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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JOURNAL OF HAZARDOUS MATERIALS
·
2024
·
IF 12.2
Jingyi Yang,Yining Zhao,Lelin Qian,Ying Yu,Yuan Zhang,Juan Zhang
In this work, a dual recognized CRISPR/Cas12a system has been proposed, in which the activation chain is cleverly divided into two parts that can serve for precise dual target recognition, and hydrazone chemistry is introduced for the formation of a whole activation chain. It has been further explored to construct a new method for the specific and sensitive detection of Staphylococcus aureus (SA) as one of the most common pathogens in infectious diseases. In virtue of proximity effect contributed by complementary base pairing, hydrazone chemistry accelerates the formation of the whole activation strand and improves the specificity of the CRISPR/Cas12a system, serving for the accurate analysis of SA. Moreover, the temporary aggregation of CRISPR/Cas12a around SA enhances its catalytical efficiency so as to further amplify signal. With high sensitivity, stability, reproducibility and specificity, the established method has been successfully applied to detect SA in complex substrates. Meanwhile, our established method can well evaluate the inhibition effect of chlorogenic acid and congo red in comparison with flow cytometry. Environmental implication Bacterial pathogens exist widely in the environment and seriously threaten the safety of human health. Staphylococcus aureus (SA) is the most common pathogen of human suppurative infection, which can cause local suppurative infection, pneumonia, and even systemic infections such as sepsis. In this work, a dual recognized CRISPR/Cas12a system mediated by hydrazone chemistry has been proposed. With high sensitivity and low detection limit, the established method can specifically detect SA and effectively evaluate the antibacterial effect of inhibitors. This method is expected to be further developed into a detection method in different scenarios such as environmental monitoring and clinical diagnosis.
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INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2025
·
IF 8.5
Fan’ge Kong,Shichao Peng,Yuchong Zhang,He Zhang,Jiasi Wang,Di Wang
Cladobotryum mycophilum , a pathogen responsible for cobweb disease, caused significant and irreversible losses in the mushroom industry. Effective monitoring and early prevention rely on the development of advanced diagnosis methods. This study introduced a novel hydrogel-based C. mycophilum detection method that integrates recombinase-aided amplification (RAA) with the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) system (CRISPR/Cas12a), referred as RCCH. The RAA reaction occurs within cross-linked PEG hydrogel, which is subsequently overlaid with a CRISPR/Cas12a-functionalized hydrogel. The porous network of the PEG hydrogel traps essential enzymes, facilitating spatial co-localization of target DNA and the CRISPR/Cas12a-crRNA complex. Upon activation of Cas12a's trans -cleavage activity, clear and countable fluorescent spots are generated for visual detection. RCCH demonstrates a limit of detection as low as 1 fg/μL, and exceptional selectivity against common fungi Trichoderma viride and T. harzianum and the host mushroom Lentinula edodes . The entire process is completed in under 40 min, indicating RCCH's potential as a rapid, accurate, and practical detection method for monitoring mushroom diseases. This innovative approach offers significant support for enhancing safety in the mushroom industry.
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CHEMICAL ENGINEERING JOURNAL
·
2025
·
IF 13.2
Yuan Zhang,Kangzheng Lv,Yu Zhang,Qin Mi,Daixin Ye,Xi Chen,Juan Zhang
The emergence and robust advancement of the CRISPR/Cas system have presented novel opportunities for the development of paper-based microfluidics. In this study, the CRISPR/Cas system is firstly ingeniously integrated into a paper-based platform as an efficient signal amplification component. The Cas12a-crRNA system is integrated with nitrocellulose (NC) membrane to prepare an anchored CRISPR-Cas12a system-boosted paper-based microfluidic device, thereby increasing the local concentration of the CRISPR/Cas12a-crRNA system and enhancing catalytic efficiency. Compared with free CRISPR/Cas12a system, our explored split anchored CRISPR/Cas12a signal amplification component demonstrates a catalytic constant that is 22 times higher and a specificity constant that is 34 times greater. Further, an anchored split CRISPR/Cas12a boosted paper-based microfluidic array (ASCPMA) has been further constructed for the on-site detection of Streptococcus mutans ( S. mutans ) to assess the early risk of oral caries. ASCPMA exhibits a wide linear range from 10 CFU/mL to 10 8 CFU/mL, with a detection limit as low as 2.08 CFU/mL, and it has been successfully applied to test 140 clinical saliva samples. With rapidity, accuracy, and portability, ASCPMA will facilitate early diagnosis and prevention of dental caries.
