ANALYTICA CHIMICA ACTA
·
2023
·
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.
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ANALYTICA CHIMICA ACTA
·
2022
·
IF 6.911
Xiaofen Zhao,Xiaoxiao Tian,Yuwei Wang,Linbin Li,Yan Yu,Shiqiao Zhao,Juan Zhang
Accurate analysis of HIV DNA is valuable for the diagnosis of AIDS. Herein, an ultrasensitive and specific fluorescence method was developed for HIV-1 DNA detection based on CRISPR-Cas12a-activated palindrome-catalytic hairpin assembly (CRISPR-Cas12a-PCHA). The presence of HIV-1 DNA activated the trans-cleavage activity of CRISPR-Cas12a, which could continuously digest the DNA fragment of hairpins connected to magnetic beads to expose single-stranded RNA . After magnetic separation, the exposed RNA triggered multiple PCHA reactions, generating many Y-shaped DNA structures that were self-assembled into the DNA superstructures via the hybridization of palindromic sticky ends, leading to the release of amounts of fluorescence signal. Different from the reported recently biosensing strategies of nucleic acid amplification technologies-activated CRISPR-Cas12a, CRISPR-Cas12a-PCHA endowed the strategy with unique advantages of simple sample pretreatment , direct duplex target detection, and ultrahigh sensitivity. The strategy was able to resist the interference of the complex matrix in real sample and distinguish between HIV patients and healthy persons. Thus, the method is a promising tool for ultrasensitive and specific detection of HIV-1 DNA for AIDS diagnosis.
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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.
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Analytical Methods
·
2025
·
IF 2.6
Jicong Hao, Xueyan Gong, Xueyuan Duan, Weiwei Qin, Haiying Ren, Xuping ShenTu, Zihong Yea ,Xiaoping Yu
The rapid and ultrasensitive detection of parvovirus B19 (B19V) DNA is critical for preventing severe complications in high-risk populations, such as fetal hydrops in pregnant women and aplastic crisis in immunocompromised patients. The absence of clinically approved vaccines or antivirals against B19V thus mandates the urgent development of accessible in vitro diagnostics to enable time-critical interventions and contain community transmission. Herein, we developed an electrochemical biosensor as a proof-of-concept for B19V by integrating CRISPR-Cas12a with a multivalent framework nucleic acids (FNAs), namely, 12 nm tetrahedral DNA nanostructures (TDNs). Target B19V DNA activates Cas12a to indiscriminately cleave the biotin-modified ssDNA protruded from the four vertexes of the TDNs, while the TDNs precisely orient ssDNA probes on electrodes, minimizing nonspecific adsorption. This method takes advantage of the target-specific cleavage ability of CRISPR-Cas12a (Cas12a-crRNA complex, 10-12 nm) and the unique structural and functional features of 12 nm TDNs. The comparable dimensions of the Cas12a-crRNA complex and the TDN suggest a potential synergistic effect, which contributes to the observed signal amplification and high detection sensitivity. The developed platform is user-friendly, has a low detection limit (2.19 fM), and shows high selectivity. This work establishes a foundational biosensing platform, demonstrating potential for ultrasensitive nucleic acid detection. By combining the accuracy of CRISPR-Cas12a and the benefits of FNAs, this method provides a more efficient, amplification-free, and reliable approach that holds promise for future development in point-of-care diagnostics and other applications.
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BIOSENSORS & BIOELECTRONICS
·
2025
·
IF 10.5
Qisheng Luo,Chuanhua Zheng,Yongning Huang,Linghuan Liang,Yuanxun Gong,Jiayi Zhang,Qianli Tang,Kai Zhang,Xianjiu Liao
Lead-halide perovskites offer bright electrochemiluminescence (ECL) but suffer from aqueous instability and Pb 2+ safety concerns. We construct a CsPbBr 3 @PDA–Au nanointerface that couples polydopamine (PDA) passivation with Au-assisted charge transfer, integrated into a peptide-to-CRISPR/Cas12a amplification scheme for ultrasensitive detection of matrix metalloproteinase-2 (MMP-2). PDA conformally coats CsPbBr 3 , suppressing trap-mediated quenching and mitigating Pb 2+ release, while providing catechol/amine anchors for a tetrahedral DNA scaffold bearing a ferrocene-labeled hairpin. Upon MMP-2 cleavage of a GPLG↓VRG peptide–DNA chimera, the released strand activates Cas12a trans-cleavage, removing the proximal quencher and switching the interface from “off” to “on.” Under unified processing (baseline subtraction and renormalization), the platform achieves a 5.6 aM detection limit with high selectivity over non-target proteases and excellent inter-device reproducibility (triplicate error bars reported). TEM/HRTEM visualize uniform nanocubes; XPS/FTIR evidence PDA catechol/quinone/amine coordination; XRD confirms phase-pure CsPbBr 3 ; and corrected CV/EIS analyses verify accelerated interfacial kinetics after Au decoration. The sensor retains ∼98 % intra-day and ≥91 % 7-day signal, supporting operational robustness. By integrating interfacial stabilization, catalytic electron transfer, and molecular amplification, this modular design advances eco-conscious, perovskite-based ECL diagnostics for low-abundance protease monitoring and broader clinical bioanalysis.
