Biomimetic Ultrasonic Vibrator with Broadband Characteristics Inspired by Leaf-Cutting Ants.

Power ultrasound is widely used in industrial production, medical equipment, aerospace, and other fields. Currently, there are two main types of commonly used power generation devices: piezoelectric ultrasonic transducers and magnetostrictive ultrasonic transducers. However, in certain situations with limited external dimensions, the applications of existing power ultrasound devices are limited. In nature, leaf-cutting ants excite vibrations through their tiny organs. Inspired by the vibratory organs of leaf-cutting ants, a new type of biomimetic ultrasonic vibrator (BUV) comprising a scraper, dentate disc, and fixture system was proposed, fabricated, and tested in this study. The experimental results showed that the BUV could operate in the frequency range of 16.8-19 kHz. Within the working frequency range, the vibration of the BUV was stable and the amplitude of the vibration displacement was greater than 22 µm. The operating frequency band of the BUV was broader than those of the piezoelectric and magnetostrictive ultrasonic transducers. In addition, the BUV can cut soft rubber and pig tissues with sufficient output power and load-carrying capacity. The BUV, as a new type of power ultrasonic excitation device, is expected to be applied in high-power micro operating scenarios, such as minimally invasive surgical instruments.

Viable Escherichia coli enumeration on a polydimethylsiloxane (PDMS) chip with vertical channel-well configuration.

The culture-based methods for viable Escherichia coli (E. coli) detection suffer from long detection time and laborious procedures, whereas the molecule tests and immune recognition technologies lack live/dead E. coli differentiation. Rapid, easy-to-use, and accessible viable E. coli detection is of benefit to bacterial infection diagnosis and risk warning of E. coli contamination of water and food, safeguarding human health. Herein, we propose a microwell chip-based solution to realize simple and rapid determination of viable E. coli. The vertical channel-well configuration is applied to develop the microwell array chip for increasing the microwell density (6200 wells/cm2), yielding a broad dynamic range from 103 to 107 CFU/mL. We incorporate an inducible enzyme assay with the developed chip and achieve the differentiation of live/dead E. coli within 4 h, significantly shortening the detection time from over 24 h in the standard method. By encapsulating single E. coli into microwells, the concentration of viable cells can be determined simultaneously through counting positive microwells. In addition, the air soluble PDMS that can store negative pressure for independent sample digitalization endows the developed chip with simple operation and less reliance on external equipment. With further developments for increasing the number of microwell and integrating more sample panels, the developed chip can become a useful tool for rapid viable E. coli enumeration with user-friendly operation, simple procedures, and accessibility in decentralized settings, thereby deploying this device for water and food safety monitoring, as well as clinical bacterial infection diagnosis.

Digital metabolic activity assay enables fast assessment of 2D materials bactericidal efficiency.

BACKGROUND: The identification and quantification of viable Escherichia coli (E. coli) are important in multiple fields including the development of antimicrobial materials, water quality, food safety and infections diagnosis. However, the standard culture-based methods of viable E. coli detection suffer from long detection times (24 h) and complex operation, leaving the unmet requirement for fast assessing the efficiency of antimicrobial materials, early alerting the contamination of water and food, and immediately treatment of infections. RESULTS: We present a digital ß-d-glucuronidase (GUS) assay in a self-priming polydimethylsiloxane (PDMS) microfluidic chip for rapid E. coli identification and quantification. The GUS expression in viable bacteria was investigated to develop a fast GUS assay at the single-cell level. Single E. coli were stochastically discretized in picoliter chambers and identified by specific GUS activity. The digital GUS assay enabled identifying E. coli within 3 h and quantifying within 4 h for different E. coli subtypes. The specificity of our method was confirmed by using blended bacteria including E. coli, Bacillus, Shigella and Vibrio. We utilized digital GUS assay to enumerate viable E. coli after incubated with antibacterial materials for assessing the antibacterial efficiency. Moreover, the degassed chip can realize automatic sample distribution without external instruments. SIGNIFICANCE: The results demonstrated the functionality and practicability of digital GUS assay for single E. coli identification and quantification. With air-tight packaging, the developed chip has the potential for on-site E. coli analysis and could be deployed for diagnosis of E. coli infections, antimicrobial susceptibility testing, and warning the fecal pollution of water. Digital GUS assay provides a paradigm, examining the activity of metabolic enzyme, for detecting the viable bacteria other than E. coli.

