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Cholesterol drives E-protein depth redistribution via a matrix-mediated helix tilt adjustment. • Escin-induced peptide localization shifts strictly require the presence of charged DMPS lipids. • Escin is hypothesized to modulate the viroporin conformation via localized helix kinking rather than matrix disruption. • Escin is able to extract cholesterol from lipid membrane. β-Escin is a well-known saponin derived from the horse chestnut tree. Saponins, including escin, exhibit a wide range of biological activities, among which membrane-modifying activity is particularly notable. The latter is key to understanding the molecular mechanisms underlying escin's biological effects, including its antiviral properties. For example, altering membrane properties can strongly influence the life cycle of enveloped viruses such as SARS-CoV-2 by modulating the activity of its envelope protein (E protein). In the present paper, we studied the localization of both escin and the E protein transmembrane domain (ETM) in model membranes with different lipid compositions. We demonstrated that escin can alter ETM localization in membranes containing the negatively charged lipid DMPS, and that escin is also capable of extracting cholesterol from model lipid membranes. Download: Download high-res image (423KB) Download: Download full-size image
Objective To evaluate the synergistic immunological protection of exosomal T-cell epitope vaccine and antibody-inducing vaccine against SARS-CoV-2 in highly humanized mice. Methods Red blood cell-derived exosomes were loaded with 27 CD8 + T-cell epitope peptides and 19 CD4 + T-cell epitope peptides followed by combined immunization with S1 protein vaccine of SARS-CoV-2 and adjuvant poly I:C in HLA-A2/DR1 double transgenic mice. After immunizations, splenocytes were assessed for epitope-specific T cell responses by intracellular cytokine staining and ELISPOT, and for functional T cell subset analysis through flow cytometry. Meanwhile, serum anti-S1 protein IgG and neutralizing antibodies were quantified via ELISA and BA.5 pseudovirus neutralization assay, respectively. Furthermore, viral challenge was performed after the combined immunization in HLA-A2/DR1/hACE2 triple transgenic mice, followed by viral load quantification, viral protein detection, and H&E staining in lungs. Results The combined immunization i) increased the titers of S1 protein-specific IgG antibodies and neutralizing antibodies as well as Tfh cell frequency as compared to the S1 protein vaccine alone; ii) induced significantly more S1 protein-specific T cells and effector memory CD4 + T cells, and inhibited T cell exhaustion and regulatory T cell differentiation, compared to the exosomal T-cell epitope vaccine alone; iii) achieved the lowest pulmonary viral loads, inflammatory cell infiltration, and histopathological damage after SARS-CoV-2 infection. Conclusion This study, for the first time, demonstrates the synergistic humoral and cellular immune responses and protective efficacy induced by the combined immunization of exosomal T-cell epitope vaccine and antibody-inducing vaccine, and provides preclinical evidence from highly humanized mice for optimizing next-generation SARS-CoV-2 vaccine protocols.
Sensitive and anti-interference detection of targeted signal(s) in body fluids is one of the paramount tasks in biosensing. Overcoming the complication and high cost of antibody/aptamer-modification, surface-enhanced Raman spectroscopy (SERS) based on antibody/aptamer-free (AAF) substrates has shown great promise, yet with rather limited detection sensitivity. Herein, we report ultrasensitive and anti-interference detection of SARS-CoV-2 spike protein in untreated saliva by an AAF SERS substrate, applying the evanescent field induced by the high-order waveguide modes of well-defined nanorods for SERS for the first time. A detection limit of 3.6 × 10 −17 M and 1.6 × 10 −16 M are obtained in phosphate buffered saline and untreated saliva, respectively; the detection limits are three orders of magnitude improved than the best records from AAF substrates. This work unlocks an exciting path to design AAF SERS substrates for ultrasensitive biosensing, not limited to detection of viral antigens.
The SARS-CoV-2 pandemic has posed a huge challenge to rapid and accurate diagnosis of SARS-CoV-2 in the early stage of infection. In this work, we developed a novel magnetic/fluorescent dual-modal lateral flow immunoassay (LFIA) based on multifunctional nanobeads for rapid and accurate determination of SARS-CoV-2 nucleocapsid protein (NP). The multifunctional nanobeads were fabricated by using polyethyleneimine (PEI) as a mediate shell to combine superparamagnetic Fe 3 O 4 core with dual quantum dot shells (MagDQD). The core-shell structure of MagDQD label with high loading density of quantum dots (QDs) and superior magnetic content realized LFIA with dual quantitative analysis modal from the assemblies of individual single nanoparticles. The LFIA integrated the advantages of magnetic signal and fluorescent signal, resulting excellent accuracy for quantitative analysis and high elasticity of the overall detection. In addition, magnetic signal and fluorescent signal both had high sensitivity with the limit of detection (LOD) as 0.235 ng mL −1 and 0.012 ng mL −1 , respectively. The recovery rates of the methods in simulated saliva samples were 91.36%–103.60% (magnetic signal) and 94.39%–104.38% (fluorescent signal). The results indicate the method has a considerable potential to be an effective tool for diagnose SARS-CoV-2 in the early stage of infection.
