**3. Classification and advances of dNAD**

In the early stage of digital detection, the used materials are 96- and 384-microwell plates. Then, due to the rapid development of microfluidic chip techniques, an increasing number of digital detection devices emerge. Also, a variety of materials have been used individually or jointly, such as silicon wafer, quartz, glass, polydimethylsiloxane (PDMS), polymethyl methacrylate, and so on. According to the approaches to partition reaction mixture, the currently launched dNAD methods can be roughly grouped into three categories: plate-based dNAD (pdNAD), droplet-based dNAD (ddNAD), and chip-based dNAD (cdNAD). On structural design, each classified dNAD has the advantages and disadvantages, and the corresponding commercial devices are also developed. In this subchapter, we are going to narrate their features and recent advances in either commercial or research aspects.

#### **3.1. pdNAD**

At present, most of the pdNADs are established as dPCR devices, but they are not hard to be developed as dINAA platforms. As the first generation of dNAD, plate-based dPCR (pdPCR) was first conducted using plenty of commercially available 96- and 384-microwell plates [23, 41]. The biggest benefit for this kind of digital platform is saving to create the plates that have been widely used in conventional PCR. Each microwell undertakes each microreaction; therefore, the high sensitivity and accuracy of detection lean entirely on the enough number of microwells. However, actually, the number is hard to be reached just using microwell plates.

Another problem causing the embarrassment is the volume of reagents required [42]. For each microwell, more than 5 μL are needed, and the cost of reagents inevitably daunts most researchers, let alone the application for POCT. To break the barriers, some researchers made modification. As shown in **Figure 2**, Morrison et al. deceased the volume of microreaction into 33 nL using a stainless steel plate (25 mm in width and 75 mm in length) in which up to 3072 microholes (320 μm in diameter) were created [43]. In contrast, the required volume was reduced to 1/64, and the throughput was increased by 24-fold, although it had the comparable sensitivity to the past. At present, this technique has been applied to commercial devices in 2009, the OpenArray RealTime PCR System from Life Technologies. However, as the number of reaction units increases, the problem turns into how to efficiently load the reagents. Consequently, it has to use some ancillary equipment-like microarray spotter or mechanical arms, which in turn raises the cost and is cumbersome.

Considering the embarrassing situation, in the second half of 2013, Life Technologies launched the next-generation digital detection device, the QuantStudio 3D dPCR system [42, 44]. It is a simple and affordable platform to provide the reliable and robust dPCR. The device used a

**Figure 2.** Plate-based chip used for the OpenArray RealTime PCR System. A rectilinear array of 3072 microholes with 320 μm in diameter was fabricated in a stainless steel plate (25×75×0.3 mm). The volume of each hole was approximate‐ ly 33 nanoliers, and to match the pitch of the wells in a 384-well microplate, the 48 groups of 64 holes are spaced at 4.5 mm. Reprinted with permission from Ref. [43]. © Copyright 2006 Oxford University Press.

special plate (10 mm in width and 10 mm in length) where a total of 20,000 hexagonal micro‐ wells are fabricated. The volume of each microwell is 0.8 nL, and each reaction well is isolated absolutely from its neighbors. At present, the system has been applied to the absolute quan‐ tification of viral load, low-level pathogen detection, sensitive genetically modified organism (GMO) detection, differential gene expression, copy number variation (CNV), NGS library quantification, and rare mutation analysis [45–50]. Although the cost of reagents is reduced, the system still calls for supporting instruments to load the reagents, amplify the sample, and read the results.

For high-throughput sample analysis, 96- and 384-microplate formats are still of use. Formu‐ latrix introduced a new commercial high-throughput pdPCR device termed as constellation dPCR. The device brings the digital analysis to a 96-sample microplate format, and the socalled high-throughput results from the preformation of dPCR on 96 samples at once and up to 384 samples per hour. As required, the number of partitions for each microwell in the plate can be easily increased, and it reaches 496 for the 96-microplate format.

#### **3.2. ddNAD**

confidence 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

In the early stage of digital detection, the used materials are 96- and 384-microwell plates. Then, due to the rapid development of microfluidic chip techniques, an increasing number of digital detection devices emerge. Also, a variety of materials have been used individually or jointly, such as silicon wafer, quartz, glass, polydimethylsiloxane (PDMS), polymethyl methacrylate, and so on. According to the approaches to partition reaction mixture, the currently launched dNAD methods can be roughly grouped into three categories: plate-based dNAD (pdNAD), droplet-based dNAD (ddNAD), and chip-based dNAD (cdNAD). On structural design, each classified dNAD has the advantages and disadvantages, and the corresponding commercial devices are also developed. In this subchapter, we are going to narrate their features and recent

