ANALYTICAL CHEMISTRY
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2024
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IF 6.7
Junpeng Lv,Di Wu,Yingming Ma,Xiyao Zhang,Wenhui Xu,Mengge Wang,Songmin Chen,Qiong Hu,Dongxue Han,Li Niu
Glycoproteins have become the most often screened tumor markers in the in vitro diagnostics. Although a large number of electrochemical methods have been proposed to sensitively detect glycoproteins, most of them involve the aid of laborious signal amplification. Herein, we report the use of glycan-evocated metallization (GlyMetal) for the amplification-free electrochemical detection of glycoproteins at low concentration levels. Briefly, the glycoproteins of interest are captured by an aptamer recognition layer, and then the glycans of targets are oxidized by NaIO4 to convert the 1,2-diol sites into aldehyde groups for the silver deposition-based metallization, followed by the electrochemical stripping assay of the deposited metallic silver for glycoprotein quantification via the established solid-state Ag/AgCl voltammetric process. As GlyMetal can enable the deposition of a large amount of metallic silver and a high signal-to-background ratio can be obtained for the solid-state Ag/AgCl voltammetric stripping assay, the developed GlyMetal-based electrochemical method is applicable to the amplification-free detection of glycoproteins. As a proof of concept, a detection limit of 1.65 pg/mL has been achieved for carcinoembryonic antigen (CEA) detection. In addition to the high selectivity, desirable results have been obtained with respect to the use of the method for CEA detection in serum samples. In consideration of the desirable simplicity, short assay time, and cost-effectiveness of the amplification-free approach, the GlyMetal-based electrochemical method shows great promise in the point-of-care detection of glycoproteins.
研究领域:
生物化学
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医学诊断
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分析化学
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产品关联
DOI
PubMed
高影响因子
ACS Nano
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2023
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IF 17.1
Dou Wang,Xuedong Wang,Feidi Ye,Jin Zou,Jiuxin Qu,Xingyu Jiang
Conventional nucleic acid detection technologies usually rely on amplification to improve sensitivity, which has drawbacks, such as amplification bias, complicated operation, high requirements for complex instruments, and aerosol pollution. To address these concerns, we developed an integrated assay for the enrichment and single molecule digital detection of nucleic acid based on a CRISPR/Cas13a and microwell array. In our design, magnetic beads capture and concentrate the target from a large volume of sample, which is 100 times larger than reported earlier. The target-induced CRISPR/Cas13a cutting reaction was then dispersed and limited to a million individual femtoliter-sized microwells, thereby enhancing the local signal intensity to achieve single-molecule detection. The limit of this assay for amplification-free detection of SARS-CoV-2 is 2 aM. The implementation of this study will establish a “sample-in-answer-out” single-RNA detection technology without amplification and improve the sensitivity and specificity while shortening the detection time. This research has broad prospects in clinical application.
研究领域:
生物技术
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分子生物学
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诊断方法
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产品关联
DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
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2025
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IF 6
Yi Tian,Jiaxuan Chen,Fengzheng Chen,Junru Xu,Lixiang Huang,Liai Peng,Honglei Li,Kai Shi
Background The threat of toxic malathion residues to human health has always been a serious food safety issue. The CRISPR/Cas system represents an innovative detection technology for pesticide residues, but its application to malathion detection has not been reported yet. In addition, the multiple-guide RNA (gRNA) powered-CRISPR/Cas biosensor has the advantages of being fast, sensitive and does not require pre-amplification. However, the reported multiple-gRNA CRISPR/Cas-based biosensors are largely only used for the detection of nucleic acid targets, and there are still certain challenges in detecting non-nucleic acid targets. Results In this work, a multiplex-gRNA-assisted CRISPR/Cas12a-based portable aptasensor (MgCPA) is developed for amplification-free and quantitative detection of malathion using a glucometer. When target malathion is present in the MgCPA strategy, it specifically binds with aptamer and then activates the trans -cleavage activity of the multiplex-gRNA CRISPR/Cas12a. The activated multiple Cas12a/gRNA complexes cut invertase-HP probes on the electrode surface to obtain glucose signals with glucometer assistance. Under optimal conditions, the developed MgCPA strategy achieves satisfactory portable quantitative and sensitive detection of malathion down to 300 fM (S/N = 3) without pre-amplification. Moreover, the satisfactory selectivity, high reproducibility, and good stability of the proposed strategy are also obtained. Due to its excellent and robust shelf life, our developed MgCPA strategy can be practically applied in detecting malathion in orange, apple, cabbage, and spinach samples. Significance Amplification-free, sensitive, portable quantitative and selective detection of malathion in food samples is achieved by employing our developed MgCPA strategy. This strategy not only opens up a new path for the non-nucleic-acid target detection using amplification-free methods based on multiple-gRNA-assisted CRISPR/Cas12a, but also has broad application prospects in ensuring food safety.
