SENSORS AND ACTUATORS B-CHEMICAL
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2021
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IF 7.46
Lijuan Yin,Ninghui Duan,Si Chen,Yuan Yao,Jifeng Liu,Long Ma
Foodborne diseases, caused by pathogenic bacteria, severely threaten global human health and cause a financial burden. Rapid, sensitive and on-site detection of pathogenic bacteria is significant. The existing methods have different defects, such as time-consuming and inconvenient. In this study, we developed a G-quadruplex-based CRISPR-Cas12a bioassay for pathogenic bacteria detection with high sensitivity and visualization capability. Salmonella was used as the detection model. Simply, the amplicons of Salmonella specific invA gene activated the trans -cleavage activity of Cas12a and triggered CRISPR-Cas12a based indiscriminate degradation of single-stranded DNAs (ssDNAs). The ssDNAs were designed with the guanine-rich sequence and formed a stable G-quadruplex DNAzyme by adding K + . This DNAzyme could catalyze the TMB-H 2 O 2 reaction in the presence of hemin, leading to an increase in absorbance at 454 nm and a color change. This change can be readily differentiated by the naked eyes as well as a smartphone with a Color Picker App. With this strategy, the limit of detection (LOD) for Salmonella was 1 CFU/mL with no cross-reactivity. A linear relationship (R 2 = 0.993) between the absorbance and the concentration of Salmonella was obtained. Furthermore, G-quadruplex-based CRISPR-Cas12a bioassay was successfully applied for sensing Salmonella in real food samples. This work not only expands the reach of CRISPR-Cas based biosensing but also provides a novel pathogenic bacteria detection method with high sensitivity, specificity and on-site capability.
研究领域:
食品科学
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分子生物学
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生物技术
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DOI
高影响因子
MICROCHEMICAL JOURNAL
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2025
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IF 5.1
Ping Qiu,Longjian Huang,Jiayi Zhang,Wenbo Li,Xudong Xin,Luchang Chen,Qianli Tang,Kai Zhang,Xianjiu Liao,Jihua Wei
We report the development of a novel split aptamer-driven electrochemical biosensor integrating entropy-driven Signal amplification (EDSA) and CRISPR-Cas12a technology for the highly sensitive and specific detection of adenosine deaminase (ADA). ADA is an important biomarker involved in immune regulation and purine metabolism, and its accurate quantification is critical for diagnosing various diseases. In our system, ADA catalyzes the deamination of adenosine to inosine, resulting in the release of Aptamer1 from a split aptamer structure. The liberated Aptamer1 initiates a strand displacement reaction within a Blocker/Bandage/Scaffold complex, triggering a cascade of entropy-driven amplification cycles that generate a DNA duplex capable of activating CRISPR-Cas12a. Activated Cas12a exhibits trans -cleavage activity, specifically cleaving a ferrocene-labeled single-stranded DNA (ssDNA) probe immobilized on a gold electrode, leading to a measurable decrease in electrochemical signal. The biosensor operates under isothermal and enzyme-free conditions, providing simplicity, rapid detection, and low cost. It achieves a low detection limit of 0.688 U·L −1 with a wide linear range from 1 to 20,000 U·L −1 . The system demonstrates excellent specificity against ADA analogues and common interfering proteins and performs reliably in diluted human serum samples. This strategy represents a versatile and modular platform for point-of-care diagnostics. By altering the aptamer sequence, the biosensor can be adapted for the detection of other clinically relevant proteins and enzymes, contributing to advances in personalized medicine.
研究领域:
生物传感技术
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分子诊断
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医学检测
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DOI
高影响因子
ANALYTICA CHIMICA ACTA
·
2023
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IF 6.2
Shiqi Xu,Songtao Wang,Ling Guo,Yuqin Tong,Lina Wu,Xingxu Huang
CRISPR-based detection was often based on the target preamplification to realize the high sensitivity. Here, we prepared a CRISPR-Cas12a system for the colorimetric detection of lead ion (Pb 2+ ) based on the assistance of DNAzyme and nanozyme instead of preamplification. The recognition between GR-5 DNAzyme and Pb 2+ could trigger the CRISPR-Cas12a system. MnO 2 nanozymes connected with magnetic beads through single stranded DNA were prepared as the colorimetric signal probes and catalyst of CRISPR-Cas12a system for the strong oxidase-like activity inducing the color change of 3,3′,5,5′-tetramethylbenzidine. The nanozyme-catalysed CRISPR-Cas12a system could be used to detect Pb 2+ through the color change with high specificity and sensitivity. The linear range of this approach was 0.8 nM–2500 nM, with a limit of detection of 0.54 nM. This method was applied for the detection of the Pb 2+ in food samples indicating good accuracy and anti-interference ability.
