Aptamer-Based Viability Impedimetric Sensor for Bacteria

The development of an aptamer-based viability impedimetric sensor for bacteria (AptaVISens-B) is presented. Highly specific DNA aptamers to live <i>Salmonella typhimurium</i> were selected via the cell-systematic evolution of ligands by exponential enrichment (SELEX) technique. Twelve rounds of selection were performed; each comprises a positive selection step against viable <i>S. typhimurium</i> and a negative selection step against heat killed <i>S. typhimurium</i> and a mixture of related pathogens, including <i>Salmonella enteritidis</i>, <i>Escherichia coli</i>, <i>Staphylococcus aureus</i>, <i>Pseudomonas aeruginosa</i>, and <i>Citrobacter freundii</i> to ensure the species specificity of the selected aptamers. The DNA sequence showing the highest binding affinity to the bacteria was further integrated into an impedimetric sensor via self-assembly onto a gold nanoparticle-modified screen-printed carbon electrode (GNP-SPCE). Remarkably, this aptasensor is highly selective and can successfully detect <i>S. typhimurium</i> down to 600 CFU mL^–1 (equivalent to 18 live cells in 30 μL of assay volume) and distinguish it from other <i>Salmonella</i> species, including <i>S. enteritidis</i> and <i>S. choleraesuis</i>. This report is envisaged to open a new venue for the aptamer-based viability sensing of a variety of microorganisms, particularly viable but nonculturable (VBNC) bacteria, using a rapid, economic, and label-free electrochemical platform

Aptamer-Based Impedimetric Sensor for Bacterial Typing

The development of an aptamer-based impedimetric sensor for typing of bacteria (AIST-B) is presented. Highly specific DNA aptamers to <i>Salmonella enteritidis</i> were selected via Cell-SELEX technique. Twelve rounds of selection were performed; each comprises a positive selection step against <i>S. enteritidis</i> and a negative selection step against a mixture of related pathogens, including <i>Salmonella typhimurium</i>, <i>Escherichia coli</i>, <i>Staphylococcus aureus</i>, <i>Pseudomonas aeruginosa</i>, and <i>Citrobacter freundii</i>, to ensure the species-specificity of the selected aptamers. After sequencing of the pool showing the highest binding affinity to <i>S. enteritidis</i>, a DNA sequence of high affinity to the bacteria was integrated into an impedimetric sensor via self-assembly onto a gold nanoparticles-modified screen-printed carbon electrode (GNPs-SPCE). Remarkably, this aptasensor is highly selective and can successfully detect <i>S. enteritidis</i> down to 600 CFU mL^–1 (equivalent to 18 CFU in 30 μL assay volume) in 10 min and distinguish it from other Salmonella species, including <i>S. typhimurium</i> and <i>S. choleraesuis</i>. This report is envisaged to open a new venue for the aptamer-based typing of a variety of microorganisms using a rapid, economic, and label-free electrochemical platform

Anti-Fab Aptamers for Shielding Virus from Neutralizing Antibodies

Oncolytic viruses are promising therapeutics that can selectively replicate in and kill tumor cells. However, repetitive administration of viruses provokes the generation of neutralizing antibodies (nAbs) that can diminish their anticancer effect. In this work, we selected DNA aptamers against the antigen binding fragment (Fab) of antivesicular stomatitis virus polyclonal antibodies to shield the virus from nAbs and enhance its in vivo survival. For the first time, we used flow cytometry and electrochemical immunosensing to identify aptamers targeting the Fab region of antibodies. We evaluated the aptamers in a cell-based infection assay and found that several aptamer clones provide more than 50% shielding of VSV from nAbs and thus have the potential to enhance the delivery of VSV without compromising the patient’s immune system. In addition, we developed a bifunctional label-free electrochemical immunosensor for the quantitation of aptamer-mediated degree of shielding and the amount of vesicular stomatitis virus (VSV) particles. Electrochemical impedance spectroscopy was employed to interrogate the level of VSV in a linear range from 5 × 10⁴ to 5 × 10⁶ PFU mL^–1 with a detection limit of 10⁴ PFU mL^–1