At present, most of the pdNADs are established as dPCR devices, but they are not hard to be developed as dINAA platforms. As the first generation of dNAD, plate-based dPCR (pdPCR) was first conducted using plenty of commercially available 96- and 384-microwell plates [23, 41]. The biggest benefit for this kind of digital platform is saving to create the plates that have been widely used in conventional PCR. Each microwell undertakes each microreaction; therefore, the high sensitivity and accuracy of detection lean entirely on the enough number of microwells. However, actually, the number is hard to be reached just using microwell plates. Another problem causing the embarrassment is the volume of reagents required [42]. For each microwell, more than 5 μL are needed, and the cost of reagents inevitably daunts most researchers, let alone the application for POCT. To break the barriers, some researchers made modification. As shown in **Figure 2**, Morrison et al. deceased the volume of microreaction into 33 nL using a stainless steel plate (25 mm in width and 75 mm in length) in which up to 3072 microholes (320 μm in diameter) were created [43]. In contrast, the required volume was reduced to 1/64, and the throughput was increased by 24-fold, although it had the comparable sensitivity to the past. At present, this technique has been applied to commercial devices in 2009, the OpenArray RealTime PCR System from Life Technologies. However, as the number of reaction units increases, the problem turns into how to efficiently load the reagents. Consequently, it has to use some ancillary equipment-like microarray spotter or mechanical

Considering the embarrassing situation, in the second half of 2013, Life Technologies launched the next-generation digital detection device, the QuantStudio 3D dPCR system [42, 44]. It is a simple and affordable platform to provide the reliable and robust dPCR. The device used a

a standard curve.

128 Lab-on-a-Chip Fabrication and Application

**3.1. pdNAD**

**3. Classification and advances of dNAD**

advances in either commercial or research aspects.

arms, which in turn raises the cost and is cumbersome.

ddNAD can go back to emulsion PCR (ePCR) [51–54]. ePCR is widely used for NGS (**Figure 3**) [55]. After generating a DNA library, the fragments of genomic DNA are attached to the beads, because their surface is modified with oligonucleotide probes whose sequences are complementary to the sequences of the fragments. When the beads are compartmentalized into water (the PCR reagent)-oil emulsion droplets, plenty of microreactors are produced. Since each bead captures single-stranded DNA fragment, in theory, ePCR can amplify it down to one DNA molecule. However, it is not easy to partition the fragments and beads into one droplet simultaneously, and then the performance of ePCR suffers from variety. Benefitting from the rapid development of microfluidic LOC techniques, ddNAD also enjoys a huge boom in recent years. dPCR is still the main part in ddNAD, but droplet-based dINAAs including dLAMP, dRPA, digital rolling circle amplification (RCA), and digital hyperbranched RCA (HRCA) are showing up more and more [34–37, 56].

**Figure 3.** ePCR used for NGS. Top left: The genomic DNA is isolated, fragmented, ligated to adapters, and separated into single strands. Top right: Fragments are bound to beads that are captured in the droplets of a PCR mixture-in-oil emulsion. Then, ePCR occurs within each droplet. Bottom right: After breaking emulsion and denaturing the DNA strands, beads with single-stranded DNA are deposited into wells of a fiber-optic slide. Bottom left: Pyrophosphate sequencing is initiated within each well after depositing smaller beads carrying immobilized required enzymes. Re‐ printed with permission from Ref. [55]. © Copyright 2005 Nature Publishing Group.

Beer et al. successfully created picoliter-scale water-in-oil droplets by using a shearing Tjunction in a fused-silica device in 2008 [57]. The NA used for the device was RNA; therefore, an off-chip valving system was integrated to stop the droplet motion, because a different thermal cycling was required for reverse transcription and subsequent PCR amplification. Each droplet contained the PCR mixture of single-copy template, primers, and reaction buffer, which was really termed as digital detection. One year later, Mazutis et al. developed a method for high-throughput dINAA platform in a 2 pL droplet-based microfluidic system [35]. The isothermal HRCA was used to perform the DNA amplification in droplets. This platform was demonstrated to allow fast and accurate digital quantification of the template. In 2011, Zhong et al. reported another picoliter-scale droplet-based multiplexing dPCR platform, breaking the one target per color barrier of qPCR [58]. The number of droplets generated reached more than 106 , which was enough for enhancing the likelihood that only one DNA molecule was amplified in each droplet. Given its great potential in application, RainDance Technologies launched the commercial digital detection system with the highest droplet throughout, the RainDrop dPCR system. Unfortunately, the system may consume up to 50 μL reagents per sample. Considering this point, the system may be not proper for rare sample detection. At the same year, Hindson et al. also established a high-throughput droplet-based dPCR (ddPCR) platform [59]. A total of 2 million droplets were generated, and the droplets were then transferred into a 96-well plate for TaqMan probe-based PCR. Finally, to read out the results, a flow cytometry-like double-channel fluorescence detection device was used in a microfluidic chip, in which droplets went through one by one. The platform was confirmed to realize the accurate measurement of germ-line CNV, discriminate the mutant molecules from the wild molecules with 105 -fold excess, and absolutely quantify circulating fetal and maternal DNA from cell-free plasma. Based on the platform, the first commercial ddPCR system was launched by QuantaLife in 2011, but in the end of that year Bio-Rad Laboratories purchased the company and launched the QX100 ddPCR. Recently, the new version, QX200 ddPCR system, is also available.

droplet simultaneously, and then the performance of ePCR suffers from variety. Benefitting from the rapid development of microfluidic LOC techniques, ddNAD also enjoys a huge boom in recent years. dPCR is still the main part in ddNAD, but droplet-based dINAAs including dLAMP, dRPA, digital rolling circle amplification (RCA), and digital hyperbranched RCA

