**5. Application of dNAD**

Compared to conventional NA detection methods, dNAD provides better sensitivity, better precision, higher tolerance, and the NA's absolute quantification. Until now, dNAD has been applied to a variety of research fields, including pathogen detection, food safety, clinical diagnosis (genetic instability estimation and early cancer), prenatal diagnosis, quantitative analysis of gene expression, and NGS library quantification. Although dPCR is still the most widespread type of dNAD, in resource-limited regions, it is confined due to the requirement of thermal cycling and robust temperature control. To overcome this awkwardness, dINAAs without thermal cycling are of great interest, which also enlarge the application of dNAD, especially for POCT. For this subchapter, the recent advances on application of dNAD will be narrated.

#### **5.1. Pathogen detection**

Honestly speaking, on pathogen detection, the advantages of most conventional NADs on both analytical sensitivity and specificity are inherited by the corresponding digital formats. In addition, dNAD has the capability of realizing NA's absolute quantification. Therefore, dNAD can be used as the ultrahigh sensitivity and more accurate method for viral load determination or bacterial quantification. Kelley et al. established a duplex ddPCR assay for high-precision methicillin-resistant *Staphylococcus aureus* (MRSA) analysis [76]. By assaying 397 clinical samples, a good agreement with the reference assay for both qPCR and ddPCR assays was indicated. Strain et al. demonstrated and estimated the application of ddPCR for a highly precise measurement of HIV DNA [77]. Total HIV DNA and episomal 2-LTR (long terminal repeat) circles in cells that were all isolated from infected patients were targeted. Compared to qPCR, ddPCR performed a significantly increased precision (5-fold for total HIV DNA and >20-fold for 2-LTR circles), making it an alternative for the measurement of HIV DNA from clinical specimens.

dNAD also enables the direct detection without NA extraction due to the ability of partitioning target NA and nontarget components (e.g., inhibitors) into different microwells. Pavšič et al. employed two dPCR platforms (QX100 ddPCR system from Bio-Rad Laboratories and the BioMark HD IFC cdPCR system from Fluidigm) for the direct quantification of two wholevirus materials of human cytomegalovirus (HCMV) without DNA extraction [78]. It was demonstrated that direct quantification by both dPCRs could provide repeatable measure‐ ments of viral DNA copy numbers, giving a closer agreement with the actual viral load than that with either dPCR or qPCR of extracted DNA.

Although dNAD certainly possesses the superiority of absolute quantitation, some reports fail to demonstrate the advantage, which calls for the requirement of optimization when estab‐ lishing dNAD. For instance, Boizeau et al. used ddPCR to provide an absolute quantitation of HBV genome molecules [79]. However, the results indicated that qPCR assays remained more sensitive than ddPCR when used for low HBV DNA levels, suggesting that optimization of ddPCR was still necessary, especially on accurately differentiating the positive from negative in samples with very low levels of target DNA molecules.

#### **5.2. Food safety**

If the precision of cdNAD is defined as the minimum difference in concentration (∆*λ*/*λ*) that is reliably detected with more than 99% true positive and more than 99% true negative rate, this situation corresponds to 4.6*σ* separation in the mean (*μ*) for two Gaussian distributions. That is to say, in the case with small ∆*λ*, ∆*μ*/∆*λ*=4.6*σ*. Then, ∂[*N*(1-*e* -*<sup>λ</sup>*)]∆*λ*/∂*λ*=*Ne* -*<sup>λ</sup>*∆*λ*=4.6[*Ne <sup>λ</sup>* (1-*e*-*<sup>λ</sup>*)]1/2, and ∆*λ*/*λ*=4.6(*e <sup>λ</sup>*-1)1/2/(*λN* 1/2). For different total chamber numbers (*N*) in chip panel,

Compared to conventional NA detection methods, dNAD provides better sensitivity, better precision, higher tolerance, and the NA's absolute quantification. Until now, dNAD has been applied to a variety of research fields, including pathogen detection, food safety, clinical diagnosis (genetic instability estimation and early cancer), prenatal diagnosis, quantitative analysis of gene expression, and NGS library quantification. Although dPCR is still the most widespread type of dNAD, in resource-limited regions, it is confined due to the requirement of thermal cycling and robust temperature control. To overcome this awkwardness, dINAAs without thermal cycling are of great interest, which also enlarge the application of dNAD, especially for POCT. For this subchapter, the recent advances on application of dNAD will be

