Analytical Methods
·
2025
·
IF 2.6
Chen Lu,Jiayue Chen,Shengsheng Zhang,Yaru Tian,Xinyu Ying,Dan Zhang,Zhidan Luo,Xinxin Si,Ming Li
Two single nucleotide polymorphisms (SNPs) in the human SLCO1B1 gene, c.388A>G (rs2306283) and c.521T>C (rs4149056), are independent determinants of the efficacy and side effects of statin drugs. Multinational clinical guidelines recommend testing for SLCO1B1 genotypes before the initial use of statins. Current SLCO1B1 SNP identification methods, primarily based on quantitative fluorescence PCR, rely on expensive equipment, are time-consuming, and require cold-chain storage for reagents, making them unsuitable for use in resource-limited healthcare settings. In this study, we developed a CRISPR/Cas12b-based amplification-free genotyping technique for SLCO1B1 SNPs. Within 30 minutes of the isothermal reaction, genotyping of the c.388A>G and c.521T>C SNPs in the SLCO1B1 gene can be observed by the naked eyes under blue light. Additionally, maltodextrin was identified as an effective vitrification stabilizer for the CRISPR/Cas12b premix. A low-cost vitrification process was optimized to prepare a glass like solid reagent via room-temperature vacuum drying. The vitrified CRISPR/Cas12b reagent retained approximately 88% of its activity after 30 days of storage at 37 °C, eliminating the need for cold-chain storage and allowing for long-term preservation at room temperature. This vitrified CRISPR/Cas12b based rapid SNP detection technique is especially suitable for genotyping drug metabolism genes in primary healthcare settings, providing effective guidance for precision medicine in clinical practice.
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SPECTROCHIMICA ACTA PART A-MOLECULAR AND BIOMOLECULAR SPECTROSCOPY
·
2024
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IF 4.3
Wen Yun,Wanshan Wang,Yiyan Lin,Ruiqi Wang,Xia Ha,Li Yang,Yue Jiang,Xin Zhang
A catalytic hairpin self-assembly (CHA) amplification method was developed for CAP detection based on cross-shaped DNA and UiO-66. MOF was used to quench the fluorescent signal of FAM labeled DNA. Cross-shaped DNA with four fluorophore group (FAM) was utilized to enhance the fluorescent intensity. CAP could open hairpin structure of H-apt and induce CHA reaction. The product of CHA hybridized with cross-shaped DNA, resulting its leaving from the surface of UiO-66 and recovery of fluorescent signal. The limit of detection (LOD) was low to 0.87 pM. This method had been successfully applied for the detection of CAP in actual samples. Importantly, the high sensitivity was attributed to the great amplification efficiency of CHA, strong fluorescent intensity of cross-shaped DNA structure and great fluorescent quenched efficiency of UiO-66.
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MICROCHIMICA ACTA
·
2024
·Vol. 191
·期 9
·1-9
·
IF 5.3
Song Yiwei,Jin Xiuyan,Zhao Yiou,Cheng Shuwen,Xu Sai,Bu Shengjun,Liu Liming,Zhou Chunyang,Pang Chunying
Single-level biomarker detection has the limitation of insufficient accuracy in cancer diagnosis. Therefore, the strategy of developing highly sensitive, multi-channel biosensors for high-throughput ctDNA determination is critical to improve the accuracy of early diagnosis of clinical tumors. Herein, in order to achieve efficient detection of up to ten targets for early diagnosis of ovarian cancer, a DNA-nanoswitch-based multi-channel (DNA-NSMC) biosensor was built based on the multi-module catalytic hairpin assembly-mediated signal amplification (CHA) and toehold-mediated DNA strand displacement (TDSD) reaction. Only two different fluorescence signals were used as outputs, combined with modular segmentation strategy of DNA-nanoswitch-based reaction platform; the multi-channel detection of up to ten targets was successfully achieved for the first time. The experimental results suggest that the proposed biosensor is a promising tool for simultaneously detecting multiple biomarkers for the early diagnosis of ovarian cancer, offering new strategies for the early screening, diagnosis, and treatment not only for ovarian cancer but also for other cancers. Graphical abstract
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CHEMICAL ENGINEERING JOURNAL
·
2024
·
IF 13.3
Jiansen Li,Chen Cao,Xinlu Zhang,Huan Li,Qin Fan,Shutong Chen,Xu Zhang,Sheng Wang
Immune checkpoint blockade therapy has made great strides in cancer treatment, but conventional immune checkpoint blockade antibodies often lead to overactivation of the immune system and severe immune-related adverse effects. Herein, we report the design of a nanomedicine (denoted as CPD@M−TDN) based on multifunctional tetrahedral DNA nanostructures (M−TDN). In this system, a bimetallic metal–organic framework (Cu/ZIF-8) is synthesized as the core; doxorubicin-loaded polydopamine acts as a shell to reduce the immunogenicity of the nanomaterials in peripheral blood. Finally, the CpG oligodeoxynucleotides and PD-L1 aptamer-based M−TDN are coupled to the core–shell structure nanomedicine through the biotin-avidin-system. Under the acidic tumor microenvironment, the decomposition of Cu/ZIF-8 and the release of copper ions and doxorubicin can lead to immunogenic death of tumor cells through chemodynamic/chemotherapy. Furthermore, the M−TDN has the dual effects of weakening the immunosuppressive microenvironment with PD-L1 aptamer and promoting dendritic cell activation with the immunostimulant CpG. Overall, the developed CPD@M−TDN can effectively enhance the response rate to immunotherapy and has excellent potential in the field of integrated tumor treatment.
