## 3 Results and Discussions  
### On-Chip Reverse Synthesis of Oligonucleotides  
Due to the fact that the coupling reaction is highly moisture-sensitive, the reverse oligonucleotide synthesis is challenging under ambient conditions [26]. To overcome the problem associated with the moisture sensitivity, we are taking advantage of a closed microfluidic system by minimizing the exposure to the atmosphere to some extent. In standard phosphoramidite chemistry, the primary 5\({}^{\prime}\)-OH group of the phosphoramidite monomer is significantly more reactive than the secondary 3\({}^{\prime}\)-OH group (or 2\({}^{\prime}\)-OH group). Contrary to standard synthesis (3\({}^{\prime}\)-5\({}^{\prime}\) direction), in (5\({}^{\prime}\)-3\({}^{\prime}\)) reverse synthesis, the 3\({}^{\prime}\)-OH group of the phosphoramidite monomer (dC\({}^{\text{Bz}}\), Figure 2) is protected with DMT, which makes the 5\({}^{\prime}\)-OH group of the monomer (dC\({}^{\text{Bz}}\), Figure 2) available to form a dimer with the less reactive secondary 3\({}^{\prime}\)-OH group of dG\({}^{\text{IBu}}\)-CPG in order to grow the sequence in the 3\({}^{\prime}\)-5\({}^{\prime}\) direction. Due to this chemistry, the coupling in reverse synthesis takes place at a slower rate than standard chemistry [8]. Therefore, to achieve a higher coupling efficiency for the reverse synthesis, optimization of the coupling and deprotection step is required in the first part. To optimize the conditions for a high coupling efficiency, oligonucleotides were synthesized by varying the synthesis conditions, such as the coupling time, deprotection time, and washing time (especially washing after the deprotection step as coupling efficiency and accuracy are increased by this wash, since premature detritylation of the incoming phosphoramidite monomer is prevented) (see Supplementary Information Figure S3). Figure S3d shows that the single intense peak at 2986.89 (m/z) corresponds to the 10-mer oligonucleotide, which was obtained as a major product with negligible small peaks of \(n\)-mer oligonucleotides (\(n<10\), truncated products), when the synthesis was performed with 4 min of coupling and 3 min washing. While the oligo synthesis was performed at a lower coupling time (see Supplementary Information, Figure S3a-c), 10-mer oligonucleotides were obtained as minor or negligible products, whereas truncated products were obtained as major products. The MS spectra obtained from the above optimization with coupling and washing time variation between 2 min and 4 min, depict that reaction propagation takes place in a stepwise manner. The longer coupling time enhances the chances of efficient coupling of the active site of the oligonucleotide chain with the incoming phosphoramidite more effectively, and therefore results in a high yield of the desired sequence (oligos yield calculation was made by trityl cation monitoring during the synthesis) with a single intense peak. The improved coupling efficiency and yield are attributed to the optimized flow conditions of the enhanced coupling and thorough washing. During the synthesis of short chain oligos, the capping step was omitted from the synthesis scheme; therefore, the effect of the capping step was also confirmed by performing synthesis of the oligonucleotide with and without the capping step (see supplementary information, Figure S4). It is clear from the results that the capping step is not as significant in the case of short chain oligos synthesis. In the next step, a comparative study of the oligonucleotide synthesis with and without inert atmosphere was performed. Figure 3 shows the MALDI-TOF/TOF MS spectra (Bruker Daltonics UltrafleXtreme, Bremen, Germany) of 10-mer oligonucleotides (TTCGAGACTG, \(m/z=3041\)) in an inert (N\({}_{2}\)) atmosphere, and (TATGACG, \(m/z=3026\)) in an air atmosphere. Specific synthesis conditions are mentioned in the graph.