Quantum dot-based fluorescence-linked immunosorbent assay for the rapid detection of lomefloxacin in animal-derived foods.

Lomefloxacin (LMF), a third-generation fluoroquinolone antibacterial agent, is often used to treat bacterial and mycoplasma infections. However, due to its prolonged half-life and slow metabolism, it is prone to residues in animal-derived foods, posing a potential food safety risk. Therefore, it is particularly urgent and important to establish a method for detecting lomefloxacin. In this study, direct and indirect competitive fluorescence-linked immunosorbent assay (dc-FLISA and ic-FLISA) based on quantum dots (QDs) was established for the detection of LMF. As for dc-FLISA, the half-maximal inhibitory concentration (IC50) and limit of detection (LOD) were 0.84 ng/mL, 0.04 ng/mL, respectively, the detection ranges from 0.08 to 9.11 ng/mL. The IC50 and LOD of ic-FLISA were 0.43 ng/mL and 0.03 ng/mL, respectively, meanwhile the detection ranges from 0.05 to 3.49 ng/mL. The recoveries of dc-FLISA and ic-FLISA in animal-derived foods (milk, fish, chicken, and honey), ranged from 95.8% to 105.2% and from 96.3% to 103.4%, respectively, with the coefficients of variation less than 8%. These results suggest that the dc-FLISA and ic-FLISA methods, which are based on QD labelling, are highly sensitive and cost-effective, and can be effectively used to detect LMF in animal-derived foods.

NIR-II photothermal conversion and imaging based on a cocrystal containing twisted components.

On account of the scarcity of molecules with a satisfactory second near-infrared (NIR-II) response, the design of high-performance organic NIR photothermal materials has been limited. Herein, we investigate a cocrystal incorporating tetrathiafulvalene (TTF) and tetrachloroperylene dianhydride (TCPDA) components. A stable radical was generated through charge transfer from TTF to TCPDA, which exhibits strong and wide-ranging NIR-II absorption. The metal-free TTF-TCPDA cocrystal in this research shows high photothermal conversion capability under 1064 nm laser irradiation and clear photothermal imaging. The remarkable conversion ability-which is a result of twisted components in the cocrystal-has been demonstrated by analyses of single crystal X-ray diffraction, photoluminescence and femtosecond transient absorption spectroscopy as well as theoretical calculations. We have discovered that space charge separation and the ordered lattice in the TTF-TCPDA cocrystal suppress the radiative decay, while simultaneously strong intermolecular charge transfer enhances the non-radiative decay. The twisted TCPDA component induces rapid charge recombination, while the distorted configuration in TTF-TCPDA favors an internal non-radiative pathway. This research has provided a comprehensive understanding of the photothermal conversion mechanism and opened a new way for the design of advanced organic NIR-II photothermal materials.

Screening and identification of B cell epitope within the major capsid protein L1 of HPV 52, using monoclonal antibodies.

The L1 protein of Human papillomavirus (HPV), the main capsid protein, induces the formation of neutralizing antibodies. In this study, HPV52 L1 protein was induced to be expressed. Monoclonal antibody (mAb) 6A7 against L1 protein were screened by cell fusion techniques. Western Blot and immunofluorescence assay (IFA) demonstrated the specificity of the mAb. The L1 protein was truncated for prokaryotic expression (N1∼N7) and Dot-ELISA showed that 6A7 recognized N3 (aa 200-350). The immunodominant regions were truncated again for expression, with 6A7 recognizing N6 (aa 251-305). The N6 proteins were further truncated and then were constructed an four-segment eukaryotic expression vector. IFA showed that 6A7 could recognize amino acid 262-279. Amino acid 262-279 was selected to be truncated into short peptides P1 and P2. Finally, Peptide-ELISA and Dot-ELISA showed that the epitope regions of mAb 6A7 were amino acid 262-273. The mAbs with defined epitopes can lay the foundation for the analysis of antigenic epitope characteristics and promote the development of epitope peptide vaccines.

Development of a double-antibody sandwich enzyme-linked immunosorbent assay for rapid detection of VZV.

