查找使用阿拉丁产品的科研文献

Aptamer-Based Activatable Tyramide Signal Amplification for Low-Background Detection of SARS-CoV-2 Nucleocapsid Protein

Zhiyong Huang,Ziyan Du,Juan Li,Da Han,Jiaxuan He,Yunben Yang,Dan Wang,Yu Liang,Yunshan Yang,Ruizi Peng,Weihong Tan

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection posed a significant threat to public health and the global economy, in vitro diagnosis of the SARS-CoV-2 nucleocapsid protein proved to be an effective way for SARS-CoV-2 infection control in the past years. Tyramide signal amplification (TSA) has been extensively utilized in tissue imaging and pathological diagnosis owing to the powerful signal enhancement. However, the elevated “ALWAYS ON” fluorescence background limited the accuracy and sensitivity of the conventional TSA assay. To achieve an activated “TURN ON” signal, herein, a small molecule, termed dichlorodihydrofluorescein tyramide (T-DCFH), was synthesized for activatable TSA. Under the catalysis of horseradish peroxidase (HRP) with hydrogen peroxide (H2O2), this T-DCFH facilitates the “TURN ON” fluorescence signal. Additionally, as a recognition tool, DNA aptamer has been used for developing in vitro diagnostic approaches. Hence, based on HRP-labeled aptamers binding with SARS-CoV-2 nucleocapsid protein, we achieved aptamers-based activatable TSA detection with a higher signal-to-noise ratio than that of fluorescent dye (FITC)-labeled aptamers, while showing lower background than traditional fluorescein tyramide with “ALWAYS ON”. The results demonstrated that the activated T-DCFH significantly enhances the fluorescence signal while diminishing the background noise. By employing multiple aptamers targeting, we offered a timely and accurate in vitro diagnostic approach for future emergent infectious diseases.

Rapid and Unamplified Detection of SARS-CoV-2 RNA via CRISPR-Cas13a-Modified Solution-Gated Graphene Transistors

Haiyang Yu,Huibin Zhang,Jinhua Li,Zheng Zhao,Minhua Deng,Zhanpeng Ren,Ziqin Li,Chenglong Xue,Mitch Guijun Li,Zhaowei Chen

The disease caused by severe acute respiratory syndrome coronavirus, SARS-CoV-2, is termed COVID-19. Even though COVID-19 has been out for more than two years, it is still causing a global pandemic. Due to the limitations of sample collection, transportation, and kit performance, the traditional reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method has a long detection period and high testing costs. An increased risk of infection is inevitable, since many patients may not be diagnosed in time. The CRISPR-Cas13a system can be designed for RNA identification and knockdown, as a promising platform for nucleic acid detection. Here, we designed a solution-gated graphene transistor (SGGT) biosensor based on the CRISPR-Cas13a system. Using the gene-targeting capacity of CRISPR-Cas13a and gate functionalization via multilayer modification, SARS-CoV-2 nucleic acid sequences can be quickly and precisely identified without the need for amplification or fluorescence tagging. The limit of detection (LOD) in both buffer and serum reached the aM level, and the reaction time was about 10 min. The results of the detection of COVID-19 clinical samples from throat swabs agree with RT-PCR. In addition, the interchangeable gates significantly minimize the cost and time of device fabrication. In a nutshell, our biosensor technology is broadly applicable and will be suitable for point-of-care (POC) testing.

Ultrasensitive detection of SARS-CoV-2 (2019-nCoV) S Protein RBD based on optical fiber plasmonic sensor

Qing Huang,Zefeng Hou,Yunong Zhao,Ruiqin Gu,Chengli Li,Minghong Yang,Huan Liu

Surface plasmon resonance (SPR) technology, boasting prominent advantages including anti-electromagnetic interference, high sensitivity, rapid response, label-free detection, and minimal sample consumption, holds broad application prospects in life sciences, clinical diagnostics, drug screening, and other related fields. However, current optical fiber biochemical sensors are generally plagued by poor probe practicality and stability, low refractive index sensitivity, and inadequate detection capability for ultra-low concentration biomolecules in clinical settings. In this study, we developed a low-cost, high-sensitivity optical fiber SPR biosensor for SARS-CoV-2 antigen detection in the standard sample, employing gold@silver‑platinum (Au@AgPt) core-shell nanospheres as the sensing enhancement module. Au@AgPt core-shell nanospheres possess unique localized surface plasmon resonance (LSPR) properties. When incident light interacts with the nanomaterial surface, it induces a localized electric field enhancement effect. This surface enhancement effect significantly amplifies the optical signal intensity, thereby effectively improving the sensor's detection sensitivity toward the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. This sensing platform achieves fg/mL-level detection sensitivity, excellent linearity, and rapid response performance, offering new technical support for expanding the application of optical fiber sensors in the quantitative detection of ultra-low concentration disease markers.

