ANALYTICA CHIMICA ACTA
·
2023
·
IF 6.2
Ruimin Wang,Shuai Wang,Wanze Guo,Tiantian Zhang,Qing Kang,Pengcheng Wang,Feimeng Zhou,Lixia Yang
A thin-layer flow cell of low internal volume (12 μL) is incorporated in a flow injection analysis (FIA) system for simultaneous and real-time photoelectrochemical (PEC) immunoassay of anti-SARS-CoV-2 spike 1 (S1) and anti-SARS-CoV-2 nucleocapsid (N) antibodies. Covalent linkage of S1 and N proteins to two separate polyethylene glycol (PEG)-covered gold nanoparticles (AuNPs)/TiO 2 nanotube array (NTA) electrodes affords 10 consecutive analyses with surface regenerations in between. An indium tin oxide (ITO) allows visible light to impinge onto the two electrodes. The detection limits for anti-S1 and anti-N antibodies were estimated to be 177 and 97 ng mL −1 , respectively. Such values compare well with those achieved with other reported methods and satisfy the requirement for screening convalescent patients with low antibody levels. Additionally, our method exhibits excellent intra-batch (RSD = 1.3%), inter-batch (RSD = 3.4%), intra-day (RSD = 1.0%), and inter-day (RSD = 1.6%) reproducibility. The obviation of an enzyme label and continuous analysis markedly decreased the assay cost and duration, rendering this method cost-effective. The excellent anti-fouling property of PEG enables accuracy validation by comparing our PEC immunoassays of patient sera to those of ELISA . In addition, the simultaneous detection of two antibodies holds great potential in disease diagnosis and immunity studies.
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VACUUM
·
2024
·
IF 3.8
Xun Zhou,Xin Yi,Zhixian Liang,Jie Zhang,Guixiang Li,Yuanliang Tang,Kun Zhou,Shaohui Zhang
In this work, the photothermal effect of aminomodified MWCNTs/TiO 2 /SiO 2 /PDMS nanocomposites was utilized as a photothermal substrate to replace the metal heating block of traditional qPCR combining of the advantages of high photothermal effect of composite materials and low sample volume of static microfluidic PCR. Meanwhile, Multiple PCR rapid thermal cycles under 808 nm laser irradiation was accomplished through a non-contact energy conversion method and the use of chamber-immobilized PCR chips as specific reaction vessels. Notably, the chip chamber architecture requires merely about 8 μL of reagents compared to traditional PCR tubes. While reducing the amount of PCR reagents used, the higher specific surface area results in a larger contact surface (SSA) between the heat source and the solution. Thereafter, the CCD image sensor is then used as a fluorescence detector to monitor the amplification of the target DNA during the PCR reaction, thus enabling simultaneous fluorescence detection. In addition, the threshold cycle (Ct) value of qPCR and standard PCR curves for SARS-CoV-2 virus have been analysed in depth. Importantly, this study has important implications for the development of low-cost materials, miniaturized and portable Point-of-Care Testing (POCT) chips/systems.
研究领域:
生物技术
·
医学检测
·
材料科学
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ACS Applied Materials & Interfaces
·
2023
·
IF 9.5
Zhongjie Fei,Ping Liu,Chu Cheng,Rongbin Wei,Pengfeng Xiao,Youfa Zhang
Although numerous approaches were proposed for the nucleic acid (NA)-based SARS-CoV-2 detection, the nonideal NA desorption efficiency of conventional magnetic beads (MBs) limits their widespread application. In this study, we developed solvent-responsive MBs (called responsive MBs), which, in the presence of buffers, modulated the absorption and desorption capacities of NA by flipping the surface −COO–. Relative to other commercial MBs, responsive MBs exhibited similar absorption profiles and markedly enhanced desorption profiles. When applied for NA detection of complex samples, responsive MBs exhibited better performance of RNA detection than DNA, with obvious advantages in sensitivity. Specifically, the RNA and DNA desorption rates of commercial MBs were ∼85 and 82.5%, while those of responsive MBs were nearly 94 and 93.5%, respectively. Furthermore, responsive MBs exhibited remarkable extraction ability in a wide range of tissues and better performance of RNA extraction than DNA. When applied for SARS-CoV-2 detection, the responsive MBs along with the simulated digital RT-LAMP (a previously established apparatus) further improved detection efficiency, yielding a precise quantitative detection as low as 25 copies and an ultimate sensibility detection of 5 copies/mL. It was also successfully employed in numerous NA-based technologies such as polymerase chain reaction (PCR), sequencing, and so on.