BACKGROUND: Varicella zoster virus (VZV) is the pathogen of varicella and herpes zoster, it is necessary to develop a rapid, sensitive and specific detection method for the prevention and control of related diseases. METHODS: We inserted the gB protein extracellular region gene (gB-ex, 1-2208 bp) of VZV into lentivirus vector, and then obtained the recombinant gB protein through mammalian expression system. BALB/c mice were immunized multiple times with purified gB protein as immunogen. Then four strains of high affinity monoclonal antibodies targeting gB protein were prepared by cell fusion technique. Monoclonal antibodies 5G4 and HRP-4E9 were selected as capture and detection antibodies respectively, and a double-antibody sandwich ELISA method was established for detection. RESULTS: The detection limit of the DAS-ELISA was 156 PFU/mL, and there was no cross-reaction with Herpes simplex virus-1/Herpes simplex virus-2/Pseudorabies virus. The coefficients of variation of intra-assay and inter-assay repeatability were less than 5%. CONCLUSIONS: In this study, a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was established for the detection of VZV. The assay has good sensitivity, specificity and repeatability, which provides strong technical support and product guarantee for the rapid clinical detection of VZV.

Label-free electrochemical immunosensor based on staphylococcal protein a and AgNPs-rGO-Nf for sensitive detection of virginiamycin M1.

Virginiamycin (VIR), a feed additive, is used to promote pig and poultry growth. However, it is hazardous to human health. This work described a label-free electrochemical immunosensor based on silver nanoparticles-reduced graphene oxide (AgNPs-rGO) nanocomposites and staphylococcal protein A (SPA) for the first time to directly detect the residual marker VIR M1. Good catalytic currents for oxygen reduction reaction were apparently obtained after the modification of nanocomposites on gold electrode. Nanocomposites were characterized using UV-Vis, X-ray diffraction (XRD) patterns, Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). SPA was targeted to immobilize VIR M1 monoclonal antibody (mAb) by binding to Fc region of antibody. The proposed immunosensor showed a wide linear range from 0.25 ng mL-1 to 100 ng mL-1, providing detection limit (LOD) of 0.18 ng mL-1 of VIR M1. Recovery rates ranged from 92.27% to 98.84%, and relative standard deviation (RSD) was not above 6.6%, indicating the immunosensor could detect VIR M1 in actual samples with high accuracy. The sensor showed good selectivity, reproducibility and stability and could be considered as a potential tool for detection of VIR M1 in feed and animal derived food.

Electrochemical immunosensor for ultrasensitive detection of human papillomaviruse type 16 L1 protein based on Ag@AuNPs-GO/SPA.

Human papillomaviruse type 16 (HPV16) is a high-risk serotype. As the main protective antigen protein, L1 protein is also the target protein for diagnosis. A simple label free electrochemical immunosensor (ECIS) was fabricated for ultrasensitive detection of HPV16 L1 protein in this work. Quasi-spherical Ag@Au core-shell nanoparticles on graphene oxide (Ag@AuNPs-GO) was developed as current response amplifier and characterized by UV-Vis Spectroscopy, Transmission Electron Microscopy and energy dispersive X-ray spectroscopy. Staphylococcal protein A was decorated on the modified electrode and utilized to immobilized the Fc portion of the monoclonal antibody specific for HPV16 L1 protein. Cyclic Voltammetry, Differential Pulse Voltammetry and Electrochemical Impedance Spectroscopy were used to verify the electrochemical performance and interfacial kinetic property. The increased concentration of HPV16 L1 protein led to slow electron transport and linearly decreased differential pulse voltammetry peak current with a detection limit of 0.002 ng mL-1 and a wide linear relationship in the range of 0.005-400 ng mL-1at a regression coefficient (R2) of 0.9948. Furthermore, this ECIS demonstrated acceptable accuracy with good reproducibility, stability and selectivity, suggesting a promising immunological strategy for HPV typing and early screening.

Electrochemical immunosensor nanoarchitectonics with the Ag-rGO nanocomposites for the detection of receptor-binding domain of SARS-CoV-2 spike protein.

