Digital Recombinase Polymerase Amplification, Digital Loop-Mediated Isothermal Amplification, and Digital CRISPR-Cas Assisted Assay: Current Status, Challenges, and Perspectives.

Digital nucleic acid detection based on microfluidics technology can quantify the initial amount of nucleic acid in the sample with low equipment requirements and simple operations, which can be widely used in clinical and in vitro diagnosis. Recently, isothermal amplification technologies such as recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), and clustered regularly interspaced short palindromic repeats-CRISPR associated proteins (CRISPR-Cas) assisted technologies have become a hot spot of attention and state-of-the-art digital nucleic acid chips have provided a powerful tool for these technologies. Herein, isothermal amplification technologies including RPA, LAMP, and CRISPR-Cas assisted methods, based on digital nucleic acid microfluidics chips recently, have been reviewed. Moreover, the challenges of digital isothermal amplification and possible strategies to address them are discussed. Finally, future directions of digital isothermal amplification technology, such as microfluidic chip and device manufacturing, multiplex detection, and one-pot detection, are outlined.

A portable, high-throughput real-time quantitative PCR device for point-of-care testing.

Nucleic acids detection has become essential in the identification of many infectious diseases and tumors. Conventional qPCR instruments are not suitable for point-of-care Moreover, current miniaturized nucleic acid detection equipment has limited throughput and multiplex detection capabilities, typically allowing the detection of a limited number of samples. Here, we present an affordable, portable, and high-throughput nucleic acid detection device for point-of-care detection. This portable device is approximately 220 × 165 × 140 mm in size and about 3 kg in weight. It can provide stable and accurate temperature control and analyze two fluorescent signals (FAM and VIC) and run 16 samples simultaneously. As a proof of concept, we used the two purified DNA samples from Bordetella pertussis and Canine parvovirus and the results showed good linearity and coefficient of variation. Moreover, this portable device can detect as low as 10 copies and has good specificity. Therefore, our device can provide advantages in real-time diagnosis of high-throughput nucleic acid detection in the field, especially for resource-limited conditions.

Adsorption-Free Self-Priming Direct Digital Dual-crRNA CRISPR/Cas12a-Assisted Chip for Ultrasensitive Detection of Pathogens.

Rapid and sensitive pathogen detection methods are critical for disease diagnosis and treatment. RPA-CRISPR/Cas12 systems have displayed remarkable potential in pathogen detection. A self-priming digital PCR chip is a powerful and attractive tool for nucleic detection. However, the application of the RPA-CRISPR/Cas12 system to the self-priming chip still has great challenges due to the problems of protein adsorption and two-step detection mode of RPA-CRISPR/Cas12. In this study, an adsorption-free self-priming digital chip was developed and a direct digital dual-crRNAs (3D) assay was established based on the chip for ultrasensitive detection of pathogens. This 3D assay combined the advantages of rapid amplification of RPA, specific cleavage of Cas12a, accurate quantification of digital PCR, and point-of-care testing (POCT) of microfluidics, enabling accurate and reliable digital absolute quantification of Salmonella in POCT. Our method can provide a good linear relationship of Salmonella detection in the range from 2.58 × 101 to 2.58 × 104 cells/mL with a limit of detection ∼0.2 cells/mL within 30 min in a digital chip by targeting the invA gene of Salmonella. Moreover, the assay could directly detect Salmonella in milk without nucleic acid extraction. Therefore, the 3D assay has the significant potential to provide accurate and rapid pathogen detection in POCT. This study provides a powerful nucleic detection platform and facilitates the application of CRISPR/Cas-assisted detection and microfluidic chips.

Recent advances in integrated microfluidics for liquid biopsies and future directions.

Liquid biopsies have piqued the interest of researchers as a new tumor diagnosis technique due to their unique benefits of non-invasiveness, sensitivity, and convenience. Recent advances in microfluidic technology have integrated separation, purification, and detection, allowing for high-throughput, high-sensitivity, and high-controllability detection of specific biomarkers in liquid biopsies. With the increasing demand for tumor detection and individualized treatment, new challenges are emerging for the ever-improving microfluidic technology. The state-of-the-art microfluidic design and fabrications have been reviewed in this manuscript, and how this technology can be applied to liquid biopsies from the point of view of the detection process. The primary discussion objectives are circulating tumor cells (CTCs), exosomes, and circulating nucleic acid (ctDNA). Furthermore, the challenges and future direction of microfluidic technology in detecting liquid biomarkers have been discussed.

