In silico evolution with an in vitro system can facilitate the development of functional aptamers with high specificity and affinity. Although a general technique known as systematic evolution of ligand by exponential enrichment (SELEX) is an efficient method for aptamer selection, it sometimes fails to identify aptamers with sufficient binding properties. We have previously developed in silico maturation (ISM) to improve functions of aptamers based on genetic algorithms. ISM represents an intelligent exploitation of a random search within a defined sequence space to optimize aptamer sequences and improve their function of interest. Here we demonstrated a successful application of ISM of aptamers to simultaneously improve specificity and affinity for Streptococcus mutans with discovery of a core sequence, which was required to form a polymerized guanine quadruplex structure for target binding. We applied ISM to aptamers selected by whole-cell SELEX and identified an aptamer with up to 16-fold improvement in affinity compared to its parent aptamers, and specificity was increased to show 12-fold more binding to S. mutans than to Lactobacillus acidophilus. Furthermore, we demonstrated a specific flow-through detection of S. mutans at a concentration range of 1 × 10(5) -10(8)  CFU/mL using the evolved aptamer immobilized on gold colloids.

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We have investigated an electrochemical method of detecting foods that cause an allergic reaction. Rat basophilic leukaemia (RBL-1) cells were sensitized with serum from a rat that was allergic to wheat. A sample containing the protein fraction of a food was added to the cells and incubated. The cells were immobilized on a membrane filter and attached to a basal-plane pyrolytic graphite electrode. When a potential was applied in the range 0-1.0 V relative to a saturated calomel electrode, an anodic peak current appeared at around 0.33 V. This peak current, attributed to serotonin, increased with time, and the maximum current (0.5 microA) was obtained 20-25 min of incubation. The response of the RBL-1 cells was specific to the protein fraction of wheat. The peak current increased linearly with increasing protein concentration in the range of 0.01-0.5 micrograms ml-1. These results suggest that the concentration of the protein bringing about the allergic reaction can be determined by cyclic voltammetry within 25 min. This method is more sensitive than the conventional skin tests.

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