BIOCHIMIE
·
2025
·
IF 3
Lin Zhang,Lvping Wu,Yiwei Guo,Enjie Wang,Jiaxing Zhang,Shengping You,Rongxin Su,Wei Qi
Recombinase polymerase amplification (RPA) is a prominent isothermal nucleic acid amplification method widely applied in molecular diagnostics. The stability and functionality of the single-stranded DNA-binding protein T4 gene 32 (gp32) crucial for pre-synaptic filament formation and D-loop stabilization, play a key role in determining RPA efficiency. In this study, V62C/T80C and Y186R mutants with improved performance were screened by rational disulfide bond construction and virtual saturation mutagenesis, respectively. The structural changes in V62C/T80C and the altered ssDNA-binding capacity in Y186R both contribute to RPA amplification by enhancing the formation of UvsX-ssDNA presynaptic filaments and stabilizing the D-loop structure during homologous recombination, respectively. The two mutants each demonstrated unique advantages in the RPA process. V62C/T80C significantly accelerates the amplification process, reducing the RPA reaction time by 47%, while Y186R showed a 123% increase in efficiency across the entire amplification cycle. Totally, this study applied a rational strategy on gp32 optimization, shortening RPA reaction times, enhancing the RPA reaction efficiency, and advancing its application in clinical and point-of-care diagnostics.
研究领域:
分子生物学
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医学诊断
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生物化学
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DOI
PubMed
高影响因子
RSC Advances
·
2019
·Vol. 9
·期 6
·3407-3415
·
IF 3.049
Xinglu Jiang,Chenggui Zhao,Xiaobo Fan,Wei Xu,Rui Zhang,Hongbo Xu,Guoqiu Wu
Cell-free DNA (cfDNA) in blood, which stems from the fetus of pregnant women and tumor in cancer patients, has gained attention in molecular diagnosis. However, cfDNA is less stable, and its amount in the serum is extremely low; these are critical barriers for the utilization of this resource. In this study, a DNA-modified polyacrylamide hydrogel (DNA-Gel) was prepared, and a specialized device was designed to simultaneously catch, purify, concentrate, and detect targeted cfDNA by electrophoresis. We demonstrated that 20–1000 bp ssDNA and dsDNA could be caught and released by the DNA-Gel-based device with high specificity and sensitivity. Upon increasing the number of cycles and electrophoresis time, higher DNA purity and density were achieved, and the separation of serum proteins, untargeted cfDNA, and other charged molecules was promoted. As low as 10 pg μL−1 of DNA could be detected using the DNA-Gel after four cycles of concentration. We also detected 1 fg μL−1 of DNA in the serum with 16 cycles of concentration, followed by 25 PCR cycles. We also designed a device to obtain DNA from the DNA-Gel. We found that the DNA loss rate was around 50%, and A260/A280 was close to 1.7. Thus, we have designed a cost-effective and highly economical device to purify DNA at low concentrations with high specificity and selectivity.
研究领域:
分子生物学
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生物医学工程
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分析化学
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遗传学
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DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2022
·
IF 12.545
Hai-Yan Jia,Hong-Li Zhao,Ting Wang,Pin-Ru Chen,Bin-Cheng Yin,Bang-Ce Ye
MicroRNAs (miRNAs) play an essential role in cancer diagnosis and prognosis. Developing a new method for sensitive detection of miRNA is constantly in demand. CRISPR/Cas12a system can nonspecifically cleave single-stranded DNA after specific recognition of target DNA, showing tremendous potential in molecular diagnostics. However, CRISPR-based detection methods require synthesizing different crRNAs for detecting different targets, which limit their widespread application. Herein, we design a versatile and sensitive miRNA detection platform based on CRISPR/Cas12a system combined with a hybridization chain reaction (HCR) circuit. In this design, the HCR circuit as the signal transducer converts each miRNA into multiple DNA duplexes, which act as the activators to activate the trans -cleavage activity of Cas12a for further signal amplification . More importantly, this platform can sensitively detect different miRNAs without changing the spacer sequence of crRNA due to the fixed activators formed by HCR. In addition, the consistency between the proposed platform and RT-qPCR in miRNA detection extracted from different cell lines validated its practicability, demonstrating the potential in clinical diagnosis of cancers and monitoring therapy.