Squeezed state in the hydrodynamic focusing regime for Escherichia coli bacteria detection.

Flow cytometry is an essential technique in single particle analysis and cell sorting for further downstream diagnosis, exhibiting high-throughput and multiplexing capabilities for many biological and biomedical applications. Although many hydrodynamic focusing-based microfluidic cytometers have been demonstrated with reduced size and cost to adapt to point-of-care settings, the operating conditions are not characterized systematically. This study presents the flow transition process in the hydrodynamic focusing mechanism when the flow rate or the Reynolds number increases. The characteristics of flow fields and mass transport were studied under various operating conditions, including flow rates and microchannel heights. A transition from the squeezed focusing state to the over-squeezed anti-focusing state in the hydrodynamic focusing regime was observed when the Reynolds number increased above 30. Parametric studies illustrated that the focusing width increased with the Reynolds number but decreased with the microchannel height in the over-squeezed state. The microfluidic cytometric analyses using microbeads and E. coli show that the recovery rate was maintained by limiting the Reynolds number to 30. The detailed analysis of the flow transition will provide new insight into microfluidic cytometric analyses with a broad range of applications in food safety, water monitoring and healthcare sectors.

Inoculum size-insensitive susceptibility determination of urine sample based on in-situ measurement of inducible enzyme activity after 20 min of antibiotic exposure.

BACKGROUND: The empirical antibiotic therapies for bacterial infections cause the emergence and propagation of multi-drug resistant bacteria, which not only impair the effectiveness of existing antibiotics but also raise healthcare costs. To reduce the empirical treatments, rapid antimicrobial susceptibility testing (AST) of causative microorganisms in clinical samples should be conducted for prescribing evidence-based antibiotics. However, most of culture-based ASTs suffer from inoculum effect and lack differentiation of target pathogen and commensals, hampering their adoption for evidence-based antibiotic prescription. Therefore, rapid ASTs which can specifically determine pathogens' susceptibilities, regardless of the bacterial load in clinical samples, are in urgent need. RESULTS: We present a pathogen-specific and inoculum size-insensitive AST to achieve the reliable susceptibility determination on Escherichia coli (E. coli) in urine samples. The developed AST is featured with an 1 h sample-to-result workflow in a filter, termed on-filter AST. The AST results can be obtained by using an inducible enzymatic assay to in-situ measure the cell response of E. coli collected from urine after 20 min of antibiotic exposure. The calculated detection limit of our AST (1.95 × 104 CFU/mL) is much lower than the diagnosis threshold of urinary tract infections. The specific expression of the inducible enzyme enables on-filter AST to correctly profile the susceptibilities of target pathogen to multi-type antibiotics without the interference from commensals. We performed the on-filter AST on 1 mL urine samples with bacterial loads varying from 105 CFU/mL to 107 CFU/mL and compared the results to that of standard method, demonstrating its insensitivity to inoculum size. SIGNIFICANCE: The developed AST is demonstrated to be of high sensitivity, specificity, and insensitive to inoculum size. With further developments for additional bacteria and clinical validation, on-filter AST is promising as a rapid and reliable surrogate of culture-based AST to promote the evidence-based prescription at the first visit and minimize the emergency of new multi-drug resistant microorganisms.

Enzymatic Antimicrobial Susceptibility Testing with Bacteria Identification in 30 min.

Rapid antimicrobial susceptibility testing (AST) with the ability of bacterial identification is urgently needed for evidence-based antibiotic prescription. Herein, we propose an enzymatic AST (enzyAST) that employs ß-d-glucuronidase as a biomarker to identify pathogens and profile phenotypic susceptibilities simultaneously. EnzyAST enables to offer binary AST results within 30 min, much faster than standard methods (>16 h). The general applicability of enzyAST was verified by testing the susceptibility of two Escherichia coli strains to three antibiotics with different action mechanisms. The pilot study also shows that the minimal inhibitory concentrations can be determined by enzyAST with the statistical analysis of enzymatic activity of the bacteria population exposed to varying antibiotic concentrations. With further development of multiple bacteria and sample treatment, enzyAST could be able to evaluate the susceptibility of pathogens in clinical samples directly to facilitate the evidence-based therapy.

Multi-color two-photon scanning structured illumination microscopy imaging of live cells.

