ANALYST
·
2022
·Vol. 147
·期 4
·625-633
·
IF 5.227
Yan Yu,Ziqing Yu,Xufeng Pan,Lei Xu,Rui Guo,Xiaohua Qian,Feng Shen
Digital polymerase chain reaction (digital PCR) can provide absolute quantification of target nucleic acids with high sensitivity, excellent precision, and superior resolution. Digital PCR has broad applications in both life science research and clinical molecular diagnostics. However, limited by current fluorescence imaging methods, parallel quantification of multiple target molecules in a single digital PCR remains challenging. Here, we present a multiplex digital PCR method using digital melting curve analysis (digital MCA) with a SlipChip microfluidic system. The self-partitioning SlipChip (sp-SlipChip) can generate an array of nanoliter microdroplets with trackable physical positions using a simple loading-and-slipping operation. A fluorescence imaging adaptor and an in situ thermal cycler can be used to perform digital PCR and digital MCA on the sp-SlipChip. The unique signature melting temperature (Tm) designed for amplification products can be used as a fingerprint to further classify the positive amplification partitions into different subgroups. Amplicons with Tm differences as low as 1.5 degrees celsius were clearly separated, and multiple amplicons in the same partition could also be distinguished by digital MCA. We further demonstrated this digital MCA method with simultaneous digital quantification of five common respiratory pathogens, including Staphylococcus aureus, Acinetobacter baumannii, Streptococcus pneumoniae, Hemophilus influenzae, and Klebsiella pneumoniae. Since digital MCA only requires an intercalation dye instead of sequence-specific hydrolysis probes to perform multiplex digital PCR analysis, it can be less expensive and not limited to the number of fluorescence channels.
研究领域:
分子生物学
·
临床诊断
·
微流控技术
✓ 已验证
产品关联
DOI
PubMed
高影响因子
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
·
医学检测
·
分子生物学
✓ 已验证
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2022
·
IF 8.008
Chunyang Wei,Chengzhuang Yu,Shanshan Li,Jiyu Meng,Tiejun Li,Jingmeng Cheng,Feng Pan,Junwei Li
Digital polymerase chain reaction (PCR) plays important roles in the detection and quantification of nucleic acid targets, while there still remain challenges including high cost, complex operation, and low integration of the instrumental system. Here, in this work, a novel microfluidic chip based on co-flow step emulsification is proposed for droplet digital PCR (ddPCR), which can achieve droplet generation, droplet array self-assembly, PCR amplification, and fluorescence detection on a single device. With the combination of single-layer lithography and punching operation, a step microstructure was constructed and it served as the key element to develop a Laplace pressure gradient at the Rayleigh–Plateau instability interface so as to achieve droplet generation. It is demonstrated that the fabrication of step microstructure is low cost, easy-to-operate, and reliable. In addition, the single droplet volume can be adjusted flexibly due to the co-flow design; thus, the ddPCR chip can get an ultrahigh upper limit of quantification to deal with DNA templates with high concentrations. Furthermore, the volume fraction of the resulting droplets in this ddPCR chip can be up to 72% and it results in closely spaced droplet arrays, makes the best of CCD camera for fluorescence detections, and is beneficial for the minimization of a ddPCR system. The quantitative capability of the ddPCR chip was evaluated by measuring template DNA at concentrations from 20 to 50 000 copies/μL. Owing to the characteristics of low cost, easy operation, excellent quantitative capability, and minimization, the proposed ddPCR chip meets the requirements of DNA molecule quantification and is expected to be applied in the point-of-care testing field.