Understanding the dynamic changes in antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for evaluating the effectiveness of the vaccine and the stage for the recovery of the COVID-19 disease. A rapid and accurate method for the detection of SARS-CoV-2-specific antibodies is still urgently needed. Here, we developed a novel fluorescent lateral flow immunoassay (LFA) platform for the detection of SARS-CoV-2-specific IgM and IgG by the aggregation-induced emission carbon dots conjugated with the SARS-CoV-2 spike protein (SSP). The aggregation-induced emission carbon dots (AIE-CDs) are one of the best prospect fluorescent probe materials for exhibiting high emission efficiency in both aggregate and solid states. The AIE-CDs were synthesized and displayed dual fluorescence emission, which provides a new perspective for the design of a high sensitivity testing system. In this work, the novel LFA platform adopted the AIE carbon dots, which are used to detect SARS-CoV-2-specific IgM and IgG conveniently. Furthermore, this sensor had a low LOD of 100 pg/ml. Therefore, this newly developed strategy has potential applications in the areas of public health for the advancement of clinical research.
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-COV-2) is a cluster of β coronaviruses. The 2019 coronavirus disease (COVID-19) caused by SARS-COV-2 is emerging as a global pandemic. Thus, early diagnosis of SARS-COV-2 is essential to prevent severe outbreaks of the disease. In this experiment, a novel label-free photoelectrochemical (PEC) immunosensor was obtained based on silver sulfide (Ag 2 S) sensitized titanium dioxide@bismuth tungstate (TiO 2 @Bi 2 WO 6 ) nanocomposite for quantitative detection of SARS-COV-2 nucleocapsid protein. The constructed TiO 2 @Bi 2 WO 6 hollow microspheres had large specific surface area and could produce high photocurrent intensity under visible light illumination. Ag 2 S was in-situ grown on the surface of thioglycolic acid (TGA) modified TiO 2 @Bi 2 WO 6 . In particular, TiO 2 @Bi 2 WO 6 and Ag 2 S formed a good energy level match, which could effectively enhance the photocurrent conversion efficiency and strength the photocurrent response. Ascorbic acid (AA) acted as an effective electron donor to effectively eliminate photogenerated holes. Under optimal experimental conditions, the constructed immunosensor presented a supersensitive response to SARS-COV-2 nucleocapsid protein, with a desirable linear relationship ranged from 0.001 to 50 ng/mL for nucleocapsid protein and a lower detection limit of 0.38 pg/mL. The fabricated sensor exhibited a wide linear range, excellent selectivity, specificity and stability, which provided a valuable referential idea for the detection of SARS-COV-2.
Quick and highly sensitive detection of the SARS-CoV-2 virus can efficiently reduce the possibility of viral spreading. In this study, a differential-phase surface plasmon resonance (SPR) biosensor is constructed to directly detect the SARS-CoV-2 virus. The SARS-CoV-2 spike protein antibodies are applied as bioprobes and functionalized on Au film, forming a sensing surface. A numerical analysis of the phase shifts of the SPR interferogram, termed the index of phase shifts (IoPS), is presented, which does not depend on a reference beam or an s-component beam. The IoPS can sensitively reflect small changes in the refractive index (as low as 3.75 × 10−10 RIU) when NaCl solution is used as an analyte. The limit of detection (LOD) of the proposed SPR biosensor in detecting the SARS-CoV-2 S protein is 1 ag mL−1. In comparison with a SARS-CoV-2 negative control (NC) sample collected from healthy people, which has an IoPS of ΔϕNC = 39°, the SPR biosensor shows a very strong response to the SARS-CoV-2 virus collected from infected people with a phase shift of ΔϕIoPS = 566°. To further improve the detection sensitivity, a sandwich structure is designed by applying SARS-CoV-2 spike protein antibody functionalized gold nanoparticles (anti-S@AuNPs). This reduces the LOD for the detection of the SARS-CoV-2 S protein to 0.1 ag mL−1.