Multi-color two-photon microscopy imaging of live cells is essential in biology. However, the limited diffraction resolution of conventional two-photon microscopy restricts its application to subcellular organelle imaging. Recently, we developed a laser scanning two-photon non-linear structured illumination microscope (2P-NLSIM), whose resolution improved three-fold. However, its ability to image polychromatic live cells under low excitation power has not been verified. Here, to improve the reconstruction super-resolution image quality under low excitation power, we increased the image modulation depth by multiplying the raw images with the reference fringe patterns in the reconstruction process. Simultaneously, we optimized the 2P-NLSIM system to image live cells, including the excitation power, imaging speed, and field of view. The proposed system could provide a new imaging tool for live cells.

Direct single-cell antimicrobial susceptibility testing of Escherichia coli in urine using a ready-to-use 3D microwell array chip.

Empirical antibiotic therapies are prescribed for treating uncomplicated urinary tract infections (UTIs) due to the long turnaround time of conventional antimicrobial susceptibility testing (AST), leading to the prevalence of multi-drug resistant pathogens. We present a ready-to-use 3D microwell array chip to directly conduct comprehensive AST of pathogenic agents in urine at the single-cell level. The developed device features a highly integrated 3D microwell array, offering a dynamic range from 102 to 107 CFU mL-1, and a capillary valve-based flow distributor for flow equidistribution in dispensing channels and uniform sample distribution. The chip with pre-loaded reagents and negative pressure inside only requires the user to initiate AST by loading samples (∼3 s) and can work independently. We demonstrate an accessible sample-to-result workflow, including syringe filter-based bacteria separation and rapid single-cell AST on chip, which enables us to bypass the time-consuming bacteria isolation and pre-culture, speeding up the AST in ∼3 h from 2 days of conventional methods. Moreover, the bacterial concentration and AST with minimum inhibitory concentrations can be assessed simultaneously to provide comprehensive information on infections. With further development for multiple antibiotic conditions, the Dsc-AST assay could contribute to timely prescription of targeted drugs for better patient outcomes and mitigation of the threat of drug-resistant bacteria.

All-In-One Escherichia coli Viability Assay for Multi-dimensional Detection of Uncomplicated Urinary Tract Infections.

The urinary tract infections by antibiotic-resistant bacteria have been a serious public health problem and increase the healthcare costs. The conventional technologies of diagnosis and antimicrobial susceptibility testing (AST) relying on multiple culture-based assays are time-consuming and labor-intensive and thus compel the empirical antimicrobial therapies to be prescribed, fueling the prevalence of antimicrobial resistance. Herein, we propose an all-in-one Escherichia coli viability assay in an enclosed 3D microwell array chip, termed digital ß-d-glucuronidase (GUS)-AST assay. It employs GUS, a specific metabolism-related enzyme, to convert the presence of E. coli into bright fluorescence. The random distribution of single bacteria in microwell array enables to quantify the E. coli concentrations by counting the positive microwells. We incorporate the most probable number with digital quantification to lower the limit of detection and expand the dynamic range to 7 orders. The digital GUS-AST assay is able to indicate the potency of antibiotics and determine the minimum inhibitory concentrations. A streamlined procedure of urine removal, bacterial separation, and digital GUS-AST is established to perform the direct analysis of bacteria population in urine. The sample-to-result workflow can be finished in 4.5 h with a limit of detection of 39 CFU/mL. With further development for additional pathogens and multiple antibiotic conditions, the digital GUS-AST assay could help physicians to prescribe timely targeted therapies for better patient outcomes and the minimum emergence of resistant bacteria.

Mechanism and Effects of Cellular Creep in a Microfluidic Filter.

Biomicroparticles such as proteins, bacterium, and cells are known to be viscoelastic, which significantly affects their performance in microfluidic applications. However, the exact effects and the quantitative study of cellular viscoelastic creep within different applications remain unclear. In this study, the cellular-deforming evolution within a filter unit was studied using a multiphysics numerical model. A general cellular creep deformation process of viscoelastic particle trapping in pores was revealed. Two featured variables, namely, the maximum surface displacement and the volumetric strain, were identified and determined to quantitatively describe the evolution. The effects of flow conditions and physical characteristics of the microparticles were studied. Furthermore, a Giardia concentration experiment was conducted using an integrated hydraulic filtration system with a porous membrane. The experimental results agreed well with the numerical analysis, indicating that, compared to pure elastic particles, it is more difficult to release cellular material matters including cells, chemical synthetic particles, and microbes from trapping due to their time-accumulated creep deformation.