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Frontiers in Microbiology
·
2020
·
IF 4.236
Cui Haoran,Zhang Leiliang
The global coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is one of seven human coronaviruses. G-quadruplexes are intrinsic obstacles to genome replication. Whether G-quadruplexes are present in human coronaviruses is unknown. In the current study, we have predicted that all seven human coronaviruses harbor G-quadruplex sequences. Conserved G-quadruplex sequences in SARS-CoV and SARS-CoV-2 were analyzed and verified by circular dichroism (CD) spectroscopy and Thioflavin T fluorescence assay. Similar to SARS-CoV, SARS-CoV-2 encodes an nsP3 protein, which is predicted to associate with G-quadruplexes. Targeting G-quadruplex sequences in the SARS-CoV-2 genome by G-quadruplex ligands could be a new way to conquer COVID-19.
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BRIEFINGS IN BIOINFORMATICS
·
2021
·Vol. 22
·期 2
·1378-1386
·
IF 11.622
Li Shasha,Liu Wenli,Chen Yangzhen,Wang Liqin,An Wenlin,An Xiaoping,Song Lihua,Tong Yigang,Fan Huahao,Lu Chenyang
Antiviral therapies targeting the pandemic coronavirus disease 2019 (COVID-19) are urgently required. We studied an already-approved botanical drug cepharanthine (CEP) in a cell culture model of GX_P2V, a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related virus. RNA-sequencing results showed the virus perturbed the expression of multiple genes including those associated with cellular stress responses such as endoplasmic reticulum (ER) stress and heat shock factor 1 (HSF1)-mediated heat shock response, of which heat shock response-related genes and pathways were at the core. CEP was potent to reverse most dysregulated genes and pathways in infected cells including ER stress/unfolded protein response and HSF1-mediated heat shock response. Additionally, single-cell transcriptomes also confirmed that genes of cellular stress responses and autophagy pathways were enriched in several peripheral blood mononuclear cells populations from COVID-19 patients. In summary, this study uncovered the transcriptome of a SARS-CoV-2-related coronavirus infection model and anti-viral activities of CEP, providing evidence for CEP as a promising therapeutic option for SARS-CoV-2 infection.
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Pharmaceuticals
·
2023
·Vol. 16
·期 5
·767
·
IF 4.6
Jie Chen,Xiang Zhou,Lifeng Fu,Haiyu Xu
Drugs that cure COVID-19 have been marketed; however, this disease continues to ravage the world without becoming extinct, and thus, drug discoveries are still relevant. Since Mprohas known advantages as a drug target, such as the conserved nature of the active site and the absence of homologous proteins in the body, it receives the attention of many researchers. Meanwhile, the role of traditional Chinese medicine (TCM) in the control of epidemics in China has also led to a focus on natural products, with the hope of finding some promising lead molecules through screening. In this study, we selected a commercial library of 2526 natural products from plants, animals and microorganisms with known biological activity for drug discovery, which had previously been reported for compound screening of the SARS CoV-2 S protein, but had not been tested on Mpro. This library contains compounds from a variety of Chinese herbs, includingLonicerae Japonicae Flos,Forsythiae FructusandScutellariae Radix, which are derived from traditional Chinese medicine prescriptions that have been shown to be effective against COVID-19. We used the conventional FRET method for the initial screening. After two rounds of selection, the remaining 86 compounds were divided into flavonoids, lipids, phenylpropanoids, phenols, quinones, alkaloids, terpenoids and steroids according to the skeleton structures, with inhibition rates greater than 70%. The top compounds in each group were selected to test the effective concentration ranges; the IC50values were as follows: (−)–gallocatechin gallate (1.522 ± 0.126 μM), ginkgolic acid C15:1 (9.352 ± 0.531 μM), hematoxylin (1.025 ± 0.042 μM), fraxetin (2.486 ± 0.178 μM), wedelolactone (1.003 ± 0.238 μM), hydroxytyrosol acetate (3.850 ± 0.576 μM), vanitiolide (2.837 ± 0.225 μM), β,β–dimethylacrylalkannin (2.731 ± 0.308 μM), melanin (7.373 ± 0.368 μM) and cholesteryl sodium sulfate (2.741 ± 0.234μM). In the next step, we employed two biophysical techniques, SPR and nanoDSF, to obtain KD/Kobsvalues: hematoxylin (0.7 μM), (−)–gallocatechin gallate (126 μM), ginkgolic acid C15:1 (227 μM), wedelolactone (0.9770 μM), β,β–dimethylacrylalkannin (1.9004 μM,), cholesteryl sodium sulfate (7.5950 μM) and melanin (11.5667 μM), which allowed better assessments of the binding levels. Here, seven compounds were the winners. Then, molecular docking experiments were specially performed by AutoDock Vina to