Theranostics
·
2022
·
IF 11.6
Bohan Yin,Qin Zhang,Xinyue Xia,Chuanqi Li,Willis Kwun Hei Ho,Jiaxiang Yan,Yingying Huang,Honglian Wu,Pui Wang,Changqing Yi,Jianhua Hao,Jianfang Wang,Honglin Chen,Siu Hong Dexter Wong,Mo Yang
Background: CRISPR-Cas12a has been integrated with nanomaterial-based optical techniques, such as surface-enhanced Raman scattering (SERS), to formulate a powerful amplification-free nucleic acid detection system. However, nanomaterials impose steric hindrance to limit the accessibility of CRISPR-Cas12a to the narrow gaps (SERS hot spots) among nanoparticles (NPs) for producing a significant change in signals after nucleic acid detection. Methods: To overcome this restriction, we specifically design chimeric DNA/RNA hairpins (displacers) that can be destabilized by activated CRISPR-Cas12a in the presence of target DNA, liberating excessive RNA that can disintegrate a core-satellite nanocluster via toehold-mediated strand displacement for orchestrating a promising “on-off” nucleic acid biosensor. The core-satellite nanocluster comprises a large gold nanoparticle (AuNP) core surrounded by small AuNPs with Raman tags via DNA hybridization as an ultrabright Raman reporter, and its disassembly leads to a drastic decrease of SERS intensity as signal readouts. We further introduce a magnetic core to the large AuNPs that can facilitate their separation from the disassembled nanostructures to suppress the background for improving detection sensitivity. Results: As a proof-of-concept study, our findings showed that the application of displacers was more effective in decreasing the SERS intensity of the system and attained a better limit of detection (LOD, 10 aM) than that by directly using activated CRISPR-Cas12a, with high selectivity and stability for nucleic acid detection. Introducing magnetic-responsive functionality to our system further improves the LOD to 1 aM. Conclusion: Our work not only offers a platform to sensitively and selectively probe nucleic acids without pre-amplification but also provides new insights into the design of the CRISPR-Cas12a/SERS integrated system to resolve the steric hindrance of nanomaterials for constructing biosensors.
研究领域:
生物技术
·
分析化学
·
分子生物学
✓ 已验证
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Fei Hu,Kaihui Liu,Yunyun Zhang,Shuhao Zhao,Tianyi Zhang,Cuiping Yao,Xing Lv,Jing Wang,Xiaolong Liu,Niancai Peng
Infectious diseases, especially respiratory infections, have been significant threats to human health. Therefore, it is essential to develop rapid, portable, and highly sensitive diagnostic methods for their control. Herein, a short-time preamplified, one-pot clustered regularly interspaced short palindromic repeats (CRISPR) nucleic acid detection method (SPOC) is developed by combining the rapid recombinase polymerase amplification (RPA) with CRISPR-Cas12a to reduce the mutual interference and achieve facile and rapid molecular diagnosis. SPOC can reduce the detection time and stably detect up to 1 copy/μL of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA without affecting the detection sensitivity. A highly sensitive one-pot assay integrated with reverse transcription RPA is achieved by wrapping paraffin with a specific melting point on the lyophilized CRISPR reagent surface. A self-heating pack is designed based on thermodynamic principles to melt the paraffin and release CRISPR reagents, enabling low-cost and time-saving detection. Notably, the designed system, coupled with RNA extraction-free technology, can achieve “sample-in-answer-out” detection of the SARS-CoV-2 Orf1ab gene within 22 min using smartphone imaging. The developed assay is validated on 12 clinical samples, and the results 100% correlate with real-time polymerase chain reaction. SPOC is time-saving, is easy to operate, and can eliminate centrifugal and complex hardware devices, satisfying the demand for point-of-care diagnostics in resource-constrained settings.
研究领域:
分子诊断学
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传染病学
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生物技术
✓ 已验证
产品关联
DOI
PubMed
高影响因子
MICROCHIMICA ACTA
·
2011
·Vol. 175
·期 3
·355-359
·
IF 2.578
Lu Wenbo,Qin Xiaoyun,Luo Yonglan,Chang Guohui,Sun Xuping
We demonstrate that CdS quantum dots (QDs) can be applied to fluorescence-enhanced detection of nucleic acids in a two-step protocol. In step one, a fluorescently labeled single-stranded DNA probe is adsorbed on the QDs to quench its luminescence. In step two, the hybridization of the probe with its target ssDNA produces a double-stranded DNA which detaches from the QD. This, in turn, leads to the recovery of the fluorescence of the label. The lower detection limit of the assay is as low as 1 nM. The scheme (that was applied to detect a target DNA related to the HIV) is simple and can differentiate between perfectly complementary targets and mismatches.