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a grave threat to human life and health, it is essential to develop an efficient and sensitive detection method to identify infected individuals. This study described an electrode platform immunosensor to detect SARS-CoV-2-specific spike receptor-binding domain (RBD) protein based on a bare gold electrode modified with Ag-rGO nanocomposites and the biotin-streptavidin interaction system. The Ag-rGO nanocomposites was obtained by chemical synthesis and characterized by electrochemistry and scanning electron microscope (SEM). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to record the electrochemical signals in the electrode modification. The differential pulse voltammetry (DPV) results showed that the limit of detection (LOD) of the immunosensor was 7.2 fg mL-1 and the linear dynamic detection range was 0.015 ~ 158.5 pg mL-1. Furthermore, this sensitive immunosensor accurately detected RBD in artificial saliva with favorable stability, specificity, and reproducibility, indicating that it has the potential to be used as a practical method for the detection of SARS-CoV-2.

Identification of a novel B-cell epitope of the African swine fever virus p34 protein and development of an indirect ELISA for the detection of serum antibodies.

African swine fever (ASF) is a viral disease caused by the African swine fever virus that can be highly transmitted and lethal in domestic pigs. In the absence of a vaccine, effective diagnosis is critical for minimizing the virus's spread. In recent years, with the decline of African swine fever virus (ASFV) virulence, antibody detection has become an important means of detection. ASFV nucleocapsid protein p34 is a mature hydrolytic product of pp220, which is highly conserved and has a high content in the structural protein of the virus. Prokaryotic cells were chosen to generate highly active and high-yield p34 protein, which was then used as an antigen for producing mouse monoclonal antibodies. The B-cell epitope 202QKELDKLQT210, which was highly conserved and found on the surface of the p34 protein, was first identified by an anti-p34 monoclonal antibody utilizing the peptide scanning technique and visualized in helix. This supported the viability of p34 protein detection even further. In addition, we established an indirect ELISA assay based on p34 to detect ASFV antibodies. The coincidence rate of this method with commercially available kits was shown to be 97.83%. Sensitivity analysis revealed that it could be detected in serum dilution as low as 1:6400, and there was no cross-reaction with other prevalent porcine epidemic diseases classical swine fever virus (CSFV), foot-and-mouth disease virus (FMDV), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine circovirus 2 (PCV2). In summary, the established ELISA method and anti-P34 monoclonal antibody have demonstrated that the p34 protein has a promising application prospect for the detection of African swine fever antibodies.

A Universal Fluorescent Immunochromatography Assay Based on Quantum Dot Nanoparticles for the Rapid Detection of Specific Antibodies against SARS-CoV-2 Nucleocapsid Protein.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the pathogenic agent leading to COVID-19. Due to high speed of transmission and mutation rates, universal diagnosis and appropriate prevention are still urgently needed. The nucleocapsid protein of SARS-CoV-2 is considered more conserved than spike proteins and is abundant during the virus' life cycle, making it suitable for diagnostic applications. Here, we designed and developed a fluorescent immunochromatography assay (FICA) for the rapid detection of SARS-CoV-2-specific antibodies using ZnCdSe/ZnS QDs-conjugated nucleocapsid (N) proteins as probes. The nucleocapsid protein was expressed in E.coli and purified via Ni-NTA affinity chromatography with considerable concentration (0.762 mg/mL) and a purity of more than 90%, which could bind to specific antibodies and the complex could be captured by Staphylococcal protein A (SPA) with fluorescence displayed. After the optimization of coupling and detecting conditions, the limit of detection was determined to be 1:1.024 × 105 with an IgG concentration of 48.84 ng/mL with good specificity shown to antibodies against other zoonotic coronaviruses and respiratory infection-related viruses (n = 5). The universal fluorescent immunochromatography assay simplified operation processes in one step, which could be used for the point of care detection of SARS-CoV-2-specific antibodies. Moreover, it was also considered as an efficient tool for the serological screening of potential susceptible animals and for monitoring the expansion of virus host ranges.

Polarization-Dependent Ultrasensitive Dynamic Wrinkling on Floating Films Induced by Photo-Orientation of Azopolymer.

Ubiquitous surface wrinkling has been well-studied theoretically and experimentally. How to modulate the stress state of a liquid-supported system for the unexploited wrinkling capabilities remains a challenge. Here we report a simple linearly-polarized-light illumination to spatiotemporally trigger ultrasensitive in situ dynamic wrinkling on a floating azo-film. The smart combination of the liquid substrate with photoresponsive azo-moieties leads to the light-induced ultrafast wrinkling evolution, accompanied by unprecedented sequential wrinkling orientation conversion (from polarization-parallel to polarization-perpendicular). The involved different polarization-dependent sequential photo-orientation for azo side chains and azo-grafted main chains of azopolymers is disclosed experimentally for the first time. Meanwhile, programmable dynamic wrinkling with all-optical switchable surface topographies is available, which has wide application potentials in photoresponsive soft photonics.