DCD-chip designed for the digital and ultraprecise quantification of copy number variation.

Copy number variation (CNV), including genomic deletions and duplication, has been associated with many kinds of diseases. It is crucial to precisely quantify the copy number variation of samples among patients, which may guide treatment. Digital PCR (dPCR) enables high-resolution CNV analysis through the ultraprecise absolute quantification of specific nucleic acid sequences. We explored a platform named digital CNV detection chip (DCD-chip), which can simultaneously and absolutely quantify the GPR146 and RPPH1 genes with amounts as low as 1.4 copies per µL. Finally, we verified that DCD-chip was more accurate than qPCR when the samples were diluted to a certain extent, which indicated the powerful quantification capacity of our DCD-chip platform.

A direct and multiplex digital PCR chip for EGFR mutation.

Digital PCR is a sensitive detection method, which has important applicability in liquid biopsy through the measurement of ctDNA. However, the current sample pre-processing of ctDNA and the multiplex detection capability of digital PCR have limitations. In view of the above two aspects, we developed a digital PCR chip with multiplex capability and established a direct amplification detection method without nucleic acid extraction. Through the design and processing of the chip, we established a self-priming multiplex digital PCR chip, which can detect 4 targets using single fluorescence. This method can be applied to most digital PCR chips. In addition, we used the plasma of lung cancer patients to establish a direct digital PCR detection method based on the chip, thereby avoiding disadvantages caused by the ctDNA extraction process. As a proof of concept, we prepared blood plasma samples with different concentration of ctDNA to prove the chip's multiplex detection capabilities and the results suggested that this multiplex digital PCR is accurate. Overall, our platform provides a novel and promising option for the detection of ctDNA.

Ginseng of different ages is affected by the accumulation of heavy metals in ginseng soil.

Heavy-metal pollution has been established to affect ginseng quality. However, this effect is still unknown in ginseng of different ages, emphasizing the need to investigate the effects of heavy metals in soils on ginseng growth. Herein, we determined the content of heavy metals (Cu, Cd, Pb, Hg, and As) in ginseng of different ages (2 to 6-year-old) and the corresponding soil samples. Then, the total ginsenosides content of ginseng and rate-limiting enzyme (HMGR, SQE, CYP450) activity in the synthesis of ginsenosides were assessed. Results from 200 differently-aged Chinese ginseng showed that increased ginsenoside content in 3 to 5-year-old ginseng was paralleled by increased heavy metal element content in ginseng and its soil. The activity of rate-limiting enzymes increased in the first four years of ginseng growth and then exhibited a steady or downward trend. Further analysis suggested that heavy metal elements in soils could directly affect ginsenoside content. Moreover, we found that Cu significantly affected the rate-limiting enzyme CYP450 activity. Further principal component analysis and correlation analysis found that heavy metals could obviously inhibit ginseng growth during the 5th and 6th years. Heavy metal content in soils has huge prospects for predicting ginsenoside, Cu and As content in ginseng. This study provided support for ginseng cultivation, quality research and quality assessment.

Blue light induces ROS mediated apoptosis and degradation of AML1-ETO oncoprotein in Kasumi-1 cells.

Light-emitting diode (LED)-based therapies, particularly blue LEDs with wavelengths of 400-500 nm, have shown beneficial results in several cancers, including melanoma, lymphoid cells, and skin tumors. In this study, the cell viability and apoptosis of Kasumi-1 cells treated by blue light (BL) irradiation have been explored. Firstly, BL can specially inhibit the proliferation and promote the apoptosis of Kasumi-1 cells. Furthermore, the apoptosis was triggered by the production of reactive oxygen species and the decline of mitochondrial membrane potential which was regulated by the ratio of Bcl-2(Bcl-xL)/Bax; BL caused the cells' final apoptosis accompanied with the increased cleavage of caspase-3 and poly-ADP-ribose polymerase. Finally, BL induced the degradation of AML1-ETO dependent on the activation of caspase-3. These results are helpful for establishing a low toxicity and high efficiency strategy of BL irradiation for clinical treatment of Kasumi-1 cells.