研究领域:
生物传感器
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分析化学
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环境科学
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食品安全
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DOI
PubMed
高影响因子
Biosensors-Basel
·
2025
·Vol. 15
·期 12
·821
·
IF 5.6
Jie Chen,Shutao Liu,Sunyi Chen,Jingwen Mai,Maiwula Abudukadi,Yao Chen,Jie Lu,Guanglei Li,Chenchen Ge
Mycoplasma hominis (MH) is a prevalent opportunistic pathogen that is strongly associated with a wide range of urogenital tract infections and severe adverse pregnancy outcomes in clinical settings. Current MH detection methods, including microbial culture and qPCR, are time-consuming and rely on complex equipment, making them unsuitable for scenarios requiring rapid or simplified testing. In this study, we developed a visual readout biosensing platform by synergistically integrating recombinase polymerase amplification (RPA), CRISPR/Cas12a-mediated target nucleic acid recognition, and lateral flow biosensors for the rapid, sensitive, and specific identification of MH. The assay specifically targets the MH-specific 16S rRNA gene, achieving a limit of detection as low as 2 copies/reaction of recombinant plasmid containing the target gene with a total assay time of 60 min. Critical reaction parameters, including Cas12a-crRNA molar ratio, volume of RPA amplicon input, and Cas12a cleavage time, were systematically optimized to maximize the biosensor’s response efficiency and detection reliability. The platform exhibited exceptional specificity, with no cross-reactivity observed against common co-occurring urogenital pathogens, and effectively minimized aerosol contamination risks via a rigorous decontamination workflow. Furthermore, this work represents the first documented implementation of a contamination-control protocol for an MH-specific CRISPR-LFA assay. Notably, testing results from 18 clinical samples demonstrated the high specificity of this assay, highlighting its promising potential for clinical application.
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PubMed
高影响因子
TALANTA
·
2025
·
IF 6.1
Mengqiu Xiong,Yao Wang,Shuhan Lu,Nasifu Lubanga,Tao Li,Zhihao Li,Bangshun He,Ying Li
Multiple infections of respiratory viruses are common in patients with clinical respiratory diseases, but current detection methods still have problems such as complex equipment and long detection time. Rapid, low-cost, and on-site detection of human respiratory viruses is crucial for both clinical diagnosis and population screening. In this research, we created a space-coded microfluidic chip (SC-Chip) for the recognition of nine respiratory viruses: influenza A virus, influenza B virus, severe acute respiratory syndrome coronavirus 2, human coronavirus OC43, human coronavirus NL63, human coronavirus HKU1, human respiratory syncytial virus, human parainfluenza virus, and human metapneumovirus. For the first time, a comprehensive sequence comparison among these viruses was performed to design the recombinase polymerase amplification (RPA) primers and Cas12a-crRNAs. The SC-Chip partitions samples amplified by RPA into spatially coded wells preloaded with CRISPR-Cas12a detection reagents, enabling the identification of all nine viral targets in a single test using a single fluorescence probe. The chip-based assay displays 9 respiratory viruses in less than 40 min with a minimum detection limit at a concentration of 10 −18 M (∼1 copy/reaction). Additionally, the efficacy of the method was assessed through its application to 35 clinical patient samples identified as being at risk for respiratory virus infection, yielding a sensitivity of 90 % and a specificity of 100 %. In summary, this space-coded microfluidic CRISPR system offers several advantages, including ease of operation, cost-effectiveness, and rapid data acquisition, thereby holding great potential for multiplexed detection of nucleic acid targets in a clinical setting.
