Image-activated pico-injection for single-cell analysis.

The addition of reagents into preformed droplets is a crucial yet intricate task in droplet-based applications where sequential reactions is required. Pico-injection offers high throughput and robustness in accomplishing this task, but the existing pico-injection techniques work in an indiscriminate manner, making it difficult to target particular groups of droplets. Here we report image-activated pico-injection (imgPico) for label-free, on-demand reagent supplementation into droplets. The imgPico detects the droplets of interest by real-time image analysis and makes decisions for the downstream pico-injection operation. We studied the performance of different algorithms for the image analysis and optimized the experimental settings of the imgPico. In the validation experiment, the imgPico successfully injected fluorescent dyes into droplets encapsulating one, two, and three cells, respectively, as expected. We further demonstrated the utility of imgPico by targeting droplets encapsulating single cells in droplet-based single-cell RNA sequencing (scRNA-seq) using exceedingly high cell density, and the results showed that the imgPico effectively reduced the presence of doublets in the scRNA-seq data. With the merits of being label-free and versatile, the imgPico represents a technical advance with potential applications in single-cell analysis.

A Robust Oxygen Microbubble Radiosensitizer for Iodine-125 Brachytherapy.

Iodine-125 (125I) brachytherapy, a promising form of radiotherapy, is increasingly applied in the clinical treatment of a wide range of solid tumors. However, the extremely hypoxic microenvironment in solid tumors can cause hypoxia-induced radioresistance to 125I brachytherapy, resulting in therapeutic inefficacy. In this study, the aim is to sensitize hypoxic areas in solid tumors using ultrasound-activated oxygen microbubbles for 125I brachytherapy. A modified emulsion freeze-drying method is developed to prepare microbubbles that can be lyophilized for storage and easily reconstituted in situ before administration. The filling gas of the microbubbles is modified by the addition of sulfur hexafluoride to oxygen such that the obtained O2/SF6 microbubbles (OS MBs) achieve a much longer half-life (>3×) than that of oxygen microbubbles. The OS MBs are tested in nasopharyngeal carcinoma (CNE2) tumor-bearing mice and oxygen delivery by the OS MBs induced by ultrasound irradiation relieve hypoxia instantly. The post-treatment results of brachytherapy combined with the ultrasound-triggered OS MBs show a greatly improved therapeutic efficacy compared with brachytherapy alone, illustrating ultrasound-mediated oxygen delivery with the developed OS MBs as a promising strategy to improve the therapeutic outcome of 125I brachytherapy in hypoxic tumors.

Water-dispersible, biocompatible and fluorescent poly(ethylene glycol)-grafted cellulose nanocrystals.

Fluorescent nanoprobe with good water dispersibility was synthesized by the coupling of fluorescent 1,8-naphthalimide dye (NANI) as well as biocompatible poly (ethylene glycol) (PEG) to cellulose nanocrystals (CNC). FTIR, TGA and XPS analysis confirmed the successful covalent conjugation of NANI and PEG. The rod-like morphology of CNC was generally retained after two-step successive grafting of NANI and PEG. The contact angle and transmittance measurements showed that the grafted PEG brushes improve the hydrophilicity of fluorescent CNC probes and their dispersibility in high-concentration NaCl solutions. The fluorescent CNC probe had good biocompatibility and was successfully used for the bioimaging of Hela cells in physiological environment at high salt concentration. Laser confocal microscopy showed that the fluorescent CNC probe can penetrate the cell membrane and disperse uniformly in the cell with good biocompatibility. The fluorescent CNC probe with nanometer size, strong fluorescence emission and high salt-tolerance possess potential application in biomedical field.

Scalable Production of Monodisperse Functional Microspheres by Multilayer Parallelization of High Aspect Ratio Microfluidic Channels.

