DMF-DM-seq: Digital-Microfluidics Enabled Dual-Modality Sequencing of Single-Cell mRNA and microRNA with High Integration, Sensitivity, and Automation.

Multimodal measurement of single cells provides deep insights into the intricate relationships between individual molecular layers and the regulatory mechanisms underlying intercellular variations. Here, we reported DMF-DM-seq, a highly integrated, sensitive, and automated platform for single-cell mRNA and microRNA (miRNA) co-sequencing based on digital microfluidics. This platform first integrates the processes of single-cell isolation, lysis, component separation, and simultaneous sequencing library preparation of mRNA and miRNA within a single DMF device. Compared with the current half-cell measuring strategy, DMF-DM-seq enables complete separation of single-cell mRNA and miRNA via a magnetic field application, resulting in a higher miRNA detection ability. DMF-DM-seq revealed differential expression patterns of single cells of noncancerous breast cells and noninvasive and aggressive breast cancer cells at both mRNA and miRNA levels. The results demonstrated the anticorrelated relationship between miRNA and their mRNA targets. Further, we unravel the tumor growth and metastasis-associated biological processes enriched by miRNA-targeted genes, along with important miRNA-interaction networks involved in significant signaling pathways. We also deconstruct the miRNA regulatory mechanisms underlying different signaling pathways across different breast cell types. In summary, DMF-DM-seq offers a powerful tool for a comprehensive study of the expression heterogeneity of single-cell mRNA and miRNA, which will be widely applied in basic and clinical research.

Novel Dielectric Barrier Discharge Trap for Arsenic Introduced by Electrothermal Vaporization: Possible Mechanism and Its Application.

In this work, a novel integrated dielectric barrier discharge (IDBD) reactor coupled to an electrothermal vaporizer (ETV) was established for arsenic determination. It is for the first time gas-phase enrichment (GPE) was fulfilled based on the hyphenation of ETV and DBD. The mechanisms of evolution of arsenic atomic and molecular species during vaporization, transportation, trapping, and release processes were investigated via X-ray photoelectron spectroscopy (XPS) and other approaches. Tentative mechanisms were deduced as follows: the newly designed DBD atomizer (DBDA) tube upstream to the air inlet fulfills the atomization of arsenic nanoparticles in vaporized aerosol, leading to free arsenic atoms that are indispensable for forming arsenic oxides; the DBD trap (DBDT) tube traps arsenic oxides under an O2-domininating atmosphere and then releases arsenic atoms under H2-dominating atmospheres. In essence, this process is a physical-chemical process rather than an electrostatic particle deposition. Such a trap and release sequence separates matrix interference and enhances analytical sensitivity. Under the optimized conditions, the method detection limit (LOD) was 0.04 mg/kg and the relative standard deviations (RSDs) were within 6% for As standard solution and real seafood samples, indicating adequate analytical sensitivity and precision. The mean spiked recoveries for laver, kelp, and Undaria pinnatifida samples were 95-110%, and the results of the certified reference materials (CRMs) were consistent with certified values. This ETV-DBD preconcentration scheme is easy and green and has low cost for As analysis in seafood samples. DBD was proved a novel ETV transportation enhancement and preconcentration technique for arsenic, revealing its potential in rapid arsenic analysis based on direct solid sampling ETV instrumentation.

High Sensitivity Analysis of Selenium by Ultraviolet Vapor Generation Combined with Microplasma Gas Phase Enrichment and the Mechanism Study.

Ultraviolet vapor generation (UVG), as an environmental/user-friendly and efficient sampling approach, was first combined with the gas phase enrichment of Se by dielectric barrier discharge (DBD) microplasma. Volatile Se species from UVG, being much more complicated than conventional hydrides, can be trapped quantitatively (∼100%) on the quartz surface of DBD tube under O2-containing atmosphere and released (∼100%) under H2-containing atmosphere. The absolute detection limit (LOD) for Se was 4 pg (injection volume = 1.2 mL), and the linear (R2 > 0.995) range was 0.05-50 µg/L. The results were in good agreement with those of certified reference materials (CRMs) of water and soil samples, and spiked recoveries for real samples were 90-102% with 1-10% relative standard deviations (RSDs). By gas phase analyte enrichment, the proposed method improved analytical sensitivity (peak height) by 16 times. The mechanism was deduced that dominating SeCO species besides H2Se generating from UVG were all trapped on the DBD quartz tube surface as SeO2 or selenite and then released/transported as atoms to the detection zone. The combination of UVG and DBD can facilitate the green uses, miniaturization, and portability revealing its promising potential in field elemental analysis.