Single Escherichia coli bacteria detection using a chemiluminescence digital microwell array chip.

Rapid and sensitive Escherichia coli (E. coli) detection is important in determining environmental contamination, food contamination, as well as bacterial infection. Conventional methods based on bacterial culture suffer from long testing time (24 h), whereas novel nucleic acid-based and immunolabelling approaches are hindered by complicated operation, the need of complex and costly equipment, and the lack of differentiation of live and dead bacteria. Herein, we propose a chemiluminescence digital microwell array chip based on the hydrolysis of 6-Chloro-4-methylumbelliferyl-ß-D-glucuronide by the ß-D-glucuronidase in E. coli to achieve fast single bacterial fluorescence detection. Taking the advantage of the picoliter microwells, single bacteria are digitally encapsulated in these microwells, thus the accurate quantification of E. coli can be realized by counting the number of positive microwells. We also show that the chemiluminescence digital microwell array chip is not affected by the turbidity of the test samples as well as the temperature. Most importantly, our method can differentiate live and dead bacteria through bacterial proliferation and enzyme expression, which is confirmed by detecting E. coli after pH and chlorination treatment. By comparing with the standard method of plate counting, our method has comparable performance but significantly reduces the testing time from over 24 h-2 h and 4 h for qualitative and quantitative analysis, respectively. In addition, the microfluidic chip is portable and easy to operate without external pump, which is promising as a rapid and on-site platform for single E. coli analysis in water and food monitoring, as well as infection diagnosis.

A Specific and Sensitive Aptamer-Based Digital PCR Chip for Salmonella typhimurium Detection.

Food poisoning and infectious diseases caused by Salmonella typhimurium (S. typhimurium) are serious public health concerns for human health and food safety. The diversity and complexity of food matrices pose great challenges for rapid and ultra-sensitive detection of S. typhimurium in food samples. A method capable of identification, detection, and quantification of S. typhimurium is essential for addressing these issues. In this study, aptamer-coated magnetic beads (Apt-MBs) are employed as capture bio-probes to specifically and selectively concentrate S. typhimurium in food samples. A self-priming chip-based digital PCR was then presented as another biosensor for on-site detection and quantification of S. typhimurium cells. The chip we developed was robust and did not require any external power for sample loading. The combination of Apt-MBs with an on-chip digital detection realized the integration into lab-on-a-chip-based biosensors for on-site monitoring of foodborne pathogens. It was possible to capture and detect S. typhimurium cells as low as 90 CFU/reaction with a capture efficiency of 94.5%. Additionally, the whole process only took about 2 h. This unique platform could also be used to monitor other target bacteria with high specificity and sensitivity by utilizing different aptamers. Furthermore, the platform has potential applications in point-of-care testing in the future.

A large-scale pico-droplet array for viable bacteria digital counting and dynamic tracking based on a thermosetting oil.

The rapid and accurate detection of viable bacteria is of great importance in food quality monitoring and clinical diagnosis. Escherichia coli (E. coli) is a major pathogenic bacterium, which causes potential threats to food safety and human health. Therefore, rapid and portable methods for preventing E. coli outbreaks are needed. Single cell analysis can be performed at the single-cell level, which has great advantages for analysis and diagnosis. Herein, we employed a thermosetting oil to generate a large-scale pico-droplet array for viable bacteria digital counting and dynamic tracking. In this array, the droplets can be solidified without any inducers due to the cross-linking reaction of the hydrosilation of vinyl silicone oil and hydrosilicone oil. Single E. coli cells were encapsulated in solidified droplets to form a microcolony. Resazurin was used as a fluorescent indicator to achieve amplification of bacterial growth signals. This method can achieve digital counting of viable E. coli cells in 4 h. We achieved real-time monitoring of E. coli cell growth and division in droplets. It is rapid, simple, and does not require a pre-enrichment process when compared to the traditional plate counting method. We successfully applied the method for the enumeration of E. coli in milk. In conclusion, the thermosetting oil enables the immobilization of droplets to achieve real-time monitoring and digital counting of bacterial growth without impairing the flexibility of droplet microfluidics, and it has the potential to provide dynamic information at high resolution in this process.

Reducing noisy annotations for depression estimation from facial images.