Direct digital polymerase chain reaction chip for the detection of EGFR T790M mutation in plasma.

Nucleic acid extraction and purification before amplification is considered an essential step for nucleic acid amplification testing. However, this may cause losses or introduce errors that can lead to inaccurate results, especially when using samples with a small nucleic acid concentration. Here, we developed a direct digital chip that enabled us to detect nucleic acid without DNA extraction and purification. We have developed a self-priming liquid-dispensing digital PCR chip that does not require any external power. This is a robust anti-evaporation digital PCR chip with fast sampling and accurate quantification performance. Using this chip, we have established an on-chip direct nucleic acid amplification method that does not require nucleic acid extraction and purification for liquid biopsy samples. In order to verify the feasibility of this chip for clinical samples, we detected the EGFR T790M mutation from plasma. Results showed that EGFR T790M mutation could be detected with an accuracy of 100% and a sensitivity of 0.01%. Without nucleic acid extraction and purification, the assay avoids complex pre-processing, thus saving time and achieving precise quantification. We expect our direct digital PCR chip to have practical applications in diagnosis, screening, and research, especially in resource-deprived regions.

Microfluidic devices for multiplexed detection of foodborne pathogens.

The global burden of foodborne diseases is substantial and foodborne pathogens are the major cause for human illnesses. In order to prevent the spread of foodborne pathogens, detection methods are constantly being updated towards rapid, portable, inexpensive, and multiplexed on-site detection. Due to the nature of the small size and low volume, microfluidics has been applied to rapid, time-saving, sensitive, and portable devices to meet the requirements of on-site detection. Simultaneous detection of multiple pathogens is another key parameter to ensure food safety. Multiplexed detection technology, including microfluidic chip design, offers a new opportunity to achieve this goal. In this review, we introduced several sample preparation and corresponding detection methods on microfluidic devices for multiplexed detection of foodborne pathogens. In the sample preparation section, methods of cell capture and enrichment, as well as nucleic acid sample preparation, were described in detail, and in the section of detection methods, amplification, immunoassay, surface plasmon resonance and impedance spectroscopy were exhaustively illustrated. The limitations and advantages of all available experimental options were also summarized and discussed in order to form a comprehensive understanding of cutting-edge technologies and provide a comparative assessment for future investigation and in-field application.

A multiplex and fast detection platform for microRNAs based on a self-priming microfluidic chip and duplex-specific nuclease.

MicroRNA expression levels highly correlate with the occurrence, diagnosis and prognosis of disease. However, challenges remain in establishing a multiplex and fast detection method. Here, we developed a multiplex and fast detection platform for microRNAs based on a self-priming microfluidic chip and duplex-specific nuclease. It can detect three types of miRNAs, including miR-100, miR-155, and Let-7a, simultaneously at 50 °C within 1 h. The probes are pre-introduced into the chip using the self-priming method and cross-contamination can be avoided by using a screw valve. The reagent consumption and cost have been largely reduced in this work compared to the traditional detection method. This chip also exhibits good quantitative performance and specificity. As a proof of concept, we detect three types of microRNAs from different cancer cell lines, which demonstrates its potential in real sample analysis. In summary, this microfluidic chip shows great advantages in multiplex, fast and simple detection of microRNAs, and possesses great potential in the early diagnosis of miRNA-related diseases, especially for point-of-care application.

A Self-Priming Digital Polymerase Chain Reaction Chip for Multiplex Genetic Analysis.

Digital PCR (polymerase chain reaction) is a powerful and attractive tool for the quantification of nucleic acids. However, the multiplex detection capabilities of this system are limited or require expensive instrumentation and reagents, all of which can hinder multiplex detection goals. Here, we propose strategies toward solving these issues regarding digital PCR. We designed and tested a self-priming digital PCR chip containing 6-plex detection capabilities using monochrome fluorescence, which has six detection areas and four-layer structures. This strategy achieved multiplex digital detection by the use of self-priming to preintroduce the specific reaction mix to a certain detection area. This avoids competition when multiple primer pairs coexist, allowing for multiplexing in a shorter time while using less reagents and low-cost instruments. This also prevents the digital PCR chip from experiencing long sample introduction time and evaporation. For further validation, this multiplex digital PCR chip was used to detect five types of EGFR (epidermal growth factor receptor) gene mutations in 15 blood samples from lung cancer patients. We conclude that this technique can precisely quantify EGFR mutations in high-performance diagnostics. This multiplex digital detection chip is a simple and inexpensive test intended for liquid biopsies. It can be applied and used in prenatal diagnostics, the monitoring of residual disease, rapid pathogen detection, and many other procedures.