In Situ Dielectric Barrier Discharge Trap for Ultrasensitive Arsenic Determination by Atomic Fluorescence Spectrometry.

The mechanisms of arsenic gas phase enrichment (GPE) by dielectric barrier discharge (DBD) was investigated via X-ray photoelectron spectroscopy (XPS), in situ fiber optic spectrometer (FOS), etc. It proved for the first time that the arsenic species during DBD trapping, release, and transportation to the atomic fluorescence spectrometer (AFS) are probably oxides, free atoms, and atom clusters, respectively. Accordingly, a novel in situ DBD trap as a GPE approach was redesigned using three-concentric quartz tube design and a modified gas line system. After trapping by O2 at 9.2 kV, sweeping for 180 s, and releasing by H2 at 9.5 kV, 2.8 pg detection limit (LOD) was achieved without extra preconcentration (sampling volume = 2 mL) as well as 4-fold enhancement in absolute sensitivity and ∼10 s sampling time. The linearity reached R2 > 0.998 in the 0.1-8 µg/L range. The mean spiked recoveries for tap, river, lake, and seawater samples were 100-106%; and the measurements of the certified reference materials (CRMs) were in good agreement with the certified values. In situ DBD trap is also suitable to atomic absorption spectrometry (AAS) or optical emission spectrometry (OES) for fast and on-site determination of multielements.

Ambient-Temperature Trap/Release of Arsenic by Dielectric Barrier Discharge and Its Application to Ultratrace Arsenic Determination in Surface Water Followed by Atomic Fluorescence Spectrometry.

A novel dielectric barrier discharge reactor (DBDR) was utilized to trap/release arsenic coupled to hydride generation atomic fluorescence spectrometry (HG-AFS). On the DBD principle, the precise and accurate control of trap/release procedures was fulfilled at ambient temperature, and an analytical method was established for ultratrace arsenic in real samples. Moreover, the effects of voltage, oxygen, hydrogen, and water vapor on trapping and releasing arsenic by DBDR were investigated. For trapping, arsenic could be completely trapped in DBDR at 40 mL/min of O2 input mixed with 600 mL/min Ar carrier gas and 9.2 kV discharge potential; prior to release, the Ar carrier gas input should be changed from the upstream gas liquid separator (GLS) to the downstream GLS and kept for 180 s to eliminate possible water vapor interference; for arsenic release, O2 was replaced by 200 mL/min H2 and discharge potential was adjusted to 9.5 kV. Under optimized conditions, arsenic could be detected as low as 1.0 ng/L with an 8-fold enrichment factor; the linearity of calibration reached R(2) > 0.995 in the 0.05 µg/L-5 µg/L range. The mean spiked recoveries for tap, river, lake, and seawater samples were 98% to 103%; and the measured values of the CRMs including GSB-Z50004-200431, GBW08605, and GBW(E)080390 were in good agreement with the certified values. These findings proved the feasibility of DBDR as an arsenic preconcentration tool for atomic spectrometric instrumentation and arsenic recycling in industrial waste gas discharge.

DMF-ChIP-seq for Highly Sensitive and Integrated Epigenomic Profiling of Low-Input Cells.

Mapping genome-wide DNA-protein interactions (DPIs) provides insights into the epigenetic landscape of complex biological systems and elucidates the mechanisms of epigenetic regulation in biological progress. However, current technologies in DPI profiling still suffer from high cell demands, low detection sensitivity, and large reagent consumption. To address these problems, we developed DMF-ChIP-seq that builds on digital microfluidic (DMF) technology to profile genome-wide DPIs in a highly efficient, cost-effective, and user-friendly way. The entire workflow including cell pretreatment, antibody recognition, pA-Tn5 tagmentation, fragment enrichment, and PCR amplification is programmatically manipulated on a single chip. Leveraging closed submicroliter reaction volumes and a superhydrophobic interface, DMF-ChIP-seq presented higher sensitivity in peak enrichment than other current methods, with high accuracy (Pearson Correlation Coefficient (PCC) > 0.86) and high repeatability (PCC > 0.92). Furthermore, DMF-ChIP-seq was capable of processing the samples with as few as 8 cells while maintaining a high signal-to-noise ratio. By applying DMF-ChIP-seq, H3K27ac histone modification of early embryonic cells during differentiation was profiled for the investigation of epigenomic landscape dynamics. With the benefits of high efficiency and sensitivity in DPI analysis, the system provides great promise in studying epigenetic regulation during various biological processes.

