Volume 27 Issue 3 - August 15, 2014 PDF
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An integrated microfluidic system for rapid screening of alpha-fetoprotein-specific aptamers
Chao-Jyun Huanga, Hsin-I Linb, Shu-Chu Shieshb,* , Gwo-Bin Leec
a Department of Engineering Science, National Cheng Kung University, Tainan 70101, Taiwan
b Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University, Tainan 70101, Taiwan
c Department of Power Mechanical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan
 
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The systematic evolution of ligands by exponential enrichment (SELEX) is a screening technique that involves the progressive selection of highly specific ligands via repeated rounds of partition and amplification from a large random pool of nucleic acid sequences. The products of this selection process are called aptamers and are either short single-stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid molecules with a high binding affinity to a large variety of target analytes. However, SELEX is a labor-intensive process requiring multiple rounds of extraction and polymerase chain reaction (PCR) amplification. In order to address these problems, this study presents a new integrated microfluidic system consisting of a magnetic bead-based microfluidic SELEX chip and a competitive assay chip to automate the aptamer screening process (Fig.1). This microfluidic system consists of a new microinjector to avoid the generation of air bubbles during the PCR reagent delivering process and an array-type microheater to improve the thermal uniformity inside the PCR reaction area which enhances the entire SELEX process. More importantly, the selected ssDNA sequences were determined to have a high affinity and specificity to the target molecules by using a new competitive assay chip. This assay chip demonstrated exceptional separation efficiency in removing weakly bound and unbound ssDNA. This facilitates the rapid identification of target-specific aptamers. With this approach, the subsequent cost of ssDNA sequencing, synthesis and surface plasmon resonance (SPR) detection can be greatly reduced. Experimental results showed that an aptamer specific to alpha-fetoprotein (AFP) was successfully selected. Furthermore, the detection of AFP by using this selected AFP-specific aptamer was also demonstrated. The selected aptamer was used as a recognition serum AFP and has a linear detection range 12.5 - 800 ng/mL.

Figure 1. (a-1) An exploded view of the microfluidic SELEX chip. It consisted of three PDMS layers for sample/reagent transport and one glass plate patterned with electrodes for nucleic acid amplification. (a-2) The layout of the microfluidic SELEX chip. (b-1) A schematic diagram of the competitive assay chip. The chips consisted of two PDMS substrates and one glass plate. (b-2) The layout of the competitive assay chip.
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