**Figure 3.** ePCR used for NGS. Top left: The genomic DNA is isolated, fragmented, ligated to adapters, and separated into single strands. Top right: Fragments are bound to beads that are captured in the droplets of a PCR mixture-in-oil emulsion. Then, ePCR occurs within each droplet. Bottom right: After breaking emulsion and denaturing the DNA strands, beads with single-stranded DNA are deposited into wells of a fiber-optic slide. Bottom left: Pyrophosphate sequencing is initiated within each well after depositing smaller beads carrying immobilized required enzymes. Re‐

Beer et al. successfully created picoliter-scale water-in-oil droplets by using a shearing Tjunction in a fused-silica device in 2008 [57]. The NA used for the device was RNA; therefore, an off-chip valving system was integrated to stop the droplet motion, because a different thermal cycling was required for reverse transcription and subsequent PCR amplification. Each droplet contained the PCR mixture of single-copy template, primers, and reaction buffer, which was really termed as digital detection. One year later, Mazutis et al. developed a method for high-throughput dINAA platform in a 2 pL droplet-based microfluidic system [35]. The isothermal HRCA was used to perform the DNA amplification in droplets. This platform was demonstrated to allow fast and accurate digital quantification of the template. In 2011, Zhong et al. reported another picoliter-scale droplet-based multiplexing dPCR platform, breaking the one target per color barrier of qPCR [58]. The number of droplets generated reached more than

, which was enough for enhancing the likelihood that only one DNA molecule was amplified in each droplet. Given its great potential in application, RainDance Technologies launched the commercial digital detection system with the highest droplet throughout, the RainDrop dPCR system. Unfortunately, the system may consume up to 50 μL reagents per

printed with permission from Ref. [55]. © Copyright 2005 Nature Publishing Group.

106

(HRCA) are showing up more and more [34–37, 56].

130 Lab-on-a-Chip Fabrication and Application

Apart from the process of droplet generation and subsequent NA amplification, other approaches to generate droplet are also reported. As shown in **Figure 4**, Shen et al. described a SlipChip to create droplet array [26]. The SlipChip was composed of two glass plates, in which elongated wells were designed to overlap and form the fluidic path for reagent loading. After sample loading, the simple slipping of the two plates broke the path, removing the overlap among wells and generating 1280 droplet array (2.6 nL for each). The device had a reservoir preloaded with oil, so each microreactor was absolutely isolated from each other

**Figure 4.** Design and mechanism of the SlipChip for dPCR. The top plate is outlined with a black solid line, the bottom plate is outlined with a blue dotted line, and red represents the sample. (a) Schematic drawing shows the design of the entire assembled SlipChip for dPCR after slipping. (b) Schematic drawing of part of the top plate. (c) Schematic draw‐ ing of part of the bottom plate. (d–f) The SlipChip was assembled such that the elongated wells in the top and bottom plates overlapped to form a continuous fluidic path. (g–i) The aqueous reagent (red) was injected into SlipChip and filled the chip through the connected elongated wells. (j–l) The bottom plate was slipped relative to the top plate such that the fluidic path was broken up and the circular wells were overlaid with the elongated wells, and aqueous drop‐ lets were formed in each compartment. (d, g, and j) Schematic of the SlipChip. (e, h, and k) Zoomed-in microphoto‐ graph of the SlipChip. (f, i, and l) Microphotograph of the entire SlipChip. Reprinted with permission from Ref. [26]. © Copyright 2010 Royal Society of Chemistry.

during thermal cycling. Finally, the results were read out using endpoint fluorescence intensity. The biggest advantage of SlipChip is the capability of realizing multistep manipu‐ lation of plenty of microvolumes to form droplet array in parallel. Attributed to the remarkable feature, until now, the SlipChip has been applied to perform immunoassays, protein crystal‐ lization, multiplex PCR, dPCR, dLAMP, dRPA, and so on [26, 34, 60–64].

To make the droplet more stable and to easily collect the amplified products, Leng et al. invented an agarose droplet-based single-molecule ePCR device [51]. The agarose performed the unique thermoresponsive sol-gel switching property, and a microfluidic chip was designed to produce uniform agarose solution droplets. Schuler et al. applied centrifugal step emulsi‐ fication to the fast and easy generation of monodisperse droplets [37]. Only by adjusting the nozzle geometry (depth, width, and step size) and interfacial tensions droplets with desirable diameters could be produced. Using this droplet device, dRPA was successfully established for the absolute quantification of *Listeria monocytogenes* DNA concentration standards within 30 min.

In ddNAD, the microreactors are generated by carefully titrating emulsions of water, oil, and chemical stabilizer; therefore, there is no requirement of the walls of microwells to separate the microreactors. Compared to pdNAD, ddNAD can easily achieve higher throughput via a microdroplet generator to produce hundreds of thousands of droplet reactions per sample. However, the workflow of ddPCR is complicated, referring to generating droplet, transferring droplet, sealing microplate, conventional PCR, and reading out the signal by other devices.

