ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Qing-Nan Li,Yun-Xi Cui,Zhi-Qi Dai,Zhi-Li Yao,Man-Ying Li,Qi-Liang Cai,De-Ming Kong
The CRISPR/Cas12a system has emerged as a powerful tool in biosensing due to its unique trans-cleavage activity. This study conducted an in-depth investigation of the modulatory capabilities of this system, particularly focusing on the 5′-end modifications of the activator strand, and found that introducing a hairpin structure (HP) at the 5′-end of the activator strand, which was designed based on the RESET effect, can effectively suppress the activator strand’s ability to activate the trans-cleavage activity of the CRISPR/Cas12a system. This suppression is independent of the HP’s relation to the activator strand and the type of linker used (DNA, RNA or peptide). Detaching the HP from the activator strand restores the system’s activity. These findings enrich the development of CRISPR/Cas12a-based biosensors, and expand their application beyond DNA-based target detection to peptide sequence-based target recognition. Based on this discovery, we constructed a sensitive biosensor for caspase-3 (Casp-3), a key executor in apoptosis, by linking the HP to the activator strand with a peptide linker containing a Casp-3 recognition site. The proposed biosensor has been validated for its sensitivity and specificity in detecting Casp-3, as well as for monitoring drug-induced apoptosis through the imaging of Casp-3 in living cells, providing a valuable tool for studying the apoptotic process, screening drugs, assessing drug efficacy, and evaluating treatment outcomes. This strategy also shows promise for detecting other peptide-based targets, broadening the horizons for early disease biomarker detection and timely therapeutic interventions.
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高影响因子
MICROCHEMICAL JOURNAL
·
2026
·
IF 5.2
Yaru Zhang,Yongchang Sun,Jing Yuan,Lihua Ding,Leiliang He,Yongjun Wu,Songcheng Yu
MicroRNAs (miRNAs), as crucial regulators of gene expression, play significant roles in cancer development. Among them, miRNA-21 has been recognized as a promising biomarker for the diagnosis and prognosis evaluation of multiple cancers. However, existing CRISPR/Cas12a-based miRNA detection methods generally require reverse transcription and pre-amplification steps, which complicate the operational workflow and are prone to introducing cross contamination. Furthermore, the dependence on single-fluorescence signal detection makes them vulnerable to environmental and instrumental fluctuations, which results in risk of false-positive outcomes. Herein, we developed a ratiometric and sensitive CRISPR/Cas12a system with trans-cleavage-triggered feedback signal amplification for amplification-free detection of miRNA-21. The strategy employs a novel multifunctional switch probe that integrates target recognition, signal transduction, and cascade amplification within a unified system. A trans-cleavage-triggered feedback signal amplification circuit was constructed to enhance detection sensitivity without the need for additional enzyme systems. Moreover, a ratiometric fluorescence signal was implemented by calculating the fluorescence intensity ratio between CY5 and Auramine O, which effectively minimizes interference from environmental and instrumental variations and substantially improves detection accuracy and reliability. Experimental results demonstrate that the method achieves a detection limit of 1.28 nM, along with excellent specificity, accuracy, and reproducibility. In real sample analysis, differential expression levels of miRNA-21 in lung adenocarcinoma cells (A549) and non-cancerous adult bronchial epithelial cells (BEAS-2B) was observed, which highlights its potential value for clinical diagnostics.