研究领域:
生物技术
·
分子生物学
·
医学检验
✓ 已验证
产品关联
DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2025
·
IF 10.5
Junjie Zeng,Yanping Li,Qinghua He,Ji Xia,Jiebiao Guo,Zhui Tu
Phage display-mediated immuno-polymerase chain reaction (PD-IPCR) combines the strengths of phage display technology and immuno-PCR. The link between genotype and phenotype has been established by displaying antibody fragments on the surface of phage particles, allowing signal transmission. In this study, we utilized aflatoxin B 1 (AFB 1 ) as a model system to investigate the efficacy of PD-IPCR. Signal amplification was achieved using both real-time fluorescence quantitative PCR (qPCR) and a custom-made droplet digital PCR (dPCR) system. Under optimized conditions, the assay using dPCR achieved a limit of detection of 0.015 ng/mL, a 6-fold improvement over qPCR. The validation of our method using major agricultural produce, such as corn, flour, and soy sauce, shows excellent recoveries ranging from 85.77 % to 108.28 %, with a total assay time of less than 2 h. Furthermore, we compared our results with a high-performance liquid chromatography (HPLC) method for AFB 1 content in corn and flour, revealing consistent outcomes (r = 0.9847). These results demonstrate the potential of nanobody-based PD-IPCR as a reliable tool for detecting mycotoxins.
研究领域:
食品安全
·
分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
BIOMEDICAL MICRODEVICES
·
2015
·Vol. 17
·期 3
·1-8
·
IF 2.877
Song Qi,Gao Yibo,Zhu Qiangyuan,Tian Qingchang,Yu Bingwen,Song Bofan,Xu Yanan,Yuan Maokai,Ma Congcong,Jin Wei,Zhang Tao,Mu Ying,Jin Qinhan
A nanoliter self-priming compartmentalization (SPC) microfluidic chip suited for the digital polymerase chain reaction (dPCR) analysis in point-of-care testing (POCT) has been developed. This dPCR chip is fabricated of polydimethylsiloxane (PDMS). After the dPCR chip is evacuated, there will be a negative pressure environment in the chip because of the gas solubility of PDMS. The negative pressure environment can provide a self-priming power so that the sample solutions can be sucked into each reaction chamber sequentially. The whole sampling process requires no external power and is valve-free. Channels that contain water are designed around each sample panel to prevent the solvent (water) from evaporating during dPCR process. A glass coverslip is also used as a waterproof layer, which is more convenient and more efficient than other waterproof methods seen in literature. This dPCR chip allows three samples to be amplified at the same time. Each sample is distributed into 1040 reaction chambers, and each chamber is only 2.08 nL. Human β-actin DNA solutions of known concentrations are used as the templates for the dPCR analyses to verify the sensitivity and accuracy of the method. Template DNA solutions diluted to concentrations of 300, 100 and 10 copies/μL are tested and shown that this simple, portable and self-priming dPCR chip can be used at any clinic as a real POCT technique.
研究领域:
分子生物学
·
医学检验
·
生物工程
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ANALYST
·
2021
·Vol. 146
·期 22
·6960-6969
·
IF 4.616
Ping Mao,Lei Cao,Zedong Li,Minli You,Bin Gao,Xianghong Xie,Zhenrui Xue,Ping Peng,Chunyan Yao,Feng Xu
Digital PCR has shown great potential for quantitative nucleic acid testing (NAT), but most existing platforms are dependent on large auxiliary equipment (e.g., vacuum pump, amplification instrument, fluorescence microscope) to achieve target dispersion, amplification, signal capture and result analysis. Such complex, expensive and bulky NAT platforms have limited their applications in resource-limited areas, especially for point-of-care testing (POCT). In this work, we designed a digital isothermal NAT platform based on a pump-free open droplet array microfluidic chip. A pump-free microfluidic chip was developed based on an open microdroplet array in the form of thousands of independent microdroplets for spontaneous sample dispersion, without the need for external power. Combined with a handheld fluorescent signal reader based on a smartphone, this digital NAT platform can accurately quantify as low as 1 copy per μL of λDNA. Therefore, our integrated NAT platform, as a potable, robust and low-cost tool for highly accurate NA quantitative analysis, holds great potential for POCT applications.