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JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2025
·
IF 6.2
Li Li,Yuanqian Xiong,Yuqian Guo,Hong Duan,Yuankui Leng,Xiaolin Huang,Gang Chen,Yonghua Xiong
DNA-templated silver nanoclusters (tDNA-AgNCs) show considerable promise as fluorescence reporters for the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system because of their ease of synthesis, strong resistance to photobleaching, and large Stokes shift. However, the weak luminous intensity and low cleavage efficiency of tDNA-AgNCs limit the sensitivity of CRISPR assays. In this study, we developed a novel approach by introducing activator DNA (aDNA) with a G-quadruplex structure to enhance the luminous intensity of the tDNA-AgNCs. As a result of the high cleavage efficiency of free aDNA by activated CRISPR/Cas12a, the G-quadruplex-enhanced tDNA-AgNCs (GED-AgNCs) were integrated into recombinase polymerase amplification and CRISPR/Cas12a system for the ultrasensitive detection of Salmonella typhimurium. By optimizing the synthesis of tDNA-AgNCs and GED-AgNCs, our developed G-quadruplex-enhanced DNA-AgNC CRISPR assay (G-DACA) platform enabled the sensitive determination of S. typhimurium with a detection limit as low as 1 CFU/mL and a wide dynamic range of 10–108 CFU/mL. Moreover, our proposed method demonstrated good accuracy and reliability for the quantitative analysis of S. typhimurium in real pasteurized milk samples, with recovery rates ranging from 81.06% to 102.33% and relative standard deviations between 7.17% and 14.84%. Overall, our innovative G-DACA platform offers an economical and versatile solution for food safety and clinical diagnostics.
研究领域:
生物传感器
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分子诊断
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纳米技术
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食品安全
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CRISPR技术
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分析化学
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DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE
·
2026
·Vol. 116
·期 3
·117523
·
IF 1.8
Huifeng Yang,Xiaoyi Li,Yimin Su,Jianxin Guo,Tengfei Wang,Zhigang Wang,Hai Fu,Zhi Chen,Zhongjian Xie,Defa Li
Purpose Acute respiratory infections caused by bacterial and viral pathogens pose a major global health burden. The recombinase polymerase amplification (RPA)-CRISPR-Cas12a system offers a promising point-of-care testing (POCT) platform, but its field deployment is limited by the instability of lyophilized reagents. This study aims to develop a composite lyoprotectant to enhance the stability of the RPA-CRISPR-Cas12a system for respiratory pathogen detection. Materials and methods A composite lyoprotectant composed of trehalose and carboxymethyl chitosan (Tre-CMC) was formulated at various mass ratios. The optimal ratio was identified by evaluating matrix microstructure, enzyme activity retention, and primer-dimer suppression. The lyophilized system was tested for sensitivity, specificity, and long-term stability against six respiratory pathogens (H1N1, IBV, Neisseria meningitidis , SARS-CoV-2, Streptococcus pneumoniae , and human adenovirus) using real-time fluorescence and gel electrophoresis. Results This study establishes a promising proof-of-concept framework for an integrated, lyophilized RPA-CRISPR–Cas12a diagnostic system. The optimal Tre: CMC ratio (2:1) produced a porous, non‑hygroscopic matrix that preserved reagent integrity. The lyophilized system achieved a detection limit of 10 copies/reaction within 30 min for all six pathogens, with 100% specificity. After six months of storage at 4°C, RPA enzyme activity remained above 89%, and Cas12a-crRNA complex functionality was fully retained. Tre-CMC significantly reduced primer-dimer formation and nonspecific background fluorescence. While clinical evaluation using 21 nasopharyngeal swab samples demonstrated 100% concordance with RT-qPCR—preliminarily supporting the system's potential diagnostic accuracy—we acknowledge that this initial methodological framework requires broader and more rigorous downstream validation with larger clinical cohorts to formally establish its robust diagnostic performance. Conclusion The Tre-CMC composite lyoprotectant effectively stabilizes the RPA-CRISPR-Cas12a system, enabling cold-chain-independent storage and reliable POCT for respiratory pathogens in resource-limited settings.