研究领域:
分子生物学
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医学诊断
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微流控技术
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DOI
PubMed
高影响因子
Synthetic and Systems Biotechnology
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2025
·
IF 4.4
Po Li,Xueying Lei,Xiaoying Niu,Wen Tian,Zhehuang Li,Songcheng Yu,Peng Zhang
MicroRNA (miRNA) is promising candidate for non-invasive diagnostic biomarker. Conventional CRISPR/Cas12a-based miRNA detection systems are constrained by reliance on reverse transcription, nucleic acid pre-amplification and costly fluorescently labeled reporters which introduce chemical modification complexity and background noise. To address these limitations, we herein developed a multifunctional switch that integrated target recognition, CRISPR/Cas12a system activation, intrinsic fluorescence signaling, and autonomous signal amplification within a single molecular architecture. As a proof of concept, this switch enabled a label-free CRISPR/Cas12a biosensing for miR-21 detection with a detection limit of 4.8 nM and robust performance in accuracy, precision, and selectivity. This proposed label-free CRISPR/Cas12a platform could be applied for real sample and is a promising candidate for point-of-care miRNA detection.
研究领域:
生物传感技术
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分子诊断
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CRISPR 基因检测
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核酸扩增
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即时检测
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DOI
PubMed
高影响因子
FOOD CHEMISTRY
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2024
·
IF 8.5
Sujun Liu,Kai Shi
The residue of clenbuterol (CLB) in food poses a potential harm to human health. Herein, we presented an electrochemical aptasensor ( E -A-CRISPR) based on employing an aptamer as a specific recognition element and asymmetric CRISPR-Cas12a as signal amplifiers for sensitive, and selective detection of CLB. In this E -A-CRISPR system, the target CLB bound to the aptamer and initiated cascade signal amplification through the DNase activity of CRISPR-Cas12a with two competitive gRNAs. Upon amplification, the active Cas12a cleaved the methylene blue-labeled hairpin probe on the electrode, reducing the peak current. Under optimal conditions, the E -A-CRISPR system showed a wide linear range (1 pM-100 nM) and a low detection limit (500 fM). This system could detect CLB in potable water, pig liver, and pork samples, showing significant potential for food safety monitoring. To our knowledge, this study is the first to use a CRISPR-Cas12a powered system for electrochemical sensing of CLB.
研究领域:
食品安全
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生物传感
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分子生物学
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DOI
PubMed
高影响因子
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.
研究领域:
微生物学
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分子生物学
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食品科学
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DOI
PubMed
高影响因子
MICROCHEMICAL JOURNAL
·
2023
·
IF 4.8
Lilin Qin,Fangxu Lou,Yan Wang,Yinhao Zhang,Shishi Liu,Xu Hun
Molecular switches formed by enzyme-DNA hybrid compounds combined with CRISPR-Cas12a cis -cleavage used for photoelectrochemical nucleic acid assay was fabricated. A compound, molecular switch, consisting of Taq DNA polymerase, aptamer, block DNA and methylene blue labeled primer (primer-Mb) is formed. Block DNA is assembled on the surface of MBs (block DNA-MBs), and hybridized with the primer-Mb. In the presence of Human Immunodeficiency Virus (HIV) target, molecular switches are turned on by hybridization of the target with block DNA. The Taq DNA polymerase is released and activated. The activated Taq DNA polymerase elongates the primer via the template, block DNA, which attached onto Magnetic beads (MBs). A dsDNA with Mb (dsDNA-Mb) was formed. With the assistance of Cas12a, a amplified photoelectrochemical signal was obtained and the HIV target DNA was qualified. Under the best conditions, the limit of detection (LOD) can be as low as 0.3 fM. This photoelectrochemical assay extends the tool kit of molecular nanotechnology.