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Food and Bioprocess Technology
·
2026
·Vol. 19
·期 5
·244
·
IF 5.6
Shen Jingqi,Zhao Yan,Duan Miaolin,Zhang Weijia,Wang Kaidi,Zhang Xiaowei,Dai Ruitong,Liu Zunying,Jia Fei
Foodborne pathogens have posed a major threat to food safety and public health, necessitating the development of accurate and sensitive methods for the detection of foodborne pathogens. In this study, a metal–organic framework (MOF) nanozyme-based CRISPR/Cas12a colorimetric biosensing system was developed and integrated with a smartphone-based portable device to meet the demand for on-site detection of Salmonella Typhimurium. Inspired by the catalytic characteristics of gold nanoparticles (AuNPs), the ZIF-8@AuNPs nanozyme with oxidase-like activity was synthesized by immobilizing AuNPs onto zeolitic imidazolate framework-8 (ZIF-8) via a self-assembly method. In this detection system, the target DNA is preamplified via the recombinase-aided amplification (RAA) procedure to achieve primary signal amplification. The amplified target DNA subsequently triggers colorimetric signal readout and secondary signal amplification through CRISPR/Cas12a-mediated specific recognition and cleavage, and the high catalytic performance of the ZIF-8@AuNPs nanozyme, thereby facilitating the biosensor to exhibit excellent detection sensitivity and specificity. The concentration of bacteria can be quantified and analyzed using a self-programmed “DeepFood” APP with the help of smartphones and 3D-printed devices. Consequently, this method allows the quantitative detection of Salmonella Typhimurium within a linear range of 10 1 to 10 7 CFU/mL, with detection limits of 11 CFU/mL for colorimetric detection and 65 CFU/mL for smartphone-based RGB detection in the range of 10 2 to 10⁷ CFU/mL. Additionally, this method facilitates sensitive, specific, and portable detection of Salmonella Typhimurium in milk samples, in comparison with traditional methods. The inherent programmability of CRISPR RNA also enables the detection of other bacterial pathogens, making this platform a valuable tool for food safety monitoring. Graphical
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ACS Sensors
·
2026
·
IF 10.9
Zhenghan Shi,Liya Ding,Li-ang Zhou,Ye Liu,Yuhang Wang,Yi Xu,Yunrui Cao,Zhinong Jiang,Qingjun Liu
Tumor necrosis factor-α (TNF-α) is an important inflammatory cytokine whose rapid and quantitative detection is essential for disease diagnosis and monitoring. However, conventional methods are often limited by bulky instrumentation and are not well suited for analysis in complex biological environments. Herein, a portable CRISPR/Cas12a-based aptasensor system was developed for detection of TNF-α by integrating aptamer recognition, Cas12a trans-cleavage signal amplification, and a miniaturized fluorescence detection device. An aptamer for TNF-α was designed to regulate the accessibility of the target sequence to the CRISPR RNA (crRNA), and the resulting change in Cas12a activity was converted into fluorescence signals. The portable device integrated light-emitting diode (LED) excitation and photodiode-based detection, with modulated signals wirelessly transmitted to a smartphone for readout. The aptasensor showed concentration-dependent responses, and the inhibition rate exhibited a linear relationship with TNF-α concentration. The applicability of the system was validated in cell culture supernatants and cervical tissue samples, including tumor and adjacent normal tissues. Distinct TNF-α levels were observed between these samples, showing good agreement with enzyme-linked immunosorbent assay (ELISA) results. This portable CRISPR/Cas12a aptasensor system provides a practical approach for point-of-care TNF-α detection and extends CRISPR-based sensing to protein biomarkers.
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SENSORS AND ACTUATORS B-CHEMICAL
·
2026
·
IF 8.3
Wenji Bao,Ailing Su,Gerile Aodeng,Lu Ga,Lan Yu,Jun Ai
A highly sensitive colorimetric sensing platform was developed based on the trans-cleavage activity of CRISPR/Cas12a and the controllable aggregation of gold nanoparticles (AuNPs) for rapid and visual detection of the foodborne pathogen Staphylococcus aureus. Following PCR or RPA, the target DNA was specifically recognized by the Cas12a/crRNA complex, thereby activating its trans-cleavage activity. This process led to the degradation of linker single-stranded DNA (ssDNA) responsible for crosslinking AuNPs probes, resulting in significant changes in both UV–vis absorption signals and solution color. Three different crRNA sequences were systematically evaluated, revealing distinct efficiencies in target recognition and signal amplification. Among them, the crRNA-3 mediated system exhibited superior analytical performance, achieving an ultralow detection limit of 1.14 fM and a good linear response over a low concentration range. Furthermore, the proposed platform demonstrated excellent anti-interference capability in complex matrices, including serum, milk, bovine serum albumin (BSA), and phosphate-buffered saline (PBS), with recovery rates ranging from 83.2% to 125.9%. This work provides a robust and visual tool for the on-site detection of S. aureus and offers new insights into the design of portable CRISPR-based diagnostic platforms.