研究领域:
生物技术
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食品安全检测
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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
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2022
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IF 8.008
Xueping Yang,Jingyun Li,Suixin Zhang,Chao Li,Jiehua Ma
CRISPR/Cas-based systems are highly attractive for developing next-generation diagnostic technologies because of their intrinsic merits such as simplicity, sensitivity, and specificity. However, currently, nucleic acid amplification procedures are still needed to achieve attomolar sensitivity in most CRISPR/Cas-based assays, which causes high cost, operation difficulty, and low efficiency. Herein, we combine the CRISPR/Cas12a-based assay and a single-microbead detection platform for one-step and amplification-free detection of DNA at the single-molecule level. By modifying DNA reporters on a biomimetic membrane-coated microbead, the activated Cas12a by targets will cleave these reporters and lighten the bead within 10 min. The method allows the detection of the target down to three copies in a 5 μL sample. Furthermore, we successfully apply this method for the specific identification of viral infection, foodborne bacteria, and DNA mutation in real samples without extra nucleic acid amplification. We believe that this approach offers new insights for developing CRISPR/Cas-based DNA assays in biomedical applications.
研究领域:
分子生物学
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生物技术
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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
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2025
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IF 6.7
Jinai Chen,Yujiao Wei,Liming Wang,Leyi Chen,Shengnan He,Honglin Liu
We present a digital surface-enhanced Raman spectroscopy (SERS) platform enabling enzyme- and amplification-free single-molecule DNA detection through Poisson distribution-driven quantification and geometrically optimized plasmonic nanostructures. Utilizing high-throughput UV lithography, we fabricated large-area nanopillar arrays (1.5 × 1.5 cm) with tunable heights (500–1300 nm) and periodicities (4–10 μm), addressing key limitations in conventional SERS methods via three innovations: (1) dynamic hotspot generation: Target DNA induces plasmonic dimerization between functionalized gold nanoprobes and Au-capped nanopillars, creating spatially resolved SERS enhancement (1326 cm–1 DTNB signal), (2) digital counting algorithms: Compensate Raman intensity variations by quantifying activated pillars as discrete molecular events, and (3) geometric confinement: Spatial confinement effects enabling statistically robust single-molecule occupancy. This platform achieved 10 fM sensitivity for Diaporthe spp. DNA detection, outperforming PCR-electrophoresis (1 pM) by 2 orders of magnitude. Applied to presymptomatic kiwifruit diagnosis, the system identified pathogen DNA in asymptomatic samples prior to visual symptom development, validated by characteristic DTNB peaks (1326 cm–1) absent in negative controls. The modular design permits multiplexed detection through probe diversification, while the absence of enzymatic steps enables field-deployable operation. By integrating single-molecule statistics with plasmonic signal amplification, this work establishes a robust framework for precision molecular diagnostics, demonstrating transformative potential for agricultural pathogen surveillance and low-abundance biomarker analysis. The technology’s batch-producible nanopillar architecture and digital quantification strategy overcome longstanding reproducibility challenges in SERS-based detection, positioning it as a versatile tool for next-generation biosensing applications.
