Encapsulation of single cells on a microfluidic device integrating droplet generation with fluorescence-activated droplet sorting

Encapsulation of single cells is a challenging task in droplet microfluidics due to the random compartmentalization of cells dictated by Poisson statistics. In this paper, a microfluidic device was developed to improve the single-cell encapsulation rate by integrating droplet generation with fluorescence-activated droplet sorting. After cells were loaded into aqueous droplets by hydrodynamic focusing, an on-flight fluorescence-activated sorting process was conducted to isolate droplets containing one cell. Encapsulation of fluorescent polystyrene beads was investigated to evaluate the developed method. A single-bead encapsulation rate of more than 98 % was achieved under the optimized conditions. Application to encapsulate single HeLa cells was further demonstrated with a single-cell encapsulation rate of 94.1 %, which is about 200 % higher than those obtained by random compartmentalization. We expect this new method to provide a useful platform for encapsulating single cells, facilitating the development of high-throughput cell-based assays.     

Antitumor Effect of Embryonic Stem Cells in a Non-Small Cell Lung Cancer Model: Antitumor Factors and Immune Responses

Research in recent years has revealed that embryonic stem cells (ESCs) could generate obvious antitumor effects in both vitro and vivo. In vitro, ESCs could secrete soluble factors that are capable of blocking cancer cells proliferation, moreover, embryonic microenvironments could effectively inhibit tumorigenesis and metastasis; while in vivo, administration of ESCs in tumor-bearing mice could generate significant antitumor effects by indirectly activating the antitumor immune system. In this study, non-small cell lung cancer cells (Lewis Lung Carcinoma cells, LLCs) and ESCs were co-injected together into mice, after that subcutaneous tumor growth was monitored, cellular and humoral immune responses were detected, and different control groups were set to compare the results in different conditions. Our results suggested that compared to be injected alone, ESCs co-injected with cancer cells could inhibit cancer cell growth more efficiently in vivo, with more CD8+ lymphocytes generated in both peripheral circulation and spleen, and with higher serum anticancer cytokine level (interleukin (IL)-2 and interferon (IFN)-γ). We conclude that the boosted antitumor effects induced by ESCs and cancer cells co-injection may be both the effects of antitumor factors secreted by ESCs and immune responses induced by ESCs in vivo.     

Lentivirus-based RNA Silencing of Nemo-like Kinase (NLK) Inhibits the CAL 27 Human Adenosquamos Carcinoma Cells Proliferation and Blocks G0/G1 Phase to S Phase

Background: The Nemo-like kinase (NLK) is a serine/threonine-protein kinase that involved in a number of signaling pathways regulating cell fate. Variation of NLK has been shown to be associated with the risk of cancer. However, the function of NLK in oral adenosquamous carcinoma cells line CAL-27 is unknown.Methods: In this study, we evaluated the function of NLK in CAL-27 cells by using lentivirus-mediated RNA silence. The targeted gene expression, cell proliferation and cell cycle are investigated by RT-PCR, western-blot, MTT method, colony forming assay and flow cytometry analysis respectively.Results: After NLK silencing, the number of colonies was significantly reduced (54±5 colonies/well compared with 262±18 colonies/well in non-infected or 226±4 colonies/well in negative control group (sequence not related to NLK sequence with mismatched bases). Using crystal violet staining, we also found that the cell number per colony was dramatically reduced. The RNA silencing of NLK blocks the G0/G1 phase to S phase progression during the cell cycle.Conclusions: These results suggest that NLK silencing by lentivirus-mediated RNA interference would be a potential therapeutic method to control oral squamous carcinoma growth.     

A novel strategy to alleviate medium acidosis for simultaneously yielding more bacterial cellulose and electricity

Bacterial cellulose (BC), a fascinating and renewable polymer, can be applied widely in various bio-based materials. However, its synthesis is generally limited by medium acidosis. Herein, we demonstrated a built-in galvanic cell within the BC fermentation medium to alleviate the acidosis, by which BC yield was promoted by 191%, and simultaneously the yield of electrical power of 0.68 W to 8.10 W during the incubation.

