REVIEW ARTICLE | MARCH 05 2024

Digital microfluidics methods for nucleic acid detection: A

mini review 
Youqiang Xing  ; Yan Wang  ; Xiang Li; Shangran Pang

Biomicrofluidics 18, 021501 (2024)

https://doi.org/10.1063/5.0180125

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Digital microfluidics methods for nucleic acid

detection: A mini review
Cite as: Biomicrofluidics 18, 021501 (2024); doi: 10.1063/5.0180125
Submitted: 7 October 2023 · Accepted: 1 February 2024 · View Online Export Citation CrossMark
Published Online: 5 March 2024

Youqiang Xing,1,a) Yan Wang,2,a) Xiang Li,3 and Shangran Pang4

AFFILIATIONS
1
School of Mechanical Engineering, Southeast University, Nanjing 211189, Jiangsu Province, People’s Republic of China
2
Clinical Laboratory, Yantai Yuhuangding Hospital, Yantai 264000, Shandong Province, People’s Republic of China
3
Bio-manufacturing Engineering Laboratory, Tsinghua Shenzhen International Graduate School, Tsinghua University,
Guangdong 518000, Shenzhen, People’s Republic of China
4
Jinzhong Normal Junior College, 189 Guang’an Street, Yuci District, Jinzhong 030600, Shanxi Province,
People’s Republic of China

a)
Authors to whom correspondence should be addressed: [email protected] and [email protected]

ABSTRACT

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Many serious infectious diseases have occurred throughout human history. Rapid and accurate detection as well as the isolation of infected
individuals, through nucleic acid testing, are effective means of containing the spread of these viruses. However, traditional nucleic acid
testing methods rely on complex machines and specialized personnel, making it difficult to achieve large-scale, high-throughput, and rapid
detection. In recent years, digital microfluidics has emerged as a promising technology that integrates various fields, including electrokinet-
ics, acoustics, optics, magnetism, and mechanics. By leveraging the advantages of these different technologies, digital microfluidic chips
offer several benefits, such as high detection throughput, integration of multiple functions, low reagent consumption, and portability. This
rapid and efficient testing is crucial in the timely detection and isolation of infected individuals to prevent the virus spread. Another advan-
tage is the low reagent consumption of digital microfluidic chips. Compared to traditional methods, these chips require smaller volumes of
reagents, resulting in cost savings and reduced waste. Furthermore, digital microfluidic chips are portable and can be easily integrated into
point-of-care testing devices. This enables testing to be conducted in remote or resource-limited areas, where access to complex laboratory
equipment may be limited. Onsite testing reduces the time and cost associated with sample transportation. In conclusion, bioassay technolo-
gies based on digital microfluidic principles have the potential to significantly improve infectious disease detection and control. By enabling
rapid, high-throughput, and portable testing, these technologies enhance our ability to contain the spread of infectious diseases and effec-
tively manage public health outbreaks.

Published under an exclusive license by AIP Publishing. https://doi.org/10.1063/5.0180125

I. INTRODUCTION polysaccharides, DNA, RNA, and other substances. Cell mem-

Throughout human history, many major diseases have been branes, proteins, and other substances may interfere with subse-
caused by viruses, leading to global security emergencies. In early quent amplification steps, so nucleic acid purification is necessary.
2020, a novel coronavirus spread globally, posing a serious threat to This can be achieved using methods such as magnetic beads and
human life.1 To control the spread of the epidemic, timely detec- concentrated salt methods to remove impurities. The second step is
tion of infected individuals or carriers of the virus and their isola- nucleic acid amplification, with the most commonly used technique
tion through nucleic acid testing is crucial. The first step in the being the polymerase chain reaction (PCR).3–7 PCR amplifies the
traditional nucleic acid testing process involves sample collection detection signal by increasing the specific target fragment in three
and nucleic acid purification.2 Samples can include blood, urine, steps: denaturation, annealing, and extension. After amplification,
saliva, and other body fluids. After collection, the cells need to be nucleic acid detection is performed using fluorescence detection,
lysed through pre-processing to release cell membranes, proteins, colorimetric methods, or turbidity detection methods.

