A Simple and Fast SELEX Using an Alternating Current Potential Modulated Microfluidic Channel and an Evaluation of Sensing Ability of Aptamers

Abstract

The systematic evolution of ligands by exponential enrichment (SELEX) is a screening technique to generate new aptamers for target molecules. The aptamers of this selection process are short single-stranded deoxyribonucleic acid (ssDNA) with a high binding affinity to a target analytes [1,2]. To overcome lengthy, labor-intensive, iterative process requiring multiple rounds (at least 8 to 20 times) of selection procedures in conventional SELEX, a new alternating current (AC) potential modulated microfluidic SELEX method was successfully developed in this study. The an electrochemical potential modulated microchannel (EPMM) device for in vitro selection was designed and fabricated using a screen printing method as previously reported [3,4]. The EPMM consisted of the conducting polymer precursor mixed carbon layer, a glass substrate, and its glass cover. The conducting polymer precursor of the channel wall was electropolymerized. The EPMM for SELEX is modified by immobilization of Plasmodium vivax dehydrogenase(PvLDH) on the conducting polymer layer as shown in Fig. 1. The modification steps of the EPMM were investigated by impedance in 0.1 M phosphate buffer solution (PBS, pH 7.4). An AC potential was applied to interact the ssDNA library with the target proteins in a microfluidic channel. AC potential modulation in the channel causes oscillation and fluctuation of ssDNA library, so that it accelerates the reaction between ssDNA library and PvLDH of the channel wall. The five different kinds of ssDNAs (LDH1, LDH2, LDH3, LDH4, and LDH5) for PvLDH, as a biomarker for malaria, were successfully generated with high binding affinity and verified by recording amperometry. The migration times for the ssDNAs were 51.6, 69.4, 92.1, and 102.0 s. After only one SELEX round, the bound ssDNAs were extracted and amplified by the PCR in order to find out the structure of DNA. Followed by cloning and sequencing, secondary structure of aptamers was simulated by the m-fold software. The dissociation constant (Kd ) of LDH1 was determined using electrochemical impedance spectroscopy and fluorescence assays. Furthermore, the amine terminated aptamers were synthesized for the detection of PvLDH. The aptamers were immobilized on pTTBA layer of the micro array electrode (MAE). Electrochemical label-free aptasensor for PvLDH was utilized by the aptamer modified sensor at various concentrations in a range of 25.0 fM to 125.0 nM using impedance spectroscopy. A linear dynamic ranges were obtained in the concentration range of 25.0 fM to 2.0 pM (R = 0.985) and 10.0 to 125.0 pM, (R = 0.994) with a LOD = 29.2 fM. Therefore, it has been demonstrated that the aptamer for PvLDH can be exploited to detect PvLDH with a high sensitivity. Using this system, selection rounds can be reduced to one cycle, which is significantly faster than other SELEX methods. Furthermore, it doesn’t need any substantial materials for anchoring target molecules on the channel unlike other microfluidic SELEX methods because the PvLDH is directly fixed by a covalent bonding on the pTTBA of EPMM. This study demonstrated that newly developed AC potential modulated microfluidic channel SELEX process provides rapid and easy to select aptamers with high specificity. The proposed microfluidic SELEX method can be applied to select aptamers for other diseases.[1] Ellington, A.; Szostak, J.; et al., Nature, 1990, 346, 818–822.[2] Lee, S.; Song, K. -M.; et al., Biosens. Bioelectron., 2012, 35, 291–296.[3] Noh, H. -B.; Shim, Y. -B.; et al., Anal.Chem., 2012, 84, 9738–9744.[4] Noh, H. -B.; Shim, Y. -B.; et al., J. Mater. Chem. A, 2016, 4, 2720–3415. Figure 1