研究领域:
分子生物学
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生物技术
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癌症研究
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诊断技术
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Songkuan Zhuang,Xiaojun Huang,Muyan Diao,Ruiling Dong,Yueyu Dong,Jun Liu,Yizhen Liu
Current one-pot CRISPR diagnostics necessitate meticulous control of nucleic acid hybridization parameters or extensive modification of CRISPR components to achieve complete enzymatic silencing, a fundamental bottleneck limiting their robustness and generalizability. Here, we resolve this challenge through dynamic crRNA reconfiguration, a paradigm-shifting strategy that exploits the intrinsic structural duality of CRISPR RNA. We present CONVERT (CRISPR One-Pot Nucleic acid detection Via Engineered crRNA Reconfiguration Technology), a universal platform where nontarget intact crRNA acts as a universal suppressor, achieving complete Cas12a inhibition during RPA (recombinase polymerase amplification) by irreversible enzyme sequestration. Target detection is initiated through programmable conversion to split crRNA activators, bypassing sequence-specific design constraints entirely. This crRNA conformational switching mechanism is implemented via photocleavable linker cleavage and subsequent assembly with presupplied truncated crRNA (tcrRNA), activating trans-cleavage signal amplification. Three transformative advances emerge: First, the endogenous crRNA engineering eliminates dependency on exogenous nucleic acid blockers or chemical modifications, reducing optimization costs and time. Second, near-total enzymatic suppression ensures zero cross-talk between amplification and detection phases. Third, the unified workflow enables contamination-resistant operation through spatiotemporal control, requiring no physical partitioning. Validated for Enterocytozoon bieneusi detection, the system demonstrates attomolar sensitivity of 1 aM, 100% diagnostic concordance with qPCR, and absolute specificity against related pathogens. By effectively decoupling CRISPR regulation from sequence-context constraints through rational crRNA structural plasticity, this work establishes a generalized framework for next-generation molecular diagnostics.
研究领域:
分子生物学
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生物工程
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诊断技术
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合成生物学
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核酸扩增
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产品关联
DOI
PubMed
高影响因子
CHEMICAL ENGINEERING JOURNAL
·
2024
·
IF 13.3
Nan Wang,Qiang Zhang,Yajie Liu,Baoyan Huang,Shuli Man,Shengying Ye,Long Ma
Foodborne pathogens endanger public health and rapid, sensitive and accurate detection of them is vital. Argonaute stands in the frontline as the next generation nucleic acid detection tool, bearing a few comparable advantages. Centrifugal microfluidic chips (CMCs) use centrifugal force to achieve liquid flowing, mixing and reaction, eliminating complicated designs of valves and pumps. In this study, for the first time, we devised a Thermus thermophilus A rgonaute ( Tt Ago)-powered centrifugal microfluidic chip reaction biosensor for S ample-in- A nswer-out detection of P athogen S. aureus (termed as ASAP) with ultrahigh rapidity and sensitivity in a one-pot fashion. The samples subjected to testing were mixed with a home-made nucleic acid fast extraction reagent and then the mixture was injected into the sample cell of CMC, which was centrifuged down into the reaction cell. The reaction cell was preloaded with both LAMP (Loop-mediated isothermal amplification) and Tt Ago systems. As such, the species-specific nuc genes were amplified by LAMP, while the Tt Ago performed site-specific cleavage to output fluorescent (FL) signals. The sample-to-answer time was 17 min, and the limit of detection (LOD) reached 1 CFU/mL. ASAP was able to perform simultaneous multiplexed detection and up to 16 samples can be detected at the same time. ASAP reduced reagent consumption and minimized the influence of carry-over and cross-contamination. ASAP was capable of detecting S. aureus in foods, but also detecting physiological samples from infect, holding great promise for practical applications. Overall, our work has enriched the Argonaute-powered biosensing and CMC biosensor technology by providing a conceptually novel bacterial detection platform.