#### **3.3. cdNAD**

The development of cdNAD is greatly attributed to the rapid progress of microfluidic techniques, which can realize the low cost, low volume, and high-throughout paralleled NA detections. In the last several decades, microreactors in cdNAD are mainly formed either by the mechanical compartmentalization of PDMS or by the succeeding isolation via immiscible liquid phase. In particular, for PDMS-based chips, the establishment of multilayer soft lithography (MSL) techniques developed by Unger et al. in 2000 also gives a huge boost, making the high-density microwells, micropumps, and microvalves easily fabricated [65]. Based on different power sources to partition reagents, cdNAD can be divided into three categories: integrated fluidic circuit (IFC) cdNAD, self-priming compartmentalization (SPC) cdNAD, and localized temporary negative pressure (LTNP)-assisted cdNAD as well as other cdNADs.

#### *3.3.1. IFC cdNAD*

The outstanding feature of IFC chip is the special design of separated and interlaced liquid and gas channels, as shown in **Figure 5**. Taking advantage of the high elasticity of PDMS, hundreds or thousands of microreaction units are formed rapidly when gas channels are added with pressure.

In 2006, Ottesen et al. used the IFC chip to achieve dPCR analysis [66]. A total of 1176 micro‐ reaction units were produced by controlling accurately the integrated microvalves and

during thermal cycling. Finally, the results were read out using endpoint fluorescence intensity. The biggest advantage of SlipChip is the capability of realizing multistep manipu‐ lation of plenty of microvolumes to form droplet array in parallel. Attributed to the remarkable feature, until now, the SlipChip has been applied to perform immunoassays, protein crystal‐

To make the droplet more stable and to easily collect the amplified products, Leng et al. invented an agarose droplet-based single-molecule ePCR device [51]. The agarose performed the unique thermoresponsive sol-gel switching property, and a microfluidic chip was designed to produce uniform agarose solution droplets. Schuler et al. applied centrifugal step emulsi‐ fication to the fast and easy generation of monodisperse droplets [37]. Only by adjusting the nozzle geometry (depth, width, and step size) and interfacial tensions droplets with desirable diameters could be produced. Using this droplet device, dRPA was successfully established for the absolute quantification of *Listeria monocytogenes* DNA concentration standards within

In ddNAD, the microreactors are generated by carefully titrating emulsions of water, oil, and chemical stabilizer; therefore, there is no requirement of the walls of microwells to separate the microreactors. Compared to pdNAD, ddNAD can easily achieve higher throughput via a microdroplet generator to produce hundreds of thousands of droplet reactions per sample. However, the workflow of ddPCR is complicated, referring to generating droplet, transferring droplet, sealing microplate, conventional PCR, and reading out the signal by other devices.

The development of cdNAD is greatly attributed to the rapid progress of microfluidic techniques, which can realize the low cost, low volume, and high-throughout paralleled NA detections. In the last several decades, microreactors in cdNAD are mainly formed either by the mechanical compartmentalization of PDMS or by the succeeding isolation via immiscible liquid phase. In particular, for PDMS-based chips, the establishment of multilayer soft lithography (MSL) techniques developed by Unger et al. in 2000 also gives a huge boost, making the high-density microwells, micropumps, and microvalves easily fabricated [65]. Based on different power sources to partition reagents, cdNAD can be divided into three categories: integrated fluidic circuit (IFC) cdNAD, self-priming compartmentalization (SPC) cdNAD, and localized temporary negative pressure (LTNP)-assisted cdNAD as well as other

The outstanding feature of IFC chip is the special design of separated and interlaced liquid and gas channels, as shown in **Figure 5**. Taking advantage of the high elasticity of PDMS, hundreds or thousands of microreaction units are formed rapidly when gas channels are added

In 2006, Ottesen et al. used the IFC chip to achieve dPCR analysis [66]. A total of 1176 micro‐ reaction units were produced by controlling accurately the integrated microvalves and

lization, multiplex PCR, dPCR, dLAMP, dRPA, and so on [26, 34, 60–64].

30 min.

132 Lab-on-a-Chip Fabrication and Application

**3.3. cdNAD**

cdNADs.

*3.3.1. IFC cdNAD*

with pressure.

**Figure 5.** An IFC chip-based 12×765 digital array from the Fluidigm. Left: Schematic diagram of a part of the IFC chip in which microchambers were connected and isolated by fluidic channels and pressure lines. Right: Optical microscop‐ ic images of the part. Reprinted with permission. © Copyright 2009 Springer.

removing the conventional microarray spotter and plates. Now, the IFC chip-based dPCR (IFC cdPCR) platform is successfully established for commercial purpose by Fluidigm. As the first vendor to commercialize dPCR device, Fluidigm provides two IFC-based systems, the BioMark HD and EP1 systems. In the two systems, the PCR reagents are mixed and partitioned automatically, the thermocycling is integrated, and the results can be read out after reaction. The BioMark HD system can offer real-time detection for each tiny reaction and eliminate false positives according to the data, so the system is also available to qPCR. Compared to BioMark HD, EP1 is just an endpoint detection machine, giving the binary output-like data whether or not the microreaction occurs. Recently, Fluidigm also combines with Olink to detect human protein biomarkers based on proximity extension assay (PEA) technology. Until now, the dPCR device from Fluidigm has been applied to single-cell analysis, early diagnosis of cancer, and prenatal diagnosis.