Three-metal-core-shell nanoparticles (Au@Pt@PdNPs) providing excellent peroxidase-like activity were applied in lateral flow immunoassay (LFIA), designated as Au@Pt@Pd-LFIA, for detecting the nucleocapsid protein (NP) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). An Au@Pt@Pd-LFIA was developed for quantitatively testing of SARS-CoV-2 NP with a range 0.12–31.25 ng/mL. The limit of detection (LOD) of Au@Pt@Pd-LFIA strip was 0.06 ng/mL, which was 16-fold or eightfold more sensitive than that of the gold lateral flow immunoassay (Au-LFIA) and the gold flower flow immunoassay (AF-LFIA) strips, respectively. For detection of clinical samples from nasal swabs using test strips, Au@Pt@Pd-LFIA had 84.09% sensitivity, 100% specificity, and 92.55% accuracy. In terms of detection time, the testing of Au@Pt@Pd-LFIA strip was 16 min similar to Au-LFIA (15 min) and AF-LFIA (10 min), but much shorter than ELISA (2 h). In conclusion, Au@Pt@Pd-LFIA is a sensitive, rapid, and simple test for quantitative detection of SARS-CoV-2 NP in nasal swab samples. Graphical Abstract
Electrochemical biosensors have shown promise in bioanalysis and diagnosis. However, they suffer from the accumulation and adhesion of biomolecules in biofluids on sensing surfaces, while the hiring of potential antifouling is limited by the cost and complexity of the construction methods. Here, an economical three-dimensional (3D) conductive antifouling coating was prepared. This coating was composed of bovine serum albumin (BSA) crosslinked glutaraldehyde (GA) and copper-gold nanowires (CuAu NWs) with excellent electrical conductivity. Furthermore, a dual-targeted aptamer (D-Apt), with a “3-in-1″ structure of two specific binding sequences and a DNA linker, was designed to specifically recognize BSA and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein, affording low nanomolar binding affinity. Importantly, the introduction of the D-Apt greatly simplified the functionalization procedure on the electrode surface and the immobilization of the biorecognition element. Then, a simple electrochemical biosensor based on the CuAu/BSA/GA and D-Apt was constructed for the detection of S protein. This sensor exhibited merits of cost-effectiveness, sensitivity and practicality for detection of S protein. The work could provide a facile avenue to construct electrochemical biosensors with low-cost antifouling for the accurate diagnosis of epidemic antigenic disease markers.
Background The COVID-19 pandemic, caused by the novel coronavirus, has had a profound impact on global health and economies worldwide. This unprecedented crisis has affected individuals, communities, and nations in diverse manners. Developing simple and accurate diagnostic methods is an imperative task for frequent testing to mitigate the spread of the virus. Among these methods, SARS-CoV-2 antigen tests in clinical specimens have emerged as a promising diagnostic method for COVID-19 due to their sensitive and accurate detection of spike (S) protein, which plays a crucial role in viral infection initiation. Results In this work, a dual-signal amplification surface enhanced Raman scattering (SERS)-based S protein biosensor was constructed based on Au NPs/COFs and enzyme-free catalytic hairpin assembly (CHA) amplification method. The approach relies on a released free DNA sequence (T), which is generated from the competition reaction between Aptamer/T and Aptamer/S protein, to trigger a CHA reaction. Due to the high binding affinity and selectivity between the S protein and its aptamer, CHA process was triggered with the maximum SERS tags (H2-conjugated Au@4-mercaptobenzonitrile@Ag) anchored onto Au NPs/COFs substrate surface. This SERS platform could detect the S protein at concentrations with high sensitivity (limit of detection = 3.0 × 10 −16 g/mL), wide detection range (1 × 10 −16 to 1 × 10 −11 g/mL), acceptable reproducibility (relative standard deviation = 7.01 %) and excellent specificity. The biosensor was also employed to detect S protein in artificial human salivas. Significance Thus, this study not only developed a novel Au NPs/COFs substrate exhibiting strong SERS enhancement ability and high reproducibility, but also proposed a promising dual-signal amplification SERS-based diagnostic method for COVID-19, holding immense potential for the detection of a wide range of antigens and infectious diseases in future applications.
In this work, we propose a Ti 3 C 2 T x -based electrochemiluminescence (ECL) sensing platform, combined with a programmable entropy-driven cycling strategy and inverted DNA tetrahedral nanomachines to detect the SARS-CoV-2 RdRp gene. The use of Ti 3 C 2 T x in the ECL sensing platform provides unique advantages that improve the assay's sensitivity. Firstly, the SARS-CoV-2 RdRp gene triggers an entropy-driven cyclic amplification reaction to obtain a large number of Output strands. Subsequently, the Output strand reacts with an inverted DNA tetrahedral swing arm strand to start the self-driven process. Finally, the ferrocene (Fc)-labeled hairpin DNA strand of the inverted DNA tetrahedron is severed to achieve the signal change. Under optimal conditions, the ECL platform integrated with entropy-driven cycle amplification reaction and inverted DNA tetrahedral nanomachines obtained a wide range of 100 aM to 10 nM and a limit of detection (LOD) of 48.3 aM for the SARS-CoV-2 RdRp gene. Moreover, the recovery rates of 97% to 103.3% and 98.34% to 103% were obtained in real environments and human pharyngeal swabs, respectively. This ECL sensing platform, which combines an entropy-driven cycle amplification reaction and inverted DNA tetrahedral nanomachines, opens up new opportunities for the detection of other substances.
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