研究领域:
分子生物学
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生物化学
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纳米技术
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病毒学
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产品关联
DOI
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2023
·
IF 12.6
Jienan Shen,Zhi Chen,Ruibin Xie,Jingfeng Li,Chunyan Liu,Yaqing He,Xiaopeng Ma,Hui Yang,Zhongjian Xie
Respiratory viruses have long been a major cause of a global pandemic, emphasizing the urgent need for high-sensitivity diagnostic tools. Typical PCR technology can only determine the type of virus in the sample, which is unable to detect different variants of the same virus without costly and time-consuming gene sequencing. Here, we introduce a simple, fully enclosed, and highly integrated microfluidic system based on CRISPR/Cas12a and isothermal amplification techniques (LOC-CRISPR) that can specifically identify multiple common respiratory viruses and their variants. The LOC-CRISPR chip integrates viral nucleic acid extraction, recombinant polymerase amplification, and CRISPR/Cas12a cleavage reaction-based detection, contamination-free detection. In addition, the LOC-CRISPR chip was designed for multiplexed detection (two-sample input and ten-result outputs), which can not only detect the presence of SARS-CoV-2, H 1 N 1 , H 3 N 2 , IVB and HRSV but also differentiate the BA.1, BA.2, and BA.5 variants of SARS-COV-2. For clinical validation, the LOC-CRISPR chip was used to analyze 50 nasopharyngeal swab samples (44 positive and 6 negative) and achieved excellent sensitivity (97.8%) and specificity (100%). This innovative LOC-CRISPR system has the ability to quickly, sensitively, and accurately detect multiple target nucleic acid sequences with single-base mutations, which will further improve the rapid identification and traceability of respiratory viruses infectious diseases.
研究领域:
分子生物学
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医学诊断
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病毒学
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产品关联
DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2024
·
IF 10.7
Xueying Lei,Lihua Ding,Xiaonan Yang,Feng Xu,Yongjun Wu,Songcheng Yu
The CRISPR/Cas12a system is increasingly used in biosensor development. However, high background signal and low sensitivity for the non-nucleic acid targets detection is challenging. Here, a p adlock activator which could i nhibit the t rans -cleavage activity of CRISPR/Cas12a system in the intact form by steric hindrance effect (PAIT effect) was designed for non-nucleic acid targets detection. The PAIT effect disappeared when padlock activator was separated into two split activators. To verify the feasibility of padlock activator, a Ca 2+ sensor was developed based on PAIT effect with the assistance of DNAzyme, activity of which was Ca 2+ dependent. In the presence of Ca 2+ , DNAzyme was activated to cleave its substrate, a padlock activator modified with adenine ribonucleotide, into split padlock activators which would trigger the trans -cleavage activity of Cas12a to generate fluorescence. There was a mathematical relationship between the fluorescence intensity and the logarithm of Ca 2+ concentration ranging from 10 pM to 1 nM, with a limit of detection of 3.98 pM. The little interference of Mg 2+ , Mn 2+ , Cd 2+ , Cu 2+ , Na + , Al 3+ , K + , Fe 2+ , and Fe 3+ indicated high selectivity. Recovery ranged from 93.32% to 103.28% with RSD s from 1.87% to 12.74% showed a good accuracy and reliability. Furthermore, the proposed sensor could be applied to detect Ca 2+ in mineral water, milk powder and urine. The results were consistent with that of flame atomic absorption spectroscopy. Thus, PAIT effect is valuable for expanding the application boundary of CRISPR/Cas12a system.
研究领域:
生物技术
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生物化学
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分析化学
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分子生物学
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DOI
PubMed
高影响因子
TALANTA
·
2024
·
IF 5.6
Hongmin Gao,Hehua Zhang,Xue Qi,Meng Miao,Longbin Que,Xin Gu,Dong Chang,Hongzhi Pan
Accurate and reliable detection of Staphylococcus aureus ( S. aureus ) is essential for preventing infections, particularly in healthcare and food safety contexts. This work presents a novel dual-mode biosensor that integrates the CRISPR/Cas12a system with an enzyme-free isothermal amplification method for detecting S. aureus . Hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA) amplify the aptamer-triggered response, significantly enhancing sensitivity. CRISPR/Cas12a′s nuclease activity is utilized in two modes: cis cleavage generates a fluorescence signal, while trans cleavage produces an electrochemical signal, enabling dual-mode detection. The biosensor demonstrates outstanding performance, with a limit of detection (LOD) as low as 5.7 CFU mL −1 in electrochemical mode and 133.7 CFU mL −1 in fluorescence mode, showcasing excellent accuracy, stability, and sensitivity. It has been successfully applied to detecting actual samples, confirming its practical applicability. This innovative approach offers a powerful tool for the swift and precise identification of S. aureus and paves the way for developing next-generation dual-mode biosensors for various analytes. Future research will aim to simplify the detection process further, making it more accessible for use in resource-limited settings.
研究领域:
分子生物学
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病原体检测
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生物传感技术
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产品关联
DOI
PubMed
高影响因子
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