CdS quantum dots (CdSQDs) can serve as an effective sensing platform for fluorescence-enhanced DNA detection. This sensing system has a detection limit of 1 nM and is capable of differentiating between complementary and mismatched sequences.
研究领域:
生物化学
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分子生物学
·
纳米技术
✓ 已验证
DOI
高影响因子
ANALYTICAL CHEMISTRY
·
2023
·
IF 7.4
Xiujuan Qiao,Zeng-Hui Qian,Wenpeng Sun,Chuan-Yong Zhu,Yanxin Li,Xiliang Luo
The construction of low-fouling biosensors for assaying biomarkers in complex biological samples remains a challenge, and the key limitation is the lack of effective anti-fouling materials. Inspired by the biomimetic process of protein phosphorylation, we herein designed a new phosphorylated peptide modified with the dihydrogen phosphate (−PO4H2) group, which significantly increased the hydrophilicity and anti-fouling capability of the peptide when compared with natural and normal peptides. Molecular simulation (MS) illustrated that, compared with the −COOH and −NH2 groups, the −PO4H2 group formed the most numbers of hydrogen bonds and stronger hydrogen bonds with water molecules. As a result, the PO4H2-oligopeptide was proved by MS to be able to attract the greatest number of water molecules, so as to form a compact layer of H2O to resist further adsorption of nonspecific biomolecules. The modification of electrodes with the designed PO4H2-oligopeptides, in addition to the adoption of neutral peptide nucleic acids (PNAs) as the sensing probes, ensured the fabrication of anti-fouling electrochemical biosensors capable of detecting nucleic acids in complex saliva. The constructed anti-fouling biosensor was able to detect the nucleic acid of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in undiluted saliva, with a wide linear response range (0.01 pM–0.01 μM) and a low limit of detection (LOD) of 3.4 fM (S/N = 3). The phosphorylation of oligopeptides offers an effective strategy to designing ultra-hydrophilic peptides suitable for the construction of promising anti-biofouling biosensors and bioelectronics.
研究领域:
生物传感器
·
分子生物学
·
生物医学工程
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2024
·
IF 5.7
Tianyi Zhang,Xiaoqin Wang,Yingtao Jiang,Yunyun Zhang,Shuhao Zhao,Jian Hu,Fei Hu,Niancai Peng
Background Nucleic acid testing is the most effective detection method currently available for the diagnosis of respiratory infectious diseases. However, the conventional real-time fluorescent quantitative PCR technique, which is regarded as the gold standard method for nucleic acid detection, presents significant challenges for implementation in home self-testing and popularization in underdeveloped regions due to its rigorous experimental standards. It is therefore clear that an easy-to-use, miniaturized nucleic acid testing technology and products for nonprofessionals are of great necessity to define the pathogens and assist in controlling disease transmission. (87) Results In this study, we propose a strategy for self-testing of respiratory pathogen nucleic acid that is oriented towards the public and user-friendly. The proposed system integrates the processes of extraction-free nucleic acid release, RPA isothermal amplification, and CRISPR fluorescence detection into a compact configuration. A microfluidic testing chip actuated by air pouches and a battery/USB-powered reusable device has been developed to enable simultaneous detection of internal reference genes and viral targets in a fully enclosed condition. The system allows for sample-in, and result-out testing in less than 30 minutes with a detection limit of 2 copies/μL. Additionally, a straightforward signal-light-based result display method has been developed to make it easy and intuitive for users to access the results. Furthermore, freeze-drying reagent is introduced to guarantee the storage and transportation of testing chips in ambient conditions. (135) Significance This work presents a miniaturized, portable, and highly sensitive nucleic acid detection system, where simple operating procedures have been designed for unskilled users. It is our belief that the testing system developed in this work is well suited for home-based self-testing and infection diagnosis in resource-limited areas, due to the above-mentioned advantages. (52)
研究领域:
分子生物学
