SENSORS AND ACTUATORS B-CHEMICAL
·
2023
·
IF 8.4
Cheng Qin,Xinlan Bian,Wei Lv,Bingzhi Li
CRISPR/Cas12a-based biosensors typically output fluorescent, colorimetric, and electrochemical signals, relying on reporters that suffer from low sensitivity, high cost, and low stability. Herein, we introduced a label-free chemiluminescent (CL) reporting system based on a DNAzyme known as “Supernova”. Supernova catalyzes the translocation of phosphate from a 1,2-dioxetane substrate (specifically, CDP-Star) to its 5′-OH ends, thereby triggering a CL reaction that produces light. Upon activation by the target, Cas12a cleaves Supernova, rendering it inactive in reacting with CDP-Star. Without pre-amplification, this biosensor can detect synthetic DNA corresponding to the SARS-CoV-2 N gene and monkeypox virus (MPXV) F3L gene with detection limits (LOD) of 1.5 and 2.4 pM, respectively. Integration with pre-amplification further enables the detection of SARS-CoV-2 and MPXV genes down to 1 copy/μL in real samples. Furthermore, the application of this CL system was expanded to the detection of non-nucleic acids with the integration of ATP aptamers, resulting in improved sensitivity in ATP biosensing compared to fluorescent assays. Overall, this work represents the first development of a label-free CL Cas12a reporting system, demonstrating its enhanced performance and broad applicability in synthetic biology-enabled sensing technology.
研究领域:
生物传感技术
·
分子诊断学
·
合成生物学
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DOI
高影响因子
ACS Applied Materials & Interfaces
·
2021
·
IF 9.229
Qiong Hu,Yilin Luo,Xiaojing Cao,Zhuohua Chen,Yanyu Huang,Li Niu
Sensing of ultralow-abundance nucleic acids (NAs) is integral to medical diagnostics and pathogen screening. We present herein an electrochemical method for the highly selective and amplified sensing of NAs, using a peptide nucleic acid (PNA) recognition probe and a bioinspired electro-RAFT polymerization (BERP)-based amplification strategy. The presented method is based on the recognition of target NAs by end-tethered PNA probes, the labeling of thiocarbonylthio reversible addition–fragmentation chain transfer (RAFT) agents, and the BERP-assisted growth of ferrocenyl polymers. The dynamic growth of polymers is electrochemically regulated by the reduction of 1-methylnicotinamide (MNA) organic cations, the redox center of nicotinamide adenine dinucleotide (NAD+, coenzyme I). Specifically, electroreduction of the MNA cations causes the fragmentation of thiocarbonylthio RAFT agents into radical species, triggering the polymerization of ferrocenyl monomers, thereby recruiting plenty of ferrocene electroactive tags for amplified sensing. It is obvious that the BERP-based strategy is inexpensive and simple in operation. Benefiting from the high specificity of the PNA recognition probe and the amplified signal by the BERP-based strategy, this method is highly selective and the detection limit is as low as 0.58 fM (S/N = 3). Besides, it is applicable to the sensing of NAs in serum samples, thus showing great promise in the selective and amplified sensing of NAs.
研究领域:
医学诊断
·
生物化学
·
分析化学
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DOI
高影响因子
Sensors & Diagnostics
·
2022
·Vol. 2
·期 1
·194-202
·
IF 0
Shiyang Qu,Lin Shi,Huan Li,Tao Li
As a nano-form of G-quadruplex (G4) based DNAzymes, spherical nucleic acid enzymes (SNAzymes) with gold nanoparticles (AuNPs) as cores have shown great promise for applications in nucleic acid detection. SNAzymes can be constructed by modifying thiolated G4 DNAs onto AuNPs through various methods. However, those constructed with label-free G4 DNAs were found to have a weak enzyme activity due to the G4 destruction on the surface of AuNPs. To address this, here we constructed label-free silicon-based SNAzymes using silica nanoparticles (SiO2) as the core for the first time. We took advantage of the versatility of SiO2 and loaded N-(4-aminobutyl)-N-ethylisoluminol (ABEI) on the surface of silicon spheres (SiO2@ABEI). Then, we found that SiO2@ABEI can achieve high-efficiency adsorption of arbitrary G4 sequences within a few minutes without high requirements for modification methods. In this way, a highly sensitive CL nanoprobe integrated with a catalyst and a chemiluminescence (CL) reagent was successfully constructed. Finally, we use the nanoprobe for the ultrasensitive chemiluminescence detection of acute myocardial infarction (AMI)-related nucleic acids, achieving a detection range of 10 fM–100 pM and an excellent detection limit of 0.8 fM.