Droplet microfluidics enables the generation of highly uniform emulsions with excellent stability, precise control over droplet volume, and morphology, which offer superior platforms over conventional technologies for material synthesis and biological assays. However, it remains a challenge to scale up the production of the microfluidic devices due to their complicated geometry and long-term reliability. In this study, we present a high-throughput droplet generator by parallelization of high aspect ratio rectangular structures, which enables facile and scalable generation of uniform droplets without the need to precisely control external flow conditions. A multilayer device is formed by stacking layer-by-layer of the polydimethylsiloxane (PDMS) replica patterned with parallelized generators. By feeding the sample fluid into the device immersed in the carrying fluid, we used the multilayer device with 1200 parallelized generators to generate monodisperse droplets (~45 µm in diameter with a coefficient of variation <3%) at a frequency of 25 kHz. We demonstrate this approach is versatile for a wide range of materials by synthesis of polyacrylamide hydrogel and Poly (l-lactide-co-glycolide) (PLGA) through water-in-oil (W/O) and oil-in-water (O/W) emulsion templates, respectively. The combined scalability and robustness of such droplet emulsion technology is promising for production of monodisperse functional materials for large-scale applications.

pH-Responsive Oxygen Nanobubbles for Spontaneous Oxygen Delivery in Hypoxic Tumors.

Tumor hypoxia is a significant factor leading to the resistance of tumors to treatment, especially for photodynamic therapy and radiotherapy where oxygen is needed to kill cancer cells. Oxygen delivery agents such as oxygen-saturated perfluorocarbon nanoemulsions and lipid oxygen microbubbles have been employed to supply oxygen to hypoxic tumors with ultrasound activation. Such oxygen delivery systems are still associated with several drawbacks, including premature oxygen release and the dependence of external stimuli. To address these limitations, we developed oxygen nanobubbles that were enclosed by the acetalated dextran polymer shells for spontaneous oxygeneration in response to a minor pH drop in the tumor microenvironment. The acetalated dextran polymer shell serves as a robust barrier against gas dissolution in the circulating blood to retain the majority of the oxygen payload, and its pH-responsive property enables an abrupt burst release of oxygen in the mild acidic tumor microenvironment. The acetalated dextran oxygen nanobubbles exhibited excellent stability and biocompatibility. In vitro and in vivo experiments were conducted to investigate the pH-responsive oxygen release. The external stimuli-free supply of oxygen by the acetalated dextran oxygen nanobubbles was evaluated on CNE2 tumor-bearing mice, and the intratumoral oxygen level increased by 6-fold after the administration of the oxygen nanobubbles, manifesting that our pH-responsive oxygen nanobubbles hold great potential as a potent oxygen delivery agent to overcome the hypoxia-induced resistance.

Lipid-Polymer Bilaminar Oxygen Nanobubbles for Enhanced Photodynamic Therapy of Cancer.

Hypoxia in solid tumors may be a hindrance to effective treatments of tumors in achieving their therapeutic potential, especially for photodynamic therapy (PDT) which requires oxygen as the supplement substrate. Oxygen delivery using perfluorocarbon emulsions or lipid oxygen microbubbles has been developed as the agents to supply endogenous oxygen to fuel singlet oxygen generation in PDT. However, such methods suffer from premature oxygen release and storage issues. To address these limitations, we designed lipid-polymer bilaminar oxygen nanobubbles with chlorin e6 (Ce6) conjugated to the polymer shell as a novel oxygen self-supplement agent for PDT. The resultant nanobubbles possessed excellent stability to reduce the risk of premature oxygen release and were stored as freeze-dried powders to avoid shelf storage issues. In vitro and in vivo experimental results demonstrated that the nanobubbles exhibited much higher cellular uptake rates and tumor targeting efficiency compared to free Ce6. Using the oxygen nanobubbles for PDT, a significant enhancement of therapeutic efficacy and survival rates was achieved on a C6 glioma-bearing mice model with no noticeable side effects, owing to the greatly enhanced singlet oxygen generation powered by oxygen encapsulated nanobubbles.

Controllable Formation of Monodisperse Polymer Microbubbles as Ultrasound Contrast Agents.

