## 4 Conclusions  
A simple PDMS microfluidic device is demonstrated for on-chip reverse chemical syntheses of oligonucleotides (5\({}^{\prime}\)-3\({}^{\prime}\)) without inert atmosphere. In this work, we have synthesized oligonucleotides up to 21 bases with optimized microfluidic synthesis conditions. The results confirm that the developed system, to some extent, is suitable for overcoming the limitation of inert atmosphere for short-chain oligo synthesis. The sealed microchamber and anhydrous conditions were most significant to achieve the adequate protection from atmospheric moisture. The design of the microchamber provides a systematic orientation to the CPG particles, which results in synthesizing oligos with more than a 75% yield by uniform interaction with synthesis reagents. The MS results also demonstrate that reverse synthesis with the developed microfluidic system provides a sufficient purity of short-chain oligonucleotides (<15-mer) with an acceptably low amount of truncated sequences. The reproducibility of synthesis by the developed system opens up the possibility of designing an automated programmable microfluidic system with multichannel synthesis in the future. However, the optimization of synthesis conditions is still required for long-chain oligos. In future work, we will also investigate methods for the analysis of synthesized oligos for precise quality and accurate yield determination with the introduction of a capping step in the synthesis cycle. We are also developing a computer controlled programmable system for on-demand syntheses of oligonucleotides and oligopeptides, which can further be used for various biosensing, biotechnology, and molecular biology applications.  
(Inset shows an end of inlet PTFE tube attached with needle). (b) Photograph of the PDMS chamber filled with CPGs, and it clearly depict a monolayer of glass particles. Figure S3: MS spectra of different oligonucleotides of 10 bases of length. Figure show the optimization of coupling time and 2 nd washing step for high coupling efficiency. (a)-(d) graph shows the effect of increasing coupling time on product yield as desired sequence produce sharp and high intensity peak as major product of the synthesis. Synthesis conditions are described in the MS spectra for each synthesis. Blue color indicates the changed parameter and green color indicate the parameters which remained same. Figure S4: MALDI-MS of 10-mer oligos with and without. Capping steps. Detailed synthesis conditions and 10-mer oligos information are mentioned in the above graph. Figure S5: MALDI-TOF/TOF MS spectra of 10 mer oligonucleotide (CCAGTCAGTC, m/z = 2987) with optimized conditions. Synthesis was performed with anhydrous reagents without molecular sieve exposure. Data shows multiple truncated products as major product while desired sequence is produced as minor product. Figure 56: Shows MALDI-TOF/TOF MS of 5-mer oligo at different laser intensities. Figure S7: Image of the glass vial for the collection of DMT cation fragment during deblocking step and washing step. Table S1: Synthesis and Flow Conditions for On-chip Synthesis of Reverse Oligonucleotide. Table S2: Shows the absorbance values obtained after trityl absorbance from 7-mer oligonucleotide synthesis.  
**Author Contributions:** R.B., and Y.T. designed the research and provided funding and intellectual support. R.B. performed the experiments and wrote the original draft of the manuscript. M.B., P.T.T., and Y.T. reviewed and edited the manuscript. All the authors analyzed the data and discussed the results.  
**Funding:** This work was supported by CREST program funded by the Japan Science and Technology Agency (JST).  
**Acknowledgments:** Akio Miyazato (JAIST) is gratefully acknowledged for their helpful comments on analysis using conventional mass spectrometry.  
**Conflicts of Interest:** The authors declare that they have no competing interests.