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医学检验
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生物工程
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Infectious Medicine
·
2022
·
IF 0
Jinrong Wang,Guowei Song,Yue Ming,Jing Pan,Ruiqing Zhang,Guohao Fan,Xinxin Shen,Xuejun Ma,Lixin Li
Background Nucleic acid amplification enhancers suitable for use in a recombinase-aided amplification (RAA) assay were studied for the first time, and amplification of a long-fragment (509 bp) was initially explored. Methods Using recombinant plasmids and clinical samples, RAA fluorescence and basic methods were used to evaluate the efficacy. The fluorescence method was evaluated by threshold time and fluorescence value, and the basic method was characterized by 2% agarose gel electrophoresis. Results Taking a previously established RAA assay for HPV18 as an example, we demonstrated that the addition of 0.2 M, 0.4 M, and 0.6 M betaine and 10% pullulan could enhance the RAA. The new RAA assays with betaine and pullulan were named B-RAA and P-RAA, respectively. Using the B-RAA and P-RAA fluorescence methods, the threshold time values could be shortened by 1.72–2.32 minutes and 2.60 minutes, respectively, and the fluorescence values could be enhanced by 8847.25–9094.37 mv and 5250 mv, respectively. Using the basic method, the sensitivity could be increased 10-fold. We successfully amplified a long-fragment of 509 bp using a P-RAA assay with a sensitivity of 10 2 copies/µL (compared with 10 3 copies/µL in the RAA assay). Conclusions Thus, we concluded that betaine and pullulan are effective additives to enhance the sensitivity of RAA assays.
研究领域:
分子生物学
·
生物化学
·
临床医学
✓ 已验证
DOI
Cell Reports Physical Science
·
2023
·
IF 8.9
Xueqi Wang,Kai Sun,Han Wang
Nucleic acid biomarkers play important roles in early diagnosis, routine management, and prognosis of diseases. However, current nucleic acid testing (NAT) approaches either require cumbersome instruments or lack sensitivity for short detection time, making point-of-care (POC) NAT challenging. Here, we report an ultrasensitive carbon fiber paper (CFP)-based electrochemical biosensor with one-step branched DNA (bDNA)-based molecular signal amplification system for rapid, sensitive, and low-cost replication-free NAT. For nucleic acid quantification, the proposed biosensor exhibits a wide linear range of 10 aM–1 μM and a low limit of detection (LOD) of 1.4 aM. For fast semi-quantitative detection, the biosensor can evaluate the concentration of nucleic acids within 10 min with a threshold as low as 1 fM. Furthermore, the biosensor demonstrates negligible batch-to-batch variation with a relative standard deviation (RSD) of 4.25% at the cost of less than $1/device. The present one-step bDNA-based electrochemical biosensor serves as a promising and universal method for rapid, sensitive, and portable NAT.
研究领域:
生物医学工程
·
分子诊断学
✓ 已验证
DOI
高影响因子
LUMINESCENCE
·
2024
·Vol. 39
·期 1
·e4675
·
IF 3.2
Gerile Oudeng,Junguo Ni,Hao Wu,Honglian Wu,Mo Yang,Chunyi Wen,Yuanwei Wang,Hui Tan
The coronavirus disease-2019 pandemic reflects the underdevelopment of point-of-care diagnostic technology. Nuclei acid (NA) detection is the “gold standard” method for the early diagnosis of the B.1.1.529 (Omicron) variant of severe acute respiratory syndrome-coronavirus disease-2. Polymerase chain reaction is the main method for NA detection but requires considerable manpower and sample processing taking ≥ 3 h. To simplify the operation processes and reduce the detection time, exonuclease III (Exo III)-aided MoS 2 /AIE nanoprobes were developed for rapid and sensitive detection of the oligonucleotides of Omicron. Molybdenum disulfide (MoS 2 ) nanosheets with excellent optical absorbance and distinguishable affinity to single-strand and duplex DNAs were applied as quenchers, and aggregation-induced emission (AIE) molecules with high luminous efficiency were designed as donor in fluorescence resonance energy transfer-based nanoprobes. Exo III with catalytic capability was used for signal amplification to increase the sensitivity of detection. The composite nanoprobes detected the mutated nucleocapsid (N)-gene and spike (S)-gene oligonucleotides of Omicron within 40 min with a limit of detection of 4.7 pM, and showed great potential for application in community medicine.
