# A Microfluidic Approach for Biosensing DNA within Forensics
## 5 COC Chips for Multiple Displacement Amplification (MDA): Biocompatibility and Real-Time Analysis  
As discussed in the previous section, COC is an attractive material for forensic microfluidic devices, since it has excellent bio(chemical)compatibility and chips of this material can be made at relatively low costs. This motivates our experimental investigation of COC for multiple displacement amplification (MDA) in microfluidic devices. It is noted that parts of the presented work are reported elsewhere as well by us [95; 96; 97]. Uniform amplification of small amounts of DNA is important for single-cell genomics, sequencing and forensic science. Due to amplification bias, the isothermal amplification method MDA is known for uneven representation of the template. Besides a reduction in the amount of reagents and sample, lowering the volume of the amplification reaction contributes to a more uniform amplification. The compatibility of COC with the MDA amplification reaction is verified by performing an on-chip reaction. Two different designs of COC chips are investigated and chips with and without BSA treatment are used to investigate the need for a coating. In SolidWorks both designs are made. Design I contains a serpentine-shaped channel (width as well as depth 1.0 mm) with a volume of ca. 55 \(\upmu\)L (Figure 2A), whereas design II comprises a straight channel (depth 0.5 mm, width 1.0 mm) with an internal volume of ca. 5 \(\upmu\)L (Figure 2B). In the vicinity of the reaction channel a channel is present for insertion of a thermocouple that is used for temperature measurement and control. Both microfluidic layouts are milled in 2 mm thick (design I) or 1 mm thick (design II) COC plates (DENZ Bio-Medical grade 6013) using a Sherline 5410 Deluxe Mill with a 1.0 mm diameter mill, followed by drilling of fluidic accesses with a 1.5 mm diameter drill into which standard pipette tips can be mounted. Post to milling and drilling, the COC chips are extensively rinsed with MilliQ water and ultrasonication (15 min) in ethanol, followed by nitrogen gas blow-drying. In order to recover the optical transparency, the surface roughness of the milled structures is lowered by following the procedure of [PERSON] et al. [98]: the chips are exposed (1 min) to cyclohex  
Figure 2: The schematic representation of the chips with the channel dimensions of (**A**) design I and (**B**) design II. Subsequently, the channel of each COC-chip is sealed by applying a piece of PCR foil (Microseal 'B' Adhesive Seals, Bio-Rad), which is a robust seal that is appropriate for biological applications [99]. Prior to performing the MDA reaction, some chips of design I are coated with BSA (1% (10 \(\upmu\)g/uL)). The MDA reactions (GenomiPhi V2 kit from GE Healthcare) are carried out in the COC chips that are incubated in a water bath of 32 \({}^{\circ}\)C for 2h. The manufacturer of the MDA kit states that this kit is optimized for whole genome amplification from at least 10 ng [100]. Next to that an input concentration of 10 ng/uL is used, since off-chip results showed an enormous increase in the DNA concentration within 2 h [97]. It is noted that in case of design I 10 \(\upmu\)L of the MDA mix (reaction buffer, sample buffer, enzyme mix, DNA and MilliQ in a ratio of 9:5:1:1:4, respectively) is injected in each chip, whereas the injected volume is 5 uL in case of design II. Post to loading of the indicated volume of MDA mixture, the fluidic accesses of the chips are closed with PCR foil. In addition, 10 \(\upmu\)L MDA mixture is pipetted into a 500 \(\upmu\)L Eppendorf polypropylene (PP) vial as a control. The concentration of double-stranded DNA (dsDNA) is determined with the Qubit \({}^{\text{TM}}\) dsDNA HS Assay Kit and the fluorescence is measured using the Qubit \({}^{\text{TM}}\) 3.0 fluorometer (both from Thermo Fisher Scientific). All samples are measured in triplicate. Table 2 summarizes the outcome of the DNA quantification of the experiments with chip design I.