Highly Multiplexing, Throughput and Efficient Single-Cell Protein Analysis with Digital Microfluidics.

Proteins as crucial components of cells are responsible for the majority of cellular processes. Sensitive and efficient protein detection enables a more accurate and comprehensive investigation of cellular phenotypes and life activities. Here, a protein sequencing method with high multiplexing, high throughput, high cell utilization, and integration based on digital microfluidics (DMF-Protein-seq) is proposed, which transforms protein information into DNA sequencing readout via DNA-tagged antibodies and labels single cells with unique cell barcodes. In a 184-electrode DMF-Protein-seq system, ≈1800 cells are simultaneously detected per experimental run. The digital microfluidics device harnessing low-adsorbed hydrophobic surface and contaminants-isolated reaction space supports high cell utilization (>90%) and high mapping reads (>90%) with the input cells ranging from 140 to 2000. This system leverages split&pool strategy on the DMF chip for the first time to overcome DMF platform restriction in cell analysis throughput and replace the traditionally tedious bench-top combinatorial barcoding. With the benefits of high efficiency and sensitivity in protein analysis, the system offers great potential for cell classification and drug monitoring based on protein expression at the single-cell level.

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.

Digital microfluidics for biological analysis and applications.

Digital microfluidics (DMF) is an emerging liquid-handling technology based on arrays of microelectrodes for the precise manipulation of discrete droplets. DMF offers the benefits of automation, addressability, integration and dynamic configuration ability, and provides enclosed picoliter-to-microliter reaction space, making it suitable for lab-on-a-chip biological analysis and applications that require high integration and intricate processes. A review of DMF bioassays with a special emphasis on those actuated by electrowetting on dielectric (EWOD) force is presented here. Firstly, a brief introduction is presented on both the theory of EWOD actuation and the types of droplet motion. Subsequently, a comprehensive overview of DMF-based biological analysis and applications, including nucleic acid, protein, immunoreaction and cell assays, is provided. Finally, a discussion on the strengths, challenges, and potential applications and perspectives in this field is presented.

Decoding Expression Dynamics of Protein and Transcriptome at the Single-Cell Level in Paired Picoliter Chambers.

Simultaneous analysis of mRNAs and proteins at the single-cell level provides information about the dynamics and correlations of gene and protein expressions in individual cells, enabling a comprehensive study of cellular heterogeneity and expression patterns. Here, we present a platform for about 1000 cellular indexing of mRNAs and membrane proteins, named multi-Paired-seq, with high cell utilization, accurate molecular measurement, and low cost. Based on hydrodynamic differential flow resistance, multi-Paired-seq largely improves cell utilization in the percentage of cells measured in population (>95%). Combined with the pump/valve structure, cell-free antibodies and mRNAs can be removed completely for highly accurate detection (R = 0.96) of protein copies. The picoliter reaction chambers allow high detection sensitivity for both mRNA transcripts and protein copies and low sequencing cost. Using multi-Paired-seq, three clusters of known breast cancer cell types are identified according to multimodal measurements, and the expression correlations between mRNAs and proteins under altered conditions are quantified. Multi-Paired-seq provides multimodal measurements at the single-cell level, which offers a new tool for cell biology, developmental biology, drug discovery, and precision medicine.

Chiral Nanoparticles with Enhanced Thermal Stability of Chiral Structures through Alloying.

Metallic chiral nanoparticles (CNPs) promisingly function as asymmetric catalysts but lack an important study in thermal stability of optical activity that stems from metastable chiral lattices. In this work, annealing is applied to silver (Ag) CNPs, fabricated by glancing angle deposition (GLAD), and causes elimination of optical activity at 200 °C, mainly ascribed to chiral-to-achiral lattice transformation. The Ag CNPs are remarkedly enhanced in thermal stability through an alloying with aluminum (Al) via layer-by-layer GLAD to generate binary Ag0.5 Al0.5 CNPs composed of solid-state liquids, whose optical activity vanishes at 700 °C. Ease in the diffusion of Al atoms in the host Ag CNPs and thermal insulation from the Al2 O3 layers partially covering the binary CNPs effectively prohibit structural relaxation of the metastable chiral lattices, accounting for the significant enhancement in thermal stability of chiral lattices. This is a pioneering work to investigate the fundamental principles determining the thermal stability of metallic CNPs in terms of chiral structures and optical activity. It paves the way toward applying metallic CNPs to asymmetric catalysis at high temperature to accelerate an asymmetric synthesis of enantiomers with designable chirality, which is one of the most important topics in modern chemistry.