analyze the mode of interactions within Mproand ligands. We finally formulated the present in silico study to predict pharmacokinetic parameters as well as drug-like properties, which is presumably the step that tells humans whether the compounds are drug-like or not. Moreover, hematoxylin, melanin, wedelolactone, β,β–dimethylacrylalkannin and cholesteryl sodium sulfate are in full compliance with the “Lipinski” principle and possess reasonable ADME/T properties, they have a greater potential of being lead compounds. The proposed five compounds are also the first to be found to have potential inhibitory effects on SARS CoV-2 Mpro. We hope that the results in this manuscript may serve as benchmarks for the above potentials.Keywords:SARS CoV-2 Mpro;natural products;high-throughput screening;lead compounds;in silico study
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Small
·
2023
·Vol. 19
·期 20
·2208167
·
IF 13.3
Mengya He,Xican Xu,Hongyu Wang,Qingyuan Wu,Linghao Zhang,Dongsheng Zhou,Yigang Tong,Xin Su,Huiyu Liu
Fluorescence-based PCR and other amplification methods have been used for SARS-CoV-2 diagnostics, however, it requires costly fluorescence detectors and probes limiting deploying large-scale screening. Here, a cut-price colorimetric method for SARS-CoV-2 RNA detection by iron manganese silicate nanozyme (IMSN) is established. IMSN catalyzes the oxidation of chromogenic substrates by its peroxidase (POD)-like activity, which is effectively inhibited by pyrophosphate ions (PPi). Due to the large number of PPi generated by amplification processes, SARS-CoV-2 RNA can be detected by a colorimetric readout visible to the naked eye, with the detection limit of 240 copies mL −1 . This conceptually new method has been successfully applied to correctly distinguish positive and negative oropharyngeal swab samples of COVID-19. Colorimetric assay provides a low-cost and instrumental-free solution for nucleic acid detection, which holds great potential for facilitating virus surveillance.
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PROTEIN EXPRESSION AND PURIFICATION
·
2022
·
IF 2.025
Yi Rong,Chaofeng Zhang,Wen-Chao Gao,Cheng Zhao
The main protease (M pro ) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a vital role in viral replication. To study the function of M pro and screen inhibitors targeting M pro , it is necessary to prepare high-purity and high-activity M pro . In this study, four types of SARS-CoV-2 M pro s containing different termini were prepared, and their activities were determined successfully. The results showed that the activity of wild-type (WT) M pro was the highest, and the additional residues at the N-terminus but not at the C-terminus had a major effect on the enzyme activity. To explain this, the alignment of structures of different forms of M pro was determined, and the additional residues at the N-terminus were found to interfere with the formation of the substrate binding pocket. This study confirms the importance of the natural N-terminus to the activity of M pro and suggests that WT-GPH 6 (M pro with eight additional residues at the C-terminus) can be used as a substitute for authentic M pro to screen inhibitors. In short, this study provides a reference for the expression and purification of new coronaviruses confronted in the future.
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TALANTA
·
2022
·
IF 6.556
Yaqin Zhang,Xiangyu Quan,Yingchun Li,Hangyu Guo,Fange Kong,Jiahui Lu,Lirong Teng,Jiasi Wang,Di Wang
The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic, highlighting the unprecedented demand for rapid and portable diagnostic methods. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) proteins-based platforms have been used for the detection of pathogens. However, in further applications and research, due to multiple steps needed, many methods showed an increased risk of cross-reactivity. The thermostable Cas12b enables the combination of isothermal amplification and CRISPR-mediated detection, which could decrease the risk of cross-contamination. In this study, we developed a portable and specific diagnostic method that combined the gold nanoparticle (AuNP) with thermal stable CRISPR/Cas12b-enhanced reverse transcription loop-mediated isothermal amplification (RT-LAMP), which is called SCAN, to distinguish the N gene of SARS-CoV-2 from flu gene. We validated our method using RNA from cells transfected by plasmids. We could easily distinguish the positive results by the naked eye based on the strong molar absorption coefficient of AuNP. Moreover, SCAN has the potential for high-throughput tests owing to its convenient operation. In sum, SCAN has broken the site and equipment restrictions of traditional detection methods and could be applied outside of hospitals and clinical laboratories, greatly expanding the test of COVID-19.