Highly Multiplexed, Efficient, and Automated Single-Cell MicroRNA Sequencing with Digital Microfluidics.

Single-cell microRNA (miRNA) sequencing has allowed for comprehensively studying the abundance and complex networks of miRNAs, which provides insights beyond single-cell heterogeneity into the dynamic regulation of cellular events. Current benchtop-based technologies for single-cell miRNA sequencing are low throughput, limited reaction efficiency, tedious manual operations, and high reagent costs. Here, a highly multiplexed, efficient, integrated, and automated sample preparation platform is introduced for single-cell miRNA sequencing based on digital microfluidics (DMF), named Hiper-seq. The platform integrates major steps and automates the iterative operations of miRNA sequencing library construction by digital control of addressable droplets on the DMF chip. Based on the design of hydrophilic micro-structures and the capability of handling droplets of DMF, multiple single cells can be selectively isolated and subject to sample processing in a highly parallel way, thus increasing the throughput and efficiency for single-cell miRNA measurement. The nanoliter reaction volume of this platform enables a much higher miRNA detection ability and lower reagent cost compared to benchtop methods. It is further applied Hiper-seq to explore miRNAs involved in the ossification of mouse skeletal stem cells after bone fracture and discovered unreported miRNAs that regulate bone repairing.

Portable and miniature mercury analyzer using direct sampling inbuilt-metal ceramic electrothermal vaporization.

In this work, a small-size inbuilt-metal ceramic heater (IMCH) was for the first time utilized as a solid sampling electrothermal vaporizer (ETV), and then a novel direct sampling mercury analyzer coupled with a miniature atomic absorption spectrometer was thereby fabricated for sensitive determination of mercury in soil. The mercury analyzer is mainly composed of an IMCH-ETV, a catalytic pyrolysis furnace (CPF) using Al2O3 particles as a new filler, an atomic absorption spectrometer (AAS) and a miniature air pump for carrier gas; herein, the mostly used gold amalgamation was canceled. The IMCH with 30 W heating fulfills the 100% vaporization of mercury from up to 80 mg soil samples using 0.1 L min-1 air carrier. Under the optimized conditions, the method detection limit (LOD) was 0.4 ng g-1 for a 50 mg sample and the RSD of 11 repeated measurements of GSS-3a soil certified reference material (CRM) was 4%. Furthermore, the measured Hg values in various soil CRMs and real soil samples were within their certified values and consistent with that using the Chinese standard method (solid sampling catalytic pyrolysis AAS with gold amalgamation); and the recoveries were 85-113%, which proved favorable analytical accuracy and precision. This fabricated instrumentation only occupies 5 kg and 200 W power consumption vs. more than 25 kg and 1000 W for the traditional Hg analyzer. Therefore, the proposed IMCH-ETV-AAS method is very suitable for portable and rapid detection of mercury in soil.

Cilo-seq: highly sensitive cell-in-library-out single-cell transcriptome sequencing with digital microfluidics.

Single-cell RNA sequencing (scRNA-seq) plays a critical role in revealing genetic expression patterns at the single-cell level for cell type identification and rare transcript detection. Although there have been great advances in scRNA-seq methodologies, existing technologies still suffer from complexity and high cost, and an integrated platform for complete library construction is still lacking. Herein we describe Cilo-seq for high-performance scRNA-seq library construction in a single device with programmed and addressable droplet handling based on digital microfluidics. The platform is simultaneously accessible for convenient single-cell isolation, efficient nucleic acid amplification, low-loss nucleic acid purification and high-quality library preparation by leveraging specific interface design, tiny reaction volume, auxiliary magnetic field control and accurate droplet control. With a closed hydrophobic interface, the platform further reduces nucleic acid loss and exogenous background interference. Cilo-seq provides excellent detection sensitivity (1.4-fold improvement over tube-based methods), accuracy (R = 0.98) and cost efficiency (10-fold decrease in cost compared to tube-based methods), and holds great promise for studies of single-cell RNA biology.