Bacterial cellulose (BC), a fascinating and renewable polymer, can be applied widely in various bio-based materials.

Bacterial cellulose (BC) is biologically synthesized by several bacteria, notably, the strain of Acetobacter xylinum.¹ This fascinating and renewable polymer possesses remarkable chemical and physical attributes (e.g. high porosity, excellent mechanical strength, and large surface area, etc.) that can be applied in a wide range of bio-based materials and products.^2–4 Its wide applications have motivated researchers to focus on the fermentation process, especially to promote the BC yield. However, the fermentation process is extensively related to many aspects, such as the employed strains, medium conditions (carbon source, nutrients, pH, and dissolved oxygen), and incubation conditions (the employed medium volume, duration, surface area).^5–8Among these factors, the medium acidosis caused by the released organic acids, such as glucuronic acid and acetic acid, in the medium will negatively affect the BC biosynthesis during bacterial metabolism, which could weaken the bacteria activities and reduce the BC yield as a consequence. Generally, the pH of the BC fermentation medium can be dramatically decreased from 5.0 to lower than 2.0 during the incubation if no pH adjustment strategy was employed.^5,9,10 Current methods for BC incubation include static, agitated, and bioreactor cultures.¹¹ For agitated and bioreactor cultures, the regulation of pH is relatively easy because of the culture system is under stirring. However, the static culture of BC is still an ideal way to obtain high-quality BC membranes for many novel applications.^12–14 Unfortunately, it is quite difficult to adjust the pH of medium during the incubation because the formation of BC film in static culture will be seriously disturbed by the mechanical agitation. Therefore, to find an ingenious way to control the medium pH in a suitable range will be very critical to the BC production in practice.The essence of medium acidosis sources from the ionized hydrogen ions (H⁺) of the released organic acids in bacterial metabolic activity. If there is a proper approach to remove the H⁺ from culture medium in a controllable way, the BC production may significantly be promoted. Nowadays, the application of batteries has greatly facilitated people''s life. This revolutionary device was originated from the prototype of “galvanic cell” (or “voltaic piles”), which was invented by Alessandro Volta in 1800.¹⁵ Even now, the galvanic cell still be employed to demonstrate the mechanism of battery for the educational aim, such as the fruit battery.^16,17 Using lemons, oranges, grapefruits, potatoes, or apples which riches in citric acid, phosphoric acid, or malic acid as the electrolyte, and two different metallic electrodes were plugged into the fruit to form a battery. The fruit battery could provide continuous electricity to run various small devices, such as light-emitting diode (LED) and a digital clock. When the two metallic electrodes was connected with those devices by wire, the ionized H⁺ of organic acids inside of the fruit will be slowly consumed in anode and producing electric energy to drive those devices.^18–20 Inspired by the principle of a galvanic cell, we assumed that the ionized H⁺ of accumulated organic acids during the BC fermentation can be consumed in the anode of the built-in galvanic cell to form H₂, which will greatly weaken the acidified conditions of culture medium. Theoretically, a suitable pH condition for bacteria synthesis will promote the BC yield in a consequent. Meanwhile, the liberated metal ions from the cathode can serve as a suitable activator to the biochemical process of bacteria if the electrodes can be selected approximately, which also can potentially promote the BC yield.²¹ Based on this conception, the BC fermentation integrated a couple of electrodes to construct a galvanic cell system as its schematic diagram in Fig. 1 (the details of this built-in galvanic cell see ESI, page S2^†). To check the possibility of this first attempt, the BC yield of this newly constructed system (GC-medium) was compared with that of the normal medium, and the performance of electricity release was investigated as well.Open in a separate windowFig. 1Schematic diagram of the fermentation apparatus and the data acquisition system.The medium pH was monitored at every 12 h during static incubation as shown in Fig. 2.²² It could be easily found that the pH of the normal medium displayed a continuous decrease as the fermentation time was prolonged to 144 h. By contrast, the pH in the GC-medium firstly increased to 4.6 after 24 h of incubation, then maintained in the range from 4.2 to 4.4. Obviously, the medium pH for BC fermentation can be stabilized via loading the galvanic cell to consume the extra acids from the medium process.Open in a separate windowFig. 