In 2011, Heyries et al. developed a megapixel dPCR platform in which 106 microunits were fabricated, and the microreaction's volume reached down to 10 pL (**Figure 6**) [67]. The density of the microreactors reached up to 440,000/mm2 , which was the highest density for IFC platform. On detection performance, this device was able to discriminate one mutant molecule from 105 wild molecules and achieve the discrimination of a 1% difference in chromosome copy number. After the platform, in 2012, Men et al. published anther dPCR platform pos‐ sessing the lowest volume (36 fL) of microreactors until now. Its density of microreactors was more than 20,000/mm2 [24]. After loading the reagents into all microreactors simultaneously, the deformation of a PDMS membrane was used to completely seal the filled microreactors. Due to the femtoliter-level microreactors fabricated, the device can greatly reduce the con‐ sumption of reagent and sample.

**Figure 6.** Schematic of megapixel dPCR device (a) and the layered device structure (b). Reprinted with permission from Ref. [67]. © Copyright 2011 Nature Publishing Group.

For dINAA, IFC chip is also combined with isothermal MDA to develop dMDA for enumer‐ ation of total NA contamination [33]. On detection of microbial genomic DNA fragments, dMDA performs higher sensitivity with orders of magnitude than qPCR.

By making the microchamber smaller or increasing its number, IFC cdNAD has a potential to be developed into a digital detection platform with higher throughout, higher density, and higher discrimination ability, but this platform still relies on the control system of integrated microvalves and micropumps to load and partition the reagents, which is hard to be applied towards POCT. Furthermore, narrowing the size of microchamber endlessly may have an impact on the efficiency of NA amplification.

#### *3.3.2. SPC cdNAD*

Targeting practical POCT devices, currently proposed plate-based, droplet-based, and IFC cdNADs are confronted with the huge challenge of demand for peripheral control instrument, for instance, external syringe pumps, droplet generation devices, and plenty of integrated microvalves and micropumps. Upon this challenge, the built-in power-driving, self-partition‐ ing, easy-to-use, and low-priced SPC cdNADs were developed by our laboratory. The builtin power results from the gas solubility and permeability of PDMS, because PDMS remains absorbing gas and letting gas go through them, although PDMS is in a solid state in chips [68].

The chip possesses the prominent feature of SPC, resulting from the used material of silicone elastomer PDMS, a relatively cheap material, which possesses high gas solubility and perme‐ ability. When the fabricated chips are evacuated, a negative pressure environment is formed due to the gas solubility of PDMS, which can service as a self-priming power to let the sample solutions be sucked into each reaction chamber and sequentially the biocompatible oil to seal and separate each filled chamber. Thus, in realizing dNAD, thousands of independent microwells can be created automatically, avoiding the external control system, which is superior to IFC cdNAD. Currently, SPC cdPCR and dINAAs [SPC chip-based dLAMP (cdLAMP) and SPC chip-based dIMSA (cdIMSA)] have been developed by our laboratory [30, 31, 69, 70].

#### *3.3.2.1. SPC cdPCR*

**Figure 6.** Schematic of megapixel dPCR device (a) and the layered device structure (b). Reprinted with permission

For dINAA, IFC chip is also combined with isothermal MDA to develop dMDA for enumer‐ ation of total NA contamination [33]. On detection of microbial genomic DNA fragments,

By making the microchamber smaller or increasing its number, IFC cdNAD has a potential to be developed into a digital detection platform with higher throughout, higher density, and higher discrimination ability, but this platform still relies on the control system of integrated microvalves and micropumps to load and partition the reagents, which is hard to be applied towards POCT. Furthermore, narrowing the size of microchamber endlessly may have an

Targeting practical POCT devices, currently proposed plate-based, droplet-based, and IFC cdNADs are confronted with the huge challenge of demand for peripheral control instrument, for instance, external syringe pumps, droplet generation devices, and plenty of integrated microvalves and micropumps. Upon this challenge, the built-in power-driving, self-partition‐ ing, easy-to-use, and low-priced SPC cdNADs were developed by our laboratory. The builtin power results from the gas solubility and permeability of PDMS, because PDMS remains absorbing gas and letting gas go through them, although PDMS is in a solid state in chips [68]. The chip possesses the prominent feature of SPC, resulting from the used material of silicone elastomer PDMS, a relatively cheap material, which possesses high gas solubility and perme‐ ability. When the fabricated chips are evacuated, a negative pressure environment is formed due to the gas solubility of PDMS, which can service as a self-priming power to let the sample solutions be sucked into each reaction chamber and sequentially the biocompatible oil to seal and separate each filled chamber. Thus, in realizing dNAD, thousands of independent microwells can be created automatically, avoiding the external control system, which is superior to IFC cdNAD. Currently, SPC cdPCR and dINAAs [SPC chip-based dLAMP

dMDA performs higher sensitivity with orders of magnitude than qPCR.

from Ref. [67]. © Copyright 2011 Nature Publishing Group.

134 Lab-on-a-Chip Fabrication and Application

impact on the efficiency of NA amplification.