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BIOELECTROCHEMISTRY
·
2025
·
IF 4.5
Licong Jia,Kai Kang,Wen Yan,Yan Jin,Kejia Xu,Jingyi Wang,Yipeng Wang,Meng Jiang,Wei Yuan,Shipeng Liu,Lingmei Niu
17β-Estradiol (E2) serves as both a reproductive accelerator and growth promoter, yet its uncontrolled application induces severe endocrine dysfunction. Point-of-care testing (POCT) has emerged as a promising analytical platform for on-site E2 quantification, due to its inherent advantages in terms of sensitive, specific, cost-effective, and instrument-free. We developed a novel strategy combining the high collateral cleavage activity of CRISPR/Cas12a with the strong amplification power of dual-cycle reaction, translating target presence into a colorimetric signal on pregnancy test strip (PTS). In the presence of E2, the dual-cycle amplification was initiated to produce HP2–HP3 (Hairpin DNA 2–Hairpin DNA 3) duplexes. These duplexes then bound to the Cas12a-crRNA complex, activating its trans-cleavage activity. The activated Cas12a subsequently cleaved the ssDNA linkers in the MBs (magnetic beads)-ssDNA (single-stranded DNA)-hCG (human chorionic gonadotropin) conjugates, releasing hCG for quantitative E2 detection through strip color intensity measurement. By integrating CRISPR/Cas12a's high cleavage efficiency with dual-cycle amplification, a visible biosensor was developed, with a linear range of 1.0 × 10 −1 –200.0 pM and a detection limit of 0.0403 pM for E2. In conclusion, an E2 detection platform characterized by prominent sensitivity and convenience was established for monitoring spiked E2 in milk and urine, providing a significant reference for the POCT detection of other targets.
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JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY A-CHEMISTRY
·
2026
·
IF 4.7
Li Zhou,Xianwei Jiao,Yuting Zhang,Changling Yan
L-selectin, a leukocyte adhesion receptor, is essential in the early step of the adhesion cascade, facilitating the migration of leukocytes into surrounding tissues during inflammation and immune surveillance. Clinical studies have identified L-selectin as a prospective treatment option for many both acute and ongoing inflammatory conditions. Clarifying the cellular signaling pathways that function in the immediate vicinity of L-selectin may yield unique treatments tailored to certain cell types or illnesses. The study describes the development of a precise and sensitive fluorescence biosensor designed for the detection of L-selectin using a split aptamer specific to L-selectin. This biosensor utilizes nicking enzyme-assisted DNA walking together CRISPR-Cas12a for dual-signal multiplication. The calibration curve for L-selectin demonstrates a detection limit of 0.45 ng/mL, with an extensive linear range spanning from 10 to 1280 ng/mL. The fluorometric sensors effectively identified L-selectin in human blood samples, producing excellent outcomes. The fluorometric sensors effectively identified L-selectin in human blood samples, producing excellent outcomes. The original appearance not only validates the concept but also possesses substantial possibilities for identifying diverse targets.
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TALANTA
·
2026
·
IF 6.7
Mingqi Yang,Jiali Hu,Zhenlin Zhang,Liting Huang,Yahan Yu,Dexing Qiu,Yexin Zu,Yanyan Liu,Zhenyu Lin
Infection with hepatitis B virus (HBV) represents a major challenge to global health, especially in areas of inadequate healthcare infrastructure, rapid and on-site detection plays a crucial role in effective disease management and control. Consequently, it is necessary to develop some simple but sensitive HBV screening techniques. In this work, a homogeneous electrochemical biosensor was constructed for sensitive and specific HBV DNA detection through the integration of CRISPR/Cas12a target recognition and magnetic bead-based separation. The sensor exploits the collateral cleavage activity of CRISPR/Cas12a protein upon recognition of target double-stranded DNA, enabling the degradation of methylene blue-labeled single-stranded DNA captured on magnetic beads (MBs). Then the MBs are separated and redissolved in the solution and the electrochemical response of the system can be tested in homogeneous solution. The electrochemical signal has relationship with the concentration of HBV DNA within a range of 10 fM - 10 nM, and the LOD is 3.74 fM. The sensor demonstrates excellent selectivity and biological stability, which has potential application in clinical diagnosis, especially in resource-limited environments.
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BIOSENSORS & BIOELECTRONICS
·
2021
·
IF 10.618
Hui Wu,Yanju Chen,Qunqing Yang,Cheng Peng,Xiaofu Wang,Mengyao Zhang,Siwenjie Qian,Junfeng Xu,Jian Wu
Vibrio parahaemolyticus ( V. parahaemolyticus ) is regarded as a major cause of seafood-associated illnesses, which has aroused widespread public concern. Here, a rapid and convenient detection method for V. parahaemolyticus detection was established by a reversible valve-assisted chip coupling with CRISPR/Cas12a. With optimized lysis buffer, loop mediated isothermal amplification (LAMP) reagents and CRISPR reagents, the whole detection process from sampling to results could be finished within 50 min. The structure of chip was simple and the cost was low. By relying on three reversible rotary valves and the rotation direction-dependent Coriolis pseudo force, the flow order of liquid and the direction of liquid flow could be precisely controlled. The LAMP amplicons were specifically and sensitively identified by CRISPR/Cas12a. Positive amplification would produce green fluorescent signal while negative amplification generated no fluorescent signal, which could be clearly distinguished by the naked eye. With 600 μL of samples processed, the limit of detection (LOD) for both pure cultured V. parahaemolyticus or spiked shrimp samples could achieve 30 copies/reaction. These illustrated the established method displayed great feasibility for real samples detection. In the future, the chip could also combine with other amplification reactions, like PCR or recombinase polymerase amplification reaction (RPA), to conduct detection by changing the corresponding lyophilized amplification reagents. Overall, the proposed detection platform displays great potential for food safety analysis and clinical diagnostics, especially in resource-limited areas.