The progressive application of single-cell RNA sequencing technology in cardiovascular diseases.

The mortality rate of cardiovascular disease ranks first in the world. Its pathogenesis involves not only internal factors such as immunity, inflammation, metabolic disorders, and self-development but also external factors such as the environment. In the last decade, the emergence of single-cell technology has greatly promoted the development of disease research. Among them, the more mature single-cell RNA sequencing can carry out high-throughput analysis of single cells while studying with single-cell resolution. This technology enables people to characterize the heterogeneity of single cells, identify rare cell types in heart and blood vessels, and construct human heart cell map. With the data analysis of bioinformatics experts, it can also reconstruct the development track of the heart, to construct a map of heart development. Single-cell sequencing plays an important role in analyzing the human physiological structure and disease progression due to its advantages of single-cell resolution. The possibility of combining other omics technologies is proposed by summarizing the existing application examples and advanced technologies like spatial transcriptome. In this review, we summarize the current single-cell sequencing technologies (plate-based and droplet-based) and describe the data analysis process. The latest findings in cardiovascular disease using single-cell RNA sequencing technology are described. Finally, we discussed the shortcomings of single-cell RNA sequencing technology. At the same time, the possibility of the combination of single-cell RNA sequencing and spatial omics technology, and how to apply it to the study of cardiovascular diseases is discussed.

Fast and High Sensitive Analysis of Lead in Human Blood by Direct Sampling Hydride Generation Coupled with in situ Dielectric Barrier Discharge Trap.

A direct sampling hydride generation (HG) system based on modified gas liquid separator (GLS) coupled with in situ dielectric barrier discharge (DBD) is first rendered to detect lead in blood samples. Herein, a triple-layer coaxial quartz tube was employed as DBD trap (DBDT) to replace the original atomizer of atomic fluorescence spectrometry (AFS) to satisfy the in situ preconcentration. After 40-fold dilution, foams generated from protein in a blood sample can be eliminated via the double-GLS set; and lead in a blood sample were generated as plumbane under 3.5% HNO3 (v:v) and 30 g/L NaOH with 8 g/L KBH4, 10 g/L H3BO3, and 5 g/L K3[Fe(CN)6]. Then, lead analyte was trapped on the DBD quartz surface by 9 kV discharging at 50 mL/min air; and subsequently released by 12 kV discharging at 110 mL/min H2. The absolute detection limit (LOD) for Pb was 8 pg (injection volume = 2 mL), and the linearity (R2 > 0.997) range was 0.05 - 50 µg/L. The results were in good agreement with that of blood certified reference materials (CRM), and spiked recoveries for real blood samples were 95 - 104% within a relative standard deviation of 5% (RSD). Via gas phase enrichment, the established method improved analytical sensitivity (peak height) by 8 times. The entire analysis time including blood sample preparation can be kept to within 10 min. The combination of modified GLS and DBDT can facilitate the quickness, accuracy, and sensitivity, revealing a promising future for monitoring lead in blood to protect humans, especially children's health.

In situ preconcentration of lead by dielectric barrier discharge and its application to high sensitivity surface water analysis.

A novel dielectric barrier discharge (DBD) reactor was utilized to in situ enrich and atomize lead in gas phase. The structure of DBD reactor was optimized to broaden the acidity window of plumbane generation from 1% to 3.5%, bringing better analytical stability and practicability deriving from hydride generation process. For the first time DBD proved effective in lead preconcentration and broadening the acidity window of plumbane generation. Pb can be trapped quantitatively (~100%) on the quartz surface of DBD tube under O2-containing atmosphere and released (~100%) under H2-containing atmosphere. The absolute detection limit (LOD) for Pb was 4.1 pg (injection volume = 1.2 mL), and the linear (R2 > 0.999) range was 0.05-100 µg/L. The results were in good agreement with those of certified reference materials (CRMs), and spiked recoveries for surface water samples were 99-104% with 2-8% RSD. By gas phase analyte enrichment, the proposed method reduced absolute LOD by 10 times. It was deduced that plumbane was changed to lead oxide species trapped on the quartz tube surface and then released, and transported in form of atoms to the detection zone.