*3.3.2. SPC cdNAD*

The chip shown in **Figure 7** is composed of three PDMS layers, two glass coverslips, and a waterproof layer [30]. The three PDMS layers include an inlet and outlet layer, a microwell array layer, and a blank layer. In the microwell array layer, a total of 5120 reaction microwells (150 μm in width, 150 μm in length, and 250 μm in height) are equally distributed in four separate panels. Each microwell contains down to 5 nL solution. The inlet and outlet layers have four 0.5-mm holes and four 2.5-mm holes in diameter punched as injection ports and suction chambers when aligning to the outlet of the microwell array layer, respectively. For mechanical stability, the blank layer coats the microwell array layer with the waterproof layer embedded. The waterproof layer is made of low permeability fluorosilane polymer, which beneficially prevents the evaporation during the step of denaturing template DNA at 95°C in PCR. One of the glass coverslips with plasma pretreated are used to seal the microwell array, and the other one is pressed on the upper surface of the SPC chip for mechanical stability at the end of microchip operation.

MSL techniques are used to fabricate the SPC chip. The chip patterns are designed by a software of CorelDRAW X4 and printed on transparency films using a high-resolution printer to create

**Figure 7.** (A) Schematic diagram of the layered device structure of the SPC chip. (B) Photograph of the prototype SPC cdPCR device. The size of the chip is 50×24×4 mm. (C) Principle and operation procedure of the SPC microfluidic de‐ vice. The red cuboids (150×150×250 μm) stand for the microwells. Reprinted with permission from Ref. [30]. © Copy‐ right 2014 Royal Society of Chemistry.

the masks of channels and microwells. The photoresist material used are SU8 serials, which are a high-contrast, epoxy-based negative photoresist. Several 4-inch silicon wafers are adopted as the mold substrate. The PDMS to replicate the SPC chip is the silicone elastomer PDMS, which is composed of PDMS base (A) and catalyst (B) at certain ratios. Because dPCR is an endpoint detection, the reaction components including PCR buffer, primers, labeled probes, and templates have to be mixed before loading into the chip. Each diluted template in the mixture is individually injected into the three panels of the chip, allowing the samples to be compartmentalized completely and each chamber contains down to 5 nL solution. A Maestro Ex In-Vivo Imaging System (CRI Maestro, USA) is used to capture the fluorescent image of the microchip after dPCR. As a new generation of microfluidic chips, SPC cdPCR has been successfully applied to the absolute quantification of β-actin DNAs and the lung cancerrelated genes.

#### *3.3.2.2. SPC chip-based dINAAs (cdINAAs)*

Although PCR is widely adopted and used as a standard analytical technique in molecular diagnosis, it is remarkably confined when applied to field and POCT due to the facts that it requires nonportable thermocycling facilities, its process of obtaining results is cumbersome, and the whole amplification takes 2 h or more. Also, SPC cdPCR confronts the same defects. Accordingly, SPC cdLAMP and SPC cdIMSA are established by our laboratory. As simple and easy world-to-chip fluidic devices, SPC cdINAAs have the great potential in POCT for the developing countries.

Similar to SPC cdPCR, SPC cdLAMP is also the completely valve-free and SPC device (**Figure 8**) [31]. It is also made mainly of PDMS and fabricated by MSL techniques. In size, the SPC chip used for dLAMP is the same as the dPCR-used chip; however, in composition, it does not contain a waterproof layer because of the absence of the DNA denaturing step in LAMP. For the microwell array PDMS layer of dLAMP-used SPC chip, a total of 4800 microwells (150 μm in width, 150 μm in length, and 300 μm in height) are fabricated and they are also equally distributed into four panels (each contains 1200 chambers), and the interval for two closed chambers is 150 μm. The big difference from the dPCR-used SPC is that the rectangular chambers are located vertically on the main channels and the branch channels link to chamber without orthogonal turning points. On performance, the SPC cdLAMP can precisely calculate the absolute DNA concentration. To conduct the data acquisition and analysis of SPC cdLAMP, the Maestro Ex In-Vivo Imaging System is employed. However, the imaging system is too cumbersome and expensive to allow the e POCT, especially in the less developed regions. Herein, an easy-to-use and cost-efficient smartphone-based dLAMP POCT device platform is also established by our laboratory.

SPC cdIMSA is an updated version of SPC cdLAMP, in which the LAMP is replaced by IMSA and a mixed dye is used to establish a label-free and sensitive dual-fluorescence detection for on-chip IMSA [70, 71]. The used SPC chip for dIMSA is the same as that for dPCR without any modifications. The SPC cdIMSA with the mixed dye for the detection of hepatitis B virus (HBV) is conducted. In contrast, the mixed dye indicating two different colors makes it easy to count the positive chamber number by visual inspection regardless of the cutoff values. Also, there Digital Nucleic Acid Detection Based on Microfluidic Lab-on-a-Chip Devices http://dx.doi.org/10.5772/62742 137

**Figure 8.** (a) Schematic diagram of the SPC chip for cdLAMP. (b) Schematic of the whole microchip and the enlarged schematic diagram of the part of the chip, with insets showing the array and microwell geometries. It contains four separate panels, each of which has an individual inlet and outlet. The blue lines (8×50 μm) are the flow channel. The red spots (150×150×300 μm) stand for the microwells. Each microwell was partitioned by oil. (c) Photograph of the pro‐ totype SPC cdLAMP device. Reprinted with permission from Ref. [31]. © Copyright 2012 Royal Society of Chemistry.

is a linear response of the counted positive chamber number to the dilution ratios of templates. Moreover, the dual fluorescence is capable of indicating more positive chamber number in contrast to single fluorescence by SYBR Green I. The mixed dye-loaded SPC cdIMSA possesses the advantage of enlarging the color changes over other currently used dyes or indicators. Similarly, the mixed dye-based dual-fluorescence detection has a potential in the POCT application of SPC cdLAMP and other SPC chip-based dINAAs.