Electrochemical Sensing of Aptamer-Facilitated Virus Immunoshielding

Oncolytic viruses (OVs) are promising therapeutics that selectively replicate in and kill tumor cells. However, repetitive administration of OVs provokes the generation of neutralizing antibodies (nAbs) that can diminish their anticancer effects. In this work, we selected DNA aptamers against an oncolytic virus, vesicular stomatitis virus (VSV), to protect it from nAbs. A label-free electrochemical aptasensor was used to evaluate the degree of protection (DoP). The aptasensor was fabricated by self-assembling a hybrid of a thiolated ssDNA primer and a VSV-specific aptamer. Electrochemical impedance spectroscopy was employed to quantitate VSV in the range of 800–2200 PFU and a detection limit of 600 PFU. The aptasensor was also utilized for evaluating binding affinities between VSV and aptamer pools/clones. An electrochemical displacement assay was performed in the presence of nAbs and DoP values were calculated for several VSV-aptamer pools/clones. A parallel flow cytometric analysis confirmed the electrochemical results. Finally, four VSV-specific aptamer clones, ZMYK-20, ZMYK-22, ZMYK-23, and ZMYK-28, showed the highest protective properties with dissociation constants of 17, 8, 20, and 13 nM, respectively. Another four sequences, ZMYK-1, -21, -25, and -29, exhibited high affinities to VSV without protecting it from nAbs and can be further utilized in sandwich assays. Thus, ZMYK-22, -23, and -28 have the potential to allow efficient delivery of VSV through the bloodstream without compromising the patient’s immune system

Electrochemical Differentiation of Epitope-Specific Aptamers

DNA aptamers are promising immunoshielding agents that could protect oncolytic viruses (OVs) from neutralizing antibodies (nAbs) and increase the efficiency of cancer treatment. In the present Article, we introduce a novel technology for electrochemical differentiation of epitope-specific aptamers (eDEA) without selecting aptamers against individual antigenic determinants. For this purpose, we selected DNA aptamers that can bind noncovalently to an intact oncolytic virus, vaccinia virus (VACV), which can selectively replicate in and kill only tumor cells. The aptamers were integrated as a recognition element into a multifunctional electrochemical aptasensor. The developed aptasensor was used for the linear quantification of the virus in the range of 500–3000 virus particles with a detection limit of 330 virions. Also, the aptasensor was employed to compare the binding affinities of aptamers to VACV and to estimate the degree of protection of VACV using the anti-L1R neutralizing antibody in a displacement assay fashion. Three anti-VACV aptamer clones, vac2, vac4, and vac6, showed the best immunoprotection results and can be applied for enhanced delivery of VACV. Another two sequences, vac5 and vac46, exhibited high affinities to VACV without shielding it from nAb and can be further utilized in sandwich bioassays

Aptamer-Based Viability Impedimetric Sensor for Viruses

The development of aptamer-based viability impedimetric sensor for viruses (AptaVISens-V) is presented. Highly specific DNA aptamers to intact vaccinia virus were selected using cell-SELEX technique and integrated into impedimetric sensors via self-assembly onto a gold microelectrode. Remarkably, this aptasensor is highly selective and can successfully detect viable vaccinia virus particles (down to 60 virions in a microliter) and distinguish them from nonviable viruses in a label-free electrochemical assay format. It also opens a new venue for the development of a variety of viability sensors for detection of many microorganisms and spores

Development of Bacteriostatic DNA Aptamers for Salmonella

<i>Salmonella</i> is one of the most dangerous and common food-borne pathogens. The overuse of antibiotics for disease prevention has led to the development of multidrug resistant <i>Salmonella</i>. Now, more than ever, there is a need for new antimicrobial drugs to combat these resistant bacteria. Aptamers have grown in popularity since their discovery, and their properties make them attractive candidates for therapeutic use. In this work, we describe the selection of highly specific DNA aptamers to <i>S. enteritidis</i> and <i>S. typhimurium</i>. To evolve species-specific aptamers, twelve rounds of selection to live <i>S. enteritidis</i> and <i>S. typhimurium</i> were performed, alternating with a negative selection against a mixture of related pathogens. Studies have shown that synthetic pools combined from individual aptamers have the capacity to inhibit growth of <i>S. enteritidis</i> and <i>S. typhimurium</i> in bacterial cultures; this was the result of a decrease in their membrane potential