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The traditional nucleic acid detection process is complex, (dLAMP) technology,13,14 which inherits the advantages of LAMP,
time-consuming, and requires skilled professionals to perform. such as low instrument complexity, high specificity, accuracy, and
This poses challenges for remote and primary hospitals with strong tolerance to inhibitors. dPCR and dLAMP15 technologies
limited resources and environmental equipment, as they struggle to are widely used in point-of-care testing (POCT). Furthermore, in
implement the traditional method and improve detection recent years, researchers have combined CRISPR technology with
efficiency. digital microfluidics technology to develop highly sensitive and spe-
Microfluidic chips, as an emerging technology in nucleic acid cific nucleic acid amplification-free digital detection methods. For
detection, offer numerous advantages in terms of high throughput, example, Shan et al. used negative pressure-driven microfluidic
high sensitivity, and automation.8 chips to generate thousands of monodisperse droplets with a size of
Using digital microfluidics technology, samples and reagents 30 μm in just 2 min.16 By confining individual target RNA recogni-
ranging from microliters to nanoliters can be automatically sepa- tion events within an isolated droplet, the activated Cas13a collat-
rated and detected with high resolution and sensitivity. It has the eral cleavage products can accumulate in a droplet. Combining
following advantages: (1) Digital microfluidics can achieve individ- microfluidic droplets with CRISPR-Cas13a, SARS-CoV-2 RNA can
ual droplet manipulation without the need for external devices be easily detected within 30 min, with a detection limit of 470 aM.
such as channels, pumps, valves, and mixers, thereby reducing the In conclusion, digital microfluidics technology has great develop-
barrier for equipment usage. (2) It allows for free manipulation of ment prospects in the field of nucleic acid detection.
droplet movement paths without the need for complex chip struc- In this review, we outline how the properties of digital micro-
tures, making it highly adaptable to different scenarios. (3) It does fluidics have been leveraged for nucleic acid detection, discuss dif-
not cause channel clogging. (4) By introducing oil-based fluids, the ferent digital microfluidics strategies (which today form part of
chip can be sealed, and droplets can be kept independent, prevent- most microfluidics-based diagnostic assays), and review the optimi-
ing sample contamination. With these advantages, digital micro- zation of digital microfluidics for POC applications, with a focus
fluidics technology has been widely applied in the construction of on assay readouts and sample preparation. We also provide an
biochemical pharmaceutical synthesis and analysis, nucleic acid overview of the emerging biomedical applications of the technology
detection, cell culture and processing, as well as immune diagnos- and discuss open challenges and opportunities.
tics platforms or systems.
In recent years, there have been many studies using digital
microfluidics technology for nucleic acid detection. For example, II. NUCLEIC ACID DETECTION

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dPCR technology can directly obtain the copy number of target In general, purified nucleic acids tend to have low concentra-
molecules, eliminating the need for standard curves or reference tions and are not easily detected directly. Therefore, amplification
materials to determine the copy number of target genes, thus of the nucleic acid signal is necessary. Based on different principles
enabling absolute quantification of target genes.9–11 The basic prin- of amplification, detection can be categorized into three groups, as
ciple of dPCR was proposed by Skes et al. in 1992.12 Although it illustrated in Fig. 1: the first group involves amplification of the
was not yet called “digital PCR” at that time, based on this publicly target fragment, wherein the detection is achieved by base pairing,
published method, the basic experimental procedure of digital PCR resulting in an increase in the target fragment; the second group is
has been established. In addition, researchers have combined digital based on CRISPR technology, which recognizes the target fragment
microfluidics technology with LAMP to develop digital LAMP and triggers protein activation in response to the reporter molecule;

FIG. 1. Three ways to test for nucleic acids.