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JOURNAL OF HAZARDOUS MATERIALS
·
2022
·
IF 14.224
Long Ma,Jiajing Wang,Yaru Li,Dan Liao,Wenlu Zhang,Xiao Han,Shuli Man
Rapid, sensitive and specific detection of bacteria is of great importance. Herein, we developed a versatile biosensing platform for ultrasensitive detection of pathogenic bacteria, termed as SCENT-Cas (Silver nanoCluster Empowered Nucleic acids Test using CRISPR/Cas12a). Simply, the species-specific invA gene of Salmonella typhimurium (S. typhi) was isothermally amplified using LAMP, which subsequently triggered the trans -cleavage of CRISPR/Cas12a. The trans -cleavage degraded any single-stranded DNA (ssDNA) non-specifically. A DNA-templated AgNCs probe was then employed, in which green fluorescence emissive AgNCs effectively converted to red fluorescence emissive AgNCs when placed in close vicinity to a pre-designed converter ssDNA. As such, the trans -cleavage was utilized for shredding converter ssDNA, enabling the green-to-red fluorescent change to form a ratiometric biosensing platform. With this strategy, target nucleic acid was dexterously converted into ratiometric fluorescence that was recorded to detect as low as 1 CFU/mL S. typhi with a dynamic range from 1 to 10 8 CFU/mL. To our knowledge, this is the first report regarding the use of ratiometric fluorescence in CRISPR/Cas-based detection, which minimizes interference and improves reliability. Lastly, this proposed strategy was challenged by detecting S. typhi contamination in real food samples. Our work enriches CRISPR/Cas toolbox in biosensing by providing a desirable method for bacterial detection.
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Theranostics
·
2022
·
IF 11.6
Bohan Yin,Qin Zhang,Xinyue Xia,Chuanqi Li,Willis Kwun Hei Ho,Jiaxiang Yan,Yingying Huang,Honglian Wu,Pui Wang,Changqing Yi,Jianhua Hao,Jianfang Wang,Honglin Chen,Siu Hong Dexter Wong,Mo Yang
Background: CRISPR-Cas12a has been integrated with nanomaterial-based optical techniques, such as surface-enhanced Raman scattering (SERS), to formulate a powerful amplification-free nucleic acid detection system. However, nanomaterials impose steric hindrance to limit the accessibility of CRISPR-Cas12a to the narrow gaps (SERS hot spots) among nanoparticles (NPs) for producing a significant change in signals after nucleic acid detection. Methods: To overcome this restriction, we specifically design chimeric DNA/RNA hairpins (displacers) that can be destabilized by activated CRISPR-Cas12a in the presence of target DNA, liberating excessive RNA that can disintegrate a core-satellite nanocluster via toehold-mediated strand displacement for orchestrating a promising “on-off” nucleic acid biosensor. The core-satellite nanocluster comprises a large gold nanoparticle (AuNP) core surrounded by small AuNPs with Raman tags via DNA hybridization as an ultrabright Raman reporter, and its disassembly leads to a drastic decrease of SERS intensity as signal readouts. We further introduce a magnetic core to the large AuNPs that can facilitate their separation from the disassembled nanostructures to suppress the background for improving detection sensitivity. Results: As a proof-of-concept study, our findings showed that the application of displacers was more effective in decreasing the SERS intensity of the system and attained a better limit of detection (LOD, 10 aM) than that by directly using activated CRISPR-Cas12a, with high selectivity and stability for nucleic acid detection. Introducing magnetic-responsive functionality to our system further improves the LOD to 1 aM. Conclusion: Our work not only offers a platform to sensitively and selectively probe nucleic acids without pre-amplification but also provides new insights into the design of the CRISPR-Cas12a/SERS integrated system to resolve the steric hindrance of nanomaterials for constructing biosensors.
