Selection and Characterization of DNA Aptamers for Egg White Lysozyme

Abstract

We have selected aptamers binding to lysozyme from a DNA library using capillary electrophoresis-systematic evolution of ligands by exponential enrichment. During the selection process the dissociation constant of the ssDNA pool decreased from the micromolar to the low nanomolar range within five rounds of selection. The final aptamer had a dissociation constant of 2.8 ± 0.3 nM, 6.1 ± 0.5 nM, and 52.9 ± 9.1 nM as determined by fluorescence anisotropy, surface plasmon resonance and affinity capillary electrophoresis respectively. The aptamers were successfully challenged for specificity against other egg white proteins. The high affinity aptamers open up possibilities for the development of aptamer based food and medical diagnostics.

Advances in Aptamer-Based Biosensors for Food Safety

“…-- (Tran et al, 2010) DNA 3 nM Electrochemical (voltammetry) 0.5 µg/mL (Cheng et al, 2007) (Kirby et al, 2004) RNAFluorescence (microarray) 70 fM (Collett et al, 2005) Section 2.3 will describe these systems briefly, while three biosensor platforms targeting pathogens in food will be discussed in Section 2.4.…”

Aptamer-Based Electrochemical Sensing of Lysozyme

“…By using electrochemical impedance spectroscopy (EIS) on screen-printed carbon electrodes [63], it was demonstrated that the lowest detection limit and wider linear range can be achieved using the aptamer proposed by Tran et al [71]. More recent studies showed that using a mixture of two aptamer sequences with different affinities allows an improved control of the sensitivity and linear range of aptasensors [72].…”

“…Based on these results, Kirby et al [70] have developed a reusable bead-based electronic tongue sensor arrays of anti-lysozyme aptamers for the detection of proteins, where fluorescence labeling is involved. Using capillary electrophoresis-SELEX, a new lysozyme aptamer characterized by an order of magnitude increased affinity was selected (Table 1) [71]. Based on these new bioreceptors, electrochemical sensors emerged as alternative devices able to offer alternative lysozyme sensing strategies.…”

“…The main characteristics of the aptamers used in lysozyme biosensors are summarized in Table 1, starting from the first examples proposed by Ellington and co-workers [68,69], followed by Tran et al [71]. By using electrochemical impedance spectroscopy (EIS) on screen-printed carbon electrodes [63], it was demonstrated that the lowest detection limit and wider linear range can be achieved using the aptamer proposed by Tran et al [71].…”

“…Figure 5A shows one example of the direct detection of lysozyme on aptamer-modified interfaces using EIS [63]. The change in the electrical properties at the sensor/solution interface was exploited for a comparison of the analytical performances of two aptamers and quantitative detection with detection limits of 25 nM for the aptamer selected by Tran et al [71] and 100 nM for that reported by Cox and Ellington [68]. To assemble the aptasensors, screen-printed graphite electrodes were modified with diazonium salts produced in situ in order to introduce carboxylic groups on the electrode surface.…”