Honestly speaking, on pathogen detection, the advantages of most conventional NADs on both analytical sensitivity and specificity are inherited by the corresponding digital formats. In addition, dNAD has the capability of realizing NA's absolute quantification. Therefore, dNAD can be used as the ultrahigh sensitivity and more accurate method for viral load determination or bacterial quantification. Kelley et al. established a duplex ddPCR assay for high-precision methicillin-resistant *Staphylococcus aureus* (MRSA) analysis [76]. By assaying 397 clinical samples, a good agreement with the reference assay for both qPCR and ddPCR assays was indicated. Strain et al. demonstrated and estimated the application of ddPCR for a highly precise measurement of HIV DNA [77]. Total HIV DNA and episomal 2-LTR (long terminal repeat) circles in cells that were all isolated from infected patients were targeted. Compared to qPCR, ddPCR performed a significantly increased precision (5-fold for total HIV DNA and >20-fold for 2-LTR circles), making it an alternative for the measurement of HIV DNA from

dNAD also enables the direct detection without NA extraction due to the ability of partitioning target NA and nontarget components (e.g., inhibitors) into different microwells. Pavšič et al. employed two dPCR platforms (QX100 ddPCR system from Bio-Rad Laboratories and the BioMark HD IFC cdPCR system from Fluidigm) for the direct quantification of two wholevirus materials of human cytomegalovirus (HCMV) without DNA extraction [78]. It was demonstrated that direct quantification by both dPCRs could provide repeatable measure‐

the precision (∆*λ*/*λ*) to expected molecules per chamber (*λ*) could be plotted.

**5. Application of dNAD**

142 Lab-on-a-Chip Fabrication and Application

narrated.

**5.1. Pathogen detection**

clinical specimens.

Food safety mainly refers to two parts: the minoring of foodborne pathogens and the detection of GMO. For example, human noroviruses (NoV) and hepatitis A virus (HAV) are two foodborne enteric viruses that have caused the vast majority of nonbacterial gastroenteritis or some fatal infectious hepatitis. Coudray-Meunier et al. conducted a comparative study of IFC RT-cdPCR and conventional RT-qPCR when quantifying the NoV and HAV from lettuce and water samples, proving that the IFC RT-cdPCR assay was more tolerant to inhibitory sub‐ stances from lettuce samples [80]. Also, the IFC RT-cdPCR may be useful for standardizing the quantification of enteric viruses in bottled water and lettuce samples. Fu et al. used the BioMark HD system equipped with a 48.770 digital array to develop cdPCR for GMO detection without pretreatment [81]. The CaMV35s promoter and the NOS terminator were selected as the targets, and nine events of GMOs (MON810, MON863, TC1507, MIR604, MIR162, GA21, T25, NK603, and Bt176) were collected to determine the specificity. The results showed that the cdPCR could achieve a discrimination of down to 0.1%, lower than the labeling threshold level of the EU, allowing highly sensitive, specific, and stable GMO screening detection. Dalmira et al. developed a duplex ddPCR assay to characterize the certified reference materials (CRMs) in terms of T-*nos*/*hmg* copy number ratio in maize [82]. After optimization using a central composite design, the duplex ddPCR method realized the absolute limit of detection (LOD) and limit of quantification (LOQ) of 11 and 23 copy number *T*-*nos*, namely, relative LOD of 0.034% and relative LOQ of 0.08%, respectively. The dynamic range of T-*nos*/*hmg* ratio ranged from 0.08% to 100%. The results indicated that the duplex ddPCR assay was useful for characterizing CRM candidates on T-*nos*/*hmg* ratio.

The bias caused by reliance on quantitative standards may have an impact on the results of qPCR, which is not beneficial for water monitoring and microbial source identification. Therefore, Cao et al. employed a duplex ddPCR to simultaneously quantify *Enterococcus* spp. and the human fecal-associated HF183 marker for evaluating the water quality [83]. The results demonstrated that ddPCR performed greater tolerance of inhibition than qPCR, with one to two orders of magnitude higher at inhibitor concentrations. Also, ddPCR brought about remarkably improved precision, although a lower upper LOQ than qPCR was indicated.

For food safety, it is indispensable to identify and quantify the meat products for unveiling species fraud and product mislabeling during food processing. Tian et al. used LTNP cdPCR to detect bovine meat in ovine meat [72]. Floren et al. successfully applied two-step ddPCR for precise quantification of cattle, horse, and pig in processed meat product, with the nuclear *F2* gene targeted [84].

