ABSTRACT
Here, we present the results of our the electrochemical aptasensing strategy for retinol binding protein-4 (RBP-4) detection based on a thiolated aptamer against RBP-4 and 6-mercaptohexanol (MCH) directly immobilized on a gold electrode surface. The most important parameters affecting the magnitude of the analytical signal generated were optimized: (i) the presence of magnesium ions in the immobilization and measurement buffer, (ii) the concentration of aptamer in the immobilization solution and (iii) its folding procedure. In this work, a systematic assessment of the electrochemical parameters related to the optimization of the sensing layer of the aptasensor was carried out (electron transfer coefficients (α), electron transfer rate constants (k0) and surface coverage of the thiolated aptamer probe (ΓApt)). Then, under the optimized conditions, the analytical response towards RBP-4 protein, in the presence of an Fe(CN)63-/4- redox couple in the supporting solution was assessed. The proposed electrochemical strategy allowed for RBP-4 detection in the concentration range between 100 and 1000 ng/mL with a limit of detection equal to 44 ng/mL based on electrochemical impedance spectroscopy (EIS). The specificity studies against other diabetes biomarkers, including vaspin and adiponectin, proved the selectivity of the proposed platform. These preliminary results will be used in the next step to miniaturize and test the sensor in real samples.
Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , Metal Nanoparticles , Biosensing Techniques/methods , Aptamers, Nucleotide/chemistry , Dielectric Spectroscopy/methods , Oxidation-Reduction , Gold/chemistry , Electrodes , Retinol-Binding Proteins , Electrochemical Techniques/methods , Limit of Detection , Metal Nanoparticles/chemistryABSTRACT
Vitamin B12 conjugates are broadly studied in biological sciences. As light offers spatiotemporal control, we decided to develop a method for the preparation of vitamin B12 conjugates that release tethered molecules upon exposure to light. Herein, we report vitamin B12 derivatives possessing a photolabile linker suitable for conjugation with amines, azides, and alkynes. The potential applications of such conjugates are broad and include the delivery of drugs, labels, and imaging agents to their place of action and spatiotemporal release.
Subject(s)
Vitamin B 12/chemistry , Alkynes/chemistry , Amines/chemical synthesis , Azides/chemistry , Catalysis , Cycloaddition Reaction , Molecular Structure , Spatio-Temporal AnalysisABSTRACT
Specifically designed, antisense oligonucleotides are promising candidates for antibacterial drugs. They suppress the correct expression of bacterial genes by complementary binding to essential sequences of bacterial DNA or RNA. The main obstacle in fully utilizing their potential as therapeutic agents comes from the fact that bacteria do not uptake oligonucleotides from their environment. Herein, we report that vitamin B12 can transport oligonucleotides into Escherichia coli and Salmonella typhimurium cells. 5'-Aminocobalamin with an alkyne linker and azide-modified oligonucleotides enabled the synthesis of vitamin B12-2'OMeRNA conjugates using an efficient "click" methodology. Inhibition of protein expression in E. coli and S. Typhimurium cells indicates an unprecedented transport of 2'OMeRNA oligomers into bacterial cells via the vitamin B12 delivery pathway.
Subject(s)
Escherichia coli/metabolism , Oligonucleotides, Antisense/chemistry , Salmonella typhimurium/metabolism , Vitamin B 12/chemistry , Alkynes/chemistry , Azides/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Catalysis , Copper/chemistry , Escherichia coli/genetics , Oligonucleotides, Antisense/metabolism , RNA/antagonists & inhibitors , RNA/genetics , RNA/metabolism , Salmonella typhimurium/geneticsABSTRACT
Vitamin B12, a natural Co-complex, catalyzes atom transfer radical addition (ATRA) of organic halides to olefins. The established conditions were found to be very selective, with atom transfer radical polymerization (ATRP) occurring only in the case of acrylates.