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ANALYTICAL CHEMISTRY
·
2023
·
IF 7.4
Zhenzhen Jia,Yujie Zhang,Chuanyu Zhang,Xueyong Wei,Mingzhen Zhang
With an increasing incidence worldwide, inflammatory bowel disease (IBD) is a chronic inflammatory disease affecting the gastrointestinal tract, which impairs the life quality of patients. Therefore, it is of great significance to construct a sensitive, simple, and convenient biosensor to analyze IBD-associated biomarkers for an auxiliary diagnosis of IBD. Intestinal alkaline phosphatase (IAP), expressed by the intestinal epithelium, is an endogenous protein that is thought to play a vital role in maintaining intestinal homeostasis and is considered a potential biomarker for IBD. Here, an IAP detection method was developed using pregnancy test strips by dephosphorylation. Initially, a double-stranded DNA (dsDNA) was designed to respond to IAP and acted as an activator of Cas12a. In the presence of IAP, the designed dsDNA was not digested by lambda exonuclease (λ exo), which hybridized to the Cas12a-crRNA duplex and resulted in the activation of the trans-cleavage of Cas12a. Further, the activated Cas12a cleaved the single-strand DNA (ssDNA) linker in the MBs-ssDNA-hCG probe, triggering the release of hCG. With magnetic separation, the released hCG could be quantitatively detected by pregnancy test strips. IAP levels were analyzed in feces from colitis and healthy mice by pregnancy test strips. The results showed that the IAP level of colitis mice (3.89 ± 1.92 U/L) was much lower than that of healthy mice (39.64 ± 24.93 U/L), indicating the correlation between IAP and intestinal inflammation. Taken together, a sensitive, user-friendly detection assay based on pregnancy test strips was constructed to monitor IAP and used as an auxiliary diagnostic approach for IBD in a clinical scene.
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ANALYTICAL CHEMISTRY
·
2023
·
IF 7.4
Zhirun Ji,Bin Zhou,Zhaoyang Shang,Sirui Liu,Xue Li,Xing Zhang,Bingzhi Li
CRISPR-Cas12a is an accurate and responsive biosensing technique, but its limited stability has restricted its widespread applications. To address this, we propose a strategy using metal–organic frameworks (MOFs) to protect Cas12a from harsh environments. After screening multiple candidate MOFs, it was found that hydrophilic MAF-7 is highly compatible with Cas12a, and the as-formed Cas12a-on-MAF-7 (COM) not only retains high enzymatic activity but also possesses excellent tolerance to heat, salt, and organic solvents. Further investigation showed that COM can serve as an analytical component for nucleic acid detection, resulting in an ultrasensitive assay for SARS-CoV-2 RNA detection with a detection limit of 1 copy. This is the first successful attempt to create an active Cas12a nanobiocomposite that functions as a biosensor without the need for shell deconstruction or enzyme release.
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ANALYST
·
2022
·Vol. 147
·期 5
·905-914
·
IF 5.227
Shumin Zhang,Minyi Liu,Haofa Cui,Muhammad Asad Ziaee,Rongwei Sun,Liting Chen,Daqi Chen,Denis Garoli,Jiahai Wang
The fabrication of nanopores with a matched pore size, and the existence of multiple interferents make the reproducible detection of small-sized molecules by means of solid-state nanopores still challenging. A useful method to solve these problems is based on the detection of large DNA nanostructures related to the existence of small-sized targets. In particular, a DNA tetrahedron with a well-defined 3D nanostructure is the ideal candidate for use as a signal transducer. Here, we demonstrate the detection of an L1-encoding gene of HPV18 as a test DNA target sequence in a reaction buffer solution, where long single-stranded DNA linking DNA tetrahedra onto the surface of the magnetic beads is cleaved by a target DNA-activated CRISPR-cas12 system. The DNA tetrahedra are subsequently released and can be detected by the current pulse in a glassy nanopore. This approach has several advantages: (1) one signal transducer can be used to detect different targets; (2) a glassy nanopore with a pore size much larger than the target DNA fragment can boost the tolerance of the contaminants and interferents which often degrade the performance of a nanopore sensor.
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