Enhanced immunogenicity induced by mRNA vaccines with various lipid nanoparticles as carriers for SARS-CoV-2 infection

Yanhao Zhang,Ji Wang,Hanlei Xing,Chao Liu,Wenhui Zha,Shuo Dong,Yuhao Jiang,Xinsong Li

mRNA vaccines have emerged as a highly promising approach for preventing cancer and infectious diseases, attributed to their superior immunogenicity, rapid development speed, and quality-controlled scale production. While homologous mRNA vaccine administration is currently the most prevalent method employed in clinical settings, heterologous administration is a promising avenue worth exploring. In this report, two types of mRNA vaccine formulations for SARS-CoV-2 infection were developed based on different lipid nanoparticle (LNP) delivery systems, and heterologous and homologous mRNA vaccinations were administered to explore the levels of immune responses comparatively. First, five novel H-series ionizable lipids were synthesized and confirmed by NMR and MS. Subsequently, six SARS-CoV-2 receptor-binding domain (RBD) mRNA-encapsulated LNP formulations were prepared using a microfluidic mixer based on H-series and MC3 lipids. These formulations exhibited spherical structures with an average diameter ranging from 90–140 nm, as characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The safety of these formulations was confirmed in vitro by the cytotoxicity assay. Moreover, transfection assay, lysosomal escape test, and western blot, and in vivo biodistribution analyses collectively demonstrated that lipids H03 and MC3 exhibited superior in vitro and in vivo delivery efficacy in comparison to other H-series lipids. Notably, H03-Fluc mRNA exhibited an approximately 2.2-fold higher in vivo bioluminescence signal intensity than MC3-Fluc mRNA. Additionally, evaluation of humoral immunity demonstrated that homologous H03-mRNA vaccination elicited an immune response that was approximately 3-fold higher than that of homologous MC3-mRNA vaccination. More significantly, the heterologous H03-mRNA/MC3-mRNA vaccination elicited an immune response that was approximately 2-3-fold higher than that of homologous H03-mRNA vaccination and 6–9-fold higher than that of homologous MC3-mRNA vaccination, without any observable adverse effects. These results suggest that heterologous mRNA vaccination is superior to homologous mRNA vaccination and may be attributed to differences in LNP carriers. Therefore, our research may inspire further exploration of different delivery systems to enhance mRNA-based therapeutics.

Capacitive Aptasensor Coupled with Microfluidic Enrichment for Real-Time Detection of Trace SARS-CoV-2 Nucleocapsid Protein

Haochen Qi,Zhiwen Hu,Zhongliang Yang,Jian Zhang,Jie Jayne Wu,Cheng Cheng,Chunchang Wang,Lei Zheng

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has lasted for almost 2 years. Stemming its spread has posed severe challenges for clinical virus detection. A long turnaround time, complicated operation, and low accuracy have become bottlenecks in developing detection techniques. Adopting a direct antigen detection strategy, we developed a fast-responding and quantitative capacitive aptasensor for ultratrace nucleocapsid protein detection based on a low-cost microelectrode array (MEA) chip. Employing the solid–liquid interface capacitance with a sensitivity of picofarad level, the tiny change on the MEA surface can be definitively detected. As a result, the limit of detection reaches an ultralow level of femtogram per milliliter in different matrices. Integrated with efficient microfluidic enrichment, the response time of this sensor from the sample to the result is shortened to 15 s, completely meeting the real-time detection demand. Moreover, the wide linear range of the sensor is from 10–5 to 10–2 ng/mL, and a high selectivity of 6369:1 is achieved. After application and evaluation in different environmental and body fluid matrices, this sensor and the detection method have proved to be a label-free, real-time, easy-to-operate, and specific strategy for SARS-CoV-2 screening and diagnosis.