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FOOD CONTROL
·
2024
·
IF 5.6
Yifei He,Jinxiang Wei,Lili Zhang,Yu Xia,Zhouping Wang,Junsong Yang
In this study, a dual-mode SERS-fluorescent aptasensor based on a DNA walker was developed for Shigella flexneri ( S . flexneri ). Fe@SiO2@QDs were connected with H1 and the walking chain (hybrid strands of aptamer and cDNA) to fabricate fluorescent probes, and Ag R6G @SiO 2 were connected with H2 to fabricate SERS tags. Aptamer (Kd = 42.6 ± 3.16 nM) selective binding of S . flexneri , left the walking chain. The Zn 2+ -specific DNAzyme in the cDNA was exposed thus driving the DNA walker. The sequence on H1 containing BHQ3 was cleaved from fluorescent probes to restore the fluorescence. The remaining H1 sequence could bind to the H2 to generate Raman signals. The detection limit of the fluorescence signal was 11 CFU/mL, and the Raman signal was 3 CFU/mL. In addition, the aptasensor was successfully applied to the detection of actual samples with recovery ranges of 98.35–107.27% and 97.57–105.91% for the fluorescence and SERS methods, respectively.
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MICROCHEMICAL JOURNAL
·
2025
·
IF 5.1
Shihua Luo,Lifeng Zhang,Xiaohe Zhang,Jiezhen Pan,Mei Wang,Ye Zhang,Aiping Qin,Wencheng Chen
The integrity of sperm DNA is essential for proper fertilization and embryonic development. Various methods exist to evaluate sperm DNA fragmentation. However, they are frequently hindered by high costs, complex procedures, and low precision. Herein, a simple and sensitive fluorescence biosensor was developed using the terminal deoxynucleotidyl transferase (TdT) and CRISPR/Cas12a system for analyzing sperm DNA fragmentation. This biosensor efficiently measures the number of 3′-OH, which correspond to the mean number of sperm DNA breakpoints (MDB). In the presence of TdT, the dATP nucleotide polymerized at the 3′-OH end of the primer, generating numerous polyA-tails, which were detected by CRISPR-guided RNA (crRNA) at the same time. Ultimately, the trans -cleavage activity of CRISPR/Cas12a was activated, which then cleaved the spherical nucleic acids (SNA) reporter, generating amplified fluorescence signal. The proposed method exhibited a broad linear detection for the quantity of 3′-OH ends in single-stranded DNA, from 1 fM to 10 pM, with a limit of detection of 2.5 fM. Moreover, the platform was successfully validated with clinical sperm samples and demonstrated a stronger correlation with sperm motility compared to traditional DNA fragmentation index (DFI) test. Therefore, the developed method offers advantages such as ease of use, elevated sensitivity, and mimimal instrumentation requirements, making it suitable for clinical application.
