**Author details**

sequencing, bringing about significantly improved amplification evenness and accuracy for the simultaneous detection of CNVs and SNVs in single-cell level [104]. Weisenberger et al. applied the Fluidigm BioMark Digital Array to establish digital bisulfite genomic DNA sequencing with high resolution and high sensitivity [105]. The results showed that IFC cdPCR was a fast and reliable method for the single-molecule-scaled detection of DNA methylation

Undoubtedly, there is growing interest in dNAD, because it allows the more precise NA quantification, the higher discrimination of rare NA mutants, and the more reproducible and less susceptible to inhibitors than the traditional NA methods. Consequently, dNAD has full potential to influence the development of biology research, clinical diagnosis, the safety of food

To enhance the impact of this promising technique and push it towards clinical application, the MIQE for dNAD (dMIQE) was also published [106]. Based on dMIQE, the experimental protocols are standardized, the efficient utilization of resources is maximized, and the data are adequately assisted. However, the promising technique of dNAD still confronts some shortcomings. On one hand, although the development of microfluidic LOC offers a lot of dNAD device platforms, these devices perform low functional integration, and the supporting detection approaches lean primarily on real-time fluorescence scanning or the endpoint analysis of CCD camera-captured images, which, to some extent, adds the real cost and also has impacts on the true detection accuracy. Therefore, in the future, it will become a general trend that dNAD devices are highly integrated with multiple functions including cell or singlecell capture, cell lysis, and NA enrichment and purification, employing more advanced supporting detection technology. Particularly, for ddNAD, the strategy of droplet generation is one of the developing directions. For instance, Tanaka et al. currently created a hands-off autonomous preparation method of monodisperse emulsion droplets using a degassed PDMS chip [107]. Jeong et al. used a specially designed three-dimensional monolithic elastomer device to create a kiloscale droplet generation [108]. According to the snap-off mechanism, Barkley et al. also invented a novel technique to generate monodisperse droplets [109].

On the other hand, dNAD is actually the digital version of NAD, thereby possessing the same disadvantages (e.g., bias or nonspecific amplification) as most sequence-based NA amplifica‐ tion methods. Based on this point, how to improve or guarantee the NA amplification fidelity in microreactors is one of the future prospects of dNAD. It should be noted that the optimized reaction conditions for NA amplification in microreactors might be different from those in bulk state, meaning that the optimization of reaction system is indispensable. Furthermore, the precision of dNAD is greatly influenced by the number and size of microchambers (for cdNAD) or droplet (for ddNAD), which in turn challenges the fabrication of microfluidic chip and the uniformity of partitioning. Accordingly, we also anticipate that, in the future, there will be some novel strategies developed to realize digital detection not only based on parti‐

information.

146 Lab-on-a-Chip Fabrication and Application

tioning reagents.

**6. Conclusion and future prospects**

and environment, and other research fields.

Xiong Ding1,2 and Ying Mu1\*

\*Address all correspondence to: muying@zju.edu.cn

1 Research Center for Analytical Instrumentation, Institute of Cyber-Systems and Control, State Key Laboratory of Industrial Control Technology, Zhejiang University, Hangzhou, P.R. China

2 College of Life Sciences, Zhejiang University, Hangzhou, P.R. China
