on the self-folding hairpin-like structure of the amplified product that continuously formed, opened, re-formed, and opened again. The target DNA was detected with a detection limit of 2.4 copies/μL.
研究领域:
生物技术
·
分子生物学
·
分析化学
·
微生物学
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产品关联
DOI
PubMed
高影响因子
Cell Reports Physical Science
·
2025
·
IF 7.3
Weidong Chen,Zelin Yang,Yuheng Luo,Jianxun Huang,Lijie Wu,Wenxiong Liu,Yifei Wang,Jiajia Wu,Yining Li,Fang Sun,Pengming Song,Baoce Sun,Qiang Xu
Although real-time quantitative polymerase chain reaction (real-time qPCR) is the gold standard for molecular diagnostics, prolonged thermal cycling constrains its rapid application. Here, we report an ultrafast real-time qPCR system utilizing dynamic water-flow thermal regulation. By integrating a flexible thin-film carrier with dynamic water flow, the system minimizes interfacial thermal resistance. Additionally, a feedforward predictive control algorithm enables precise temperature control using low-cost solenoid valves. Consequently, the system completes 45 cycles in under 10 min, with heating and cooling ramps of ∼3.7 and ∼0.7 s, respectively. In Mycoplasma pneumoniae validation, the device exhibits superior performance, achieving 10 copies/μL sensitivity, 96.8% efficiency, and R 2 > 0.99 linearity. This work integrates speed, precision, and low cost, demonstrating significant promise for point-of-care diagnostics and large-scale genetic screening.
研究领域:
分子诊断
·
基因工程
·
生物医学工程
·
微流控技术
·
热控系统
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产品关联
DOI
高影响因子
Advanced Healthcare Materials
·
2026
·
IF 11
Yun Cheng,Xi Luan,Rokshana Parvin,Dingmeng Hu,Nan Wu,Ke Lin,Gangling Wang,Huaqiong Li,Lexiang Zhang,Fangfu Ye
Microneedle (MN) technology is an appealing route for treating skin cancers, but remains many challenges, such as accommodating multiple theranostic functionalities and configuring personalized sensing, particularly given that MNs typically collect sample volumes from microliters to milliliters, necessitating suitable trace analysis techniques. In this study, we developed a versatile, multilayer, detachable MN administration system capable of simultaneous photocontrolled drug delivery therapy that operated based on real-time in situ conditions monitored by droplet-based PCR (dPCR). The detachable MN consisted of an innermost poly (ethylene glycol) diacrylate extraction layer, an outer gelatin methacryloyl drug-loaded layer containing Vemurafenib and black phosphorus (BP), and a polyvinyl alcohol connection layer designed for thermal detachment. The outer layer enabled light-responsive drug release through BP's photothermal properties, achieving 78% release within 24 h. Subsequently, the significant mechanical strength and swelling characteristics facilitated the effective extraction of approximately 26 µL of interstitial fluid within 10 min. Both in vitro and in vivo studies on melanoma demonstrated the platform's capability to enhance therapeutic efficacy while minimizing systemic toxicity. It enabled dPCR-based monitoring of MCAM and BRAF genes, including the drug-resistant V600E polymorphism, with detection limits of 223 copies/µL, along with digital proximity ligation assay detection of the protein markers IL-6, VEGF, and Ki-67 at 0.64 pg/mL. This well-designed biosystem highlighted its capability to interact with the pathophysiological environment, providing a preclinical proof-of-concept for minimally invasive theranostics in superficial tumors.
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DOI
PubMed
高影响因子
WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY
·
2022
·Vol. 38
·期 6
·1-10
·
IF 4.253
Liang YaoYao,Luo Juan,Yang Chenhao,Guo Shuning,Zhang Bowen,Chen Fengqianrui,Su Kairui,Zhang Yulong,Dong Yi,Wang Zhihao,Fu Hongda,Sui Guangchao,Wang Pengchao
Hydroxy-mandelic acid (HMA) is widely applied in pharmaceuticals, food and cosmetics. In this study, we aimed to develop an allosteric transcription factors (aTFs) based biosensor for HMA. PobR, an aTF for HMA analog 4-hydroxybenzoic acid, was used to alter its selectivity and create novel aTFs responsive to HMA by directed evolution. We established a PobR mutant library with a capacity of 550,000 mutants using error-prone PCR and Megawhop PCR. Through our screening, two mutants were obtained with responsiveness to HMA. Analysis of each missense mutation indicating residues 122–126 were involved in its PobR ligand specificity. These results showed the effectiveness of directed evolution in switching the ligand specificity of a biosensor and improving HMA production.
研究领域:
生物技术
·
分子生物学
·
化学生物学
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产品关联
DOI
PubMed
高影响因子
Analytical Methods
·
2019
·Vol. 11
·期 26
·3286-3293
·
IF 2.378
Zhiqiang Jiao,Lei Zhao,Chu Tang,Hongyan Shi,Fu Wang,Bo Hu
Recently, 3D printing technology has been widely applied in microfluidics due to its capacity of three-dimensional construction, rapid and automatic fabrication, and less time consumption. However, a droplet-based PCR in 3D-printed microfluidic chips is rarely reported. In this study, a 3D-printed microfluidic chip was designed and fabricated for droplet-based PCR detection of miRNA-21. In this chip, the capacity of monodisperse droplet generation was demonstrated. Moreover, the temperature of the chip was uniform and sufficiently stable, meeting the requirement for the PCR process. In addition, the working conditions of the droplet-based PCR process in the 3D-printed chip were explored. Finally, the droplet-based PCR detection of miRNA-21 levels in the MCF-10A cells and MDA-MB-231 cells was successfully achieved using the 3D-printed chip. We expect that the enormous potential of 3D printing technology in the research area of microfluidics will be explored in the future.
研究领域:
微流控技术
·
分子生物学
·
生物医学工程
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DOI
高影响因子
危险品化学品经营许可证（带存储）