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and the third group is nucleic acid detection based on local surface between nucleic acid concentration and refractive index, quantitative
plasmon vibration technology. measurements can be obtained. This assay does not rely on tradi-
tional fluorescence detection equipment and requires only a small
A. Amplification sample size. However, the application of this method for detecting
nucleic acids in complex samples still poses challenges.
Currently, amplification is the most commonly used method
for nucleic acid detection. Based on the temperature requirements,
these methods can be divided into two categories. The first category III. DIGITAL MICROFLUIDICS
is polymerase chain reaction (PCR), which is considered the gold Nucleic acid detection can be accomplished through various
standard for nucleic acid detection. In the PCR system, polymerase, methods such as amplification, CRISPR-based technology, or SPR
primers, and templates are key components. The process involves technology. The above methods are more difficult to realize the
denaturation of the double-stranded DNA at approximately 95 °C, detection of single molecules. Digital microfluidics, on the other
followed by primer binding to the template at 60 °C, and extension hand, can separate single molecules by dilution so that each reac-
of the primers along the template at 72 °C through the action of tion chamber contains 0 or ≥1 copy of the target DNA. A Poisson
polymerase, leading to amplification. However, PCR reactions are correction can be added to the results to account for reaction
dependent on temperature changes and are not suitable for chambers containing multiple molecules and to estimate the abso-
point-of-care testing (POCT) assays. As a result, an alternative lute number of target sequences.31–35
method emerged, known as isothermal amplification. This includes
techniques like loop-mediated isothermal amplification
(LAMP),17,18 recombinase polymerase amplification (RPA),19 and A. Statistical analysis
helicase-dependent amplification (HDA).20,21 LAMP requires a The theory of digital microfluidics is based on the theory of
temperature of 65 °C and typically involves the use of 4–6 primer Poisson distribution in statistics.36 The probability function of the
pairs. It offers good specificity and holds great potential for POCT Poisson distribution can be expressed as follows:
when combined with microfluidic technology. RPA, on the other
hand, can be performed at a temperature range of 37–42 °C and λk λ
can utilize the surface temperature of the human body as a heat P(X ¼ k) ¼ e , k ¼ 0, 1, . . . , (1)
k!
source, making it suitable for wearable devices.
where λ is the average number of random events per unit time (or

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unit area). When the model is applied to describe the distribution
B. CRISPR-based methods for detection
of nucleic acid molecules, λ is the ratio of the number of positive
Since its discovery in 1980, the CRISPR system has found molecules to the total number of molecules,
widespread use in gene editing and other applications.22 CRISPR/
Cas systems can be classified into two categories based on the b
λ¼ , (2)
nature of the ribonucleoprotein effector complexes. Class I systems n
consist of complexes composed of multiple effector proteins, while
Class II systems involve a single crRNA-binding protein that can where b represents the number of positive molecules and n repre-
be used for nucleic acid detection. Cas9, guided by both crRNA sents the number of the total molecules. When k equals 0, the
and tra-crRNA, can specifically cleave DNA sequences, relying on meaning of P(k = 0) is the probability of negative molecules, which
pre-amplification (1). Cas12, guided solely by crRNA, can also spe- can be deduced from the following relationship:
cifically cleave DNA sequences and has non-specific DNA cleavage b
¼ en :
b
activity. Likewise, Cas13 can specifically cleave RNA sequences P(X ¼ 0) ¼ 1  (3)
n
with crRNA guidance and exhibit non-specific RNA cleavage activ-
ity. Both Cas12 and Cas13 systems have been applied to nucleic Then, the relationship between the concentration c and the
acid detection in various ways. They can be combined with pre- volume of each chamber v can then be established as follows:
amplification techniques,23 such as DETECTR,24 SHERLOCK,25
CARMEN,26 and others. Additionally, these systems can be used b
c¼ : (4)
for direct amplification-free nucleic acid diagnosis.27,28 Fozouni nv
et al.29 developed a device that enables the direct detection of
SARS-CoV-2 using cell phones, without the need for amplification. The above equations can be combined to calculate the concen-
tration formula as follows:
C. Surface plasmon resonance for detection  
b
Surface plasmon vibration technology is an optical detection  ln 1 
n
method used for real-time monitoring of biomolecular interac- c¼ : (5)
v
tions.30 Typically, an oligonucleotide probe is attached to the sensor
surface, and if the sample being tested contains the target fragment, In summary, when quantifying nucleic acids using digital
it will bind to the surface probe, resulting in a change in the refrac- microfluidics, the concentration of nucleic acids in the correspond-
tive index of the sensor surface. By establishing a correlation ing sample can be calculated simply by knowing the volume of