Microbubbles have been widely used as ultrasound contrast agents in clinical diagnosis and hold great potential for ultrasound-mediated therapy. However, polydispersed population and short half-life time (<10 min) of the microbubbles still limit their applications in imaging and therapy. To tackle these problems, we develop a microfluidic flow-focusing approach to produce monodisperse microbubbles stabilized by Poly(lactic-co-glycolic acid) (PLGA) as the polymer shell. The size of PLGA microbubbles can be tightly controlled from ∼600 nm to ∼7 µm with a coefficient of variation less than 4% in size distribution for ensuring highly homogeneous echogenic behavior of PLGA polymer microbubbles in ultrasound fields. Both in vitro and in vivo experiments showed that the monodisperse PLGA microbubbles had excellent echogenicity and elongated sonographic duration time (>3 times) for ultrasound imaging in comparison with the commercial lipid microbubbles.

Suppressing Ice Nucleation of Supercooled Condensate with Biphilic Topography.

Preventing or minimizing ice formation in supercooled water is of prominent importance in many infrastructures, transportation, and cooling systems. The overall phase change heat transfer on icephobic surfaces, in general, is intentionally sacrificed to suppress the nucleation of water and ice. However, in a condensation frosting process, inhibiting freezing without compromising the water condensation has been an unsolved challenge. Here we show that this conflict between anti-icing and efficient condensation cooling can be resolved by utilizing biphilic topography with patterned high-contrast wettability. By creating a varying interfacial thermal barrier underneath the supercooled condensate, the biphilic structures tune the nucleation rates of water and ice in the sequential condensation-to-freezing process. Our experimental and theoretical investigation of condensate freezing dynamics further unravels the correlation between the onset of droplet freezing and its characteristic radius, offering a new insight for controlling the multiphase transitions among vapor, water, and ice in supercooled conditions.

Tunable Confinement for Bridging Single-Cell Manipulation and Single-Molecule DNA Linearization.

DNA linearization by nanoconfinement has offered a new avenue toward large-scale genome mapping. The ability to smoothly interface the widely different length scales from cell manipulation to DNA linearization is critical to the development of single-cell genomic mapping or sequencing technologies. Conventional nanochannel technologies for DNA analysis suffer from complex fabrication procedures, DNA stacking at the nanochannel entrance, and inefficient solution exchange. In this work, a dynamic and tunable confinement strategy is developed to manipulate and linearize genomic-length DNA molecules from a single cell. By leveraging pneumatic microvalve control and elastomeric collapse, an array of nanochannels with confining dimension down to 20 nm and length up to sub-millimeter is created and can be dynamically tuned in size. The curved edges of the microvalve form gradual transitions from microscale to nanoscale confinement, smoothly facilitating DNA entry into the nanochannels. A unified micro/nanofluidic device that integrates single-cell trapping and lysis, DNA extraction, purification, labeling, and linearization is developed based on dynamically controllable nanochannels. Mbp-long DNA molecules are extracted directly from a single cell and in situ linearized in the nanochannels. The device provides a facile and promising platform to achieve the ultimate goal of single-cell, single-genome analysis.

High aspect ratio induced spontaneous generation of monodisperse picolitre droplets for digital PCR.

Droplet microfluidics, which involves micrometer-sized emulsion droplets on a microfabricated platform, has been demonstrated as a unique system for many biological and chemical applications. Robust and scalable generation of monodisperse droplets at high throughput is of fundamental importance for droplet microfluidics. Classic designs for droplet generation employ shear fluid dynamics to induce the breakup of droplets in a two-phase flow and the droplet size is sensitive to flow rate fluctuations, often resulting in polydispersity. In this paper, we show spontaneous emulsification by a high aspect ratio (>3.5) rectangular nozzle structure. Due to the confinement and abrupt change of the structure, a Laplace pressure difference is generated between the dispersed and continuous phases, and causes the thread thinning and droplet pinch-off without the need to precisely control external flow conditions. A high-throughput droplet generator was developed by parallelization of a massive number of the basic structures. This device enabled facile and rapid partition of aqueous samples into millions of uniform picolitre droplets in oil. Using this device, on-chip droplet-based digital polymerase chain reaction (PCR) was performed for absolute quantification of rare genes with a wide dynamic range.