研究领域:
农业科学
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分子诊断学
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纳米技术
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产品关联
DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
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2023
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IF 6.2
Yuwan Du,Shuaifeng Ji,Qingyang Dong,Jiang Wang,Dianpeng Han,Zhixian Gao
Nucleic acid markers have been widely used in the detection of various virus-related diseases, including hepatitis B virus (HBV), which is spreading worldwide. The trans -activated CRISPR-Cas system has shown excellent sensitivity and specificity in nucleic acid detection. However, nucleic acid testing usually requires amplification of the target nucleic acid for more accurate and specific detection; furthermore, current nucleic acid assays are time-consuming, costly, and are limited by non-specific cross-reactivity. We developed an amplification-free viral DNA biosensor-based diagnostic method that uses a clustered regularly interspaced short palindromic repeats-associated system (CRISPR/Cas)-based approach with surface enhanced Raman spectroscopy . This method can specifically identify the target site by changing the crRNA sequence. In addition, the incubation period and development of the disease can be determined by quantitative detection of viral DNA. This system could achieve rapid and highly sensitive detection of HBV DNA within 50 min and vast detection range from 0.1 pM to 1 nM. Therefore, a combined CRISPR/Cas12a-SERS-based assay would improve the sensitivity of detection in assays using multiple biomarkers. In conclusion, our CRISPR/Cas12a-based biosensor would enable rapid, simple, and sensitive detection of HBV nucleic acids.
研究领域:
病毒学
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分子诊断
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生物医学工程
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF HAZARDOUS MATERIALS
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2023
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IF 13.6
Long Ma,Wenlu Zhang,Lijuan Yin,Yaru Li,Jianwen Zhuang,Liang Shen,Shuli Man
The pandemic of COVID-19 creates an imperative need for sensitive and portable detection of SARS-CoV-2. We devised a SERS-read, CRISPR/Cas-powered nanobioassay, termed as OVER-SARS-CoV-2 (One-Vessel Enhanced RNA test on SARS-CoV-2), which enabled supersensitive, ultrafast, accurate and portable detection of SARS-CoV-2 in a single vessel in an amplification-free and anti-interference manner. The SERS nanoprobes were constructed by conjugating gold nanoparticles with Raman reporting molecular and single-stranded DNA (ssDNA) probes, whose aggregation-to-dispersion changes can be finely tuned by target-activated Cas12a though trans -cleavage of linker ssDNA. As such, the nucleic acid signals could be dexterously converted and amplified to SERS signals. By customizing an ingenious vessel, the steps of RNA reverse transcription, Cas12a trans -cleavage and SERS nanoprobes crosslinking can be integrated into a single and disposal vessel. It was proved that our proposed nanobioassay was able to detect SARS-CoV-2 as low as 200 copies/mL without any pre-amplification within 45 min. In addition, the proposed nanobioassay was confirmed by clinical swab samples and challenged for SARS-CoV-2 detection in simulated complex environmental and food samples. This work enriches the arsenal of CRISPR-based diagnostics (CRISPR-Dx) and provides a novel and robust platform for SARS-CoV-2 decentralized detection, which can be put into practice in the near future.
研究领域:
分子诊断
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纳米技术
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病毒学
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生物医学工程
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
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2023
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IF 7.4
Jianwen Wan,Yiyi Liang,Qiong Hu,Zhiwen Liang,Wenxing Feng,Yiyan Tian,Shiqi Li,Zhuojun Ye,Mingru Hong,Dongxue Han,Li Niu
The rapid quantification of therapeutic monoclonal antibodies (mAbs) is of great significance to their pharmacokinetics/pharmacodynamics (PK/PD) research and the personalized medication for disease treatment. Taking advantage of the direct decoration of tens of redox tags to the target of interest, we illustrate herein an amplification-free ratiometric electrochemical aptasensor for the point-of-care (POC) detection of trace amounts of therapeutic mAbs. The POC detection of therapeutic mAbs involved the use of the methylene blue (MB)-conjugated aptamer as the affinity element and the decoration of therapeutic mAbs with ferrocene (Fc) tags via the boronate crosslinking, in which the MB-derived peak current was used as the reference signal, and the peak current of the Fc tag was used as the output signal. As each therapeutic mAb carries tens of diol sites for the site-specific decoration of the Fc output tags, the boronate crosslinking enabled the amplification-free detection, which is cost-effective and quite simple in operation. In the presence of bevacizumab (BevMab) as the target, the resulting ratiometric signal (i.e., the IFc/IMB value) exhibited a good linear response over the range of 0.025–2.5 μg/mL, and the limit of detection (LOD) of the electrochemical aptasensor was 6.5 ng/mL. Results indicated that the aptamer-based affinity recognition endowed the detection of therapeutic mAbs with high selectivity, while the ratiometric readout exhibited satisfactory reproducibility and robustness. Moreover, the ratiometric electrochemical aptasensor is applicable to the detection of therapeutic mAbs in serum samples. Taking together, the amplification-free ratiometric electrochemical aptasensor holds great promise in the POC detection of therapeutic mAbs.