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MICROCHEMICAL JOURNAL
·
2025
·
IF 5.1
Ruyue Cong,Kaiyue Hu,Dandan Hu,Jingran Sun,Jinglu Yao,Zhiyong Guo,Sui Wang,Yufang Hu
The detection of UDG activity is essential for understanding DNA damage repair mechanisms, assessing cellular genetic stability, and advancing diagnostics and treatments for related diseases. An advanced electrochemical biosensor based on a silver-enhanced copper metal–organic framework (Ag@Cu-MOF) has been developed for the sensitive and selective detection of uracil-DNA glycosylase (UDG) activity in cellular environments. This design incorporates a prism substrate to stabilize the liquid phase on the electrode surface, with DNA probes immobilized on the prism surface. Upon UDG-catalyzed uracil excision, the probe undergoes structural changes, initiating a cascade reaction. The abundance of free DNA ends facilitates the approach of MOF materials to the electrode interface, creating a stable environment conducive to H 2 O 2 reduction, thereby amplifying the electrochemical signal. Comparative catalytic analyses revealed that the Ag@Cu-MOF-modified electrode, with its enhanced conductivity and high surface area, achieved efficient catalysis compared to Cu-MOF. The biosensor exhibited a linear response to UDG concentrations ranging from 0.00002 to 0.5 U/mL, with an impressively low detection limit of 0.00002 U/mL, highlighting its capability for quantitative analysis. The sensor demonstrated high selectivity, minimal interference from other biomolecules, and stable performance across multiple cycles, underscoring its robustness. Furthermore, UDG activity was successfully detected at varying cell densities and under hypoxic conditions, providing valuable insights into cellular DNA repair mechanisms and responses to stress. In summary, the Ag@Cu-MOF-based biosensor offers a promising platform for detecting UDG activity in cancer diagnostics and cellular research, with potential applications in monitoring DNA repair activities under both physiological and pathological conditions.
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ANALYTICAL CHEMISTRY
·
2024
·
IF 6.7
Mengxin Zhang,Yingyu Zhang,Xianwei Zhang,Kangbo Liu,Lifeng Li,Zhidan Yu,Jingya Yuan,Wancun Zhang
Due to the potential off-tumor signal leakage and limited biomarker content, there is an urgent need for stimulus-responsive and amplification-based tumor molecular imaging strategies. Therefore, two tetrahedral framework DNA (tFNA-Hs), tFNA-H1AP, and tFNA-H2, were rationally engineered to form a polymeric tFNA network, termed an intelligent DNA network, in an AND-gated manner. The intelligent DNA network was designed for tumor-specific molecular imaging by leveraging the elevated expression of apurinic/apyrimidinic endonuclease 1 (APE1) in tumor cytoplasm instead of normal cells and the high expression of miRNA-21 in tumor cytoplasm. The activation of tFNA-H1AP can be achieved through specific recognition and cleavage by APE1, targeting the apurinic/apyrimidinic site (AP site) modified within the stem region of hairpin 1 (H1AP). Subsequently, miRNA-21 facilitates the hybridization of activated H1AP on tFNA-H1AP with hairpin 2 (H2) on tFNA-H2, triggering a catalytic hairpin assembly (CHA) reaction that opens the H1AP at the vertices of tFNA-H1AP to bind with H2 at the vertices of tFNA-H2 and generate fluorescence signals. Upon completion of hybridization, miRNA-21 is released, initiating the subsequent cycle of the CHA reaction. The AND-gated intelligent DNA network can achieve specific tumor molecular imaging in vivo and also enables risk stratification of neuroblastoma patients.
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MICROCHEMICAL JOURNAL
·
2025
·
IF 5.1
Ming Li,Junhu Zhao,Meiyan Liao,Heng Zheng,Fei Cheng,Youhang Zhang,Hanfei Zhang,Pier-Luc Tremblay,Tian Zhang
The accurate detection of circulating tumor DNA (ctDNA) in patients’ biological fluids can facilitate cancer diagnosis and prognosis. This study reports the development of a differential pulse voltammetric (DPV) biosensor for the detection of ctDNA coding for the key T790M lung cancer mutation of the epidermal growth factor receptor (EGFR). The biosensor was built with a working electrode (WE) made of optimized electroconductive particles comprising covalent organic frameworks (COF) displaying Au nanoparticles (Au NPs) modified with polyethyleneimine (COF@Au-PEI). Au NPs accelerated electron transfer while COFs and PEI maximized the architecture of the WE to improve electrochemical reactions and molecular probe binding. Next, hairpin DNA probes (haiDNA) specific to the EGFR T790M ctDNA analyte were bonded to COF@Au-PEI. Lastly, Au@Pt nanohybrids were attached to the WE to enhance its electrochemical performance. In this system, the long polynucleotide EGFR T790M ctDNA analyte interferes with electrochemical reactions upon binding to the haiDNA probes, resulting in a proportional decrease of the DPV signal. The device detected EGFR T790M ctDNA in blood plasma and exhibited good sensitivity, accuracy, stability, and reproducibility. The sensor was also highly selective with no DPV signal for the EGFR wildtype allele harboring a difference of only one base pair.