Microfluidic production of nanoscale perfluorocarbon droplets as liquid contrast agents for ultrasound imaging.

Liquid perfluorocarbon (PFC) nanodroplets may have a better chance to extravasate through inter-endothelial gaps (400-800 nm) into tumor interstitium for extravascular imaging, which holds promise as an innovative strategy for imaging-guided drug delivery, early diagnosis of cancer and minimally invasive treatment of cancer. Currently available emulsion technologies still face challenges in reducing droplet sizes from the microscale to the nanoscale. To control size and ensure monodispersity of PFC nanodroplets, we developed a flame-shaped glass capillary and polydimethylsiloxane (PDMS) hybrid device that creates a concentric flow of the dispersed phase enclosed by the focusing continuous phase at the cross-junction. Through adjustment of the pressure applied, a stable tip-streaming mode can be obtained for PFC nanodroplet generation. Using this device, we synthesized various kinds of PFC nanodroplets as small as 200 nm in diameter with polydispersity index (PDI) <0.04. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) were carried out for the characterization of the PFC nanodroplets. Finally, ultrasound imaging was conducted to demonstrate that the liquid PFC nanodroplets can be used for enhancing the ultrasound contrast upon vaporization.

The synergy between natural polyphenol-inspired catechol moieties and plant protein-derived bio-adhesive enhances the wet bonding strength.

Novel soybean meal-based biomimetic (STP) adhesives were fabricated via soybean meal (SM) and enhanced by tannic acid (TA) and polyetheylenimine (PEI) (TAPI) co-crosslinking network based on natural polyphenol-inspired chemistry. The multiple physico-chemical interactions (including intermolecular H-bonding and covalent bonding) between the TAPI co-crosslinking system and SM matrices were examined by the Fourier transform infrared spectroscopy, solid-state 13C nuclear magnetic resonance, X-ray diffraction, thermogravimetric analysis, and scanning electron microscopy. The results showed that a dense, robust, and water-resistant adhesive layer was constructed between network-bound catechol moieties in the TAPI and SM system, endowing the STP adhesive with high wet bonding strength for plywood. As expected, TAPI-modified SM adhesives showed a 156.1% increase in wet bonding strength compared to the control SM adhesive. The adhesion meets standard requirements for interior-use plywood. Both the solid content and residual mass analysis also confirmed that the enhancement in the STP adhesive was attributable to the network crosslinking density and stiffness after integrating the TAPI system. Moreover, the thermal stability of the resultant STP adhesive exhibited a significant improvement. The proposed STP adhesive may be a promising cost-effective and wet-resistant bio-adhesive for the application in the wood composites industry.

Development of Eco-friendly Soy Protein Isolate Films with High Mechanical Properties through HNTs, PVA, and PTGE Synergism Effect.

This study was to develop novel soy protein isolate-based films for packaging using halloysite nanotubes (HNTs), poly-vinyl alcohol (PVA), and 1,2,3-propanetriol-diglycidyl-ether (PTGE). The structural, crystallinity, opacity, micromorphology, and thermal stability of the resultant SPI/HNTs/PVA/PTGE film were analyzed by the Attenuated total reflectance-Fourier transformed infrared (ATR-FTIR) spectroscopy, X-ray diffraction (XRD), UV-Vis spectrophotometry, scanning electron microscopy (SEM), and thermo-gravimetric analysis (TGA). The SPI/HNTs/PVA/PTGE film illustrated that HNTs were uniformly dispersed in the SPI matrix and the thermal stability of the film was enhanced. Furthermore, the tensile strength (TS) of the SPI/HNTs/PVA/PTGE film was increased by 329.3% and the elongation at the break (EB) remained unchanged. The water absorption (WA) and the moisture content (MC) were decreased by 5.1% and 10.4%, respectively, compared to the unmodified film. The results highlighted the synergistic effects of SPI, HNTs, PVA, and PTGE on the mechanical properties, water resistance, and thermal stability of SPI films, which showed excellent strength and flexibility. In short, SPI films prepared from HNTs, PVA, and PTGE showed considerable potential as packaging materials.