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ANALYTICA CHIMICA ACTA
·
2020
·
IF 5.977
Lei Peng,Jin Zhou,Lijuan Yin,Shuli Man,Long Ma
For the first time, three 2-input elementary AND, OR, INHIBIT logic gates have been constructed by using CRISPR-Cas12a system. These logic gates utilised the intrinsic advantages of programmability, sequence specificity and high base resolution of CRISPR-Cas12a system. Among them, the AND gate owned the potentials as a built-in biosensor that responded rapidly to external pathogenic bacteria such as Staphylococcus aureus with high sensitivity and specificity. We applied the CRISPR-Cas12a based bacterial detection after a target-amplification using PCR. The total sample-to-answer time was appropriately 2.0 h, the limit of detection (LOD) was 10 3 CFU/mL, and the dynamic range was 10 3 –10 7 CFU/mL. Also, the sequence addressability enabled this AND logic gate to accurately trace back and distinguish input genes. These above-mentioned features were highly ideal to incur a rapid response to pathogenic bacteria for decision making. Our results not only validated the possibility of using CRISPR-Cas systems for constructing bio-computing devices but also provided a prototype of biosensor for rapid and intelligent pathogenic bacteria detection.
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Device
·
2024
·
IF 0
Siyi Zou,Jingnan Li,Sheng Lu,Danyu Li,Yuxuan Liu,Weiguo Zhang,Xiaolin Cui,J. Justin Gooding,Kang Tian,Guozhen Liu
Inflammation quantification through the detection of cytokines and inflammatory factors in a point-of-care scenario is crucial for managing and preventing diseases. Achieving early-stage inflammation quantification in osteoarthritis (OA), before the radiographic findings become apparent, may provide an opportunity for accurate diagnosis and effective therapy, potentially preventing joint degeneration. Here, we developed a glass-fiber-based universal system with CRISPR-Cas12a-based immunosensors (GUSCI) for simultaneous detection of multiple inflammation biomarkers using a portable fluorescence detection device. GUSCI enables the detection of five inflammation biomarkers (interleukin-4 [IL-4], IL-6, tumor necrosis factor alpha [TNF-α], interferon γ [IFN-γ], and matrix metalloproteinase 3 [MMP-3]) with picomolar sensitivity in a 10-μL sample while having less than 75 min of assay time and a smartphone signal readout. A cohort study of 35 synovial samples from OA patients revealed differences in inflammation levels. This “sample in, answer out” platform not only aids in understanding OA progression but also offers a versatile strategy for point-of-care diagnosis of a wide range of diseases in resource-limited settings.
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MICROCHEMICAL JOURNAL
·
2024
·
IF 4.9
Xuan Wu,Ning-Ning Pi,Fei Deng,Shi-Yang Tang,Cheng-Chen Zhang,Lu Zhu,Fang-Fang Sun,Xiao-Yao He,Han-Qing Li,Shi-Long Zhao,Rong Xiang,Yi Li
Periodontitis not only leads to tooth loss but is also associated with various systemic symptoms. Porphyromonas gingivalis ( Pg ) is the microorganism most closely linked to periodontitis. A rapid and accurate Pg detection method holds significant potential health benefits for patients. While several nucleic acid amplification-based diagnostic methods have been developed, there is a current lack of rapid diagnostic tools for Pg , particularly at chair-side level. Here, two detection methods for the Pg 16S gene based on the CRISPR/Cas12a system have been developed. To achieve ultra-high sensitivity in the developed CRISPR/Cas12a assays without pre-amplification, we designed a circular DNA molecule (termed as Cir-amplifier) which amplifies the signal output of the Cas12a reactions in a cascade response manner. The Cir-amplifier enhanced Fluorescence-Cas12a reaction achieved a minimum detectable limit of 0.39 CFU of Pg within 80-minute under room-temperature. In addition, the Cir-amplifier enhanced Lateral Flow Detection (LFD)-Cas12a reaction has demonstrated the ability to detect a minimum of 3.9 CFU of Pg in 90 min, with results observable by the naked eye. The potential of the two methods for Pg detection in clinical samples has been demonstrated, showing 100 % sensitivity compared to the traditional real-time PCR method. In summary, we have established two efficient detection methods for Pg that eliminate the need for pre-amplification, hence no amplicon contaminations, making them promising candidates for chair-side Pg diagnosis. Furthermore, the Cir-amplifier has been demonstrated to be adaptable to CRISPR/Cas systems producing various signal formats. The Cir-amplifier is expected to become a universal CRISPR/Cas enhancer.