Depression has been considered the most dominant mental disorder over the past few years. To help clinicians effectively and efficiently estimate the severity scale of depression, various automated systems based on deep learning have been proposed. To estimate the severity of depression, i.e., the depression severity score (Beck Depression Inventory-II), various deep architectures have been designed to perform regression using the Euclidean loss. However, they do not consider the label distribution, and they do not learn the relationships between the facial images and BDI-II scores, which can be resulting in the noisy labeling for automatic depression estimation (ADE). To mitigate this problem, we propose an automated deep architecture, namely the self-adaptation network (SAN), to improve this uncertain labeling for ADE. Specifically, the architecture consists of four modules: (1) ResNet-18 and ResNet-50 are adopted in the deep feature extraction module (DFEM) to extract informative deep features; (2) a self-attention module (SAM) is adopted to learn the weights from the mini-batch; (3) a square ranking regularization module (SRRM) to create high partitions and low partitions is proposed; and (4) a re-label module (RM) is used to re-label the uncertain annotations for ADE in the low partitions. We conduct extensive experiments on depression databases (i.e., AVEC2013 and AVEC2014) and obtain a performance comparable to the performances of other ADE methods in assessing the severity of depression. More importantly, the proposed method can learn valuable depression patterns from facial videos and obtain a performance comparable to the performances of other methods for depression recognition.

Surface disinfection with silver loaded pencil graphite prepared with green UV photoreduction technique.

Carbon-based materials have been studied for their antimicrobial properties. Previously, most antimicrobial studies are investigated with suspended nanoparticles in a liquid medium. Most works are often carried out with highly ordered pyrolytic graphite. These materials are expensive and are not viable for mass use on high-touch surfaces. Additionally, highly antimicrobial silver nanoparticles are often incorporated onto substrates by chemical reduction. At times, harmful chemicals are used. In this work, low-cost graphite pencils are mechanically exfoliated and transferred onto Si substrates. The sparsely-covered graphite flakes are treated by either plasma O2or UV irradiation. Subsequently, Ag is photo reduced in the presence of UV onto selected graphite flake samples. It is found that graphite flake surface topography and defects are dependent on the treatment process. High surface roughness and (defects density,ID/IG) are induced by plasma O2follows by UV and pristine graphite flake as follows: 6.45 nm (0.62), 4.96 nm (0.5), 3.79 nm (0.47). Antimicrobial tests withE. colireveal high killing efficiency by photoreduced Ag-on-graphite flake. The reversible effect of Ag leaching can be compensated by repeating the photoreduction process. This work proposes that UV treatment is a promising technique over that of plasma O2in view that the latter treated surface could repel bacteria resulting in lower bacteria-killing efficiency.

Label-free E. coli detection based on enzyme assay and a microfluidic slipchip.

An enzyme assay based method in a microfluidic slipchip was proposed for the rapid and label-free detection of E. coli. The specific target analyte of E. coli was ß-d-glucuronidase (GUS) which could catalyze the substrate 6-chloro-4-methyl-umbelliferyl-ß-d-glucuronide (6-CMUG) to release the fluorescent molecule 6-chloro-4-methyl-umbelliferyl (6-CMU). E. coli culture, lysis and enzymatic reaction steps could be conducted in a microfluidic slipchip without any pumps and valves, which was tailored for fluorescence detection using a commercial plate reader, to achieve a rapid E. coli test. A mixture of the culture broth, enzyme inducer and E. coli was injected into the chambers on the top layer. A mixture of the substrate and lysis solution was injected into the chambers on the bottom layer. Then, the slipchip was slid to make each chamber independent. E. coli was cultured in the chamber in the LB broth for 2.5 h. After that, the slipchip was slid again to introduce the lysis solution into the culture solution for GUS release and enzyme reaction, and then incubated in the plate reader at 42 °C for another 2.5 h. During incubation, the fluorescence intensity of each chamber was recorded. This proposed label-free method can directly detect E. coli with a low concentration of 8 CFU per chamber within 5 h, thus showing great potential in on-site E. coli detection.

On-Chip Optical Detection of Viruses: A Review.

