Three-dimensional (3-D) microfluidic-channel-based DNA biosensor for ultra-sensitive electrochemical detection Young Jo Kim a , John E. Jones a , Hao Li a , Helen Yampara-Iquise b , Guolu Zheng c , Charles A. Carson b , Michael Cooperstock d , Michael Sherman d , Qingsong Yu a,⇑ a Center for Surface Science and Plasma Technology, Department of Mechanical and Aerospace Engineering, University of Missouri, Columbia, MO 65211, USA b Department of Veterinary Pathobiology, University of Missouri, Columbia, MO 65211, USA c Cooperative Research Programs, Lincoln University in Missouri, Jefferson City, MO 65101, USA d Department of Child Health, University of Missouri, Columbia, MO 65211, USA article info Article history: Received 12 November 2012 Received in revised form 5 April 2013 Accepted 11 April 2013 Available online 1 May 2013 Keywords: Anodic aluminum oxide Microfluidic channel DNA sensor Cyclic voltammetry abstract A three-dimensional (3-D) microfluidic-channel-based electrochemical DNA biosensor was constructed using anodic aluminum oxide (AAO) filtration membranes as the designing templates by taking advan- tage of their well-defined cylindrical fluidic channels. By electroless gold plating and through thio chem- istry, single stranded DNA sequences with 19 base pairs specified to Bacteroides thetaiotaomicron were immobilized into the AAO fluidic channels to form a probe DNA electrode for electrochemical sensing. Cyclic voltammetry (CV) was used to measure the electrochemical response of the sensor using two dif- ferent redox indicators, [Fe(CN) 6 ] 4 and/or [Ru(NH 3 ) 6 ] 3 before and after hybridization with the comple- mentary target DNA sequences. The microfluidic-channel-based DNA biosensor showed detection sensitivity amplified to 10 19 M, which is one order of magnitude higher than attomolar (10 18 M) range. Such enhanced detection sensitivity is attributed to the well-defined fluidic channel structure that gives high surface area for probe DNA immobilization and enables target DNA solutions to pass through its flu- idic channels for rapid and efficient hybridization. Ó 2013 Elsevier B.V. All rights reserved. 1. Introduction Detection of DNA sequences is of particular importance and has wide applications in genetics, pathology, criminology, pharmaco- genetics, food and water safety, environmental monitoring, de- fense against bioterrorism, and many other fields. Detecting single strand DNA (ss-DNA) fragments by utilizing their hybridiza- tion with complementary sequences immobilized on a surface is at the heart of the DNA-based biosensors [1]. Various signal transduc- tion techniques, including fluorescence [2], surface-enhanced Ra- man [3], surface plasmon resonance [4], interferometric [5] etc., have been used for identification of DNA hybridization process. Currently, two of the most promising approaches in DNA biosensor development are optical biosensors and electrochemical biosen- sors. Optical biosensors utilizing fluorescence readout are highly sensitive with detection limits approaching 10 7 molecules/cm 2 , and arrays containing thousands of unique sequences have been constructed [6]. These fluorescence-based DNA biosensors require chemical DNA modification, labeling, expensive instrumentation and sophisticated numerical algorithms to interpret the data. These requirements limit their use to laboratories. In contrast, electrochemical DNA biosensors combine the ana- lytical power of electrochemical methods with the specificity of the nucleic acid recognition process via DNA hybridization. Since 1990s, extensive research conducted in this field has demonstrated the promise of developing sensitive electrochemical DNA biosen- sing devices [7]. Because the electrochemical redox reactions for DNA sequence recognition give an electric signal directly, electro- chemical DNA sensors use electronic transduction are character- ized by greater simplicity, inexpensive instrumentation, and ease of data processing. In other words, electrochemical DNA sensors provide fast, inexpensive, portable, efficient and simple nucleic acid assays without the need of expensive equipment and complex data reading and processing, and thus are well suited for rapid and direct detection of DNA sequences combining high sensitivity, small dimensions, low cost, and operation simplicity [8]. Electro- chemical DNA biosensors have been investigated and developed using various electrochemical techniques [9–13]. Electrochemical DNA biosensors rely on the immobilization of ss-DNA probe sequences. The most commonly used one-dimen- sional (1-D) probe DNA macroelectrode, such as that constructed on a flat Au surface, limits the distribution, packing density, and 1572-6657/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jelechem.2013.04.021 ⇑ Corresponding author. Address: E2403D Lafferre Hall, Department of Mechan- ical and Aerospace Engineering, University of Missouri, Columbia, MO 65211, USA. Tel.: +1 573 882 8076; fax: +1 573 884 5090. E-mail address: YuQ@missouri.edu (Q. Yu). Journal of Electroanalytical Chemistry 702 (2013) 72–78 Contents lists available at SciVerse ScienceDirect Journal of Electroanalytical Chemistry journal homepage: www.elsevier.com/locate/jelechem