(388f) Evaluation of Aptamer Technology for Detection of Quorum Sensing Molecules Produced By Pseudomonas Aeruginosa

Aptamers are single-stranded DNA molecules that selectively form a complex with cognate targets by folding into three-dimensional structures. They are becoming increasingly popular as therapeutic agents, but their potential as diagnostic agents remains untapped. Zao et. al., 2013 evaluated eight aptamer sequences, which inhibit biofilm formation of Pseudomonas aeruginosa by binding to quorum sensing molecules (QSMs) produced by the bacteria. They identified that ALSAP-5 aptamer had a high affinity (K_D ~20 nM – 35 nM) for a QSM called 3-oxo-dodecanoyl L-homoserine lactone (3O-C12-HSL). Binding of ALSAP-5 to 3O-C12-HSL arrested the growth of P. aeruginosa in LB cultures. In this study, we further evaluate the binding affinity of ALSAP-5 for 3O-C12-HSL using three orthogonal biosensing techniques viz. Surface Plasmon Resonance (SPR), Quartz Crystalline Microbalance (QCM), and Electrochemistry with the intent to evaluate the performance of aptamer-based diagnostic technologies.

A high affinity binding chemistry of streptavidin-biotin (K_D ~ 10¹⁴ M) was exploited for attaching the aptamer onto the gold sensor surface. For both SPR and QCM, thiolated biotin (biotin-SH) was bound to the gold sensor surface by gold-thiol bond. Streptavidin was then bound to the biotin surface. Subsequently, ALSAP-5 aptamer was flowed through the microfluidics on to the sensor surface where it bound to streptavidin using the biotin attached at its 5’ end. The binding of biomolecules on the gold sensor surface causes change of the refractive index in SPR, which is measured as a change in resonance angle. Similarly, binding of biomolecules on the gold coating of the quartz crystal sensor results in a reduction in the oscillation frequency of quartz in QCM.

For electrochemical sensing, the 5’ and 3’ ends of ALSAP-5 were functionalized with a thiol group and methylene blue, respectively. ALSAP-5 was immobilized on the surface of the gold working electrode using gold-thiol bond formation between gold surface and thiol group at the 5’ end of the aptamer. Electrochemical sensing using square wave voltammetry is based on principle of change in current produced by the redox active molecule methylene blue on the 3’ end of the aptamer upon binding of 3O-C12-HSL to the aptamer surface. Binding of 3O-C12-HSL to ALSAP-5 results in a conformational change in aptamer, thereby changing the interactions between the methylene blue and the gold surface, which results in a concentration dependent current change.

Aptamer and 3O-C12-HSL concentrations from 100 nM – 10 µM were tested, and linearity, accuracy and precision, limit of quantitation (LoQ), and limit of detection (LoD) were determined for all three test methods. The results obtained using all three biosensing techniques were comparable.