研究领域:
生物化学
·
医学诊断
·
纳米技术
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DOI
ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Kai Shi,Wenjie Luo,Ying Cheng,Honglei Li,Liai Peng,Xiangrui Luo,Yu Hu,Jiaheng Zhang,Jiaxuan Chen
CRISPR/Cas12a-powered biosensors with guanine (G)-rich sequence reporters (e.g., G-quadruplex and G-triplex) are widely used in detection applications due to their simplicity and sensitivity. However, when these biosensors are employed for molecular detection in complex samples, they may encounter difficulties such as high background signal and susceptibility to interference because of the “turn-off” signal output. Herein, we explore, for the first time, a set of phosphorothioate (ps)-modified G-quadruplex (G4) and G-triplex (G3) sequences that can bind with thioflavin T (ThT) in an active split CRISPR/Cas12a system (SCas12a) to generate a “turn-on” fluorescent signal. To apply this new phenomenon, we develop a universal SCas12a-powered biosensor for “turn-on” fluorescent detection of nucleic acid (miRNA-21) and non-nucleic acid (kanamycin) targets by using ps-modified hairpin G3 as a reporter (SCas12a/psHG3). Target recognition activates SCas12a’s trans-cleavage activity, leading to cleavage at the loop region of the psHG3 reporter. The released prelocked psG3 DNA binds ThT to produce a strong fluorescence signal. Without preamplification, this strategy can detect miRNA-21 with a detection limit of 100 fM. Moreover, the SCas12a/psHG3 system was further utilized for detecting kanamycin by incorporating its aptamers, enabling the detection of kanamycin at concentrations as low as 100 pM. This work is the first to develop a “turn-on” SCas12a/psHG3 system, showcasing its improved performance and wide range of applications in synthetic biology-based sensing technology.
研究领域:
生物技术
·
分子生物学
·
合成生物学
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产品关联
DOI
PubMed
高影响因子
CELLULOSE
·
2022
·Vol. 29
·期 4
·2479-2495
·
IF 6.123
Tang Ruihua,Li Min,Yan Xueyan,Xie Mingyue,Liu Li Na,Li Zedong,Xu Feng
Cheap, rapid, simple and equipment-free nucleic acid extraction (NAE) is highly preferred for implementing nucleic acid detection at point-of-care (POC). Paper-based NAE materials have been extensively utilized due to their low cost, abundance, portability, biocompatibility and ease of chemical modification. However, it is challenging for users to choose the proper one from existing paper-based NAE materials for specific POC applications, which is determined by their physical and chemical properties. Additionally, building the relationship between the physical and chemical properties and the NAE efficiency of paper-based materials is instructive for development of new paper-based NAE materials. In this study, we first systematically compared the physical and chemical properties of six widely used paper-based NAE materials (namely Whatman filter paper #1, FTA card, FTA elute card, Fusion 5, silica membrane and polyethersulfone (PES) membrane), and then evaluated their NAE efficiency. The obtained results indicated that pore uniformity, wet strength, porosity and functional groups are key parameters to affect the efficiency of NAE. The NAE performance of FTA card is the best with high concentration and purity. Finally, we envision that more cost-effective paper-based NAE materials will be developed for POCT application in the future. Graphic abstract
研究领域:
生物技术
·
医学检验
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2015
·
IF 4.513
Yingfeng Qin,Yefei Ma,Xue Jin,Liangliang Zhang,Gaojie Ye,Shulin Zhao
As an important antitumor drug, bleomycin (BLM) is widely used in the treatment of a variety of cancers. In addition, nucleases play a crucial role in DNA replication, recombination and repair which are associated with cancer development. Thus, the development of BLM and nuclease detection methods is of great significance in cancer therapy and related biological mechanism research. Here, a WS 2 nanosheet-based turn-on fluorescent sensing platform for simple, fast and sensitive detection of BLM and nuclease was reported. WS 2 nanosheet exhibits different affinity toward ssDNA with different length and excellent fluorescence quenching ability. A fluorescein (FAM)-labeled long ssDNA could be adsorbed on the surface of WS 2 nanosheet and the fluorescence was therefore quenched. In the presence of BLM·Fe(II) or S1 nuclease (a ssDNA-specific nuclease which was used as a model enzyme), an irreversible scission of long ssDNA was underwent through the BLM-induced oxidation cleavage or S1 nuclease-induced enzymatic hydrolysis. Short FAM-linked oligonucleotide fragments which could not be adsorbed on the nanosheet surface were then produced, resulting in a weak fluorescence quenching after mixing WS 2 nanosheets. Thus, the fluorescence signal was restored. The proposed sensor displays a wide linear range and a high sensitivity with a detection limit of 0.3 nM for BLM and 0.01 U mL −1 for S1 nuclease. It also exhibits a good performance in complex biological samples. This method not only provides a strategy for BLM or S1 nuclease assay but also offers a potential application in biomedical and clinical study.