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each test chamber and the proportion of chambers with positive B. Digital microfluidic methods for nucleic acid
molecules to the total chambers. detection
The theoretical analysis above was validated using specific data. The increased specific surface area of microdroplets reduces
The same microstructure was divided into 56, 400, 5000, and 20 000 reaction time and improves the performance of biochemical perfor-
pores, and the same number of target molecules were injected mance of analytical assays, reducing sample and reagent consump-
into the microstructure.37 As more holes are added, the size of the tion. In contrast to conventional macroscopic. Compared with
holes is gradually partitioned into smaller volumes as illustrated in conventional macroscopic systems, microfluidics manipulates
Fig. 2(a). Calculating the Poisson distribution for each of the four droplet systems at the micro- and nano-levels and has unique prop-
types separately, it is found that 99.5% of the wells are digitized as erties compared with ordinary droplets.39,40 (1) Microdroplets can
0 or 1 as the number of holes increases as illustrated in Fig. 2(b). be considered as a large number of small-sized isolated units,

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FIG. 2. Data processing. (a) Micro-structures with 56, 400, and 20 000 wells. As the number of well increases, the size of well gets partitioned into smaller volumes.
(b) Poisson probability distribution when injecting 2000 copy genes in 56 (A), 400 (B), 5000 (C), and 20 000 wells (D). Reproduced with permission from Lee et al.,
Genom. Inform. 19(3), e34 (2021). Copyright 2021 Author(s), licensed under a Creative Commons Attribution (CC BY) license.38

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FIG. 3. Construction and operation principle of the MiCA-emulsifier. (a) Assembly of a container with the MiCA. The main body was made of PEEK with a PTFE gasket
ring. (b) The components. (c) The swing buckets with microcentrifuge tubes and MiCA inserts will flip centripetally when spinning. (d) During spinning, the centrifugal force
is perpendicular to the MiCA plate, breaking the solution into small droplets, which then form emulsion in the receiving oil. (e) The emulsion stably sits at the bottom of a
microcentrifuge tube after centrifugation. (f ) Microphotograph of emulsion droplets after 40 thermal cycles of PCR. (g) Fluorescence microphotograph indicating the digital
amplification within the emulsion. Scale bars: 100 μm. Reproduced with permission from Chen et al., Lab Chip 17(2), 235–240 (2017). Copyright 2017 The Royal Society
of Chemistry.42

which are very suitable for single-cell or single-molecule analysis.41 1. Chip-in-a-tube

(2) Rapid mixing and negligible thermal inertia make micro- Using benchtop centrifuges, highly efficient and productive
droplets very suitable for single-cell or single-molecule analysis. production of monodisperse emulsion droplets is achieved. The
(3) The two immiscible liquids and the interface between them process involves centrifugal rotation, where the continuous aqueous
offer the possibility of new reactions. For digital microfluidics, phase is dispersed into monodisperse droplet jets in the air through
the most important thing is to generate stable and homoge- a microchannel array (MiCA). These droplets are then immersed
neous droplets. However, at the same time, the volume of each in oil, resulting in the formation of a stable emulsion,42 as depicted
chamber needs to be satisfied to be small enough when using in Fig. 3. This method ensures effective droplet dispersion while
this formula to calculate the concentration, which is currently a minimizing the risk of cross-contamination. The MiCA, which is a
difficulty in the field of digital microfluidics. Here, we classify disk with a hydrophobic monolayer beneath it, plays a crucial role.
the currently available digital microfluidic chips for nucleic acid As the water phase is propelled toward the bottom of the tube by
detection into the following three types based on the microdroplet centrifugal force, it continuously undergoes pinching at the MiCA
travel principle. nozzle, aligning itself as water droplets that subsequently enter the