#### **5.3. Clinical diagnosis**

At present, clinical diagnosis including genetic instability estimation and early cancer is also the main application field for dNAD. It has been clearly confirmed that the genetic instability of human cells is one of the causes of cancer, including somatic mutation, allelic imbalance, loss of heterozygosity, CNVs, and single nucleotide variations (SNVs). Accordingly, how to discriminate the rare mutant gene from abundant normal NAs attracts great concerns of researchers. Interestingly, it is also the question that initiates the concept formation of dNAD.

As *de novo* CNV may be caused by reprogramming somatic cells into induced pluripotent stem cells, Abyzov et al. used qPCR and ddPCR to detect and estimate this phenomenon. The results showed that, in parental fibroblasts, at least half of the CNVs are indicated as low-frequency somatic genomic variants [85]. Boettger et al. successfully applied a ddPCR approach to analyze father-mother-offspring trios from HapMap at specific sites within region 1 on the investigation of inference of complex CNV and single nucleotide polymorphism (SNP) haplotypes at the human 17q21.31 locus [86].

Detecting biomarkers associated with tumor formation, development, and drug evaluations on gene expression is the useful approach for early cancer diagnosis. Up to now, various biomarkers have been identified, and dNAD reflects the unique advantage of absolute quantitation of their gene expression [87]. Zhu et al. applied SPC cdPCR to the detection of three lung cancer-related genes (PLAU, ENO2, and PLAT). cdPCR yielded comparable results to qPCR, illustrating that the established platform had the ability of realizing absolute quantitation for gene expression [30]. Floren et al. employed ddPCR to detect the BRAF-V600E and V600K mutations in melanoma circulating tumor with high sensitivity [84]. The study demonstrated that ddPCR performed 200-fold increased sensitivity than competitive allelespecific PCR (castPCR), giving an LOD of 0.0005% when combined with whole-genome amplification (WGA). Through noninvasive analysis of circulating free plasma DNA, Geven‐ sleben et al. determined the presence of oncogenic amplification by developing a plasma DNA ddPCR assay targeting HER2 [88]. In the independent validation cohort, ddPCR could reach a positive and negative predictive value of 70% and 92%, respectively. The results suggested that ddPCR had the potential to the analysis of any locus amplified in cancer, not only in metastatic breast cancer. Beaver et al. also employed a ddPCR assay for the detection of circulating plasma tumor DNA (ptDNA) in patients with early-stage cancer [89]. A total of 30 tumors were first analyzed by Sanger sequencing for common PIK3CA mutations, and ddPCR was then used to analyze their extracted DNA for the same mutations. This ddPCR-based accurate mutation detection platform was demonstrated to be of great use for early-stage breast cancer.

As a new generation of cancer biomarkers, abnormally DNA methylation in the gene's regulatory regions can affect identical residues that may cause the cancer. The analysis of methylated genes therefore becomes more and more important in cancer research. For example, Li et al. developed a sensitive bead-based ddPCR for the quantification of DNA methylation, which could detect down to one methylated DNA molecule in approximately 5000 unmethylated DNA molecules from plasma or fecal samples [20]. Weisenberger et al. developed an improved DNA methylation detection with single-molecule high-resolution based on IFC cdPCR and successfully identified the breast cancer-specific hypermethylation phenomenon in the CpG islands of RUNX3, CLDN5, and FOXE1 [90].

#### **5.4. Prenatal diagnosis**

precise quantification of cattle, horse, and pig in processed meat product, with the nuclear *F2*

At present, clinical diagnosis including genetic instability estimation and early cancer is also the main application field for dNAD. It has been clearly confirmed that the genetic instability of human cells is one of the causes of cancer, including somatic mutation, allelic imbalance, loss of heterozygosity, CNVs, and single nucleotide variations (SNVs). Accordingly, how to discriminate the rare mutant gene from abundant normal NAs attracts great concerns of researchers. Interestingly, it is also the question that initiates the concept formation of dNAD. As *de novo* CNV may be caused by reprogramming somatic cells into induced pluripotent stem cells, Abyzov et al. used qPCR and ddPCR to detect and estimate this phenomenon. The results showed that, in parental fibroblasts, at least half of the CNVs are indicated as low-frequency somatic genomic variants [85]. Boettger et al. successfully applied a ddPCR approach to analyze father-mother-offspring trios from HapMap at specific sites within region 1 on the investigation of inference of complex CNV and single nucleotide polymorphism (SNP)