研究领域:
分子生物学
·
生物医学工程
·
医学诊断
✓ 已验证
DOI
PubMed
高影响因子
ADVANCED FUNCTIONAL MATERIALS
·
2022
·Vol. 32
·期 48
·2207879
·
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
·
分子诊断
·
纳米技术
·
生物医学工程
✓ 已验证
产品关联
DOI
高影响因子
ANALYTICAL CHEMISTRY
·
2023
·
IF 7.4
Liping Xia,Juxin Yin,Jianjian Zhuang,Weihong Yin,Zheyu Zou,Ying Mu
Rapid and sensitive pathogen detection methods are critical for disease diagnosis and treatment. RPA-CRISPR/Cas12 systems have displayed remarkable potential in pathogen detection. A self-priming digital PCR chip is a powerful and attractive tool for nucleic detection. However, the application of the RPA-CRISPR/Cas12 system to the self-priming chip still has great challenges due to the problems of protein adsorption and two-step detection mode of RPA-CRISPR/Cas12. In this study, an adsorption-free self-priming digital chip was developed and a direct digital dual-crRNAs (3D) assay was established based on the chip for ultrasensitive detection of pathogens. This 3D assay combined the advantages of rapid amplification of RPA, specific cleavage of Cas12a, accurate quantification of digital PCR, and point-of-care testing (POCT) of microfluidics, enabling accurate and reliable digital absolute quantification of Salmonella in POCT. Our method can provide a good linear relationship of Salmonella detection in the range from 2.58 × 101 to 2.58 × 104 cells/mL with a limit of detection ∼0.2 cells/mL within 30 min in a digital chip by targeting the invA gene of Salmonella. Moreover, the assay could directly detect Salmonella in milk without nucleic acid extraction. Therefore, the 3D assay has the significant potential to provide accurate and rapid pathogen detection in POCT. This study provides a powerful nucleic detection platform and facilitates the application of CRISPR/Cas-assisted detection and microfluidic chips.
研究领域:
分子生物学
·
生物技术
·
微生物学
·
分析化学
·
即时诊断
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Small
·
2022
·Vol. 18
·期 16
·2107858
·
IF 15.153
Lexiang Zhang,Parvin Rokshana,Yunru Yu,Yuanjin Zhao,Fangfu Ye
Digital PCR (dPCR) surpasses the performance of earlier PCR formats because of highly precise, absolute quantification and other unique merits. A simple thermocycling approach and durable microcarrier are of great value for dPCR advancement and application. Herein, a near-infrared (NIR) controlled thermocycling approach by embedding magnetic graphene oxide (GO) composite into the agarose microcarriers is developed. The core-shell composite is constructed by sequentially encapsulating GO and silica outside the magnetic nanocores. Benefiting from these additives, the resultant composite agarose gains appealing features as light-driven temperature changing, switchable gel–sol phase transforming, biocompatibility, and magnetic traction. By further emulsifying into droplets via the microfluidics method, the influence of typical parameters including material loading amount, laser intensity, and droplet diameter at various ranges is investigated for assembling microcarriers with different responsiveness. Then a paradigm of the NIR program can be easily tailored for PCR thermocycling. Finally, the feasibility of the approach is verified by detecting statistically diluted Klebsiella pneumoniae DNA samples, from 0.1 to 2 copies per drop. It is anticipated that this method has promising prospects for dPCR-based and other temperature-controlled applications.