研究领域:
生物化学
·
分子生物学
·
分析化学
·
癌症研究
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产品关联
DOI
PubMed
高影响因子
ACS Synthetic Biology
·
2022
·
IF 5.249
Dongying Wei,Yueyao Wang,Dongfan Song,Ze Zhang,Juan Wang,Jia-Yu Chen,Zhe Li,Hanyang Yu
Threose nucleic acid (TNA) is considered a potential RNA progenitor due to its chemical simplicity, base pairing property, and capability of folding into a functional tertiary structure. However, it is unknown whether the functional property can be maintained during transition from TNA to RNA. Here, we use a toggle in vitro selection to identify nucleic acid catalyst sequences that are active in both TNA and RNA backbones. One such nucleic acid enzyme with exchangeable backbone (CAMELEON) catalyzes an RNA cleavage reaction when prepared as TNA (T) and RNA (R). Further biochemical characterization reveals that CAMELEON R and T exhibit different catalytic behaviors such as rate enhancement and magnesium dependence. Structural probing and mutagenesis experiments suggest that they likely fold into distinct tertiary structures. This work demonstrates that the catalytic activity can be preserved during backbone transition from TNA to RNA and provides further experimental support for TNA as an RNA precursor in evolution.
研究领域:
生物化学
·
分子进化
·
核酸化学
✓ 已验证
DOI
PubMed
高影响因子
TALANTA
·
2020
·
IF 5.339
Xiaoyu Cai,Huimin Zhang,Xiufeng Yu,Wei Wang
MicroRNAs (miRNAs) are considered as the potential biomarkers for many cancers. To determine miRNAs in cancer cells is significant for realizing these diseases. In this work, a microfluidic paper-based laser-induced fluorescence sensor based on duplex-specific nuclease (DSN) amplification was developed and applied to selectively and sensitively determine miRNAs in cancer cells. An interface for laser-induced fluorescence detection was firstly applied to perform the sample detection on the paper-based chip. Under the optimal conditions, DSN (3 μL 0.10 U) and Taqman probes (2 μL 2.5 × 10 −7 M) were preserved on the circles (Diameter 4 mm) of the folded paper chip. When miRNA solution was added, the mixed solution could trigger fluorescence signal amplification by cyclically digesting hybrids of miRNAs and Taqman probes by DSN. The whole determination, including sample heating process, could be accomplished within 40 min. The detection limits for miRNA-21 and miRNA-31 were 0.20 and 0.50 fM respectively, corresponding to only 1.0 and 1.5 zmol consumption of miRNAs. The testing of mismatched miRNAs showed that the method had good specificity. Finally, the method was applied to determine miRNA-21 and miRNA-31 in lysates of cancer cells of A549 and HeLa, and hepatocyte LO2. MiRNA-21 and miRNA-31 could be successfully found from the two cancer cells. The concentrations for miRNA-21 and miRNA-31 were 1.74 × 10 −13 M and 6.29 × 10 −14 M in HeLa cell lysate (3.75 × 10 4 cells/mL), 3.07 × 10 −15 M and 3.28 × 10 −15 M in A549 cell lysate (8.33 × 10 6 cells/mL) respectively. The recoveries ranged from 87.30% to 111.83%, indicating the results were reliable. The developed method was effective, selective and sensitive in the determination of miRNAs in cancer cells.
研究领域:
生物医学工程
·
分子生物学
·
癌症研究
✓ 已验证
DOI
PubMed
高影响因子
NATURE BIOTECHNOLOGY
·
2022
·Vol. 40
·期 11
·1601-1609
·
IF 68.164
Chen Ji,Chen Mengyin,Zhu Ting F.
Mirror-image aptamers made from chirally inverted nucleic acids are nuclease-resistant and exceptionally biostable, opening up opportunities for unique applications. However, the directed evolution and selection of mirror-image aptamers directly from large randomized l -DNA libraries has, to our knowledge, not been demonstrated previously. Here, we developed a ‘mirror-image selection’ scheme for the directed evolution and selection of biostable l -DNA aptamers with a mirror-image DNA polymerase. We performed iterative rounds of enrichment and mirror-image polymerase chain reaction (PCR) amplification of l -DNA sequences that bind native human thrombin, in conjunction with denaturing gradient gel electrophoresis (DGGE) to isolate individual aptamers and l -DNA sequencing-by-synthesis to determine their sequences. Based on the selected l -DNA aptamers, we designed biostable thrombin sensors and inhibitors, which remained functional in physiologically relevant nuclease-rich environments, even in the presence of human serum that rapidly degraded d -DNA aptamers. Mirror-image selection of biostable l -DNA aptamers directly from large randomized l -DNA libraries greatly expands the range of biomolecules that can be targeted, broadening their applications as biostable sensors, therapeutics and basic research tools.