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ACS Sensors
·
2026
·
IF 10.9
Yinghui Sun,Rui Wang,Hongxiao Sun,Jiadong Chen,Chengwei Liang,Zhiyang Zhang,Yujie Feng,Lingxin Chen,Xiaokun Wang
MicroRNAs (miRNAs) are emerging biomarkers for early hepatocellular carcinoma (HCC) detection, but most CRISPR−Cas12a assays rely on reverse transcription and preamplification and often lack simple visual readouts. Here, we develop a reverse transcription-free, cleavage-guided strategy that converts miRNA recognition into visual outputs. In the presence of the target miRNA, a scaffold RNA forms an active crRNA that triggers Cas12a trans-cleavage using a preformed dsDNA activator, enabling two readout formats. In a homogeneous non-crosslinking colorimetric assay (mC-NCA), Cas12a cleavage regulates π−π-stacking-mediated gold nanoparticle (AuNP) aggregation to produce a rapid visual colorimetric response. In a heterogeneous versatile lateral flow assay (mC-vLFA), the cleavage of partially hybridized DNA−magnetic bead probes generates a target-dependent test line while maintaining a built-in control line. Using miRNA-21 and miRNA-122 as targets, mC-NCA shows calculated limits of detection (LODs) of 1.62 and 1.64 fM with a linear range of 50 fM−500 pM, whereas mC-vLFA shows calculated LODs of 1.59 and 1.40 fM with a linear range of 10 fM−10 nM. In a preliminary clinical evaluation, both formats show good agreement with reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and clinical assessment while enabling faster detection with minimal instrumentation. Overall, this dual-readout platform couples RT-free CRISPR−Cas12a miRNA recognition with cleavage-directed visual signal transduction, enabling rapid and low-instrument-dependence CRISPR diagnostics for miRNAs.
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MICROCHEMICAL JOURNAL
·
2026
·
IF 5.2
Wenjia Dong,Xiaoyan Wu,Hongsheng Li,Junjie Yu,Siqi Xu,Yayun Jiang
Methicillin-resistant Staphylococcus aureus (MRSA) infections progress rapidly, posing a significant threat to patient health. Culture-based drug susceptibility testing and nucleic acid amplification methods are time-consuming and complex, requiring specialized instruments and technicians. To address these limitations, we present a novel, visual POCT platform for MRSA detection with the naked eye. This visualization method uses CRISPR/ Ca s12a to accurately identify target genes, cleave the strand displacement amplification ( SD A) template, and regulate the aggregation of DNA-functionalized gold nanoparticles ( Au NPs), with the resulting color change serving as a signal (CaSD@Au). The presence of the target gene activates CRISPR/Cas12a enzymatic activity, which cleaves the SDA template. This cleavage suppresses the SDA reaction, prevents DNA-AuNP aggregation, and maintains nanoparticle dispersion, resulting in a visible red color. In the absence of the target gene, the SDA reaction proceeds, and the gold nanoparticles aggregate, turning purple. Sensitivity analysis confirmed that CaSD@Au detects MRSA with a limit of detection of 5 cfu mL −1 . When combined with recombinase polymerase amplification (RPA), CaSD@Au showed detection performance comparable to qPCR in the actual detection of 20 clinical strains. Furthermore, this platform enables results to be determined simply by observing color changes with the naked eye, without the need for complex instruments. The CaSD@Au platform offers reliable, sensitive, and specific detection, enhancing the applicability of CRISPR/Cas12a-based assays for pathogen detection, especially in resource-limited settings.
