DNA hybridization enhancement using piezoelectric microagitation through a liquid coupling medium

Abstract

In conventional DNA microarray hybridization, delivery of target cDNAs to surface-bounded probes depends solely on diffusion, which is notoriously slow, and thus typically requires 6-20 h to complete. In this study, piezoelectric microagitation through a liquid coupling medium is employed to enhance DNA hybridization efficiency and the results are compared with the standard static hybridization method. DNA hybridization was performed in a sealed aluminium chamber containing DNA microarray glass chip, coupling medium and piezoelectric transducers. 3×SSC (Saline Sodium Citrate) was used as a coupling medium to prevent overheating of the piezoelectric transducers and to effectively transmit ultrasonic wave to the glass chip. Flow visualization using fluidic dye and velocimetry (PTV) technique was applied to observe fluid transport in the hybridization chamber. It was revealed that the dye solution was homogeneously distributed within 10 min under dynamic agitation while it took over 1 h to reach the same level of homogeneity in static condition. Plasmodium falciparum DNA microarrays and total RNA extracted from parasite cells were used as a model for DNA microarray experiments. It was found that the required hybridization time may be substantially reduced from 16 h to 4 h by the use of dynamic hybridization scheme. With the same hybridization time of 16 h, dynamic hybridization resulted in higher fluorescent signals of ∼33% and ∼24% compared to static hybridization in Cy3 and Cy5 channels, respectively. Additionally, good/effective spots, some of which were not formed by static method, were enhanced and distributed more uniformly over the microarray. Therefore, the developed dynamic hybridization with integrated piezoelectric microagitation platform is highly promising for DNA analysis in molecular biology and medical applications.

Investigation of acoustic streaming patterns around oscillating sharp edges

“…[27][28][29][30][31][32] In particular, acoustically driven, oscillating bubbles have been used to achieve fast and homogeneous mixing by generating acousticallyinduced microvortices. [33][34][35] Bubble-based acoustic mixers have been utilized for enzyme reaction characterization, 36 DNA hybridization enhancement, 37,38 chemical gradient generation, 39 and optofluidic modulators. 40,41 However, bubble-based acoustic mixers have also proven to be somewhat challenging due to bubble instability, heat generation, and hard-to-control bubble-trapping processes.…”

An acoustofluidic micromixer based on oscillating sidewall sharp-edges

“…This phenomenon results in a more prominent perturbation of the surrounding fluids, greatly facilitating the mass transport of fluids. Thus far, bubble-based acoustic mixers [51][52][53][54] have been used for characterizing enzyme reactions, 2 enhancing DNA hybridization, 51,55 generating chemical gradients, 56 and developing advanced optofluidic devices. 57 Although acoustically driven, bubble-based micromixers have shown tremendous potential in a wide variety of applications, there are many concerns regarding bubble instability, 53,57 heat generation, 48 and inconvenient bubble-trapping processes.…”

Enhancement of DNA hybridization under acoustic streaming with three-piezoelectric-transducer system

“…The reasons for choosing 3xSSC solution as a coupling fluid were that it is closely matched in acoustic impedance (z) with the silica glass, it is low in heat transfer and it is non-contaminant to the hybridization process. 22 The 3-PZT design was proposed in order to improve the non-uniformity problem found from our previous design, in which microarrays were placed on top of two PZTs. It was found that signal intensity in the middle of the hybridization chamber tended to be low compared with other regions, which may have been a result of an outward flow of acoustic wave from the center of the microarray.…”

“…Recently, we proposed a simple and low cost device for DNA hybridization based on acoustic streaming induced by two piezoelectric transducers with a coupling fluid. 22 Each piezoelectric disk, placed directly beneath each hybridization chamber, generates acoustic waves that are effectively transferred to microarrays via a coupling fluid. The DNA targets were observed to move in both vertical and horizontal directions under the influence of acoustic streaming facilitating hybridization with their complementary DNA probes.…”