Single-Cell Sequencing Methodologies: From Transcriptome to Multi-Dimensional Measurement.

Cells are the basic building blocks of biological systems, with inherent unique molecular features and development trajectories. The study of single cells facilitates in-depth understanding of cellular diversity, disease processes, and organization of multicellular organisms. Single-cell RNA sequencing (scRNA-seq) technologies have become essential tools for the interrogation of gene expression patterns and the dynamics of single cells, allowing cellular heterogeneity to be dissected at unprecedented resolution. Nevertheless, measuring at only transcriptome level or 1D is incomplete; the cellular heterogeneity reflects in multiple dimensions, including the genome, epigenome, transcriptome, spatial, and even temporal dimensions. Hence, integrative single cell analysis is highly desired. In addition, the way to interpret sequencing data by virtue of bioinformatic tools also exerts critical roles in revealing differential gene expression. Here, a comprehensive review that summarizes the cutting-edge single-cell transcriptome sequencing methodologies, including scRNA-seq, spatial and temporal transcriptome profiling, multi-omics sequencing and computational methods developed for scRNA-seq data analysis is provided. Finally, the challenges and perspectives of this field are discussed.

Mapping Gene Expression in the Spatial Dimension.

The main function and biological processes of tissues are determined by the combination of gene expression and spatial organization of their cells. RNA sequencing technologies have primarily interrogated gene expression without preserving the native spatial context of cells. However, the emergence of various spatially-resolved transcriptome analysis methods now makes it possible to map the gene expression to specific coordinates within tissues, enabling transcriptional heterogeneity between different regions, and for the localization of specific transcripts and novel spatial markers to be revealed. Hence, spatially-resolved transcriptome analysis technologies have broad utility in research into human disease and developmental biology. Here, recent advances in spatially-resolved transcriptome analysis methods are summarized, including experimental technologies and computational methods. Strengths, challenges, and potential applications of those methods are highlighted, and perspectives in this field are provided.

Narrowband Near-Infrared Perovskite/Polymer Hybrid Photodetectors.

The distinct visible-blind narrowband near-infrared (NIR) photodetection behaviors in the perovskite/polymer hybrid photodetectors (PDs) have been investigated. The narrowband NIR response in the hybrid PDs is realized through the buildup of the space charges at the perovskite/polymer interface. The semiconducting perovskite layer acts as an internal NIR bandpass due to its high absorption to the visible light and high transparency to the NIR light. It also acts as an excellent hole-transporting layer, facilitating the efficient extraction of the holes generated in the low band gap NIR light-absorbing polymer blend layer. The hybrid PDs thus demonstrated have a -3 dB cutoff frequency of 300 kHz, providing an exciting option for a plethora of applications in bioimaging, environmental detection, and security monitoring.

The high efficient catalytic properties for thermal decomposition of ammonium perchlorate using mesoporous ZnCo2O4 rods synthesized by oxalate co-precipitation method.

Mesoporous ZnCo2O4 rods have been successfully prepared via oxalate co-precipitation method without any template. The nano-sized spinel crystallites connected together to form mesoporous structure by annealing homogeneous complex oxalates precursor at a low rate of heating. It is found that the low anneal rate plays an important role for the formation of mesoporous ZnCo2O4 rods. The effects of the heat temperature on the phase, morphology and catalytic properties of the products were studied. The XRD, SEM TEM, and N2 absorption/desorption have been done to obtain compositional and morphological information as well as BET surface area of the as-prepared sample. Catalytic activities of mesoporous ZnCo2O4 rods toward the thermal decomposition of ammonium perchlorate (AP) were investigated with differential scanning calorimetry (DSC) and thermogravimetry (TG) techniques. The results show that the addition of ZnCo2O4 rods to AP dramatically reduces the decomposition temperature. The ZnCo2O4 rods annealed at 250 °C possesses much larger specific area and exhibits excellent catalytic activity (decrease the high decomposition temperature of AP by 162.2 °C). The obtained mesoporous ZnCo2O4 rods are promising as excellent catalyst for the thermal decomposition of AP.