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BIOSENSORS & BIOELECTRONICS
·
2025
·
IF 10.5
Wen Tian,Tao Liu,Xiaoying Niu,Po Li,Zhehuang Li,Songcheng Yu,Peng Zhang
DNA methylation serves as a critical epigenetic biomarker for early disease screening and clinical diagnostics, exemplified by the FDA-approved detection of methylated Septin9 gene in peripheral blood for colorectal cancer (CRC) screening. Conventional CRISPR/Cas12a-based detection systems rely on fluorophore-quencher-labeled ssDNA probes to transduce trans-cleavage activity into fluorescence signal, which yet suffers from limitations including complex probe synthesis, background noise induced by incomplete quenching and high cost. In this study, we developed a cascaded and label-free CRISPR/Cas12a system for Septin9 methylation detection using DNAzyme and split DAP-10. The method employed methylation-sensitive Aci I digestion to selectively hydrolyze non-methylated DNA, leaving methylated Septin9 DNA fragment intact to activate CRISPR/Cas12a system. Activated Cas12a triggered trans-cleavage of DNAzyme, a bifunctional molecule that acted as both trans-cleavage substrate of Cas12a and hydrolysis regulator of linker DNA that facilitated split DAP-10 reassembly to light-up Auramine O (AO) fluorescence proportionally to methylation levels. The assay achieved a linear range from 2 to 200 nM with a detection limit of 1.74 nM and robust performance in accuracy, precision, and selectivity. The proposed method was successfully applied in methylated Septin9 detection in cell lines, clinical tissues and blood samples, which indicated application potential for CRC screening.
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Journal of Materials Chemistry B
·
2025
·
IF 5.7
Siyue Wang,Xiaotian Guan,Shuqing Sun
C-reactive protein (CRP) is a well-established biomarker whose plasma levels increase significantly during inflammatory and infectious episodes. It plays a vital role in the diagnosis of bacterial infections, autoimmune diseases, and cardiovascular risk assessment. However, conventional detection techniques often struggle to balance high sensitivity with a broad dynamic range. Here, we developed and validated a novel fluorescence-based aptasensor named AuCA (AuNP-enhanced CRISPR Aptasensor), which integrates nucleic acid aptamers, magnetic beads (MBs), gold nanoparticles (AuNPs), and the CRISPR/Cas12a system for robust CRP quantification. In this system, MBs functionalized with aptamer 1 selectively captured the target protein, while AuNPs co-modified with aptamer 2 and a Cas12a activation sequence (Trigger) enabled efficient signal transduction. Upon target binding, the Trigger activated the Cas12a/crRNA complex, initiating trans-cleavage of fluorescent reporters and producing a markedly amplified signal. The optimized AuCA platform achieved a low detection limit of 60 ng/mL and a quantifiable range of 0.1 -150 µg/mL. It exhibited excellent specificity and resistance to biological interference, ensuring reliable measurements even in complex sample matrices. When applied to clinical human plasma, AuCA demonstrated strong concordance with results from a commercial immunoturbidimetric assay. AuCA allowed the simultaneous detection of both standard and hypersensitive CRP (hsCRP), supporting comprehensive full-range CRP (frCRP) analysis with strong potential for clinical application.
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SENSORS AND ACTUATORS B-CHEMICAL
·
2026
·
IF 8.3
Weiwei Jiang,Shuyu Zhou,Shaoqi Dong,Xinyi Gu,Liuyu Pan,Zhaohui Qiao,Danting Yang
Nucleic acid amplification-free detection of Salmonella Typhimurium ( S. Typhimurium ) remains challenging for decentralized food safety monitoring. Herein, we report a multivalent aptamer–mediated competitive electrochemical CRISPR platform for amplification-free detection of S. Typhimurium . A hybridization chain reaction (HCR) was employed to construct HCR-multiApt, a multivalent aptamer scaffold containing repeated crRNA-complementary activator regions. In the absence of bacteria, HCR-multiApt interacts with the Cas12a–crRNA complex and activates trans -cleavage of surface-immobilized electrochemical reporters. Upon target binding, S. Typhimurium competitively occupies the aptamer domains and reduces the accessibility of the crRNA-complementary activator regions, thereby suppressing Cas12a activation and modulating the electrochemical signal. A three-dimensional reduced graphene oxide/gold nanoparticle (3D-rGO/AuNPs) interface was used to enhance probe immobilization and electron transfer, while Hg 2 + -mediated T–Hg²⁺–T coordination enabled conformational regulation of the methylene blue-labeled reporter for improved signal transduction. Mechanistic studies, including microscale thermophoresis, enzyme kinetic analysis, and electrophoretic mobility shift assay, supported the target-dependent competitive regulation mechanism and indicated that HCR-multiApt enhanced the apparent Cas12a activation–cleavage efficiency mainly by increasing productive activation rather than directly altering the intrinsic catalytic activity of activated Cas12a. Under optimized conditions, the platform exhibited a linear detection range of 10 0 –10 8 CFU mL −1 with a detection limit of 3.75 CFU mL −1 . Selective discrimination against non-target bacteria and acceptable recoveries of 83.0–105.7% in spiked milk samples were achieved, with results consistent with conventional plate counting. This competitive multivalent EC-CRISPR strategy provides a programmable framework for amplification-free electrochemical pathogen detection.
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