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ANALYST
·
2012
·Vol. 138
·期 1
·197-203
·
IF 4.23
Sai Bi,Yangyang Cui,Li Li
An ultrasensitive and highly selective method for polymerase chain reaction-free (PCR-free) messenger RNA (mRNA) expression profiling is developed through a novel cross-rolling circle amplification (C-RCA) process based on DNA-rotaxane nanostructures. Two species of DNA pseudorotaxane (DPR) superstructures (DPR-I and DPR-II) are assembled by threading a linear DNA rod through a double-stranded DNA (dsDNA) ring containing two single-stranded gaps. In this assay, cDNA that is specific for β-actin (ACTB) mRNA is taken as a model analyte. Upon the introduction of the target cDNA, the cDNA and the biotin-modified primer are hybridized to the single-stranded regions of the DNA rod and the gap-ring, respectively. As a result, the DPR-I dethreads into free DNA macrocycle and a dumbbell-shaped DNA nanostructure. In the presence of DNA polymerase/dNTPs, two release-DNA on the DPR-I are replaced by polymerase with strand-displacement activity, which can act as the input of the DPR-II to trigger the dethreading of DPR-II and the RCA reaction, releasing another two specified release-DNA strands those in turn serve as the “mimic cDNA” for DPR-I. The C-RCA reaction then proceeds autonomously. To overcome the high background induced by hemin itself, the biotinylated rolling circle products are captured by streptavidin-coated MNPs, achieving a detection limit as low as 0.1 zmol cDNA. The assay also exhibits an excellent selectivity due to its unique DNA nanostructure fabricated through base pairing hybridization. The ACTB mRNA expression in mammary cancer cells (MCF-7) is successfully detected.
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BIOSENSORS & BIOELECTRONICS
·
2012
·
IF 5.602
Huanshun Yin,Yunlei Zhou,Zhenning Xu,Lijian Chen,Di Zhang,Shiyun Ai
DNA methylation is one of important epigenetics events, and responsible to transcription, genomic imprinting and cellular differentiation . Aberrant DNA methylation is always contacted with various diseases. Methyl binding domain (MBD) proteins can specifically bind to the methylated CpG dinucleotides . Conventional assay for DNA methylation normally need bisulfide treatment, methylated nucleotide labeling or PCR amplification. Here, we fabricated a novel electrochemical biosensor for detection of DNA methylation, assay of DNA methyltransferase (MTase) activity and screening of MTase inhibitor based on MBD protein and coomassie brilliant blue G250 (CBB-G250), where the electrochemical signal of CBB-G250 was used to monitor the methylation event. After the hybrids of DNA S1 and DNA S2 were treated with M. Sss I MTase in the presence of S -adenosylmethionine, the MBD proteins were specifically conjugated to the methylation site of CpG dinucleotides, and then, the MBD proteins were stained with CBB-G250. The electrochemical signal of CBB-G250 increased linearly with increasing M. Sss I MTase concentration in the range from 0.1 to 40 unit/mL. Furthermore, the inhibition investigation demonstrates that fisetin and chlorogenic acid can inhibit the M. Sss I MTase activity with the IC 50 value of 153.12 and 137.07 μM, respectively. Therefore, we think that this study may provide a sensitive platform for screening of DNA MTase inhibitors.
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ANALYST
·
2012
·Vol. 137
·期 19
·4539-4545
·
IF 4.23
Cuie Wang,Xue-Mei Jia,Chuan Jiang,Guang-Nan Zhuang,Qin Yan,Shou-Jun Xiao
Microarrays hold considerable promise in large-scale biology on account of their analytical, massive and parallel nature. In a step toward further enabling such a capability, we describe the application of rolling circle amplification (RCA) for a sensitive and multiplex detection of nucleic acid targets on oligonucleotide-conjugated polymer brushes covalently grown from porous silicon. Both RCA and polymer brushes have been taken to increase the loading quantity of target molecules and thus improve the detection sensitivity without loss of multiplexing. Besides, polymer brushes were employed to protect porous silicon and to provide biologically simulated environments, making the attached biomolecules maintain bioactivity. This approach can reach a detection limit of 0.1 nM target analytes and three orders of magnitude dynamic range of 0.1–100 nM, with a fluorescence scanner. A two-colour DNA microarray was achieved via RCA of two kinds of circular DNA targets on one chip simultaneously. The porous silicon chip-based RCA technique is promising for the multiplex detection of deoxynucleic acids on microarrays.
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