A portable and field optical emission spectrometry coupled with microplasma trap for high sensitivity analysis of arsenic and antimony simultaneously.

In this work, a portable and reliable optical emission spectrometric (OES) instrument based on solid acid hydride generation (HG) and subsequent in situ dielectric barrier discharge (DBD) preconcentration was first developed for simultaneous and field analysis of ultratrace As and Sb in environmental water. In situ DBD fulfilled both gas phase enrichment (GPE) and excitation; effective enrichment made it possible to use a low-cost charge coupled device (CCD) as detector. To simplify field protocol, solid tablet made from sulfamic acid was first used to replace hydrochloric acid for co-generation of As and Sb hydrides. Moisture interference was eliminated by carrier gas sweeping without any desiccant. After calculating peak volume for emission data handling, detection limits (LODs) were 0.5 µg L-1 for As and 0.2 µg L-1 for Sb, respectively, with <3% relative standard deviations (RSDs) at 10 µg L-1; linear dynamic ranges (R2>0.995) were 2-200 µg L-1 for As and 1-200 µg L-1 for Sb, respectively. The results agreed with certified values of CRMs and recoveries were 87-97% vs. inductively coupled plasma mass spectrometry. The running costs can be controlled within one dollar per use. This HG-in situ DBD trap-OES scheme, with demonstrated advantages in sensitivity, low-cost, power (<60 W), size (0.6 m × 0.5 m × 0.3 m), weight (15 kg), gas consumption (300 measurements per 4 L tank), and multi-element capability, was implemented in a miniature spectrometer for field analysis.

A novel 3D printed negative pressure small sampling system for bubble-free liquid core waveguide enhanced Raman spectroscopy.

Liquid core waveguide (LCW) is well-known as an effective fiber enhanced approach for Raman spectroscopy with features of long optical path and small sampling size. However, inevitable air bubbles introduced in the LCW tube possibly caused light scattering, refraction and reflection so as to further hamper the quantitative analysis. In this work, to eliminate air bubbles, a novel negative pressure system combined with 3D printing was first utilized for the enhanced Raman spectroscopy on the principle of the gas permeability of LCW tube. After optimization, the LCW tube made of Teflon-AF was inserted into a D-shaped support with an internal channel manufactured by 3D printing to create a sealed space; then, air pressure outside the LCW tube was reduced to create a negative pressure via diaphragm pump and magnetic valve controlled by computer. Under adjustable negative pressure, not only can liquid sample be introduced into the LCW tube automatically, but air bubbles can also be removed through the tube wall simply and completely. For real samples, the assembled apparatus was employed as the small sampling system of Raman spectrometer to measure rhodamine B and ethanol in solutions, with the highest 82-fold (ethanol) enhancement of analytical sensitivity vs. the traditional colorimetric ware. The limit of detections (LODs) were 0.7 µg/mL rhodamine B and 0.03% (v:v) ethanol with only 250 µL sample consumption; their linear correlation coefficients (r) were 0.998 and 0.999 in the range from 2 µg/mL to 25 µg/mL (rhodamine B) and 0.1%-5% (ethanol), respectively. It is worth mentioning that the intraday stability and 7-days reproducibility can be both controlled within 7%, which is extremely superior to the previous enhanced Raman spectroscopy. For another, 3D printing enables the LCW detection system more integrated and easier to assemble. So, the proposed method proves many advantages, such as stability, sensitivity, and quickness, in addition of effective physical enhancement, low sample consumption, and long light path. Considering the flexibility of LCW tube, as a versatile module, the negative pressure LCW system should be further suitable to ultraviolet, fluorescence and other detectors, which reveals a favorable application prospect for the fast testing instruments.

On-line microplasma decomposition of gaseous phase interference for solid sampling mercury analysis in aquatic food samples.