The current outbreak of the coronavirus disease-19 (COVID-19) pandemic worldwide has caused millions of fatalities and imposed a severe impact on our daily lives. Thus, the global healthcare system urgently calls for rapid, affordable, and reliable detection toolkits. Although the gold-standard nucleic acid amplification tests have been widely accepted and utilized, they are time-consuming and labor-intensive, which exceedingly hinder the mass detection in low-income populations, especially in developing countries. Recently, due to the blooming development of photonics, various optical chips have been developed to detect single viruses with the advantages of fast, label-free, affordable, and point of care deployment. Herein, optical approaches especially in three perspectives, e.g., flow-free optical methods, optofluidics, and surface-modification-assisted approaches, are summarized. The future development of on-chip optical-detection methods in the wave of emerging new ideas in nanophotonics is also briefly discussed.

Immobilized Droplet Arrays in Thermosetting Oil for Dynamic Proteolytic Assays of Single Cells.

Matrix metalloproteinases (MMPs) play an important role in tumor progression. The study of dynamic MMPs activity at the single-cell level can dissect tumor heterogeneity in the time domain and facilitate finding out more efficient clinical solutions for tumor treatment. Due to the fluidity of the carrier oil, the existing droplet-based methods for single-cell MMP analysis rarely have the capability to track proteolytic assays in droplets continuously. Therefore, we describe a thermosetting oil for real-time monitoring of MMP assays in droplets, which can immobilize droplets by transforming into solid after droplet generation. The solidification of this oil can be accomplished in 33 min at 37 °C, basing on the hydrosilation of vinyl silicone oil and hydrosilicone oil without other inducers (e.g. UV, Ca2+). Through monitoring the MMP assays of single cells, the reaction rates can be calculated according to real-time fluorescent curves, showing significant cell heterogeneity in MMP activity. Moreover, the dynamic MMP activity reveals that some of the A549 cells transiently secrete MMP. In conclusion, the thermosetting oil enables immobilize droplets to achieve real-time monitoring of single-cell proteolytic activity without impairing the flexibility of droplet microfluidics and has a potential in other cell-based assays for providing dynamic information at high resolutions.

A self-powered bidirectional partition microfluidic chip with embedded microwells for highly sensitive detection of EGFR mutations in plasma of non-small cell lung cancer patients.

Circulating tumor DNA (ctDNA) is a promising biomarker for tumor genotyping and therapy monitoring. Herein, we developed a digital PCR chip with embedded microwell and bidirectional partition network for highly sensitive ctDNA analysis. The embedded microwell contributes to increasing microreaction density (up to 7000 microwells/cm2) and reducing evaporation during amplification. The bidirectional partition network can achieve fast and random distribution of targets, ensuring the precise quantification of nucleic acid. We used plasmids, artificial samples and 32 clinical blood samples from non-small cell lung cancer patients to test the performance of this platform. The results demonstrated that our chip has not only comparable quantification performance to commercial counterpart but also the ability to detect EGFR mutations with as low as 0.01% mutation rate and 20 alter molecules in 27 ng genomic DNA. The identification of EGFR mutations in plasma using developed chip exhibited 85.71% sensitivity and 94.44% specificity for L858R mutation and 100% sensitivity and 86.96% specificity for T790 M mutation. Moreover, the monitoring of mutant allele in plasma was accomplished in this work. In conclusion, the developed chip has a potential in lung tumor genotyping and therapy monitoring for precision medicine, even other tumors.

Rapid In Situ Photoimmobilization of a Planar Droplet Array for Digital PCR.

Digital PCR (dPCR) is a powerful technique capable of absolute quantification of nucleic acids with good accuracy. Droplet-based dPCR (ddPCR), among others, is one of the most important dPCR techniques. However, the surface tension-controlled droplets may suffer from fusion/fission due to the vigorous temperature change in PCR thermal cycling. Besides, the free movement of droplets makes them unsuitable for real-time fluorescence monitoring. In this paper, we first developed a photoimmobilized planar droplet array (PIPDA) by using a photocurable polyurethane as the continuous oil phase. It is found that uniform water-in-oil droplets of various sizes can be readily generated, and more importantly, the oil phase can be rapidly solidified in just a few seconds upon exposure to UV irradiation. This process will leave the droplets immobilized in the accommodation chamber as a stable planar array and, thus, effectively prevent the movement, coalescence, and breakup of droplets. In addition, a novel multilayered chip design has been proposed, which can thoroughly overcome the evaporation issue that commonly exists in polydimethylsiloxane (PDMS)-based dPCR chips. With these two innovations, the ddPCR experiment could be performed in a robust manner, and shows a promising potential in the development of real-time ddPCR technique. These features may therefore enable the wide application of PIPDA-based ddPCR in various fields.