KIZ/GM114 Balances the NF-ĸB Signaling by Antagonizing the Association of TRAF2/6 With Their Upstream Adaptors.

NF-κB signaling is a pivotal regulator of the inflammatory response and it must be tightly controlled to avoid an excessive inflammatory response that may lead to human chronic inflammatory and autoimmune diseases. Thus, how NF-κB signaling is precisely controlled is a long-standing question in the field. TRAF family proteins function as key adaptors to mediate NF-κB signaling induced by various receptors. Here, we characterize KIZ/GM114 as a negative regulator balancing the NF-κB signaling. Mechanistically, KIZ/GM114 binds TRAF6/2 by targeting the TRAF domains to antagonize the TRAF6-IRAK1 association or the TRAF2-TRADD association, consequently reducing the IL-1ß/LPS/TNFα-induced NF-κB activation. Importantly, upon dextran sulfate sodium treatment, Gm114 deficiency induces a stronger inflammatory response, more severe acute colitis and lower survival rate in mice compared with control mice. Collectively, our study not only identifies KIZ/GM114 as a negative regulator to balance the NF-κB signaling, but it also implies a new strategy for limiting excessive inflammatory response.

Corrigendum: ZDHHC11 Positively Regulates NF-κB Activation by Enhancing TRAF6 Oligomerization.

[This corrects the article DOI: 10.3389/fcell.2021.710967.].

ZDHHC11 Positively Regulates NF-κB Activation by Enhancing TRAF6 Oligomerization.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a RING domain ubiquitin ligase that plays an important role in nuclear factor-κB (NF-κB) signaling by regulating activation of the TAK1 and IKK complexes. However, the molecular mechanisms that regulate TRAF6 E3 activity remain unclear. Here, we found that ZDHHC11, a member of the DHHC palmitoyl transferase family, functions as a positive modulator in NF-κB signaling. ZDHHC11 overexpression activated NF-κB, whereas ZDHHC11 deficiency impaired NF-κB activity stimulated by IL-1ß, LPS, and DNA virus infection. Furthermore, Zdhhc11 knockout mice had a lower level of serum IL6 upon treatment with LPS and D-galactosamine or HSV-1 infection than control mice. Mechanistically, ZDHHC11 interacted with TRAF6 and then enhanced TRAF6 oligomerization, which increased E3 activity of TRAF6 for synthesis of K63-linked ubiquitination chains. Collectively, our study indicates that ZDHHC11 positively regulates NF-κB signaling by promoting TRAF6 oligomerization and ligase activity, subsequently activating TAK1 and IKK complexes.

Carbon Nanotubes and Polydopamine Modified Poly(dimethylsiloxane) Sponges for Efficient Oil-Water Separation.

In this paper, effective separation of oil from both immiscible oil-water mixtures and oil-in-water (O/W) emulsions are achieved by using poly(dimethylsiloxane)-based (PDMS-based) composite sponges. A modified hard template method using citric acid monohydrate as the hard template and dissolving it in ethanol is proposed to prepare PDMS sponge composited with carbon nanotubes (CNTs) both in the matrix and the surface. The introduction of CNTs endows the composite sponge with enhanced comprehensive properties including hydrophobicity, absorption capacity, and mechanical strength than the pure PDMS. We demonstrate the successful application of CNT-PDMS composite in efficient removal of oil from immiscible oil-water mixtures within not only a bath absorption, but also continuous separation for both static and turbulent flow conditions. This notable characteristic of the CNT-PDMS sponge enables it as a potential candidate for large-scale industrial oil-water separation. Furthermore, a polydopamine (PDA) modified CNT-PDMS is developed here, which firstly realizes the separation of O/W emulsion without continuous squeezing of the sponge. The combined superhydrophilic and superoleophilic property of PDA/CNT-PDMS is assumed to be critical in the spontaneously demulsification process.

Ionic Hydrogen-Bonded Organic Frameworks for Ion-Responsive Antimicrobial Membranes.