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NANO LETTERS
·
2021
·
IF 11.189
Hua Kang,Xuejun Wang,Mingquan Guo,Changhao Dai,Renzhong Chen,Lei Yang,Yanling Wu,Tianlei Ying,Zhaoqin Zhu,Dapeng Wei,Yunqi Liu,Dacheng Wei
The fast spread of SARS-CoV-2 has severely threatened the public health. Establishing a sensitive method for SARS-CoV-2 detection is of great significance to contain the worldwide pandemic. Here, we develop a graphene field-effect transistor (g-FET) biosensor and realize ultrasensitive SARS-CoV-2 antibody detection with a limit of detection (LoD) down to 10–18 M (equivalent to 10–16 g mL–1) level. The g-FETs are modified with spike S1 proteins, and the SARS-CoV-2 antibody biorecognition events occur in the vicinity of the graphene surface, yielding an LoD of ∼150 antibodies in 100 μL full serum, which is the lowest LoD value of antibody detection. The diagnoses time is down to 2 min for detecting clinical serum samples. As such, the g-FETs leverage rapid and precise SARS-CoV-2 screening and also hold great promise in prevention and control of other epidemic outbreaks in the future.
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BIOSENSORS & BIOELECTRONICS
·
2021
·
IF 10.618
Ying Peng,Yanhong Pan,Zhaowei Sun,Jinlong Li,Yongxiang Yi,Jie Yang,Genxi Li
The pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) is continuously worsening globally, herein we have proposed an electrochemical biosensor for the sensitive monitoring of SARS-CoV-2 RNA. The presence of target RNA firstly triggers the catalytic hairpin assembly circuit and then initiates terminal deoxynucleotidyl transferase-mediated DNA polymerization. Consequently, a large number of long single-stranded DNA products can be produced, and these negatively charged DNA products will bind a massive of positively charged electroactive molecular of Ru(NH 3 ) 6 3+ due to the electrostatic adsorption. Therefore, significantly amplified electrochemical signals can be generated for sensitive analysis of SARS-CoV-2 RNA in the range of 0.1–1000 pM with the detection limit as low as 26 fM. Besides the excellent distinguishing ability for SARS-CoV-2 RNA against single-base mismatched RNA, the proposed biosensor can also be successfully applied to complex matrices, as well as clinical patient samples with high stability, which shows great prospects of clinical application.
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ACS Applied Materials & Interfaces
·
2021
·
IF 9.229
Zhong Wei Jiang,Ting Ting Zhao,Chun Mei Li,Yuan Fang Li,Cheng Zhi Huang
A reliable and sensitive detection approach for SARS-CoV 2 is essential for timely infection diagnosis and transmission prevention. Here, a two-dimensional (2D) metal–organic framework (MOF)-based photoelectrochemical (PEC) aptasensor with high sensitivity and stability for SARS-CoV 2 spike glycoprotein (S protein) detection was developed. The PEC aptasensor was constructed by a plasmon-enhanced photoactive material (namely, Au NPs/Yb-TCPP) with a specific DNA aptamer against S protein. The Au NPs/Yb-TCPP fabricated by in situ growth of Au NPs on the surface of 2D Yb-TCPP nanosheets showed a high electron–hole (e–h) separation efficiency due to the enhancement effect of plasmon, resulting in excellent photoelectric performance. The modified DNA aptamer on the surface of Au NPs/Yb-TCPP can bind with S protein with high selectivity, thus decreasing the photocurrent of the system due to the high steric hindrance and low conductivity of the S protein. The established PEC aptasensor demonstrated a highly sensitive detection for S protein with a linear response range of 0.5–8 μg/mL with a detection limit of 72 ng/mL. This work presented a promising way for the detection of SARS-CoV 2, which may conduce to the impetus of clinic diagnostics.
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