研究领域:
生物技术
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分子生物学
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微生物学
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食品安全
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生物传感器
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DOI
高影响因子
TALANTA
·
2021
·
IF 6.057
Xianfeng Wang,Xiaolong Chen,Chengxiang Chu,Yuanyi Deng,Mei Yang,Danqun Huo,Faliang Xu,Changjun Hou,Jiayi Lv
Accurate and effective detection of single-stranded nucleic acids is vital in both disease diagnosis and pathological studies. Hence, we develop a PAMmer-assisted CRISPR/Cas9 system mediated G4-EXPAR (Cas-G4EX) strategy for site-specific detection of ssRNA and ssDNA . PAMmer-assisted CRISPR/Cas9 executes the site-specific cleavage of target ssRNA or ssDNA and released product fragment with the desired sequence at the 3′-terminal. This fragment serves as a primer to activate subsequent sequence-dependent exponential amplification reaction (EXPAR). The G-rich EXPAR products assembles with hemin to form a G-Quadruplex (G4/hemin). G4/hemin catalyzes ABTS-H 2 O 2 system with the appearance of vivid green color, realizing naked-eye analysis. Cas-G4EX integrates the superiority of CRISPR/Cas9 and EXPAR, presenting outstanding site-specific recognition and high-performance amplification efficiency. Meanwhile, the programmability of CRISPR/Cas9 system makes the proposed method become a universal detection paradigm for any ssRNA or ssDNA. Cas-G4EX assay shows the linear relationship from 250 aM to 2.5 nM for ssRNA detection with the actual LOD of 250 aM, and that ranges from 100 aM to 1 nM for ssDNA detection with the actual LOD of 100 aM. Additionally, the acceptable recoveries of 101.48%–109.61% for ssRNA and 93.25%–111.98% for ssDNA in real detection of human serum are obtained for detection of single-strand nucleic acid in real samples. Cas-G4EX also exhibits the excellent discrimination for single-base mutation of single-stranded nucleic acids. Therefore, Cas-G4EX assay provides a promising platform in the applications of molecular diagnosis and pathological analysis.
研究领域:
生物技术
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分子生物学
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诊断医学
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病理学
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产品关联
DOI
PubMed
高影响因子
Journal of Analysis and Testing
·
2025
·
IF 7
Ren Dandan,Dong Wenpei,Shen Shiqi,Zhao Wenchen,Qian Tianwei,Xia Xinyi,Li Yuqiu,Xu Fuxin,Tang Xiaoya,Li Xingchen,Chen Yu,Zhang Jue,Cheng Xia,Yang Jianjun,Yu Ping
Colorectal cancer (CRC) is a gastrointestinal malignancy with high morbidity and mortality rates. Timely and effective early diagnosis is crucial for improving the survival rate of CRC patients. However, current histopathological biopsy techniques are not only invasive but also fail to fully capture the biological characteristics and dynamic changes of CRC, making them ineffective for early identification. Since dysregulated microRNA (miRNA) signatures are associated with various diseases, including cancers, miRNA analysis in liquid biopsies offers a promising approach for cancer diagnosis, prognosis and monitoring. Nonetheless, single biomarker profiling is no longer sufficient to meet the specificity required for early CRC screening. It is expected that multiple miRNA analyses will enable both early CRC identification and dynamic monitoring. In this work, we synthesized a fluorescent metal-organic framework (UiO-66-NH 2 ) with desirable optical properties and successfully developed a ratiometric fluorescence sensor. This method enabled the highly sensitive and precise simultaneous quantification of CRC-related miRNA-21, miRNA-17 and miRNA-92a. It has also shown excellent performance in clinical samples. The integrated detection of multiple CRC-related miRNAs enhanced data accuracy through unique probe designs, offering significant potential for widespread applications.