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ANALYTICAL AND BIOANALYTICAL CHEMISTRY
·
2025
·
IF 3.8
Lai Shanshan,Guo Jiahong,Li Xin,Yu Xiaoxiao,Lai Guosong
Fluorescence biosensors hold significant importance for testing antibiotic residues which seriously endanger public health. However, how to adopt appropriate strategies to address the false result disadvantage involved in traditional single-channel biosensors is still a great challenge. Meanwhile, too much attention focused on designing signal amplification strategies of biosensors unavoidably decreases their detection efficiency. Herein, we combined the designed dual DNA recycling amplification strategy with CRISPR/Cas12a-mediated dual-channel signal output mode to successfully develop a novel ratiometric fluorescence biosensor for testing kanamycin (Kana) residues in complex sample matrices. The first recycling was formed from an exonuclease-assisted aptamer recognition reaction, which also triggered another cascade DNA recycling to amplify the release of the Cas12a activator. With the non-discrimination cleavage of Cas12a to cause reverse fluorescence changes of copper nanoclusters and an AMAC-labeled signal DNA, the ratiometric signal transduction strategy was constructed. Under optimal conditions, this biosensor could be applied for ultrasensitive testing of Kana antibiotics in a five-order of magnitude wide linear range with a low detection limit of 17.2 fg mL −1 . Benefiting from the self-correction function of the ratiometric signal transduction mode, it showed promising practicality in lake water and milk samples with the relative error less than 4.9% to the standard ELISA results. Besides CRISPR/Cas12a-based fluorescence output efficiency improvement, this biosensor also excluded the complicated manipulations and expensive instruments required in traditional methods. Therefore, it provides a good choice for expanding the application of fluorescence biosensing technology for practical analysis application. Graphical
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MICROCHEMICAL JOURNAL
·
2025
·
IF 5.1
Jiayi Zhang,Zichun Song,Jiuying Cui,Wenbo Li,Song Huang,Bo Ling,Kai Zhang,Xianjiu Liao,Jihua Wei
The human gastrointestinal system contains the conditional pathogen Escherichia coli ( E. coli ), which is a significant cause of vomiting and diarrhea—both of which can be fatal in extreme cases. Given the surge in hospital infections and water pollution, the development of early detection and diagnosis procedures is imperative. This study proposes an electrochemical biosensor for E. coli detection, utilizing nucleic acid aptamers as recognition elements and using E. coli-2571 as an example. It is based on the CRISPR/Cas gene-editing technology. Specifically, the collateral cleavage activity of Cas12a shifts the electrochemical signal after loading the DNA sequence tagged with an electrochemical signal onto the gold electrode, which ultimately enables the identification of E. coli . The biosensor has a detection limit of 13.1 CFU/mL and exhibits a good linear relationship in the concentration range of 10 2 ––10 7 CFU/mL. It was effectively used for the quantitative detection of E. coli in milk and pure water, with a recovery rate ranging from 93.12 % to 98.93 %. The biosensor offers a rapid, precise, affordable, and sensitive tool for E. coli detection. It is anticipated to become a powerful instrument for identifying microorganisms that pose a risk to public health and human health.
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Biosensors-Basel
·
2025
·Vol. 15
·期 10
·700
·
IF 5.6
Yu Qin,Jiali Xie,Shujun Zhen
Fluorescence anisotropy (FA) has been widely used for analyzing biomolecules due to its high throughput, homogeneous detection, and strong resistance to photobleaching. However, the traditional FA method suffers from low sensitivity when the target molecules are small and rotate rapidly, often producing insignificant changes in the FA value. In this study, by combining double signal amplification through the trans-cleavage of CRISPR-Cas12a and DNA tetrahedron assemblies with a large molecular size, a new, fast, simple and highly sensitive FA method was constructed to achieve the quantitative detection of hepatitis B virus DNA (HBV-DNA). The experimental results showed that the linear range of this method was 0.5–9 nmol/L, and the detection limit (LOD = 3σ/k) was 48 pmol/L. In addition, the method demonstrated excellent selectivity and anti-interference, and it was successfully applied to detect HBV-DNA in human serum, indicating that this method has the potential for clinical diagnosis.
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