Optimising biochemical reactions

Acoustic micro-agitation is a powerful tool to overcome artifacts caused by the geometry of small reaction volumes. Effective agitation prevents diffusion limitation of reaction kinetics increasing, such as the sensitivity and signal to-noise ratio of DNA- or protein microarrays.

Microarray technology has become a vital tool of molecular biology due to its high degree of parallelisation and small sample volume. Highly developed spotting and scanning systems and powerful analysing software enable even small labs to use the method. However, miniaturising assay formats has some pitfalls: the geometry of today's chip formats makes diffusion a key factor in binding reactions. A quick glance at the relevant physical parameters illustrates how dramatic diffusion limitation really is: oligo-nucleotides need about 100 minutes to travel a distance of 1 millimetre on a DNA-chip. For typical PCR products it will even take 30 hours to be displaced by the same distance.

Thus, on a chip measuring several square centimetres probe molecules of the different spots will interact with only a fraction of the target molecules during practical incubation times. Because of the local depletion of target molecules it would take months to establish reaction equilibrium. As a result typical microarray data is in-homogeneous and rarely reflects the true reaction kinetics of target and probe molecules.

The diffusion limit can be overcome by agitating the sample during incubation. While this is a trivial task in the macroscopic world, the physics of microscopic reaction volumes renders mechanical stirring or shaking techniques useless.

In addition the laminar flow condition of the capillary gap geometry prevents turbulence - the most effective mixing mechanism.

Advalytix uses surface acoustic waves (SAW) to agitate the hybridisation solutions. SAW pumps work without any mechanically moving parts directly inside the reaction chamber and do not introduce any dead volume into microscopic systems. Fig.1 shows the principle of SAW-fluidics: a specially designed metal electrode (interdigital transducer) on a piezoelectric substrate generates mechanical waves when fed with a resonant radio frequency signal. This wave propagates on the surface of the substrate very much like earthquakes do. If the piezo crystal is in contact with a fluid, part of the wave's energy is coupled into the fluid causing it to flow in the direction of the sound wave.

By varying some properties of the signal fed to the electrodes (e.g. frequency) different streaming patterns can be induced in the liquid. This superposition of different streaming patterns, called quasi-chaotic streaming, effectively agitates even macroscopic reaction chambers.

Fig. 2 shows the acoustic agitation at work on the area of a microscope slide (25x75mm). The reagent is confined to a capillary gap of 100µm. Four holes drilled into the cover slip were used to introduce drops of dye into the gap. The lower part of the time sequence shows the dye islands remaining essentially immobile. Activating the three SAW mixing chips (upper sequence) spreads dye across the whole area in about 15 minutes.

four chambers

The Advalytix ArrayBooster incubation system (Fig. 3) uses SAW technology in four individually programmable incubation chambers. The reaction geometry is similar to the classical cover slip method. A spring-loaded mechanism holds a glass card with integrated SAW mixer chips (Adva-Card) on top of the microarray slide. A spacer on the AdvaCard forms a uniform capillary gap of 60µm. For arrays of different spotting areas AdvaCards come in three standard sizes ensuring the use of minimal sample volume.

To analyse the influence of incubation time on the hybridisation signal Advalytix incubated conventionally fabricated chips (50 genes of the rat genome, represented by 50-mer oligos, two replicas each, six identical sub-arrays) at 42°C. As a target 25µg total RNA were reverse transcribed and used as cDNA for hybridisation after fluorescence labelling. Fig. 4 shows the background-corrected mean signal intensities for experiments with (red) and without (blue) SAW-agitation. All hybridisations were carried out with an identical amount of DNA. The hybridisation volume of the ArrayBooster experiment was 70µl, the cover slip experiment was 40µl.

In the case of passive hybridisation the signal remains more or less constant after several hours of incubation. After this time the target solution in the vicinity of the spot is depleted so the signal can increase only marginally.

Under micro-agitation, the signal develops in a completely different fashion: the intensity rises strongly over the first 10 hours. The rate decreases but the signal is not saturated after 48 hours of incubation. The total gain in signal intensity amounts to a factor of 16 after 48 hours, even though the ArrayBooster experiment was carried out with approximately half the target concentration of the cover slip hybridisation. The figure also shows that SAW agitation can be used to shorten incubation time without compromising on signal-to-noise ratio.

The homogeneity of the array is optimised by acoustic mixing, too. Comparing the CV-values of different sub-arrays the advantage is approximately a factor of two in comparison with manual hybridisation (10-20% vs. 30-40%).

The relative gain in intensity as well as the point in time after which the hybridisation becomes diffusion limited depends on the biological system under investigation. Key parameters are target concentration and the length of probe molecules.

To enable researchers to investigate the various phenomena governing hybridisation, such as depletion, diffusion, cross reactions and inhomogeneities of replicas under controlled conditions, Alopex has developed the Prime Array kit. The kit contains two oligo-arrays, ready-to-use hybridisation solution with Cy3-labelled oligos as well as washing solutions. The kit can be used to evaluate hybridisation protocols and compare the affectivity of hybridisation stations.

marked differences

Active (ArrayBooster) and passive (cover slip) hybridisation of the Prime Array containing 400 replicas of a 16-mer oligo on an area of 18x18mm were compared. After incubation with a matching target the array should show identical signals. Fig. 5 shows the colour-coded scans and enlargements of two spots from the centre of the array. All scans were acquired using the same scanner settings.

The passive hybridisation shows pronounced inhomogeneities after 60 minutes of incubation. The relative variation of intensities is 98%. SAW agitation reduces the variation to 20%. At the same time the ArrayBooster increases signal intensity by a factor of 10 without affecting background fluorescence. This results in an increase of SNR by the same amount. The enlarged insets show that the typical 'doughnut' artifact present after passive hybridisation is completely eliminated by SAW micro-agitation.

Fig. 6 shows a sub-array of the Prime Array chip used for analysing the specificity of the hybridising event. Probe oligos are spotted with one,two or three mismatches with respect to the target molecule that is hybridised against the array. Under passive hybridisation conditions a single mismatch is reflected by a difference of a factor of 2 in signal intensity (incubation time: 60 minutes at 44°C).

By micro-agitation this contrast in intensities can be increased to 10:1. Increasing allele discrimination is of special importance in medical diagnostics since more and more point mutations of the genome (SNPs - single nucleotide polymorphisms) can be correlated with specific diseases and SNP analyses can yield clues to the effectiveness of certain drugs.

Advalytix' analyses show that the sensitivity and discrimination of microarrays can be enhanced significantly by SAW agitation. Based on the ArrayBooster experiments it will be possible to analyse a clinically relevant number of SNPs on a chip with sufficient statistical confidence.

The acoustic micro-mixer is an important tool for the miniaturisation of analytical systems. Its use is not limited to the microarray geometry. In co-operations the technology has been adapted to various micro-assay formats. Fig. 7 shows the use of acoustic waves for the agitation of standard 96-well microtitre plates.

The acoustic agitator is silent and vibration-free so it can easily be integrated into pipetting or spotting robots. It doesn't carry the risk of well-to-well contamination associated with mechanical shakers. As well size decreases, the acoustic mixer is the only viable technology for effective agitation. Rapid mixing in 384-well and 1536-well type plates has been demonstrated. The acoustic microtitre plate agitator is scheduled for next year.