研究领域:
肿瘤学
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生物传感器
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纳米材料
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分子诊断
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分析化学
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产品关联
DOI
高影响因子
ANALYTICA CHIMICA ACTA
·
2025
·
IF 6
Zhaleh Asadi Fakhr,Wei Xie,Su Zeng,Sheng Cai
Background CRISPR-Cas13a biosensing enables rapid, amplification-free RNA diagnostics, yet assay sensitivity varies widely because guide RNAs (gRNAs) differ in their ability to activate the enzyme. Two factors, including the gRNA-target binding affinity and the structural accessibility of the target site, have been proposed to govern activation efficiency, but their relative importance remains unclear. In this study, we systematically disentangle these contributions by measuring binding affinities for gRNAs that span a spectrum of site accessibilities and by comparing their Michaelis–Menten kinetic parameters. Results Three ciRS-7–specific gRNAs were designed with high, intermediate, and low spacer accessibility. Isothermal titration calorimetry (ITC) quantified site accessibility through entropy changes (ΔS = –862, –813, and –615 cal/mol/K), confirming greater structural exposure for less structured spacers, and also determined binding affinity for each gRNA–target pair. Michaelis–Menten analysis showed k cat values of 1.39, 1.31, and 1.16 s -1 for the high, intermediate, and low-accessibility guides, respectively, establishing a clear relationship between structural accessibility and catalytic turnover. Importantly, the most structured gRNA exhibited lower activation efficiency compared with the gRNA that had higher site accessibility and lower binding affinity, demonstrating that site accessibility drives Cas13a activation. Detection-limit experiments also confirmed these results, showing that gRNAs with greater spacer accessibility yielded stronger signals and superior sensitivity. Significance Our data establish site accessibility as a critical determinant of Cas13a activation for amplification-free RNA sensing. Prioritizing unstructured spacer regions enables improved enzyme activation efficiency, providing a clear design rule for next-generation CRISPR diagnostics. This accessibility-driven strategy will facilitate the development of faster, simpler, and more sensitive point-of-care assays for diverse RNA biomarkers.
研究领域:
分子诊断
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CRISPR技术
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RNA生物学
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生物化学工程
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即时检测
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产品关联
DOI
PubMed
高影响因子
MICROCHEMICAL JOURNAL
·
2025
·
IF 5.1
Minqiao Feng,Chengyan Yin,Hailiang Qin,Wei Liu,Yunqi Wang,Ruth Antwi-Baah,Yao Rao,Shuxia Xu
Terminal deoxynucleotidyl transferase (TdT) is actively expressed in leukemia patients and plays an important role in maintaining the genetic diversity of an organism. Therefore, developing simple and sensitive analytical methods for the accurate detection of TdT in serum is of significant importance. Herein, an electrochemical biosensor based on a toluidine blue (TB)-loading mesoporous silica nanoparticles (MSN) was established for the detection of TdT in serum. First, MSN with amino group was synthesized and subsequently was carbonylated to obtain carboxyl-functionalized MSN (MSN-COOH), which was used to load TB. Thereafter, MSN-COOH was sealed with the substrate strand S(rA)-NH 2 of the Mg 2+ -dependent DNAzyme to form S(rA)-NH 2 /TB/MSN-COOH. In the presence of TdT, it can add deoxyadenosine triphosphate (dATP) to the 3’-OH end of Enc-5 A, activating its cleavage activity on the substrate chain S(rA)-NH 2 , thus releasing TB encapsulated in MSN-COOH. By measuring the change in the electrochemical signal of TB, the TdT concentration can be determined. The linear range is from 5 to 400 U/mL and the limit of detection (LOD) is 0.369 U/mL (3 σ/K). Since DNAzyme can be extended by TdT without the need of a template, the proposed biosensor demonstrates excellent selectivity towards TdT in the presence of other DNAzyme, such as T4 DNA ligase, phi29 DNA polymerase, DreamTaq DNA polymerase, and Klenow fragment ( exo -). Furthermore, the proposed biosensor was successfully applied to the detection of TdT in serum with the recovery rates ranged from 91.0 % to 94.7 %, indicating its potential for early detection of leukemia and other related diseases.
研究领域:
生物分析化学
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生物传感器
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分子诊断
·
纳米生物技术
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肿瘤学
·
临床化学
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产品关联
DOI
高影响因子
MICROCHEMICAL JOURNAL
·
2025
·
IF 5.1
Qiu-Long Zhang,Xue Yang,Xuan-Ting Lin,Sha-Sha Chen,Zhu,Guang-Xiang Luan,Kun-Quan Chen,Zhi Zeng,Jian-Min Chen
In this study, a universal fluorescent aptasensor was developed for the sensitive detection of ochratoxin A (OTA) and aflatoxin B1 (AFB1). The design integrated a non-competitive transduction platform with highly efficient RNA-cleaving DNAzymes, forming the core of the sensing system. When OTA or AFB1 is present in the sample, the target mycotoxin specifically recognizes and binds to its corresponding aptamer. The strand displacement reaction is significantly hastened by this binding event, and in response, the DNAzyme is released as a consequence. The activated DNAzyme then triggers an amplified fluorescent signal, enabling quantitative detection of the target. The proposed aptasensor displayed sensitivity with a detection limit of 0.26 nM for AFB1 and 0.17 nM for OTA respectively. Beyond single-target detection, the platform also enabled simple and flexible implementation of logical operations between the two mycotoxins, along with the design of orthogonal experiments for multi-target analysis. Furthermore, specificity tests confirmed that other co-existing mycotoxins had negligible interference on the detection of AFB1 or OTA, verifying the sensor's high selectivity. To assess practical applicability, the aptasensor was utilized for the detection of AFB1 and OTA in actual samples, yielding recoveries of 93 %–101 % in milk and 101 %–107 % in corn flour, demonstrating the sensor's reliability in complex matrices. Collectively, this developed fluorescent aptasensor features portability, simplicity, cost-effectiveness, and broad adaptability, making it a promising alternative tool for detecting various mycotoxins in food safety monitoring and related fields.