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receiving oil, aligning further as an anhydrous emulsion. Droplets However, it is important to consider the pressure applied when
of various sizes can be generated by adjusting the x-rotation speed, introducing the oil phase using this method. The second method
the number of channels, or the size of the MiCA. The notable is based on mobile microfluidics, which typically involves two
advantage of this approach lies in the core design of MiCA, which sliding plates. In this approach, a mixture of liquid components is
can be easily mass-produced using existing technology and is applied, and the droplets are mixed by sliding the plates to
highly reproducible. Additionally, this method utilizes a benchtop achieve cross-dispersion-free droplets,43–46 as demonstrated in
centrifuge, a widely available instrument in biological and chemical Fig. 4(c). The third method relies on the hydrophilic and hydro-
laboratories. Its seamless integration allows for the efficient gen- phobic properties of the substrate,47 as depicted in Fig. 4(d). By
eration of emulsions in a highly parallel manner with excellent modifying the hydrophilic array on a hydrophobic substrate,
scalability. Moreover, the fabrication of MiCA plates is cost- droplets can adhere to the hydrophilic array. Additionally, the
effective compared to PDMS and glass-based microfluidic chips, size of the microchamber can be adjusted by changing the size of
thanks to the high throughput capabilities of microchannel plate the hydrophilic array. Using microstructures for digital microflui-
technology. In conclusion, this approach offers a low-cost and dic droplet dispersion offers several advantages. First, it allows for
controllable digital microfluidic method that can be readily precise control over the size of the chamber. Second, the loss of
scaled up for various applications. the mixture is minimized, resulting in more accurate nucleic acid
quantification. Third, it helps prevent aerosol contamination.
Finally, integrating most procedures on the same device enhances
2. Microstructure the portability of the chamber-based microfluidic chip. For
This method involves the fabrication of numerous uniform example, by incorporating a heater and a signal reader, this digital
microchambers on a substrate, with soft photolithography or etching microfluidics approach can be integrated into a portable machine,
techniques commonly employed in current research. PDMS is the further enhancing its portability.
most frequently used material, as it enables the processing of tiny In addition to microstructures that can be realized using
chambers through photolithography. Once a large number of PDMS materials, PCTE can also be used, which is a commercially
microchambers have been created, as depicted in Fig. 4(a), the key available membrane with numerous pores on its surface containing
next step is to disperse the solution into each individual chamber a high density of uniform micro-/nanopores. Each pore serves as
while preventing cross-contamination. One approach to achieve an individual nano-reactor for DNA amplification48 as depicted in
this is oil-phase cutting separation, as illustrated in Fig. 4(b), Fig. 5. One significant advantage of this method is the cost-

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which relies on the immiscibility between the oil and water phases. effectiveness of the disposable PCTE membrane, which is priced at

FIG. 4. Microstructure microfluidic chips. (a) Photolithography production process; (b) oil-phase cutting separation chips; (c) slip chip; (d) hydrophilic and hydrophobic chip.

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less than $0.10 per piece. To our knowledge, this is the most afford- C. Result analysis
able way to perform digital LAMP. This membrane system opens In nucleic acid detection, the application of digital microflui-
up possibilities for point-of-care users and general laboratories by dics involves dispersing nucleic acid molecules from a sample into
allowing them to conduct digital quantification, single-cell analysis, a high-density array of microchamber reactions. Positive chambers
and other bioassays in an inexpensive, flexible, and streamlined
containing target molecules exhibit amplification or the CRISPR
manner.
system, while negative chambers do not contain target molecules.
By quantifying the number of positive and negative chambers and
3. Dielectric wetting applying a Poisson distribution for data correction, the concentra-
In this technique, the movement, merging, splitting, and gen- tion of the target sample can be accurately determined. This
eration of microdroplets with volumes ranging from picoliters to method of digital microfluidics offers several advantages: it does
microliters can be achieved by applying a voltage to the droplets on not rely on internal reference genes, enables absolute quantification
a hydrophobic surface and altering the contact angle between the of DNA or RNA molecules without the need for a standard curve,
droplets and the surface.49,50 Furthermore, the addition of nano- allows for detection of nucleic acid molecules even at a single-copy
particles to the droplet causes a favorable recovery of the electro- level, and exhibits increased tolerance to variations in the external
spreading characteristics of a soft surface by realizing an alteration environment. Based on the statistical analysis discussed earlier, it is
in the effective dielectric constant of the interfacial region. This evident that this approach facilitates precise concentration
technology may open up new vistas in droplet-based microflui- determination.
dics.51,52 The digital microfluidic method based on the dielectric According to the aforementioned that the digital microfluidic
wetting principle shares similar advantages with traditional flow method can achieve the dispersion of droplets, then the result anal-
channel microfluidics, including low reagent consumption, efficient ysis needs to be done. From Sec. III B, the nucleic acid concentra-
heat transfer, and easy integration. In addition to these benefits, tion of the sample to be tested can be calculated by the following
this method presents several unique advantages: (a) Digital micro- equation:
fluidics can achieve individual droplet manipulation without exter-  
nal devices such as channels, pumps, valves, and mixers, which b
 ln 1 
reduces the threshold of using devices. (b) Free manipulation of n
c¼ : (6)
droplet movement paths can be achieved without the need for v
complex chip structures, which is highly applicable to different sce-

