# A Microfluidic Approach for Biosensing DNA within Forensics
## 5 COC Chips for Multiple Displacement Amplification (MDA): Biocompatibility and Real-Time Analysis
As can be seen, the concentration of DNA in uncoated chips is well above 200 ng/uL (after a 2 h run), which is in agreement with the control vials, runs in a thermocyler (see [95; 97] for more details) and literature [100; 101; 102]. Remarkable is that use of a BSA coating in the COC microfluidic channels clearly negatively affects the MDA reaction, in fact it results in inhibition, whereas such coating is known as method to prevent non-specific binding to COC surfaces [103; 104; 46; 105]. The adsorption of polymerase on the channel walls can be prevented by the addition of BSA to the amplification mix or by coating the channel walls with BSA [106; 107; 108]. [PERSON] et al. suggest to use a concentration of 2.5 \(\upmu\)g/uL (0.25%) [107]. [PERSON] et al. state that high BSA concentrations (>0.15%) result in a lower yield compared to absence of BSA in the amplification mixture and that increasing the BSA concentration will finally result in total inhibition of the PCR reaction. The optimum BSA concentration for their silicon chip is found to be to be 0.05% resulting in a 2-fold increase in yield [108]. [PERSON] et al. conclude that 2 \(\upmu\)g/uL (0.2%) of BSA does not have any negative influence on the PCR reaction [106]. [PERSON] et al. recommend to use 0.2-0.4 \(\upmu\)g/uL (0.02-0.04%) to relieve the inhibition of humic acids in PCR [109]. [PERSON] et al. studied the optimal pH and BSA concentration to coat their Norland Optical Adhesive (NOA) chip. They report that the absorption capacity of the BSA to the NOA surface does not increase any further after 1 h. An acidic environment (pH 4) of the BSA solution shows a higher PCR efficiency than neutral pH. A BSA concentration of 5 \(\upmu\)g/uL (0.5%) gives the best results. Using a lower concentration might not be sufficient, while a higher concentration can lead to PCR inhibition [110]. According to [PERSON] et al. [17], another reason for obtaining a lower DNA concentration upon using a BSA coating could be that salts present in the amplification mixtures (e.g., KCl, MgCl\({}_{2}\), MgSO\({}_{4}\) or NaOH) negatively affect the coating. By using X-ray photo electron spectroscopy and contact angle measurements [PERSON] et al. determined the mode of surface passivation by BSA. On a hydrophilic substrate BSA surface passivation shows two modes: it covers the surface and it can interact with \begin{table}
\begin{tabular}{c c c c c c} \hline \hline  & **Run 1** & **Run 2** & **Run 3** & **Run 4** & **Average \({}^{\text{a}}\)** \\  & **(ng/uL)** & **(ng/uL)** & **(ng/uL)** & **(ng/uL)** & **(ng/uL)** \\ \hline Eppendorf vial & 204 \(\pm\) 7 & 218 \(\pm\) 8 & 265 \(\pm\) 13 & 200 \(\pm\) 16 & 222 \(\pm\) 30 \\ Chip without BSA coating & 118 \(\pm\) 6 & 209 \(\pm\) 6 & 295 \(\pm\) 13 & 233 \(\pm\) 20 & 214 \(\pm\) 73 \\ Chip with BSA coating & 79 \(\pm\) 3 & \({}^{\text{b}}\) & 86 \(\pm\) 2 & 62 \(\pm\) 3 & 76 \(\pm\) 12 \\ \hline \hline \end{tabular} \({}^{\text{a}}\) The standard deviation was taken from the average values of the four runs. \({}^{\text{b}}\) The seal of the chip was not secured properly and came off, hence no quantification measurement could be carried out.  
\end{table} 
Table 2: The concentration of double-stranded DNA (dsDNA) obtained after 2 h of amplification in the Eppendorf vials and chips of design I with and without BSA coating. an initially deposited layer for further adsorption. Surface passivation of a hydrophobic surface is reached in one step. The measured contact angle is the same after completion of the adsorption reaction [111]. Additionally, the BSA surface coverage is at maximum 53% for a complete monolayer on hydrophobic surfaces, while on hydrophilic surfaces an almost full monolayer can be present [112]. COC is hydrophobic, probably resulting in an incomplete monolayer of BSA. In this research 10 \(\upmu\)g/\(\upmu\)L (1%) is used, which is higher than recommended concentrations [108; 109; 106], which probably causes (partial) inhibition of the MDA reaction.