## 3 Results and Discussions
### On-Chip Reverse Synthesis of Oligonucleotides
The MS data of oligonucleotides synthesized in the inert atmosphere [Figure 3a] and in the air atmosphere [Figure 3b] show the single intense peak of the desired product at \(m/z=3043.8\) and \(m/z=3026.2\), respectively, corresponding to 10-mer oligonucleotides. Some other peaks are also observed in Figure 3a,b with extremely low intensities and can be assigned to truncated products (products with one or more missing nucleotides from the desired sequence) with a negligibly low amount compare to the target sequence. The MS results (Figure 3) confirm the successful synthesis of 10-mer oligonucleotides as major products in both cases. The overall yield of the 10-mer oligonucleotides was calculated to be 95% in the N\({}_{2}\) atmosphere and >91% in the air atmosphere from the stepwise yield using trityl cation absorbance. From the MS spectra and calculated yield, it is explicit that reverse syntheses of oligonucleotides in inert atmosphere and in air atmosphere obtained comparable results. This achievement was made possible by applying anhydrous conditions to all the freshly prepared solutions using molecular sieves, and optimized synthesis conditions. Figure S5 (see supplementary information) confirms this explanation as we synthesized the same length of oligonucleotides with anhydrous reagents without molecular sieve treatment. Before the experiment, reagents were kept in closed vials for at least 24 h, and identical synthesis conditions were applied. The result clearly shows that the major products obtained from this synthesis were truncated products with an unacceptable low amount of the desired  
Figure 3: MALDI-TOF/TOF MS spectra of on-chip synthesized reverse oligonucleotides of 10-mer length (**a**) in inert atmosphere and (**b**) in air-atmosphere (without inert atmosphere) with optimized conditions. Specific synthesis conditions are described for each synthesis. Data shown are baseline corrected using flex analysis software. sequence. The above experimental results clearly show that this microfluidic system and anhydrous conditions allow the reverse synthesis of oligonucleotides without inert atmosphere by providing sufficient protection from atmospheric moisture. The on-chip reverse synthesis conditions for oligonucleotides (>10 bases) were optimized further with an increased flow time of the deprotection step. Figure 4 shows the MALDI-TOF/TOF MS spectra of the 12-mer oligonucleotides synthesized in an air atmosphere before and after changing the flow time of the deprotection step. Detailed microfluidic syntheses conditions are summarized in Table S1, unless otherwise specified (see Supplementary Information Table S1). Figure 4a shows the MS spectra of the 12-mer oligonucleotide (CTGGCTTTAGTA, \(m/z=3649\)) with a deprotection time of 2 min, and the peak of the desired sequence corresponds to 3651.62 (m/z), which was obtained with a <70% yield (calculated). It also shows the peaks of truncated sequences (n-1 mer, n = 12) with a peak intensity comparable to the demanded sequence. It is clear from the MS spectra that optimized conditions are not acceptable for synthesizing 12-mer oligos with a high quality. To improve the quality of oligo sequences (>10-mer), the time of the deprotection step was varied from 2 min to 2.5 min, and 3 min, which resulted in an improved quality, as shown in Figure 4b (data with deprotection time of 2.5 min not shown here). Figure 4b shows that the MS-spectra of the synthesized 12-mer oligonucleotide (CCAGTCGACCGA, \(m/z=3613\)) with a deprotection time of 3 min corresponds to the intense peak at 3615.13 (m/z). With increased deprotection time, we become successful at achieving an improved quality of 12-mer oligonucleotides with a 85% yield (synthesized in an air atmosphere). The MS spectra (Figure 4b) also demonstrate peaks at 2972.96 and 1752.64 (m/z), which correspond to (n-2) and (n-6) truncated products, respectively, but these sub-sequences with a minor amount can be ignored compared to the amount of desired sequence.