研究领域:
生物工程
·
生物技术
·
分子生物学
·
分析化学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
TALANTA
·
2021
·
IF 6.057
Xu Cui,Tianbao Hu,Qiang Chen,Qiang Zhao,Yin Wu,Tengbao Xie,Pengyong Liu,Xi Su,Gang Li
Digital bioassays are powerful methods to detect rare analytes from complex mixtures and study the temporal processes of individual entities within biological systems. In digital bioassays, a crucial first step is the discretization of samples into a large number of identical independent partitions. Here, we developed a rapid and facile sample partitioning method for versatile digital bioassays. This method is based on a detachable self-digitization (DSD) chip which couples a reversible assembly configuration and a predegassing-based self-pumping mechanism to achieve an easy, fast, and large-scale sample partitioning. The DSD chip consists of a channel layer used for loading the sample and a microwell layer used for holding the sample partitions. Benefitting from its detachability, the chip avoids a lengthy oil flushing process used to remove the excess sample in loading channels and can compartmentalize a sample into more than 100,000 wells of picoliter volume with densities up to 14,000 wells/cm 2 in less than 30 s. We also demonstrated the utility of the proposed method by applying it to digital PCR and digital microbial assays.
研究领域:
生物技术
·
分析化学
·
分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
LAB ON A CHIP
·
2021
·Vol. 21
·期 18
·3429-3437
·
IF 6.799
Tengbao Xie,Ping Wang,Lei Wu,Bangyong Sun,Qiang Zhao,Gang Li
In this work, we present a simple and equipment-free system for discretizing samples into tens of thousands of discrete volumes in tens of seconds. Unlike conventional sample discretization systems that require bulky syringe pumps, pressure controllers, or vacuum equipment, our system requires only a sheet of water-soluble film, a hand-operated syringe, and a microfluidic device containing a high-density microchamber array. In this system, the water-soluble film seals the device inlet to form a closed channel–chamber system, while the syringe is used to create a vacuum in the closed system. Benefitting from the high negative pressure created by syringe-vacuum and the dissolution-triggered gating mechanism of the sealing water-soluble film, the aqueous sample loaded into the device inlet can be rapidly partitioned into tens of thousands of isolated chambers without the need for any expensive pumping systems. We demonstrated the utility of this system by exploiting it for digital PCR. We believe that this simple discretization system will find broad applications, such as in digital bioassays, single-cell analysis, and point-of-care diagnostics.
研究领域:
微流控技术
·
生物技术
·
即时诊断
·
数字生物分析
·
单细胞分析
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ACS Sensors
·
2025
·
IF 9.1
Lijuan Huang,Bing Du,Xiaohu Cui,Meiqi Liu,Zhoufei Li,Xiaoyun Cui,Ziying Xu,Zihui Yu,Shuo Zhao,Yanling Feng,Hanqing Zhao,Jianhai Long,Jing Yuan
Klebsiella pneumoniae (Kpn) is a common opportunistic pathogen, with hypervirulent strains (hvKpn) posing significant risks even to healthy individuals. Traditional clinical diagnostics often use real-time quantitative polymerase chain reaction (PCR) with TaqMan probes, which are costly and unstable due to their reliance on fluorophore and quencher molecules. This study introduces a novel probe using silicon-doped carbon quantum dots (Si-QDs) and single-layer graphene oxides (GOs), forming the Si-QD-probe-GO. This new probe offers excellent selectivity, sensitivity, stability, low photobleaching, and cost-effectiveness. A sensor array targeting khe, peg-344, and iucA genes is developed to detect Kpn and differentiate between classical Kpn (cKpn) and hvKpn. Using a partial least-squares discriminant analysis (PLS-DA) model, the system achieves 100% accuracy in distinguishing hvKpn from cKpn. The study also employs droplet digital PCR (ddPCR) with microfluidic technology to enhance sensitivity and quantification, achieving a detection limit of 5 CFU/mL. In a mouse pneumonia model, the PLS-DA model accurately identifies hvKpn in lung samples. The ddPCR quantification using Si-QD-probe-GO shows strong correlation with traditional culture methods. This research presents a promising ddPCR system for precise detection and virulence identification of Kpn, offering a new direction for clinical diagnostics.
研究领域:
微生物学
·
分子诊断
·
纳米技术
·
生物医学工程
·
传染病学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）