Contents lists available at ScienceDirect Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios Automated DNA hybridization transfer with movable super-paramagnetic microbeads in a microflow reactor Robert Penchovsky Department of Genetics, Faculty of Biology, Sofia University “St. Kliment Ohridski”, 8 Dragan Tzankov Blvd., 1164, Sofia, Bulgaria ARTICLE INFO Keywords: Microfluidics Super-paramagnetic beads DNA hybridization under flow conditions DNA selection under isothermal conditions Fluorescent DNA detection ABSTRACT An automated DNA hybridization transfer in a microflow reactor is demonstrated by moving paramagnetic beads between two spatially separate solutions with different pH values. The microbeads-based microfluidic platform is fully automated and programmable. It employs a robust chemical procedure for specific DNA hybridization transfer in microfluidic devices under isothermal conditions based on reversible pH alterations. The method takes advantage of high-speed DNA hybridization and denaturation on beads under flow conditions, high fidelity of DNA hybridization, and small sample volumes. The microfluidic platform presented is saleable and applicable to many areas of modern biotechnology such as DNA hybridization chip microarrays, molecular computation, on-chip selection of functional nucleic acids, high-throughput screening of chemical libraries for drug discovery, and DNA amplification and sequencing. 1. Introduction Micro-and-nano-fluidics have been used for the development of various lab-on-a-chip platforms for many different applications in modern biotechnology. Such applications include real-time detection of gene expression (Yan et al., 2016), microRNA analysis (Shamsi et al., 2016), cyanobacteria identification (Olcer et al., 2015), single cell ar- rays (Zhao et al., 2016), DNA sequencing and SNP analyses (Penchovsky, 2013b), PCR-based amplification (Pekin and Taly, 2017), (Yu et al., 2017) virus detection (Na et al., 2018), and many others. Therefore, it is important to engineer various microfluidic devices that can be used as functional elements in building complex microfluidic platforms. Here we demonstrate the application of one microfluidic module for automated DNA selection by hybridization and denatura- tion on movable paramagnetic microbeads under isothermal condi- tions. We applied already established a chemical procedure for re- versible pH alterations for multiple DNA selections on beads placed in cascade microchambers (Penchovsky and McCaskill, 2002) (Penchovsky, 2013b). Here, we employ different microfluidic design using switching paramagnetic beads between microchannels with hy- bridization or denaturation solutions. The method is fully automated, programmable, and reusable since the beads can be moved, im- mobilized with different DNA oligomers, and replaced automatically into the microreactor. It takes advantages of small reaction volume needed, fast DNA hybridization kinetics on beads under flow condi- tions, the instant DNA denaturation by NaCl under flow conditions (Penchovsky, 2013b). The approach is applicable to many biotechno- logical applications, including DNA detection, selection, and amplifi- cation. It can be integrated into complex microfluidic designs for var- ious applications such as DNA/RNA computing (Penchovsky and Ackermann, 2003), (Penchovsky and Breaker, 2005), (Penchovsky, 2012), molecular diagnostics (Penchovsky, 2012), (Penchovsky, 2013b), selection of functional DNA molecules (Penchovsky, 2014), and drug discovery (Blount et al., 2006), (Penchovsky and Stoilova, 2013), (Penchovsky, 2013a), (Penchovsky and Traykovska, 2015). The appli- cation of DNA selection under isothermal conditions can allow us to integrate more selection modules on a single-wafer than that using a temperature gradient because silica is a very good thermo-conductor. 2. Materials and Methods 2.1. Synthetic DNA oligomers, DNA immobilization to super-paramagnetic beads, and PCR amplification The deoxyoligonucleotides were obtained from IBA-NAPS (Göttingen, Germany). All of them were purified by HPLC. Carboxyl- coated PVA beads, diameter = 15 μm ± 3, with a high magnetic con- tent (50%), were purchased from Chemagen (Baesweiler, Germany). The beads were essentially monodispersed. Amino-modified (5′-end) deoxyoligonucleotides at a concentration of 10 μM were immobilized to the carboxyl-coated beads in the presence of 50 mM EDAC (Ethylene Di Amid Carbodiimide), 100 mM MES buffer, pH 6.1, and 100 mM NaCl in https://doi.org/10.1016/j.bios.2019.04.014 Received 16 September 2018; Received in revised form 24 March 2019; Accepted 6 April 2019 E-mail address: robert.penchovsky@hotmail.com. Biosensors and Bioelectronics 135 (2019) 30–35 Available online 11 April 2019 0956-5663/ © 2019 Elsevier B.V. All rights reserved. T