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.

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.

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 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.

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.

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.

Study on a 65-mer peptide mimetic enzyme with GPx and SOD dual function.

Excessive reactive oxygen species (ROS) levels are harmful to the body. The peroxidase, GPx, and the superoxide dismutase, SOD, are important antioxidant enzymes for preventing ROS-induced damage. Se-CuZn-65P is an enzyme mimetic with dual GPx and SOD antioxidant function. However, currently, its production is mainly based on the cysteine auxotrophic expression technique, which is inefficient, expensive, and time consuming. In this study, we combined protein engineering and the chemical mutation method to synthesize Se-CuZn-65P. The DNA sequence encoding the 65 amino acid peptide with the desired sequence transformations to incorporate the SOD and the GPx catalytic sites was cloned and expressed in a soluble protein expression vector. The protein yield increased up to 152 mg/L, which is 10 times higher than in previous studies. The SOD and GPx activity of Se-CuZn-65P was high (1181 U/mg and 753 U/µmol, respectively). The binding constant of glutathione was 5.6 × 104  L·mol-1 , which shows that Se-CuZn-65P efficiently catalyzed hydrogen peroxide reduction by glutathione. Mitochondrial damage experiments confirmed the double protective role of the Se-CuZn-65P peptide and demonstrated functional synergy between the SOD and the GPx domains, which indicates its potential to be used in the treatment of ROS-related diseases. Our research may give a new thought to increase the yield of mimic.

Study on the Correlation between Gene Expression and Enzyme Activity of Seven Key Enzymes and Ginsenoside Content in Ginseng in Over Time in Ji'an, China.

Panax ginseng is a traditional medicine. Fresh ginseng is one of the most important industries related to ginseng development, and fresh ginseng of varying ages has different medicinal properties. Previous research has not systematically reported the correlation between changes in key enzyme activity with changes in ginsenoside content in fresh ginseng over time. In this study, for the first time, we use ginseng samples of varying ages in Ji'an and systematically reported the changes in the activity of seven key enzymes (HMGR, FPS, SS, SE, DS, CYP450, and GT). We investigated the content of ginsenoside and gene expression of these key enzymes. Ginsenoside content was measured using HPLC. HPLC, GC-MS, and LC-MS were combined to measure the enzyme activity of the key enzymes. Quantitative PCR was used in the investigation of gene expression. By analyzing the correlation between the enzyme activity and the transcription level of the key enzymes with ginsenoside content, we found that DS and GT enzyme activities are significantly correlated with the ginsenoside content in different ages of ginseng. Our findings might provide a new strategy to discriminate between ginseng of different years. Meanwhile, this research provides important information for the in-depth study of ginsenoside biosynthesis.

The Small Glutathione Peroxidase Mimic 5P May Represent a New Strategy for the Treatment of Liver Cancer.

Glutathione peroxidase (GPx) is an antioxidant protein containing selenium. Owing to the limitations of native GPx, considerable efforts have been made to develop GPx mimics. Here, a short 5-mer peptides (5P) was synthesized and characterized using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Enzyme coupled assays were used to evaluate GPx activity. The cell viability and apoptosis of H22 cells were tested, and mice bearing H22 cell-derived tumors were used to determine the effects of 5P on tumor inhibition. In comparison with other enzyme models, 5P provided a suitable substrate with proper catalytic site positions, resulting in enhanced catalytic activity. In our mouse model, 5P showed excellent inhibition of tumor growth and improved immunity. In summary, our findings demonstrated the design and synthesis of the small 5P molecule, which inhibited tumor growth and improved immunity. Notably, 5P could inhibit tumor growth without affecting normal growth. Based on these advantages, the novel mimic may have several clinical applications.

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.

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.

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.

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.

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.

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.

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 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.

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.

Diverse autophagy and apoptosis in myeloid leukemia cells induced by 20(s)-GRh2 and blue LED irradiation.

Autophagy is an important mechanism for cell death regulation. To improve the anticancer effect during the treatment of leukemia and promote the apoptosis of leukemic cells, it is important to define the relationship between autophagy and apoptosis. A key bioactive compound in traditional Chinese medicine, 20(s)-Ginsenoside (GRh2), demonstrated an advancement in leukemia treatment. Blue LED therapy (BL) is a physical treatment method that can induce leukemic cell death. In this study, we tested the effect of 20(s)-GRh2, BL, and their combination (BL-GRh2) on the activation of leukemic cell apoptosis and autophagy. Both treatments, whether used individually or simultaneously, induce apoptosis through the induction of reactive oxygen species (ROS), disrupted mitochondrial membrane potential (MMP) and regulated the expression of apoptosis-related genes and proteins. Furthermore, using western blotting to analyze the autophagy markers LC3B and P62, we detected the activation of autophagy. In cells treated with autophagy inhibitor 3-MA, both autophagy and apoptosis were inhibited, either by BL alone or by BL-GRh2. However, apoptosis in 20(s)-GRh2-treated cells was enhanced. In cells treated with apoptosis suppressor Z-VAD-FMK, autophagy was inhibited in the BL and BL-GRh2-treated cells, although it was enhanced in cells treated with 20(s)-GRh2 alone. Moreover, we observed a stronger induction of apoptosis by BL-GRh2 in myeloid leukemia cells. Our data indicate that autophagy induced by different factors can play diverse roles on the same cells. Our results also indicate that the combination of traditional Chinese medicine with physical therapy may be a new strategy for anti-cancer therapy.