Quantification of receptor targeting aptamer binding characteristics using single‐molecule spectroscopy

Abstract

This experimental design presents a single molecule approach based on fluorescence correlation spectroscopy (FCS) for the quantification of outer membrane proteins which are receptors to an aptamer specifically designed to target the surface receptors of live Salmonella typhimurium. By using correlation analysis, we also show that it is possible to determine the associated binding kinetics of these aptamers on live single cells. Aptamers are specific oligonucleotides designed to recognize conserved sequences that bind to receptors with high affinity, and therefore can be integrated into selective biosensor platforms. In our experiments, aptamers were constructed to bind to outer membrane proteins of S. typhimurium and were assessed for specificity against Escherichia coli. By fluorescently labeling aptamer probes and applying FCS, we were able to study the diffusion dynamics of bound and unbound aptamers and compare them to determine the dissociation constants and receptor densities of the bacteria for each aptamer at single molecule sensitivity. The dissociation constants for these aptamer probes calculated from autocorrelation data were 0.1285 and 0.3772 nM and the respective receptor densities were 42.27 receptors per µm(2) and 49.82 receptors per µm(2). This study provides ample evidence that the number of surface receptors is sufficient for binding and that both aptamers have a high-binding affinity and can therefore be used in detection processes. The methods developed here are unique and can be generalized to examine surface binding kinetics and receptor quantification in live bacteria at single molecule sensitivity levels. The impact of this study is broad because our approach can provide a methodology for biosensor construction and calculation of live single cell receptor-ligand kinetics in a variety of environmental and biological applications.

Aptamers against pathogenic microorganisms

“…LOD is 0.2 CFU/ml in bacterial suspension.Zelada-Guillén et al (2009)  Mixture of OMPs of   S. typhimurium DNAAptamers, immobilized on magnetic beads, were applied for the capturing of the pathogen in biological samples with subsequent PCR detection. LOD is 10 CFU/g in biological samples.Book et al (2011), Joshi et al (2009); Jyoti et al (2011), and Singh et al (2012)  Electrochemical aptasensor. LOD is 3 CFU/ml in bacterial suspension.Ma et al (2014)   S. typhimurium whole   live cellsDNASandwich-type system with fluorescent detection.…”

“…These aptamers, immobilized on magnetic beads, were applied to capture the pathogen from biological samples with subsequent PCR detection; the detection limit of the system was 10 CFU/g of biological sample. Fluorescence correlation spectroscopy analysis of the binding affinity of aptamers 33 and 45 revealed that both aptamers form tight complexes with bacterial targets with sub-nanomolar K d (0.1285 nM and 0.3772 nM, respectively), and the receptor density for these aptamers is about 40 receptors per µm 2 (Book et al, 2011). Therefore, both aptamers were considered to be suitable for the development of detection systems.…”

Aptamer: A Novel Therapeutic Oligonucleotide

“…In that study, Aptamers were designed to bind to outer membrane proteins of S. typhimurium and were measured for specificity against Escherichia coli. Aptamer probes were fluorescently labeled and exposed to FCS so that they could study the diffusion dynamics of bound and unbound aptamers and could link them to determine the dissociation constants and receptor densities of the bacteria for each aptamer at very low concentration (at single molecule level) [35].…”

“…In that study, Aptamers were designed to bind to outer membrane proteins of S. typhimurium and were measured for specificity against Escherichia coli. Aptamer probes were fluorescently labeled and exposed to FCS so that they could study the diffusion dynamics of bound and unbound aptamers and could link them to determine the dissociation constants and receptor densities of the bacteria for each aptamer at very low concentration (at single molecule level) [35].Aptamers have properties of high specificity and affinity for a particular protein that can be used for protein purification (downstream processing). Histidine (His)-tagged proteins were separated using affinity chromatography by immobilizing Histagged aptamers on magnetic beads linked by covalent bonds.…”

Selective Nanoparticles in Microextraction

“…In this way, we can find electrochemical biosensors, based on CNTs (58) or gold electrodes (59) for the sensitive and robust determination of human α-thrombin with anti-thrombin aptamer, lysozime, (60) several small molecules, (61) or even whole cells in human blood. (62) Another versatile alternative is the light-based biosensors that include chemiluminescence, (63) fluorescence, (64) and colorimetric sensors. (65) Colorimetric biosensors have gained especial interest because of its simplicity: the results can be seen with naked eye without the need of an external apparatus.…”