#### *3.3.3. LTNP cdNAD*

the masks of channels and microwells. The photoresist material used are SU8 serials, which are a high-contrast, epoxy-based negative photoresist. Several 4-inch silicon wafers are adopted as the mold substrate. The PDMS to replicate the SPC chip is the silicone elastomer PDMS, which is composed of PDMS base (A) and catalyst (B) at certain ratios. Because dPCR is an endpoint detection, the reaction components including PCR buffer, primers, labeled probes, and templates have to be mixed before loading into the chip. Each diluted template in the mixture is individually injected into the three panels of the chip, allowing the samples to be compartmentalized completely and each chamber contains down to 5 nL solution. A Maestro Ex In-Vivo Imaging System (CRI Maestro, USA) is used to capture the fluorescent image of the microchip after dPCR. As a new generation of microfluidic chips, SPC cdPCR has been successfully applied to the absolute quantification of β-actin DNAs and the lung cancer-

Although PCR is widely adopted and used as a standard analytical technique in molecular diagnosis, it is remarkably confined when applied to field and POCT due to the facts that it requires nonportable thermocycling facilities, its process of obtaining results is cumbersome, and the whole amplification takes 2 h or more. Also, SPC cdPCR confronts the same defects. Accordingly, SPC cdLAMP and SPC cdIMSA are established by our laboratory. As simple and easy world-to-chip fluidic devices, SPC cdINAAs have the great potential in POCT for the

Similar to SPC cdPCR, SPC cdLAMP is also the completely valve-free and SPC device (**Figure 8**) [31]. It is also made mainly of PDMS and fabricated by MSL techniques. In size, the SPC chip used for dLAMP is the same as the dPCR-used chip; however, in composition, it does not contain a waterproof layer because of the absence of the DNA denaturing step in LAMP. For the microwell array PDMS layer of dLAMP-used SPC chip, a total of 4800 microwells (150 μm in width, 150 μm in length, and 300 μm in height) are fabricated and they are also equally distributed into four panels (each contains 1200 chambers), and the interval for two closed chambers is 150 μm. The big difference from the dPCR-used SPC is that the rectangular chambers are located vertically on the main channels and the branch channels link to chamber without orthogonal turning points. On performance, the SPC cdLAMP can precisely calculate the absolute DNA concentration. To conduct the data acquisition and analysis of SPC cdLAMP, the Maestro Ex In-Vivo Imaging System is employed. However, the imaging system is too cumbersome and expensive to allow the e POCT, especially in the less developed regions. Herein, an easy-to-use and cost-efficient smartphone-based dLAMP POCT device platform is

SPC cdIMSA is an updated version of SPC cdLAMP, in which the LAMP is replaced by IMSA and a mixed dye is used to establish a label-free and sensitive dual-fluorescence detection for on-chip IMSA [70, 71]. The used SPC chip for dIMSA is the same as that for dPCR without any modifications. The SPC cdIMSA with the mixed dye for the detection of hepatitis B virus (HBV) is conducted. In contrast, the mixed dye indicating two different colors makes it easy to count the positive chamber number by visual inspection regardless of the cutoff values. Also, there

related genes.

136 Lab-on-a-Chip Fabrication and Application

developing countries.

also established by our laboratory.

*3.3.2.2. SPC chip-based dINAAs (cdINAAs)*

As another POCT-oriented LOC device, LTNP chip resembling SPC chip also uses the gas solubility of PDMS to load solution into chambers (**Figure 9**) [29]. However, the measure of evacuating the LTNP chip is different from the SPC chip. For the former, the gas solubility and permeability of the chip itself and another PDMS layer coating on the chip services simulta‐ neously as the real-time power source of evacuating with a syringe filter. For the latter, preevacuating by a vacuum pump is indispensable, which calls for robust and efficient approaches of sealing and packaging chips [30].

The LTNP cdPCR has been already exploited [29]. The chip consists of three parts, which are a lamina-chip layer (LCL), a vaporproof layer (VPL), and a syringe filter-like microfluidic device (μfilter) with helical channels. The μfilter has two parts. One is designed for generating the LTNP through pulling the plug of the syringe connected at one end of helical channels. The helical channel in the μfilter is 200 μm in width and 40 μm in depth. The other is used for sample and oil loading via the punched ports (0.5 mm in diameter) aligning at the ones in the LCL. The area diameter of the region of helical channel in the two μfilters is 20 mm, covering entirely the area of the whole chambers in the LCL. Sandwiched by the two parts of the μfilter, LCL contains 650 chambers in a square array. The chamber is cylindrical with 200 μm in diameter and 200 μm in depth, and the distance between two closed chambers is also 200 μm. VPL on the LCL is also a thin circular layer with chambers at 100 mm in diameter, and its area is 17.0 mm in diameter, entirely mulching the LCL.

