Influence of the Flow Velocity on the Hybridization Rate in Convection-driven DNA Microarray Analysis

 

F. Detobel1, K.Pappaert1, P. Van Hummelen2, De Tandt C.3, Ranson W.3 and G. Desmet1

1 Department of Chemical Engineering, Vrije Universiteit Brussel, Belgium

2 MicroArrayFacility Lab, Flemish Institute for Biotechnology (VIB), Belgium

3 Laboratory for Micro- & Photonelectronics, Vrije Universiteit Brussel, Belgium

tel.: (+).32.2.629.36.17, fax.: (+).32.2.629.32.48, e-mail: fdetobel@vub.ac.be


 

DNA hybridization in micro-arrays is a strongly diffusion limited process, very often requiring overnight waiting before a sufficient amount of probe DNA molecules has had the time to diffuse towards the target spot carrying their matching counterpart. To speed up the analysis, it seems straightforward to organize some form of a convective transport across the surface of the microarray. In the past few years, several convection driven-systems have hence been developed and some of them have even been commercialized. These systems mainly differentiate themselves by the driving force that is used to generate the transport of the DNA sample, including pressure forces, acoustic waves, centrifugal forces and viscous drag forces.

 

One of the fundamental issues related to the design of such systems is the compromise that needs to be made between the creation of a sufficiently strong convective transport and supply of probe strands (device scale problem), without disturbing the molecular binding events (nano-scale problem). The present study has investigated this compromise using a rotating shear driven hybridization system (Figure 1). At a particular rotational speed of the circular channel, the sample spots on the microarray slide are subjected to a different flow velocity, depending on their radial position. Using this system, the influence of a whole range of radial velocities on the hybridization process can be analyzed in a limited set of experiments, consuming only limited quantities of DNA sample.

 

In the present study the microarrays consist of three rows of probe DNA (Nras, PolA and Rad 52), spotted in the center longitudinal direction of the slide. For each experiment, 90 µl of a fluorescent labeled complementary strand of Nras, used as target DNA with a concentration of 0.36 ng/μl, was brought into the channel with a depth of 6.3µm. After rotation of the circular channel during 30 minutes at a particular rotation speed (0.25 - 4 rpm), the intensities of the different spots on the microarray were analyzed and plotted as a function of the mean local fluid velocity (Figure 3).

 

Initially, when the flow velocity is raised, there is an increase of the fluorescent signal intensity as a result of the improved transport of the target DNA to its matching counterpart. At a certain point, the convective transport has reached its maximal gain and the spot intensities are no longer influenced by the flow velocity over a broad range of velocities. At the end of this range (running up to 3 mm/s) the hybridization signal starts to decrease again. This effect however is caused by a system failure, rather than by a disturbance of the molecular binding events: scratches appear into the microarray spots, caused by small glass particles which are created in the contact region between the microarray and the channel spacers at high rotation speeds, hence reducing the average spot intensity. An improved experimental set-up should prevent these system problems.

 

In conclusion, this preliminary study shows that with the used experimental conditions only limited flow velocities are necessary to obtain the maximal gain of convective transport on the hybridization rate. Although there is no information yet for high flow velocities, no negative influence of the flow rate on the hybridization process could be observed. These results may not be valid for other (lower) channel depths.

Figure 1: Schematic overview of the rotating shear-driven hybridization system. In this system, the hybridization chamber is composed of an etched circular glass channel, sealed with a plastic holder, containing the microarray slide. Rotation of the circular channel, while keeping the plastic holder stationary, induces a shear driven convective flow. This flow ensures a continuous supply of sample DNA out of a reservoir, created by the plastic holder at both sides of the microarray slide.

 

Text Box: Figure 2: To obtain insight in how the convective transport overcomes the creation of depletion zones, a limited set of numerical flow and reaction-diffusion calculations has been made using CFD-software. The concentration profile of target-DNA (C0 = 1 nM) above a DNA-spot (Ø = 100 µm, H0 = 10-9 mol/m²) after 100 s in a 2D hybridization channel is given for pure diffusion transport (a) and for shear-driven convective transport with a wall velocity of 2.10-5 m/s (b) and 2.10-4 m/s (c). The color scaling is proportional to the target-DNA concentration (M) in the channel and is case-dependent for clarity reasons. The kinetic parameters for the hybridization reaction were ka = 105 M-1.s-1 and kd =5.10-3 s-1.

 

 

 


 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 3: Hybridization signal as a function of the mean flow velocity. The data points were obtained using different rotation speeds: 0 rpm (♦), 0.4 rpm (♦), 0.8 rpm (♦), 1 rpm (♦), 1.2 rpm (♦), 1.6 rpm (♦), 2.5 rpm (♦), 3 rpm (♦) and 4 rpm (♦).

 


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