研究领域:
分子生物学
·
生物技术
·
纳米技术
·
治疗学
·
传感器
✓ 已验证
DOI
PubMed
高影响因子
RSC Advances
·
2021
·Vol. 11
·期 52
·32898-32903
·
IF 3.361
Lancheng Wang,Huimin Zhou,Kun Yan,Peng Xu,Bin Di,Chi Hu,Mengxiang Su
The binding of small molecules to their DNA aptamers can modulate their susceptibility to digestion by exonucleases, however, absolute differentiation between digestion and inhibition has never been reported. Here, we show that the digestion of aptamers by T7 exonuclease can be completely inhibited upon binding of small-molecule targets and exploit this finding for the first time to achieve sensitive, label-free small-molecule detection. We use a quinine-binding aptamer to show that target binding entirely halts T7 exonuclease digestion, leaving behind an intact double-stranded product that retains strong target affinity. On the contrary, digestion of nontarget-bound aptamer produces a single-stranded product incapable of target binding. Exonuclease I efficiently eliminates these single-stranded products but is unable to digest the target-bound double-stranded product. The remaining products can be fluorescently quantified with SYBR Gold to determine target concentrations, giving a limit of detection of 100 nM with the linear range from 0 to 8 μM. We demonstrate the first example of a dual-exonuclease-mediated approach capable of producing a concentration-dependent response in terms of aptamer digestion modules, therefore improving performance of the current aptamer-based assay for small-molecule detection.
研究领域:
生物化学
·
分子生物学
·
分析化学
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产品关联
DOI
PubMed
高影响因子
Science of Advanced Materials
·
2014
·Vol. 6
·期 9
·1936-1942
·
IF 2.908
Li Xiang,Ding Xuelian,Wang Gongke,Zhou Yixuan,Hou Ruipeng,Gao Shuyan,Lu Yan
In this work, we have developed a simple and highly sensitive fluorescent biosensor for the detection of HIV-1 RNase H activity and inhibition by using graphene oxide (GO) as sensing platform. In our approach, a DNA-RNA substrate was prepared which a carboxy fluorescein (FAM) was labeled
at the 3 termini of single strand DNA. The FAM labeled DNA-RNA substrate preserves most of the fluorescence when mixed with GO. However, in the present of RNase H, RNase H can cleavage the RNA into fragments, resulting in the dissociation of ssDNA and mononucleotides. The fluorescence intensity
was greatly quenched after the addition of GO. Because of the super quenching ability of GO, the as-proposed method provides a wide linear range from 1.0 to 26.3 units mL–1 and a low detection limit of 0.6 units mL–1 for HIV-1 RNase H activity analysis. Additionally,
this approach shows potential for high-throughput screening of HIV-1 RNase H inhibitors. The method not only provides a universal platform for monitoring activity and inhibition of RNase H but also shows great potential in biological process researches, drug discovery, and clinic diagnostics.
研究领域:
生物技术
·
分子生物学
·
纳米技术
·
病毒学
·
药物发现
✓ 已验证
DOI
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2022
·
IF 9.221
Fu-Ting Wang,Hu Luo,Yang-Yang Hou,Yu Ya,Ke-Jing Huang,Shu-Peng Li,Lan-Rui Wang,Ya-Lin Song,Xuecai Tan
We present a dual-microRNAs-driven enzyme biofuel cells (EBFCs) based visual self-powered biosensing system with a capacitor assistance significant amplification of the output signal for the sensitive detection of microRNA-21 and microRNA-141. When microRNA-21 is captured by the aptamer (Atp1–21), the glucose oxidase (GOD)-modified aptamer (Atp2–21-GOD) is immobilized on the electrode surface by base-complementary pairing. When microRNA-21 and microRNA-141 are captured by the dual aptamer recognition system, the enzyme load is more effectively increased and the input voltage of the EBFCs is significantly enhanced. In the linear range of 0.0005–100 PM and 0.001–1000 PM, the detection limits for microRNA-21 and microRNA-141 are 0.16 and 0.36 fM, respectively. Furthermore, the EBFCs are connected to the capacitor integrated circuit, and the input voltage of the EBFCs is converted to an enhanced instantaneous current and charging to the capacitor which is then directly detected by a multimeter , and the sensitivity of sensing system is improved by 29.48 times. In order to improve the reproducibility and accuracy of self-powered biosensing system, the reading of the multimeter is obtained in real time by software that opens new door to the visualization of detection signal. The method provides an efficient strategy for visualization of multimeter-based detection signal and opens the possibility for medical diagnosis and therapeutic device power in the biosensing device.
研究领域:
生物化学
·
分子生物学
·
医学诊断
✓ 已验证
产品关联
DOI
高影响因子
危险品化学品经营许可证（带存储）