研究领域:
生物医学工程
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分析化学
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药物分析
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产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2025
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IF 7.7
Yue Wang,Shuning Lei,Youjun Zeng,Wei Ni,Jie Zhou,Guo-Jun Zhang
MicroRNA(miRNA) has emerged as a particularly promising biomarker for the early diagnosis of Alzheimer's disease (AD). However, current detection methods for miRNA remain challenges in terms of sensitivity and pre-amplification steps. This study proposes a novel surface plasmon resonance imaging (SPRi) biosensor that combines tetrahedral DNA nanostructures (TDN)-based surface organization with CRISPR-Cas13a-mediated molecular recognition, enabling amplification-free, sensitive, and specific detection of AD-related miRNA-137. The biotin-labeled RNA reporters are anchored to TDN framework on the gold film surface, ensuring the orientation of the probe. In the presence of target miRNA-137, the CRISPR-Cas13a system activates, cleaves these reporters and prevents subsequent binding of streptavidin-HRP, thereby inhibiting precipitate formation from the enzymatic reaction. Under optimal condition, this sensor, integrated with the self-developed highly sensitive intensity-modulated SPRi system, displays a good linear response to miRNA-137 ranging from 0.5 pM to 500 nM with a detection limit of 212 fM. The platform demonstrates excellent specificity of discriminating single-base mutation, and reliable analysis capability in clinical plasma samples. Notably, our sensor confirms significantly reduced miRNA-137 levels in AD patients compared to healthy people. This innovative assay provides a powerful tool for the detection of AD-related miRNAs, promising to advance early diagnosis research for AD.
研究领域:
分子诊断
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神经生物学
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生物传感技术
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纳米医学
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转化医学
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分子生物学
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临床检验
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产品关联
DOI
高影响因子
MICROCHEMICAL JOURNAL
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2024
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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.
研究领域:
分子生物学
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微生物学
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诊断学
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生物技术
✓ 已验证
DOI
高影响因子
CHEMICAL ENGINEERING JOURNAL
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2024
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IF 13.3
Jiayuan Zheng,Jinhua Li,Tianci Lin,Zhanpeng Ren,Fucheng Wang,Zhonghao Shi,Haiyang Yu,Wei Jiang,Wei Tang
Staphylococcus aureus ( S. aureus ) is one of the leading causes of foodborne illnesses worldwide, posing a significant risk to food quality and safety. However, conventional detection methods often require complex amplification and labeling procedures, which are time consuming and expensive. In this study, an extremely sensitive electrochemical biosensor using a solution-gated graphene transistor (SGGT) was constructed for the amplification-free and label-free rapid detection of S. aureus by utilizing specific single-stranded DNA (ssDNA) to modify the gold (Au) sensing gate. This modified ssDNA was designed to hybridize with the target S. aureus DNA sequence. Interface engineering of the ssDNA-modified sensing gate via optimization of specific target and probe sequences was proposed to improve its hybridization efficiency. This enabled the sensitive quantitative detection of S. aureus DNA. The resulting biosensor exhibited excellent specificity in distinguishing non-complementary, 3-base/9-base DNA mismatch targets of S. aureus DNA and the specific DNA sequences of other common pathogenic bacteria. It also enabled the rapid detection of extracted target DNA sequences in solution (within 27 min) and had a low limit of detection (LoD) of 10 −17 M over a wide linear detection range (10 −17 –10 −8 M). Furthermore, the newly-developed biosensor facilitated the real-time and on-site detection of the S. aureus genome (ATCC 6538) with LoD of 10 −17 M (10 3 CFU/mL) in a portable smart-sensing system. This suggests a promising future for the development of rapid, label-free, and amplification-free detection of foodborne illnesses using low-cost, highly sensitive SGGT DNA biosensors with appropriate functionalization and interface engineering of the sensing electrode.
研究领域:
生物技术
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纳米技术
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食品安全
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电化学
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生物传感器
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产品关联
DOI
高影响因子
危险品化学品经营许可证（带存储）