Ultra-Fast Impedimetric Immunoassay for Detection of Streptococcus agalactiae Using Carbon Electrode with Nanodiamonds Film.

This publication presents the results of work on the development of a quick and cheap electrochemical immunosensor for the diagnosis of infections with the pathogen Streptococcus agalactiae. The research was carried out on the basis of the modification of the well-known glassy carbon (GC) electrodes. The surface of the GC (glassy carbon) electrode was covered with a film made of nanodiamonds, which increased the number of sites for the attachment of anti-Streptococcus agalactiae antibodies. The GC surface was activated with EDC/NHS (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-Hydroxysuccinimide). Determination of electrode characteristics after each modification step, performed using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS).

Low-volume label-free SARS-CoV-2 detection with the microcavity-based optical fiber sensor.

Accurate and fast detection of viruses is crucial for controlling outbreaks of many diseases; therefore, to date, numerous sensing systems for their detection have been studied. On top of the performance of these sensing systems, the availability of biorecognition elements specific to especially the new etiological agents is an additional fundamental challenge. Therefore, besides high sensitivity and selectivity, such advantages as the size of the sensor and possibly low volume of analyzed samples are also important, especially at the stage of evaluating the receptor-target interactions in the case of new etiological agents when typically, only tiny amounts of the receptor are available for testing. This work introduces a real-time, highly miniaturized sensing solution based on microcavity in-line Mach-Zehnder interferometer (µIMZI) induced in optical fiber for SARS-CoV-2 virus-like particles detection. The assay is designed to detect conserved regions of the SARS-CoV-2 viral particles in a sample with a volume as small as hundreds of picoliters, reaching the detection limit at the single ng per mL level.

Performance of electrochemical immunoassays for clinical diagnostics of SARS-CoV-2 based on selective nucleocapsid N protein detection: Boron-doped diamond, gold and glassy carbon evaluation.

The 21st century has already brought us a plethora of new threats related to viruses that emerge in humans after zoonotic transmission or drastically change their geographic distribution or prevalence. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first spotted at the end of 2019 to rapidly spread in southwest Asia and later cause a global pandemic, which paralyzes the world since then. We have designed novel immunosensors targeting conserved protein sequences of the N protein of SARS-CoV-2 based on lab-produced and purified anti-SARS-CoV-2 nucleocapsid antibodies that are densely grafted onto various surfaces (diamond/gold/glassy carbon). Titration of antibodies shows very strong reactions up to 1:72 900 dilution. Next, we showed the mechanism of interactions of our immunoassay with nucleocapsid N protein revealing molecular recognition by impedimetric measurements supported by hybrid modeling results with both density functional theory and molecular dynamics methods. Biosensors allowed for a fast (in less than 10 min) detection of SARS-CoV-2 virus with a limit of detection from 0.227 ng/ml through 0.334 ng/ml to 0.362 ng/ml for glassy carbon, boron-doped diamond, and gold surfaces, respectively. For all tested surfaces, we obtained a wide linear range of concentrations from 4.4 ng/ml to 4.4 pg/ml. Furthermore, our sensor leads to a highly specific response to SARS-CoV-2 clinical samples versus other upper respiratory tract viruses such as influenza, respiratory syncytial virus, or Epstein-Barr virus. All clinical samples were tested simultaneously on biosensors and real-time polymerase chain reactions.

Antibody Modified Gold Electrode as an Impedimetric Biosensor for the Detection of Streptococcus pyogenes.

Streptococcus pyogenes is a known cause of a wide spectrum of diseases, from mild and acute to severe invasive infections. This paper concerns the development of a novel impedimetric biosensor for the detection of the mentioned human pathogen. The proposed biosensor is a gold disk electrode modified with commercially available antibodies attached to the surface of the electrode by carbodiimide chemistry. The conducted tests confirmed the specificity of the antibodies used, which was also demonstrated by the results obtained during the detection of S. pyogenes using electrochemical impedance spectroscopy. The developed sensor successfully detected the presence of S. pyogenes in the sample and the detection limit was calculated as 9.3 cfu/mL. The results obtained show a wide linear range for verified concentrations of this pathogen in a sample from 4.2 × 102 to 4.2 × 106 cfu/mL. Furthermore, the optimal experimentally determined time required to perform pathogen detection in the sample was estimated as 3 min, and the test did not lead to the degradation of the sample.

Presenting Influenza A M2e Antigen on Recombinant Spores of Bacillus subtilis.

Effective vaccination against influenza virus infection is a serious problem mainly due to antigenic variability of the virus. Among many of investigated antigens, the extracellular domain of the M2 protein (M2e) features high homology in all strains of influenza A viruses and antibodies against M2e and is protective in animal models; this makes it a potential candidate for generation of a universal influenza vaccine. However, due to the low immunogenicity of the M2e, formulation of a vaccine based on this antigen requires some modification to induce effective immune responses. In this work we evaluated the possible use of Bacillus subtilis spores as a carrier of the Influenza A M2e antigen in mucosal vaccination. A tandem repeat of 4 consensus sequences coding for human-avian-swine-human M2e (M2eH-A-S-H) peptide was fused to spore coat proteins and stably exposed on the spore surface, as demonstrated by the immunostaining of intact, recombinant spores. Oral immunization of mice with recombinant endospores carrying M2eH-A-S-H elicited specific antibody production without the addition of adjuvants. Bacillus subtilis endospores can serve as influenza antigen carriers. Recombinant spores constructed in this work showed low immunogenicity although were able to induce antibody production. The System of influenza antigen administration presented in this work is attractive mainly due to the omitting time-consuming and cost-intensive immunogen production and purification. Therefore modification should be made to increase the immunogenicity of the presented system.

A system of vectors for Bacillus subtilis spore surface display.

BACKGROUND: Bacterial spores have been utilized as platforms for protein display. The best studied display systems are based on Bacillus subtilis spores in which several coat proteins have successfully been used as anchors for heterologous protein. Increasing knowledge about spore coat structure enables selection of new anchor proteins such as CotZ and CgeA. Here we describe a system of vectors for display of proteins on the surface of B. subtilis spores. RESULTS: We have designed and constructed a set of 16 vectors for ectopic integration which can be used for spore surface display of heterologous proteins. There is a selection of five coat proteins: CotB, CotC, CotG, CotZ and CgeA which can be used for construction of fusions. Three of these (CotB, CotC and CotG) enable obtaining N-terminal and C-terminal fusions and other two (CotZ and CgeA) are designed to produce C-terminal fusions only. All the vectors enable introduction of an additional peptide linker between anchor and displayed protein to enhance surface display. As a selection marker trophic genes are used. Additionally we describe an example application of presented vector system to display CagA protein of Helicobacter pylori in fusion with CgeA spore coat protein. CONCLUSIONS: Described system of vectors is a versatile tool for display of heterologous proteins on the surface of B. subtilis spores. Such recombinant spores can be further used as for example biocatalysts or antigen-carriers in vaccine formulations. The lack of antibiotic resistance genes in the system makes such spores an interesting option for applications in which a possible release to the environment can occur.