Ratiometric fluorescence immunoassay of SARS-CoV-2 nucleocapsid protein via Si-FITC nanoprobe-based inner filter effect

Mao Guobin,Yang Yang,Cao Shijie,Ye Silu,Li Yifang,Zhao Wei,An Hongwei,Liu Yingxia,Dai Junbiao,Ma Yingxin

The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has necessitated rapid, easy-to-use, and accurate diagnostic methods to monitor the virus infection. Herein, a ratiometric fluorescence enzyme-linked immunosorbent assay (ELISA) was developed using Si-fluorescein isothiocyanate nanoparticles (FITC NPs) for detecting SARS-CoV-2 nucleocapsid (N) protein. Si-FITC NPs were prepared by a one-pot hydrothermal method using 3-aminopropyl triethoxysilane (APTES)-FITC as the Si source. This method did not need post-modification and avoided the reduction in quantum yield and stability. The p-nitrophenyl (pNP) produced by the alkaline phosphatase (ALP)-mediated hydrolysis of p-nitrophenyl phosphate (pNPP) could quench Si fluorescence in Si-FITC NPs via the inner filter effect. In ELISA, an immunocomplex was formed by the recognition of capture antibody/N protein/reporter antibody. ALP-linked secondary antibody bound to the reporter antibody and induced pNPP hydrolysis to specifically quench Si fluorescence in Si-FITC NPs. The change in fluorescence intensity ratio could be used for detecting N protein, with a wide linearity range (0.01–10.0 and 50–300 ng/mL) and low detection limit (0.002 ng/mL). The concentration of spiked SARS-CoV-2 N protein could be determined accurately in human serum. Moreover, this proposed method can accurately distinguish coronavirus disease 2019 (COVID-19) and non-COVID-19 patient samples. Therefore, this simple, sensitive, and accurate method can be applied for the early diagnosis of SARS-CoV-2 virus infection.

Lateral Flow Immunoassay Coupled with Copper Enhancement for Rapid and Sensitive SARS-CoV-2 Nucleocapsid Protein Detection

Tao Peng,Xueshima Jiao,Zhanwei Liang,Hongwei Zhao,Yang Zhao,Jie Xie,You Jiang,Xiaoping Yu,Xiang Fang,Xinhua Dai

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory coronavirus 2 (SARS-CoV-2) is still raging all over the world. Hence, the rapid and sensitive screening of the suspected population is in high demand. The nucleocapsid protein (NP) of SARS-CoV-2 has been selected as an ideal marker for viral antigen detection. This study describes a lateral flow immunoassay (LFIA) based on colloidal gold nanoparticles for rapid NP antigen detection, in which sensitivity was improved through copper deposition-induced signal amplification. The detection sensitivity of the developed LFIA for NP antigen detection (using certified reference materials) under the optimized parameters was 0.01 μg/mL and was promoted by three orders of magnitude to 10 pg/mL after copper deposition signal amplification. The LFIA coupled with the copper enhancement technique has many merits such as low cost, high efficiency, and high sensitivity. It provides an effective approach to the rapid screening, diagnosis, and monitoring of the suspected population in the COVID-19 outbreak.Keywords:SARS-CoV-2;nucleocapsid protein;signal amplification;copper deposition

Design and Evaluation of a Novel Peptide–Drug Conjugate Covalently Targeting SARS-CoV-2 Papain-like Protease

Na Liu,Yichi Zhang,Yingshou Lei,Rui Wang,Meimiao Zhan,Jianbo Liu,Yuhao An,Yaoqi Zhou,Jian Zhan,Feng Yin,Zigang Li

Coronavirus disease 2019 (COVID-19) pandemic, a global health threat, was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 papain-like cysteine protease (PLpro) was recognized as a promising drug target because of multiple functions in virus maturation and antiviral immune responses. Inhibitor GRL0617 occupied the interferon-stimulated gene 15 (ISG15) C-terminus-binding pocket and showed an effective antiviral inhibition. Here, we described a novel peptide–drug conjugate (PDC), in which GRL0617 was linked to a sulfonium-tethered peptide derived from PLpro-specific substrate LRGG. The EM-C and EC-M PDCs showed a promising in vitro IC50 of 7.40 ± 0.37 and 8.63 ± 0.55 μM, respectively. EC-M could covalently label PLpro active site C111 and display anti-ISGylation activities in cellular assays. The results represent the first attempt to design PDCs composed of stabilized peptide inhibitors and GRL0617 to inhibit PLpro. These novel PDCs provide promising opportunities for antiviral drug design.

