Biosensors and Bioelectronics 21 (2006) 1443–1450 Two-dimensional micro-bubble actuator array to enhance the efficiency of molecular beacon based DNA micro-biosensors Peigang Deng a , Yi-Kuen Lee a,∗ , Ping Cheng b a Department of Mechanical Engineering, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong b School of Mechanical and Power Engineering, Shanghai Jiaotong University, Shanghai 200030, PR China Received 23 March 2005; received in revised form 14 June 2005; accepted 17 June 2005 Available online 11 August 2005 Abstract Two-dimensional micro-bubble actuator arrays were developed and studied in detail to enhance the hybridization kinetics of a DNA micro-biosensor. The hybridization between a molecular beacon, a kind of oligonucleotide probe, and its complement was investigated in a millimeter-sized PDMS based reaction chamber, where various 2D micro-heater arrays were distributed on the bottom for micro-bubble generation. The hybridization assay without the micro-bubble actuation revealed that the fluorescence increased fast at the beginning and slowed down after that. However, a uniform fluorescence increase was observed when periodic micro-bubble agitation was introduced in the static hybridization solution. A comparison of hybridization assays with and without micro-bubble agitation revealed that the hybridization time could be effectively shortened by 33% with 10 cycles of micro-bubble agitation from a 2 × 1 bubble actuator array, and by 43% with 10 cycles of micro-bubble agitation from a 2 × 2 bubble actuator array. © 2005 Elsevier B.V. All rights reserved. Keywords: Micro-bubble actuator; Molecular beacon; Flow perturbation; DNA hybridization; Micro-biosensor 1. Introduction DNA hybridization is the underlying principle of DNA biosensors. In DNA hybridization, the oligonucleotide probe (DNA probe) specifically recognizes, and binds to, a nucleic acid target (target DNA), which forms a double-stranded hybrid with its nucleic acid complement with high efficiency and specificity. The main types of hybridization used today are solution hybridization, filter hybridization, the poly- merase chain reaction, in situ hybridization and hybridization on DNA chips (Tenover, 1993; Anderson, 1999; Freeman et al., 2000; Sosnowski et al., 2002). Hybridization in solution is believed to be a two-step process involving nucleation and zipping up, in which nucleation is the rate-limiting step, and a second-order reaction equation can be used to describe the process (Anderson, 1999). ∗ Corresponding author. Tel.: +852 2358 8663; fax: +852 2358 1543. E-mail address: meyklee@ust.hk (Y.-K. Lee). Generally, the hybridization of DNA probes is diffusion- limited, i.e. the signal intensity is determined by the number of target molecules reaching the probe. However, diffusion is such an intrinsically slow process for large molecules that in micro-arrays even after overnight hybridization the system does not reach equilibrium. Furthermore, the slow diffusive transport could result in poor binding efficiency as less than 1% in micro-arrays (Pappaert et al., 2003). The diffusion lim- itation is a particular problem for a micro-scale biological reaction, where only small amounts of analyte are available and the required reaction volume limits the analyte con- centration. The obvious solution to overcome the diffusion limitation of hybridization experiments is to agitate the sam- ple solution. Recently, efforts have been devoted to increase the mix- ing and diffusion for DNA hybridization in a micro-scale confined space. Nanogen (San Diego, CA, USA) developed microchip-based hybridization arrays (NanoChip TM ) that uti- lized electric fields as an independent parameter to control DNA transport and enhance hybridization (Edman et al., 0956-5663/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2005.06.007