**1. Introduction**

It has been clearly investigated that nearly all of the diseases possess a series of biomarkers associated with nucleic acid (NA) molecules during the development of biological researches [1–5]. Determining these NA molecules and their intercellular and extracellular changes is a well-worked strategy for estimating therapy efficacy, monitoring minimal residual diseases, unveiling the mechanisms of cellular signal transduction, and so on [6–8]. To reflect individu‐ al genetic differences, single-molecule level quantitation of NAs has been increasingly con‐

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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 in modern biological researches and diagnosis fields.

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.

decades based on differently designed microsystems, including femtoliter array, spinning disk, SlipChip, droplet, microfluidic formats, and so on [24–30]. Some even have been successfully paced into industrial phase because of the superiority of testing and the promising application. Currently, several vendors in the biological industry, such as Fluidigm, Bio-Rad Laboratories, Life Technologies (ThermoFisher), RainDance Technologies, and Formulatrix, have launch individually their commercialized dPCR devices (**Figure 1**).

Apart from dPCR, digital isothermal NA amplification (dINAA) devices also arouse great concern. Unlike dPCR, dINAA leans on the isothermal NA amplification, which can be carried out at a consistent temperature, obviating the requirement of highly stable thermocycling devices. Thus, when targeting practical point-of-care testing (POCT) devices, dINAA is superior to dPCR. However, viewed from the principle of realizing digital detection, the concept of dINAA is the same as that of dPCR, just replacing PCR with isothermal amplifica‐ tion. In particular, due to loop-mediated isothermal amplification (LAMP) displaying as the best promising method among a lot of isothermal NA amplifications, digital LAMP (dLAMP) is the first dINAA developed [31, 32]. Later, other dINAAs have been reported, such as digital multiple displacement amplification (dMDA), digital isothermal multiple-self-matchinginitiated amplification (dIMSA), digital recombinant polymerase amplification (dRPA), and so on [33–38]. However, the development of dINAA devices is still in the research stage, as the commercial products have not been launched yet.

As of now, more and more researchers are enthusiastic about the potential of digital NA detection (dNAD) based on microfluidic Lab-on-a-Chip (LOC) devices, since an increasingly significant role has been played in single-cell analysis, early diagnosis of cancer, prenatal diagnosis, and so on. In this chapter, we will concentrate on the principle, classification and advances, analysis and evaluation, application, and future prospects of dNADs that are accomplished either through commercialized LOC devices or the devices our laboratory or other laboratories have established.
