**2. Principle of dNAD**

cernedrecentlydue to its superiorityonanalytical sensitivityandaccuracy[9, 10].Furthermore, single NA molecule detection is also highly preferred as the calibration strategy for nextgeneration sequencing (NGS) to better serve recently proposed ambitious plans of precision medicine [11–13]. Thus, testing NAs, especially in single-molecule level, plays an essential role

At present, the widely used approaches in detecting NA molecules are quantitative polymer‐ ase chain reaction (qPCR) and quantitative reverse transcription-PCR (qRT-PCR). Apart from the capability of real-time monitoring the amplification, they can be applied to quantify the target NA molecules through two common strategies: relative quantification and absolute quantification. The former is based on internal reference genes (namely, housekeeping genes) to normalize and reflect fold differences in expression levels of mRNA, which is commonly interpreted as cDNA [14, 15]. The latter can provide the exact number of targeted molecules using an established standard curve of the change in quantification cycles with known molecule number of NA standards [16–19]. However, qPCR is compromising the ability of single-molecule quantitation analysis [20, 21]. Alternatively, when PCR meets microfluidic or nanofluidic chips, a highly sensitive NA quantification technique [digital PCR (dPCR)]

emerges, estimating NAs advantageously at a single-molecule analysis level [22].

At the end of 20th century, the first concept of dPCR was proposed by Vogelstein and Kinzler [23]. Since the concept was proposed, many dPCR platforms have been launched for several

**Figure 1.** Some vendors and their launched microfluidic chips and dPCR devices. The pictures are all from the web‐ sites of the corresponding companies or reprinted with permission from Ref. [59]. © Copyright 2011 American Chemi‐

cal Society.

in modern biological researches and diagnosis fields.

126 Lab-on-a-Chip Fabrication and Application

According to the strategies of amplifying NA, dNAD or single-molecule NA detection can be divided into dPCR and dINAA. However, both of them share the same principle.

Generally speaking, the principle of dNAD is composed of three core steps [39]. First, the original sample should be partitioned into thousands or hundreds of thousands of individual microreactions, endeavoring to make each contain nearly one target molecule. Second, the number of "positive" microreactors indicated either in a real-time reaction or in an endpoint reaction is counted and analyzed. Third, the concentration of nonpartitioned sample is calculated using certain statistical methods. In theory, if the number of microreaction is more enough or the number of target molecules is less enough, one reaction unit with positive signal represents one target molecule. However, in fact, a positive partition may contain more than one molecule. Therefore, in calculating the target's true concentration, a Poisson distribution is adopted in hope to correct the results. Therefore, dNAD can be considered as a binary output (present or absent like "1" or "0" in computer science) measurement, giving a direct and highconfidence NA molecule's measurement method [10, 40]. Compared to conventional tubebased NA detection, digital analysis is superior in realizing the absolute quantification with high sensitivity, high precision, and low ambiguity, avoiding the requirement of establishing a standard curve.