In this work, dielectric barrier discharge (DBD) was first utilized to eliminate gaseous phase interference from complicated solid sample. So, a novel solid sampling Hg analyzer was first designed using a coaxial DBD reactor to replace catalytic pyrolysis furnace for sensitive mercury determination in aquatic food samples. The Hg analyzer mainly comprised an electrothermal vaporizer (ETV), a DBD reactor to decompose gaseous interfering substances including volatile organic compounds (VOCs), a gold-coil Hg trap to eliminate matrix interference and an atomic fluorescence spectrometer (AFS) as detector. These units were connected by a manifold integrating air and Ar/H2 (v/v = 9 : 1), fulfilling on-line decomposition of up to 12 mg dried aquatic food powder at ambient temperature. The proposed method detection limit (LOD) was 0.5 µg/kg and the relative standard deviations (RSDs) were within 5% for Hg standards as well as within 10% for real samples, indicating adequate analytical sensitivity and precision. In addition, the on-line DBD reactor consumes only 40 W, which is obviously lower than that (>300 W) of the commercial Hg analyzers; including the sample pre-treatment, the overall analysis could be completed within 5 min. This method is easier, greener and safer for Hg analysis in real samples obviating chemical reagents. The new DBD apparatus can facilitate the miniaturization and portability with low power consumption and instrumental size revealing its promising potential in direct Hg analysis instrumentation development.

A novel gas liquid separator for direct sampling analysis of ultratrace arsenic in blood sample by hydride generation in-situ dielectric barrier discharge atomic fluorescence spectrometry.

A novel direct sampling (DS-HG) system consisting of an enlarged gas liquid separator (GLS) coupled with a foam breaker was firstly utilized for the in-situ dielectric barrier discharge atomic fluorescence spectrometer (DBD-AFS). After direct dilution using 5% HCl (v:v), a prepared blood sample was introduced into the DS-HG with a UV digestion unit, of which arsenic hydrides directly generated from sample under 5% HCl (v:v) and 5 g/L KBH4 in 1.5 g/L KOH. Herein, the newly designed DS-HG is capable of effectively eliminating foam generation deriving from protein in blood sample. Then, arsenic hydrides were trapped by 11 kV discharging at 110 mL/min air, and released by 13 kV at 180 mL/min H2 orderly. Under the optimized conditions, the linearity ranged from 0.05 to 50 ng/mL with a regression coefficient (R2) = 0.996. The method detection limit (LOD) was 7 pg arsenic (0.14 ng/mL), and relative standard deviation (RSD) of 10 repeated measurements for a real blood sample was 4.2%, indicating a good precision. The spiked recoveries for real samples were in the range of 97%-102%. Furthermore, arsenic presence in real blood samples measured by the proposed method were consistent (P > 0.05) with the microwave digestion ICP-MS. The whole analytical time can be controlled within 8 min including sample dilution. As a matter of fact, it is a favorable progress for DBD technique to eliminate matrix interferences of real samples based on the gas phase enrichment (GPE) principle, with advantages such as excellent sensitivity, digestion-free, fast and simple operation. Thus, the recommended DS-HG-in-situ DBD-AFS are suitable to the fast analysis of ultratrace arsenic in blood samples to protect human's health.

Evaluation of a hydride generation-atomic fluorescence system for the determination of arsenic using a dielectric barrier discharge atomizer.

A new atomizer based on atmospheric pressure dielectric barrier discharge (DBD) plasma was specially designed for atomic fluorescence spectrometry (AFS) in order to be applied to the measurement of arsenic. The characteristics of the DBD atomizer and the effects of different parameters (power, discharge gas, gas flow rate, and KBH(4) concentration) were discussed in the paper. The DBD atomizer shows the following features: (1) low operation temperature (between 44 and 70 degrees C, depending on the operation conditions); (2) low power consumption; (3) operation at atmospheric pressure. The detection limit of As(III) using hydride generation (HG) with the proposed DBD-AFS was 0.04 microgL(-1). The analytical results obtained by the present method for total arsenic in reference materials, orchard leaves (SRM 1571) and water samples GBW(E) 080390, agree well with the certified values. The present HG-DBD-AFS is more sensitive and reliable for the determination of arsenic. It is a very promising technique allowing for field arsenic analysis based on atomic spectrometry.