研究领域:
食品安全
·
分析化学
·
生物传感器
·
分子诊断
·
环境监测
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产品关联
DOI
高影响因子
ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Yueyue Li,Yisheng Wang,Yingying Xiong,Xuejiao Liu,Yang Li,Ying Zhang,Haojie Lu
Human serum albumin (HSA), the most abundant plasma protein predominantly synthesized and secreted by the liver, harbors atypical N-glycosylation modifications at Asn68 and Asn123. Despite their potential biological relevance, the comprehensive characterization of these modifications remains limited. Here, we systematically characterized atypical N-glycosylation on HSA using a sialic acid linkage-specific derivatization strategy coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), identifying 35 N-glycans including 23 sialylated N-glycans distinguished sialic acid linkage isomers. LC-ESI-MS analysis further resolved 25 site-specific N-glycopeptides, establishing the most detailed HSA N-glycosylation profile to date and elucidating its structural diversity. Given the liver-specific origin of HSA, we further explored the clinical relevance of HSA glycosylation in serum samples from healthy controls (HC), liver cirrhosis (LC), and hepatocellular carcinoma (HCC) patients and found that HSA glycopeptides exhibited exceptional diagnostic accuracy in distinguishing LC from HCC patients (AUC = 0.906). These findings not only decode the complexity of HSA glycosylation but also validate its clinical utility as a liver-specific biomarker for HCC detection.
研究领域:
糖蛋白质组学
·
临床蛋白质组学
·
癌症生物标志物
·
肝脏疾病
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质谱分析
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分子诊断
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Yuqian Guo,Yuanqian Xiong,Weipeng Tong,Xiaolin Huang,Yonghua Xiong
Ultrasensitive signal transduction systems are essential for developing rapid, robust, and sensitive nucleic acid tests using the amplification-free CRISPR-Cas12a system. In this study, we introduce a novel platform termed CRISPR-DART (dynamic light scattering Assisted Rapid Test), which combines CRISPR-Cas12a with a dynamic light scattering (DLS) signal readout. We systematically evaluated the effects of nanoparticle size (20, 60, and 100 nm) and morphology (spherical, cubic, and flower-like) on DLS sensing performance. Results demonstrate that larger and more structurally complex nanoparticles significantly enhance scattering intensity, allowing stable DLS signals at lower concentrations and improving sensitivity for detecting low-abundance nucleic acid targets. By utilizing the high specificity and trans-cleavage activity of CRISPR-Cas12a, target-triggered cleavage of single-stranded DNA linkers modulates nanoparticle aggregation, enabling quantitative nucleic acid detection. Notably, CRISPR-DART based on 100 nm gold nanoflowers with large size and complex morphology achieves a detection limit (LOD) of 32 aM, an improvement of 5 orders of magnitude over conventional CRISPR-Cas12a assays using fluorophore-quencher as signal output. Furthermore, the amplification-free CRISPR-DART platform also achieves a LOD of 92 CFU/mL for Listeria monocytogenes in food samples, and after a brief preincubation step, successfully detects 1 CFU in 25 g of food sample. In summary, the CRISPR-DART platform provides a straightforward, highly sensitive, and specific tool for rapid on-site diagnostics and food safety monitoring.
研究领域:
分子诊断
·
食品安全监测
·
纳米生物技术
·
CRISPR技术
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生物传感
✓ 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）