**Figure 9.** (A) Schematic diagram of the LTNP chip. (B) Schematic of the reagent-loaded chip. It contains reagent-loaded lamina chip (red), water-loaded VPL (blue), PDMS on the coverglass, and optical adhesive cover. (C) Digital image of the prototype LTNP cdPCR device. Reprinted with permission from Ref. [29]. © Copyright 2015 Royal Society of Chemistry.

Currently, LTNP cdPCR has been used for the detection of keratin 19 in A549 lung carcinoma cells [29]. Additionally, the integrated LTNP chip with NA enrichment, isolation, and digital detection functions has been developed and successfully applied to detect bovine DNA in ovine meat for food adulteration detection [72, 73].

#### *3.3.4. Other cdNADs*

Gansen et al. described a self-digitization (SD) cdLAMP device shown in **Figure 10** [32]. In this device, the reagents were partitioned into the microchambers based on an inherent fluidic phenomenon that the interplay between fluidic forces and interfacial tension could cause the self-dispersion of an income aqueous fluid into an array of chambers prefilled with an immiscible fluid. Therefore, the sample loading could be realized with manual or automated syringe pumps or external air pressure, removing the hydraulic valves or mechanical action. Less than 2 μL sample was used for the accurate quantification of relative and absolute DNA concentrations. To improve the efficiency of partitioning samples, a new generation of SD chip was invented with close to 100% efficiency in 2013 [74]. In 2014, based on the SD chip, digital RT-PCR was developed to absolutely quantify mRNA from single cells [75]. Due to the simplicity and robustness of the SD chip, the SD cdNAD is an inexpensive and easy-to-operate digital detection device.

sample and oil loading via the punched ports (0.5 mm in diameter) aligning at the ones in the LCL. The area diameter of the region of helical channel in the two μfilters is 20 mm, covering entirely the area of the whole chambers in the LCL. Sandwiched by the two parts of the μfilter, LCL contains 650 chambers in a square array. The chamber is cylindrical with 200 μm in diameter and 200 μm in depth, and the distance between two closed chambers is also 200 μm. VPL on the LCL is also a thin circular layer with chambers at 100 mm in diameter, and its area

**Figure 9.** (A) Schematic diagram of the LTNP chip. (B) Schematic of the reagent-loaded chip. It contains reagent-loaded lamina chip (red), water-loaded VPL (blue), PDMS on the coverglass, and optical adhesive cover. (C) Digital image of the prototype LTNP cdPCR device. Reprinted with permission from Ref. [29]. © Copyright 2015 Royal Society of

Currently, LTNP cdPCR has been used for the detection of keratin 19 in A549 lung carcinoma cells [29]. Additionally, the integrated LTNP chip with NA enrichment, isolation, and digital detection functions has been developed and successfully applied to detect bovine DNA in

Gansen et al. described a self-digitization (SD) cdLAMP device shown in **Figure 10** [32]. In this device, the reagents were partitioned into the microchambers based on an inherent fluidic phenomenon that the interplay between fluidic forces and interfacial tension could cause the self-dispersion of an income aqueous fluid into an array of chambers prefilled with an immiscible fluid. Therefore, the sample loading could be realized with manual or automated syringe pumps or external air pressure, removing the hydraulic valves or mechanical action. Less than 2 μL sample was used for the accurate quantification of relative and absolute DNA

is 17.0 mm in diameter, entirely mulching the LCL.

138 Lab-on-a-Chip Fabrication and Application

ovine meat for food adulteration detection [72, 73].

Chemistry.

*3.3.4. Other cdNADs*

**Figure 10.** Design of the SD cdLAMP device. (A) Schematic diagram of the individual components of a fully assembled chip. Air pressure was delivered via a removable adapter, which was connected to an external pressure source. (B) Layout of the microfluidic network. A dense array of rectangular side chambers was connected to a thin main channel. The whole array was surrounded by a separate water reservoir to saturate the PDMS during incubation at 65°C. Scale bar, 5 mm. (C) Geometry of the side chamber array and main channel. All dimensions are in micrometers. (D) Sequen‐ tial images showing the initial filling of the side-chamber array with aqueous solution. (E) Sequence of images show‐ ing the SD of aqueous sample in the side chambers. Reprinted with permission from Ref. [32]. © Copyright 2012 Royal Society of Chemistry.

In 2010, Sundberg et al. designed a spinning disk platform to achieve dPCR [25]. The disk was an inexpensive and disposable plastic disk-like chip. Differing from other approaches, 1000 nL microwells were generated by passive compartmentalization through centrifugation, and the volume of each well was 33 nL in average. The whole process, including disk loading, thermocycling, and fluorescent imaging, only costs less than 35 min. However, this kind of cdNAD performed some defects, such as the tedious plastic disk manufacturing and the probable NA adsorption to the disk.