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.

Advanced "lab-on-a-chip" to detect viruses - Current challenges and future perspectives.

Massive viral outbreaks draw attention to viruses that have not been thoroughly studied or understood. In recent decades, microfluidic chips, known as "lab-on-a-chip", appears as a promising tool for the detection of viruses. Here, we review the development of microfluidic chips that could be used in response to viral detection, specifically for viruses involved in more recent outbreaks. The advantages as well as the disadvantages of microfluidic systems are discussed and analyzed. We also propose ideas for future development of these microfluidic chips and we expect this advanced technology to be used in the future for viral outbreaks.

A "sample-in-multiplex-digital-answer-out" chip for fast detection of pathogens.

Point-of-care (POC) testing offers rapid diagnostic results. However, the quantification of current methods is performed using standard curves and external references, and not direct and absolute quantification. This paper describes an integrated multiplex digital recombinase polymerase amplification (ImdRPA) microfluidic chip which combines DNA extraction, multiplex digital RPA and fluorescence detection together in one chip, creating a "sample-in-multiplex-digital-answer-out" system. Multi-layer soft lithography technology was used, with polydimethylsiloxane (PDMS) as the chip material and a glass slide as the substrate. This microfluidic chip has a six-layer structure and screw microvalve control function. The sample preparation for the chip involved magnetic bead-based nucleic acid extraction, which was completed within 15 min without any instrument dependence. The dRPA region was divided into 4 regions (3 positive detection areas and 1 negative control area) and included a total of 12 800 chambers, with each chamber being able to contain a volume of 2.7 nL. The screw valve allowed for the reaction components of each specific goal to be pre-embedded in different regions of the chambers. The reagents were passively driven into the dRPA region using vacuum-based self-priming introduction. Furthermore, we successfully demonstrated that the chip can simultaneously detect three species of pathogenic bacteria within 45 min and give digital quantitative results without the need to establish a standard curve in contaminated milk. Moreover, the detection limit of this ImdRPA microfluidic chip was found to be 10 bacterial cells for each kind of pathogen. These characteristics enhance its applicability for rapid detection of foodborne bacteria at the point-of-care (POC). We envision that the further development of this integrated chip will lead to rapid, multiplex and accurate detection of foodborne bacteria in a feasible manner.

A fast nucleic acid extraction system for point-of-care and integration of digital PCR.

Digital PCR is a powerful amplification method for absolute quantification of nucleic acids. The systems that integrated the nucleic acid extraction and amplification can reduce detection time, improve accuracy, and reduce labor costs. However, current nucleic acid extraction systems cannot be integrated well with integrated fluidic circuit (IFC) dPCR or droplet digital PCR chips perfectly and limit the application of digital PCR. In this study, a polytetrafluoroethylene (PTFE)-based nucleic acid extraction (PNE) system, which was able to achieve fully closed extraction for micro samples and was able to be integrated with IFC dPCR or droplet digital dPCR (ddPCR) chips perfectly is proposed. For this system, PTFE tubing with an inner diameter of 1 mm was used to load the reagents and superparamagnetic particles (PMPs) were used to extract nucleic acids. The system can extract nucleic acids from cells and blood in 5 minutes. Meanwhile, when nucleic acid extraction was completed, PNE was able to be directly combined with IFC dPCR or ddPCR chips without any intermediate steps. Therefore, the PNE system can realize sample-in-digital-answer-out. It will be highly useful in point-of-care (POC) and promote the development and application of dPCR.

Investigation of the Presence of 22 Organochlorine Pesticide Residues in Ginseng from Jilin Province, China.