研究领域:
生物医学工程
·
分子生物学
·
病毒学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2010
·
IF 5.214
Juan Hu,Chun-yang Zhang
Detection of specific DNA sequences is important to molecular biology research and clinical diagnostics. To improve the sensitivity of surface-enhanced Raman scattering spectroscopy (SERS), a variety of signal amplification methods has been developed, including Raman-active-dye, polymerase chain reaction (PCR) technology, molecular beacon, SERS-active substrates, and SERS-tag. However, the combination of rolling circle amplification (RCA) with SERS for nucleic acid detection has not been reported. Herein, we describe a new approach for nucleic acid detection by the combination of RCA reaction with SERS. Because of the binding of abundance repeated sequences of RCA products with gold nanoparticle (Au NP) and Rox-modified detection probes, SERS signal is significantly amplified and the detection limit of 10.0 pM might be achieved. The sensitivity of RCA-based SERS has increased by as much as 3 orders of magnitude as compared to PCR-based SERS and is also comparable with or even exceeds that of both RCA-based electrochemical and RCA-based fluorescent methods. This RCA-based SERS might discriminate perfect matched target DNA from 1-base mismatched DNA with high selectivity. The high sensitivity and selectivity of RCA-based SERS makes it a potential tool for early diagnosis of gene-related disease and also offers a great promise for multiplexed assays with DNA microarrays.
研究领域:
分子生物学
·
临床诊断学
✓ 已验证
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2014
·
IF 5.825
Yi Zhang,Lu Zhang,Jiashu Sun,Yulei Liu,Xingjie Ma,Shangjin Cui,Liying Ma,Jianzhong Jeff Xi,Xingyu Jiang
This report demonstrates a straightforward, robust, multiplexed and point-of-care microcapillary-based loop-mediated isothermal amplification (cLAMP) for assaying nucleic acids. This assay integrates capillaries (glass or plastic) to introduce and house sample/reagents, segments of water droplets to prevent contamination, pocket warmers to provide heat, and a hand-held flashlight for a visual readout of the fluorescent signal. The cLAMP system allows the simultaneous detection of two RNA targets of human immunodeficiency virus (HIV) from multiple plasma samples, and achieves a high sensitivity of two copies of standard plasmid. As few nucleic acid detection methods can be wholly independent of external power supply and equipment, our cLAMP holds great promise for point-of-care applications in resource-poor settings.
研究领域:
分子生物学
·
医学诊断
·
生物技术
✓ 已验证
DOI
PubMed
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
✓ 已验证
DOI
ANALYTICAL CHEMISTRY
·
2021
·
IF 6.986
Zhen Song,Yihui Ma,Min Chen,Adriano Ambrosi,Caifeng Ding,Xiliang Luo
Biofouling caused by the accumulation of biomolecules on sensing surfaces is one of the major problems and challenges to realize the practical application of electrochemical biosensors, and an effective way to counter this problem is the construction of antifouling biosensors. Herein, an antifouling electrochemical biosensor was constructed based on electropolymerized polyaniline (PANI) nanowires and newly designed peptides for the detection of the COVID-19 N-gene. The inverted Y-shaped peptides were designed with excellent antifouling properties and two anchoring branches, and their antifouling performances against proteins and complex biological media were investigated using different approaches. Based on the biotin–streptavidin affinity system, biotin-labeled probes specific to the N-gene (nucleocapsid phosphoprotein) of COVID-19 were immobilized onto the peptide-coated PANI nanowires, forming a highly sensitive and antifouling electrochemical sensing interface for the detection of COVID-19 nucleic acid. The antifouling genosensor demonstrated a wide linear range (10–14 to 10–9 M) and an exceptional low detection limit (3.5 fM). The remarkable performance of the genosensor derives from the high peak current of PANI, which is chosen as the sensing signal, and the extraordinary antifouling properties of designed peptides, which guarantee accurate detection in complex systems. These crucial features represent essential elements for future rapid and decentralized clinical testing.
研究领域:
生物医学工程
·
分子生物学
✓ 已验证
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）