Functionalization of hydrogen-bonded organic frameworks (HOFs) for specific applications has been a long-lasting challenge in HOF materials. Here, an efficient way to integrate functional species in the HOF structure through constructing an anionic framework is presented. The obtained HOFs, taking PFC-33 (PFC = porous materials from FJIRSM,CAS) as an example, integrate a porphyrin photosensitizer as a porous backbone and a commercial biocide as counterions in the structure. The permanent channels and the electrostatic interaction between the framework and the counterions provide PFC-33 ion-responsive biocide-release behavior in various physiological environments, thus exhibiting synergistic photodynamic and chemical antimicrobial efficiency. The unbonded carboxyl groups residing on the HOF surface further allow for manipulating the interfacial interaction between the PFC-33 and the polymer matrix for membrane fabrication. Therefore, a polyHOF membrane with high stability, desired flexibility, and good permeability is obtained, which demonstrates noticeable bacterial inhibition toward Escherichia coli. This study may shed light on the functionalization of HOF materials for broad application potentials.

Path-Guided Hierarchical Surface Relief Gratings on Azo-Films Induced by Polarized Light Illumination through Surface-Wrinkling Phase Mask.

Surface relief gratings (SRGs) with hierarchical microstructures are highly needed owing to their diverse applications in various fields. Here, we introduce surface-wrinkling templates as novel nonlithographic phase masks to direct the generation of hierarchical well-prescribed SRGs on nonconformally contacted azo-films by a simple single-beam illumination. The light-induced SRGs have controlled microstructures including single/double/triple wavelengths and single/double orientations as well as their organizations. These microstructures can be well tailored by the wavelength of the surface-wrinkling phase masks and the polarization direction of incident light relative to the wrinkling patterns in the phase masks. Interestingly, we find that the larger wavelength is induced prior to the smaller ones, offering another new strategy to tailor the microstructures of SRGs through simple manipulation of the illumination duration. In particular, path-guided SRGs with unprecedented well-organized hierarchical microstructures have been available in the case of controlled moving of the light illumination through the surface-wrinkling phase mask. As demonstrated, the obtained hierarchical SRGs with the capability of multiple optical inscription/erasure have great application potentials in fields such as confidential information (or pattern) records and encryption/decryption.

All-Optical Reversible Azo-Based Wrinkling Patterns with High Aspect Ratio and Polarization-Independent Orientation for Light-Responsive Soft Photonics.

Azobenzene-containing polymers (azopolymers) can serve as building blocks for an emerging class of soft photonics. Using their photoresponses for the micro/nanofabrication of smart surface is a key but still a challenging step. Here, we report a simple visible-light-illumination strategy to trigger diverse configurations of surface wrinkling on azopolymer-based film/substrate systems, which can be switched between flat and wrinkled states by controlling the intensity of the incident light. Different photoresponsive characteristics of azobenzene are involved in driving the wrinkling/dewrinkling switch. For the first time, we achieve the controlled wrinkling with an unexpected high aspect ratio and surprisingly polarization-independent ordered orientation by exploiting the unique photosoftening effect of azobenzene. Theoretical analysis reveals that an in situ photoinduced reversible soft/hard-contrast boundary determines the wrinkling orientation, which is used to fabricate diverse on-demand hierarchical wrinkles. These photoresponsive systems find broad photonic applications that are not easily accessible to other systems, e.g., optically reversible smart display, information security, and well-regulated optical devices.

Parkin negatively regulates the antiviral signaling pathway by targeting TRAF3 for degradation.

Chronic neuroinflammation is a characteristic of Parkinson's disease (PD). Previous investigations have shown that Parkin gene mutations are related to the early-onset recessive form of PD and isolated juvenile-onset PD. Further, Parkin plays important roles in mitochondrial quality control and cytokine-induced cell death. However, whether Parkin regulates other cellular events is still largely unknown. In this study, we performed overexpression and knockout experiments and found that Parkin negatively regulates antiviral immune responses against RNA and DNA viruses. Mechanistically, we show that Parkin interacts with tumor necrosis factor receptor-associated factor 3 (TRAF3) to regulate stability of TRAF3 protein by promoting Lys48-linked ubiquitination. Our findings suggest that Parkin plays a novel role in innate immune signaling by targeting TRAF3 for degradation and maintaining the balance of innate antiviral immunity.