The purpose of this study was to investigate the status of organochlorine pesticide residues in 186 ginseng samples collected in Jilin, People's Republic of China. Based on the Chinese Pharmacopoeia 2015 method for detection of organochlorine pesticide residues in ginseng, 22 organochlorine pesticide residues were identified. Chlordane, aldrin, epichlorohydrin, and dieldrin and their isomers were not detected in ginseng from this region. Heptachlor was detected in only one ginseng sample, and the concentration did not exceed the maximum residual limit (MRL) prescribed in the Pharmacopoeia (0.05 mg/kg). Benzene hexachloride was detected in two samples, one of which was above the MRL. Hexachlorobenzene and pentachloronitrobenzene (quintozene) were found in 11.8 and 52.1% of the samples, respectively, and the residues in these samples exceeded the MRL by 4.3 and 8.6%, respectively.

Integrated microfluidic systems with sample preparation and nucleic acid amplification.

Rapid, efficient and accurate nucleic acid molecule detection is important in the screening of diseases and pathogens, yet remains a limiting factor at point of care (POC) treatment. Microfluidic systems are characterized by fast, integrated, miniaturized features which provide an effective platform for qualitative and quantitative detection of nucleic acid molecules. The nucleic acid detection process mainly includes sample preparation and target molecule amplification. Given the advancements in theoretical research and technological innovations to date, nucleic acid extraction and amplification integrated with microfluidic systems has advanced rapidly. The primary goal of this review is to outline current approaches used for nucleic acid detection in the context of microfluidic systems. The secondary goal is to identify new approaches that will help shape future trends at the intersection of nucleic acid detection and microfluidics, particularly with regard to increasing disease and pathogen detection for improved diagnosis and treatment.

Proximity ligation assays for precise quantification of femtomolar proteins in single cells using self-priming microfluidic dPCR chip.

The quantification of low concentration proteins can facilitate the discovery of some significant biomarkers, and provide us a more profound understanding of cell heterogeneity when applied to single cell analysis. However, most state-of- art single cell protein detection platforms are bulky, expensive and complicated. Here we report a simple and low cost microfluidic dPCR (digital polymerase chain reaction) chip-based proximity ligation assay (PLA) for the quantification of low concentration proteins. First, standard hCSTB (human cystatin B) protein was used to optimize the related experimental conditions. Comparing to ordinary PLA tests, the results showed that our method achieved femtomolar limit of detection (LOD) with a linear dynamic range over three to four orders of magnitude. Then human CD147 protein, a reported biomarker for hepatoma carcinoma, was detected in single HepG2 and L02 cells. The results showed that there were wide disparities in single cell CD147 abundance for both of the two cell lines. And the average CD147 protein content in single HepG2 cells displayed 2-fold increase in comparison to that in single L02 cells. Comparing to the research findings obtained at bulk level, our method can provide more useful information for diagnosis and targeted therapy of tumors.

Power-free polydimethylsiloxane femtoliter-sized arrays for bead-based digital immunoassays.

A novel microfluidic chip employing power-free polydimethylsiloxane (PDMS) femtoliter-sized arrays was developed for the detection of low concentrations of protein biomakers by isolating individual paramagnetic beads in single wells. Arrays of femtoliter-sized wells were fabricated with PDMS using well-developed molding techniques. Paramagnetic beads were functionalized with specific antibodies to capture the antigens. These antigens were labeled with enzymes via conventional multistep immunosandwich approach. After suspending in aqueous solutions of enzyme substrate, the solutions were delivered to the arrays using a conventional micropipette. The aqueous solutions were introduced into the microwells by capillarity and the beads were loaded into microwells by gravity. A fluorocarbon oil was then flowed into the chip to remove excess beads from the surface of the array and meanwhile isolated the femtoliter-sized wells. All processes were achieved by conventional micropipette, without external pumping systems and valves. Finally, the arrays were imaged using standard fluorescence imaging after incubation 30 min for digital counting enzyme molecules. It was demonstrated that the chip platform possessed the performance of digital counting with a linear dynamic range from 1 aM to 1 fM for the detection of biotinylated ß-galactosidase (BßG), achieving a limit of detection (LOD) of 930 zM. Using this chip, a digital immunoassay to detect Tumor Necrosis Factor α (TNF- α) was developed. Since the chip fabrication is low-cost and circumvents the surface modification, we expect it can become a new chip-based digital immunoassay platform for ultrasensitive diagnostic of biomarkers.