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narios. (c) The open chip structure allows solid samples to be pro- Taking log on both sides, we get the following formula:
cessed without blocking channels. (d) The chip can be closed by   
adding oil-based liquid, and the droplets are independent of each b
lg(v) ¼ lg  ln 1   lgc: (7)
other to prevent contamination between samples. With the above n
advantages, this technology has been widely used in recent years to
build platforms or systems for biochemical and pharmaceutical Therefore, the intercept is used to calculate the
synthesis and analysis, cell culture and processing, and immune concentration.54–56 For example, the DNA concentration is calcu-
diagnosis.53 lated using this equation (Fig. 6).

FIG. 5. PCTE. (a) PCTE membrane dispersion sample liquid process. (b) Mechanism for excess sample removal.

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FIG. 6. Fluorescence image results of different concentrations of λDNA by using the fully integrated NA detection platform. (a) Fluorescence images of digital LAMP with
serially diluted λDNA (concentrations ranging from 1/2 to 1/10000 of the stock sample concentration [1 × 104 copies per μl]). (b) A regression curve obtained by plotting
the fraction of positive droplets against the expected λDNA concentration (CPD) according to the Poisson distribution. (c) Comparison of measured λDNA concentrations
to the expected concentrations. The measured λDNA concentrations match well with the expected λDNA concentrations according to Poisson statistics. Reproduced with
permission from Mao et al., Analyst 146(22), 6960–6969 (2021). Copyright 2021 The Royal Society of Chemistry.47

IV. CONCLUSIONS AND PROSPECTS systems. Tian et al.63 introduced a Cas13a assay inspired by the
In recent years, digital microfluidics has gained widespread confinement effect for single-molecule RNA diagnostics, eliminat-
application in nucleic acid detection, including dPCR,57–62 among ing the requirement for NAA and RT. Shinoda et al.64 have devel-
others. Digital microfluidics not only complements traditional oped a platform called “CRISPR-based amplification-free digital
nucleic acid amplification methods but also integrates with CRISPR RNA detection (SATORI)” that combines CRISPR-Cas13-based

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RNA detection with microchamber-array technology. Digital AUTHOR DECLARATIONS

microfluidics offers several advantages over traditional molecular Conflict of Interest
diagnostic methods, as outlined below.
Digital microfluidic technology is highly integrable and can The authors have no conflicts to disclose.
integrate multi-functions on a chip. Moreover, digital microfluidic
technology has the potential of automation, which can be widely Author Contributions
used in the field of immediate detection. Besides, digital microflui-
dic technology can realize highly sensitive detection, which is con- Youqiang Xing: Conceptualization (lead); Funding acquisition
ducive to early diagnosis. (lead); Writing – original draft (lead); Writing – review & editing
Due to these advantages, digital microfluidics has been widely (equal). Yan Wang: Conceptualization (equal); Investigation
used in the development of platforms or systems for biochemical (equal); Writing – original draft (equal); Writing – review &
and pharmaceutical synthesis and analysis, cell culture and process- editing (lead). Xiang Li: Investigation (equal); Resources (equal);
ing, as well as immune diagnosis in recent years. Writing – original draft (equal). Shangran Pang: Resources
However, despite the potential of digital microfluidics for (supporting); Writing – original draft (supporting).
nucleic acid detection, its application in the clinical setting is still
limited to the laboratory stage. Currently, the Quant Studio 3D is DATA AVAILABILITY
the only commercially available instrument, highlighting the need
for further advancements and improvements in this field. The data that support the findings of this study are available
First, there has been significant progress in increasing the within the article.
number of microchambers that can be integrated on a chip.
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