Chemiluminescent Nanogels as Intensive and Stable Signal Probes for Fast Immunoassay of SARS-CoV-2 Nucleocapsid Protein

Fajin Qu,Jiangnan Shu,Shanshan Wang,Mohammad A. Haghighatbin,Hua Cui

It is highly desired to exploit good nanomaterials as nanocarriers for immobilizing chemiluminescence (CL) reagents, catalysts and antibodies to develop signal probes with intensive and stable CL properties for immunoassays. In this work, N-(4-aminobutyl)-N-ethylisoluminol (ABEI) and Co2+ bifunctionalized polymethylacrylic acid nanogels (PMAANGs-ABEI/Co2+) were synthesized via a facile strategy by utilizing carboxyl group-rich PMAANGs as nanocarriers to immobilize ABEI and Co2+. The obtained PMAANGs-ABEI/Co2+ showed extraordinary CL performance. The CL intensity is 2 orders of magnitude higher than that of previously reported ABEI and Cu2+–cysteine complex bifunctionalized gold nanoparticles with high CL efficiency. This was attributed to the excellent catalytic ability of Co2+ and polymethylacrylic acid nanogels, as well as the improved CL catalytic efficiency from a decreased spatial distance between ABEI and the catalyst. The as-prepared nanogels also possess abundant surface reaction sites and good CL stability. On this basis, a sandwich immunoassay for the nucleocapsid protein of SARS-CoV-2 (N protein) was developed by using magnetic bead connected primary antibody as a capture probe and PMAANGs-ABEI/Co2+ connected secondary antibody as a signal probe. The linear range of the proposed method for N protein detection was 3.16–316 ng/mL, and its detection limit was 2.19 ng/mL (S/N = 3). Moreover, the developed immunoassay was performed with a short incubation time of 5 min, which greatly reduced the detection time for N protein. By using corresponding antibodies, the developed strategy might be applied to detect other biomarkers.

Development of Ultrasensitive New Sensor Based on Functionalized Triangular Gold Nanosheets Layers for SARS-CoV-2 Detection

Bing Xu,Xiaoying Xiang,Zhihui Luo,Decheng Wang

Quercetin: A promising drug candidate against the potential SARS-CoV-2-Spike mutants with high viral infectivity

Boyu Pan,Senbiao Fang,Liangjiao Wang,Zhanyu Pan,Min Li,Liren Liu

The emergence of SARS-CoV-2-Spike mutants not only enhances viral infectivity but also lead to treatment failure. Gaining a comprehensive understanding of the molecular binding mode between the mutant SARS-CoV-2-Spike and human ACE2 receptor is crucial for therapeutic development against this virus. Building upon our previous predictions and verifications regarding heightened viral infectivity of six potential SARS-CoV-2-Spike mutants, this study aims to further investigate the potential disruption of the interaction between these mutants and ACE2 by quercetin, a Chinese herbal compound. Molecular docking and dynamics simulations results reveal that the binding sites of quercetin particularly enriched around a specific “cavity” at the interface of Spike/ACE2 complex, indicating a favorable region for quercetin to interfere with Spike/ACE2 interaction. Virus infection assay confirms that quercetin not only attenuates wild-type virus infectivity but also suppresses the infectivity of all six tested SARS-CoV-2-Spike mutants. Therefore, quercetin represents a promising therapeutic candidate against both wild-type and potential future variants of SARS-CoV-2 exhibiting high viral infectivity.

Combining hybrid nanoflowers with hybridization chain reaction for highly sensitive detection of SARS-CoV-2 nucleocapsid protein

Wen Yin,Ji Hu,Fang Chen,Li Zhu,Yingxin Ma,Nuo Wang,Hongping Wei,Hang Yang,Shan-Ho Chou,Jin He

Background COVID-19 (coronavirus disease 2019) pandemic has had enormous social and economic impacts so far. The nucleocapsid protein (N protein) is highly conserved and is a key antigenic marker for the diagnosis of early SARS-CoV-2 infection. Results In this study, the N protein was first captured by an aptamer (Aptamer 58) coupled to magnetic beads (MBs), which in turn were bound to another DNA sequence containing the aptamer (Aptamer 48-Initiator). After adding 5′-biotinylated hairpin DNA Amplifier 1 and Amplifier 2 with cohesive ends for complementary hybridization, the Initiator in the Aptamer 48-Initiator began to trigger the hybridization chain reaction (HCR), generating multiple biotin-labeled DNA concatamers. When incubated with synthetic streptavidin-invertase-Ca 3 (PO 4 ) 2 hybrid nanoflower (SICa), DNA concatamers could specifically bind to SICa through biotin-streptavidin interaction with high affinity. After adding sucrose, invertase in SICa hydrolyzed sucrose to glucose, whose concentration could be directly read with a portable glucometer, and its concentration was positively correlated with the amount of captured N protein. The method is highly sensitive with a detection limit as low as 1 pg/mL. Significance We believe this study provided a practical solution for the early detection of SARS-CoV-2 infection, and offered a new method for detecting other viruses through different target proteins.