## 3 Results and Discussions
### On-Chip Reverse Synthesis of Oligonucleotides
From the MS spectra (Figure 4), it is clear that oligo synthesis (<10 bases) with 2 min is insufficient, which leads to incomplete deprotection. It interferes in the next synthesis cycle, which results in the production of truncated sequences with remaining protected sites from the previous cycle. The improved result of the 12-mer oligonucleotides using PDMS chips in an air atmosphere confirms that the extended exposure of the deprotection reagents along with optimized coupling and washing times leads to a pure product with a high yield. In this work, we extended the oligonucleotide chain up to 18 mer and 21 mer using optimized conditions for the on-chip reverse synthesis. Figure 5a,b show the MALDI-TOF/TOF MS spectra of the 18-mer (CAGTCTGAGTCAGCTGAT, \(m/z=5511\)) and 21-mer (GCGGCTGAAGACGGCCTATGT, \(m/z=6480\)) oligo sequences, respectively. Both spectra show the intense peak of the corresponding sequences with a maximum yield of 75% in each case (average yield of >98%). The MS spectra (Figure 5) also demonstrate multiple peaks of sequences other than the target sequence. Some of these correspond to the truncated products of the respective target sequence, but there are still plenty number of peaks which correspond to unknown sequences. We are not able to analyze these peaks through m/z with respect to the synthesized sequence. The exact reasons for these peaks are not clear yet, but it is expected that the peaks are obtained from the sequences with the deletion of nucleotides during the synthesis. The most probable reason for those unexpected peaks could be because of omitting  
Figure 4: MALDI-TOF/TOF MS spectra comparison of 12-mer oligonucleotide synthesis without inert atmosphere (**a**) before and (**b**) after modified synthesis conditions. MS data clearly shows the effect of deprotection step on longer synthesis. Specific synthesis conditions are described for each synthesis. Data shown are baseline corrected using flex analysis software. the capping steps from the synthesis cycle. Although we proved in Figure S5 (see supplementary information) that the capping step is not significant, long-chain oligo synthesis (>12 bases) is expected to have a large impact from the omitting of the capping step as capping blocks the remaining active site from the previous cycle. Another reason could be the effect of long exposure of the deprotection reagent on the growing oligo chain, which results in the deporination of bases and thus creates another site for a side reaction. Therefore, a study on the deporination of bases with an optimized deprotection time is required in the future to understand the origin of these peaks. Finally, the random fragmentation of the oligo sequence during ionization, most probably related to the high laser intensity, could also not be neglected, which led to multiple peaks at random \(m/z\) that are difficult to analyze (see supplementary information also, Figure S6). Figure 5: MALDI TOF/TOF MS spectra of (**a**) 18-mer and (**b**) 21-mer oligonucleotide with optimized conditions. Figure 5b inset shows the PAGE image of differently synthesized 21-mer oligonucleotides with running conditions at the bottom. The optimized synthesis conditions are described for both syntheses. The data shown are baseline corrected using flex analysis software (Version 2.4, Bruker Daltonics, Bremen, Germany). For analyzing the quality of synthesized oligos (21-mer), we also performed a denatured polyacrylamide gel electrophoresis (PAGE) analysis with a known marker to confirm the length of the synthesized product. Three different oligonucleotide sequences with the same length of 21 bases were synthesized separately using the same conditions and checked using gel electrophoresis. The oligonucleotides were dissolved in Milli-Q water, and sample solutions of 10 pmol/\(\upmu\)L were prepared using the NanoDrop system for the PAGE analysis. Figure 5b (inset) shows a denatured gel image with all four samples of 21 mer (lanes 2-5) and a 5 bp ladder (lane 1) for a standard marker.