Detecting biomarkers associated with tumor formation, development, and drug evaluations on gene expression is the useful approach for early cancer diagnosis. Up to now, various biomarkers have been identified, and dNAD reflects the unique advantage of absolute quantitation of their gene expression [87]. Zhu et al. applied SPC cdPCR to the detection of three lung cancer-related genes (PLAU, ENO2, and PLAT). cdPCR yielded comparable results to qPCR, illustrating that the established platform had the ability of realizing absolute quantitation for gene expression [30]. Floren et al. employed ddPCR to detect the BRAF-V600E and V600K mutations in melanoma circulating tumor with high sensitivity [84]. The study demonstrated that ddPCR performed 200-fold increased sensitivity than competitive allelespecific PCR (castPCR), giving an LOD of 0.0005% when combined with whole-genome amplification (WGA). Through noninvasive analysis of circulating free plasma DNA, Geven‐ sleben et al. determined the presence of oncogenic amplification by developing a plasma DNA ddPCR assay targeting HER2 [88]. In the independent validation cohort, ddPCR could reach a positive and negative predictive value of 70% and 92%, respectively. The results suggested that ddPCR had the potential to the analysis of any locus amplified in cancer, not only in metastatic breast cancer. Beaver et al. also employed a ddPCR assay for the detection of circulating plasma tumor DNA (ptDNA) in patients with early-stage cancer [89]. A total of 30 tumors were first analyzed by Sanger sequencing for common PIK3CA mutations, and ddPCR was then used to analyze their extracted DNA for the same mutations. This ddPCR-based accurate mutation detection platform was demonstrated to be of great use for early-stage breast

As a new generation of cancer biomarkers, abnormally DNA methylation in the gene's regulatory regions can affect identical residues that may cause the cancer. The analysis of methylated genes therefore becomes more and more important in cancer research. For example, Li et al. developed a sensitive bead-based ddPCR for the quantification of DNA

gene targeted [84].

cancer.

**5.3. Clinical diagnosis**

144 Lab-on-a-Chip Fabrication and Application

haplotypes at the human 17q21.31 locus [86].

Prenatal diagnosis is usually carried out by invasive or noninvasive approaches [91]. The invasive method (e.g., amniocentesis) refers to inserting needles into the uterus, which is timeconsuming (several weeks required) and risky to the fetus [92]. In contrast, noninvasive method seems to be more rapid and safer. Currently, noninvasive prenatal diagnosis mainly involves ultrasonography of the womb or detecting cell-free fetal DNA (cffDNA) in maternal serum and plasma. The latter is also termed as noninvasive prenatal testing (NIPT). Appling dNAD to NIPT is the emerging noninvasive approach with high sensitivity and high precision [93–97]. Gu et al. employed the QX100 ddPCR system from Bio-Rad Laboratories to detect cffDNA for the risk of methylmalonic acidemia, confirming that ddPCR is a cost-effective and noninvasive prenatal method when diagnosing known mutations associated with Mendelian disorders [98]. Pornprasert and Prasing implemented a ddPCR for the deletion of α(0) thalassemia Southeast Asian (SEA)-type deletion [99]. The study showed that ddPCR might be an alternative technology to routine clinical diagnosis. Barrett et al. adopted 12×765 digital array chips to establish an IFC cdPCR to analyze the cffDNA for NIPT of sickle cell anemia [94]. The results suggested that the built IFC cdPCR is a useful method to determine the genotype of fetuses at risk for sickle cell anemia. Meantime, the report also illustrated that it was essential to optimize the fractional fetal DNA concentration.

Because most cffDNA fragments were approximately 200 bp in size and, in the early gestation, the cffDNA occupies low percentage (mostly <10%) in maternal plasma, efficient methods for the extraction of cffDNA are in great need. Holmberg et al. thereby estimated two commercial platforms to extract cffDNA: the Akonni Biosystems TruTip technology and the Circulating Nucleic Acids Kit from Qiagen [100]. Determined by QX100 ddPCR system and qPCR, the extracted products from two platforms performed similar results.

#### **5.5. NGS library quantification**

As known to all, the establishment of NGS libraries still leans mainly on manual bench-top procedures, which is slow and inefficient [101]. To solve it, Kim et al. invented an automated digital microfluidic LOC-based sample preparation for the NGS. Compared to the conven‐ tional methods, digital microfluidic LOC platform is cost-efficient and has high throughout [102]. Similarly, Thaitrong et al. integrated a droplet-based microfluidic LOC system with a unit of capillary-based reagent delivery and the quantitative CE module to develop an automated quality-control platform for NGS [103]. Besides, White et al. confirmed that dPCR is able to provide sensitive and absolute calibration for NGS, enabling direct sequencing without titration runs and with sufficient precision [11]. Fu et al. developed a picoliter-scaled droplet digital WGA (ddWGA) platform for realizing uniform and accurate single-cell 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 information.
