Surface Area Effects on DNA Amplification

The high surface area to volume ratio present in microfluidic systems, as compared to conventional PCR tubes, has been shown to have a dramatic effect on the ability to perform DNA amplification. Erill et al. carried out a systematic analysis of the interaction between glass-silica surfaces and PCR reagents and found that at high surface area to volume ratios DNA polymerase adsorption occurs resulting in inhibition of PCR.122 _Glass

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tubes which mimicked the dimensions of a conventional 0.2 ml polypropylene PCR tube were produced in house by the University of Hull glassblower. Performing DNA amplification in both tube types using the Peltier heating system showed that there was no difference in PCR efficiency. Having kept the surface area to volume ratio the same and varying the material used this confirms that PCR is not inhibited by the material alone.

In order to further investigate surface area to volume ratio effects glass capillaries [Blaubrand® _{intraMARK, Germany], with varying internal diameters, were used. PCR was} performed using the bench-top Peltier heating system for 28 cycles in the glass capillaries and the samples analysed using slab-gel electrophoresis to see if the DNA amplification reactions were successful. Inhibition of PCR was found to occur at surface area to volume ratios greater than 10:1 (Table 4.3).

Table 4.3: Analysis of the effect of surface area to volume ratio (given as mm2_{:µl) of}

glass capillaries on the inhibition of DNA amplification.

Analysis of the surface area to volume ratio of the microfluidic devices used here for DNA amplification (Figures 4.1a. 4.1b and 5.1) are shown in Figure 4.4. Using an etch depth of 300 µm results in a surface area to volume ratio that does not exhibit PCR inhibition but requires a relatively large volume. Such volumes do not offer any considerable advantage over conventional systems in terms of cost of analysis and also will suffer from slower

Surface Area (mm2) Volume (µl) Ratio PCR

158.95 25 6:1 Yes

138.50 20 7:1 Yes

98.16 10 10:1 Yes

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thermal transition therefore an etch depth of 100 µm is more appropriate. However, at this depth the surface area to volume ratio results in PCR inhibition and therefore surface passivation was required to overcome this.

Figure 4.4: Graph depicting the success (green) or failure (red) of DNA amplification when performing PCR at different surface area to volume ratios based on the capillary results in Table 4.3. The surface area and volume details for the different microfluidic systems tested here are shown ( ) with etch depths of 100 µm (‡) and 300 µm (*), respectively.

Dynamic passivation was achieved using a previously reported combination of 0.2 µg/µl BSA, 0.01% (w/v) PVP and 0.1% (v/v) Tween-20.167 _{While the use of dynamic passivation} agents, such as BSA, results in competitive adsorption to the surface in place of the DNA polymerase some enzyme will still be adsorbed due to dynamic equilibrium. Whilst increasing the concentration of BSA can further reduce the effects of PCR inhibition, there

14:1

10:1

7:1 6:1

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is a certain concentration threshold (≥ 5 µg/µl) above which adverse effects on the DNA amplification process occur due to an increased viscosity.122

A wide number of static passivation techniques have been reported in the literature for a variety of applications and a number have been evaluated here for use in the microfluidic DNA amplification chamber. As silanisation produces a more hydrophobic surface, the change in contact angle of water with the internal glass surface was used to assess the efficiency of each of these techniques (Figure 4.5).

Figure 4.5: Graph showing the contact angles for water in the channel of microfluidic devices (Figure 4.1a) which had undergone various static passivation protocols. Uncoated microfluidic devices were used as a contact angle control. Error bars shown represent the standard deviation (n=4).

The greater the contact angle, the more hydrophobic the internal glass surface and so theoretically the less interaction there will be with the DNA polymerase and the more efficient the PCR reaction. Four silanisation techniques for treatment of the internal surfaces of the microfluidic device were investigated using SigmaCoteTM_{, Safetycoat}TM_{, OTS} and TPS. Analysis of the efficiency of each of the techniques showed very little difference

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in the contact angles obtained. As the contact angles were highest with OTS, this static passivation technique was initially chosen for preparation of microfluidic devices for PCR. It was soon found, however, that the silanisation treatment was subject to apparent degradation during the PCR thermal cycling process, as evident from an observed decrease in the contact angle (from ~115° to ~60°) following 40 cycles of DNA amplification. Such degradation of silanisation coatings has been reported by Felbel et al., who showed that chlorotrimethylsilane, dichlorodimethylsilane, hexamethyldisilazane and trichloropropylsilane were all affected, albeit to different extents, by the DNA amplification process, resulting in cleavage of the silyl-ether bond.118 _Further investigation was conducted, therefore, in order to determine how each of the surface treatments were affected by the thermal cycling process. Following static passivation, the microfluidic devices were thermally cycled, using Peltier heating, and the contact angle recorded every 5 cycles (Figure 4.6).

Figure 4.6: Graph showing the contact angles for water in the channel of microfluidic devices which have undergone thermal cycling. Static passivation

methods applied were SigmaCoteTM _{( ), SafetyCoat}TM _{( ), OTS ( ) and}

TPS ( ). Uncoated microfluidic devices ( ) were used as a contact angle

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Analysis of the contact angles showed that SigmaCoteTM_{, SafetyCoat}TM _{and OTS silanisation} treatments were all susceptible to degradation during the thermal cycling process as observed by a decrease in the contact angles recorded. While TPS did not exhibit the highest contact angle initially, it was found to display the most consistent results during the thermal cycling process and so was chosen for passivation of microfluidic devices in all subsequent experiments. It has been hypothesised that enhanced stability of silanisation reagents, during DNA amplification can be achieved by formation of multi-layers by cross polymerisation of additional chlorine substituents.118

By comparing at the chemical structures of the different silanisation reagents it is hypothesised that TPS (CF3(CF2)5(CH2)2SiCl3) exhibits the greatest stability due to the presence of fluorine atoms, which will form more stable bonds due to higher electronegativity than chlorine atoms, and a flexible chain reducing steric hindrance. OTS (C18H37Cl3Si) is the next most stable silanisation coating and contains in total three chlorine atoms. SigmaCoteTM _{(Cl[Si(CH3)2O]3Si(CH3)2Cl) and SafetyCoat}TM _{((CH3)3SiCl) do} not have additional chlorine substituents and also have bulky chemical structures increasing steric hindrance and therefore reduces the number of molecules which can be attached to the microfluidic surface. In addition, the structure of TPS shares similar properties with Teflon® _{which has been demonstrated to be a contained adsorption} material i.e. one which exhibits rapid saturation of DNA polymerase adsorption.175 _In order to provide the optimum conditions for performing DNA amplification on the microfluidic device a combination of static passivation, by TPS, and dynamic passivation, by a mixture of BSA, PVP and Tween-20 was chosen.