2Changes of medium pH during the incubation. Roman numerals of (I), (II), and (III) and Arabic numerals of (1) and (2) represent the stage of pH changes during the incubation in GC-medium and normal medium, respectively.Correspondingly, the BC yield from the GC-medium was 0.358 g L⁻¹, which was 2.9-fold higher than that of the normal medium (Fig. 3). Apparently, the pH adjustment by the galvanic cell can work well to yield more BC, verifying the possibility of the proposed conception. Besides, the residual glucose in the GC-medium was lower than that of the normal medium, as well as the residual glucuronic acid. These results again proved that more carbon source was converted into BC. When the BC film started to form at the air–liquid interface, the bacteria would be embedded into the film and fixed, which can result in a clearer medium. The relatively lower OD₆₁₀ in the GC-medium indicated that the more bacteria were fixed result from the thicker BC film that produced in GC-medium.²³ Moreover, the relatively higher DO (dissolved oxygen) was remained in the GC-medium after 6 days incubation also demonstrated that the conditions for cellulose biosynthesis of bacteria were better than that of the normal medium,²⁴ which also suggested more sugar will be consumed for BC synthesis once the galvanic cell was built during the fermentation process.Open in a separate windowFig. 3Medium conditions after 6 days incubation. BC referred to the yield of bacterial cellulose (g L⁻¹); OD₆₁₀ is the optical density of medium at 610 nm; DO is dissolved oxygen (mg L⁻¹); RG is residual glucose (g L⁻¹); GA is glucuronic acid (g L⁻¹). Analysis of variance (ANOVA): ***p < 0.001, * 0.01 < p < 0.05.The formed current with 2 Ω-load of the built-in galvanic cell can be detected by the digital multi-meter. As shown in Fig. 4a, the current decreased sharply from 2013 μA to 878 μA in the first 2 h, this result was related to the rapid consumption of the H⁺ that contained in the strain when the built-in galvanic cell was activated. When the bacteria started to self-replicate in the first 72 h, more organic acid was produced and released to the medium (refer to Fig. 2), which slowed down the rate of current reduction in the following 22 h. The prominent increase of current appeared in 24–72 h, proving that the medium was still rich in H⁺, meanwhile, the number of bacteria grew exponentially. These results also could be observed in Fig. 2, the changes of pH were well matched with the adjustment of the built-in galvanic cell. This correlativity between the medium pH and the formed current of GC has the potential to be designed as a fully automatic pH control system (ESI, Fig. S1^†).Open in a separate windowFig. 4Electricity generation performance during the incubation. (a) Current monitoring with 2 Ω resistor; (b) cumulative energy output in the whole BC fermentative production.Afterward, the current was maintained around 1163 μA during the following 3 days, which can demonstrate the amount of Acetobacter xylinum in GC-medium reach a stable period. Meanwhile, the pH of GC-medium was still within a suitable range of 4.2–4.4 while the pH of the normal medium has been reduced to around 2.7–2.8, which was already negative to the bacterial metabolic activities (Fig. 2). Generally, the death period of bacteria was mainly attributed to the medium acidosis.¹⁰ Hence, the mechanism of improving the BC yield by this built-in galvanic cell was to prevent the medium acidosis and maintained the activity of bacteria, which can stabilize the BC biosynthesis successfully.The cumulative energy output was calculated and showed in Fig. 4b, although the initial pH of GC-medium was relatively lower than that after 48 h, the energy output had a remarkable promotion. This result proved the major factors for the energy output was not only affected by the apparent medium pH but also related to the ability to provide H⁺ within bacteria metabolism. This ability was mainly contributed by the number of bacteria and the bacterial activity, which would be in a better condition if there was a built-in galvanic cell. Previous studies have suggested that the H⁺ concentration in the electrolyte, metal type of electrode, and the number of galvanic cells to be assembled in series are the key factors that influenced the output voltage of the galvanic cell.¹⁷ In this study, the magnesium (Mg) anode, copper (Cu) cathode, and the H⁺-rich culture medium formed an integrated galvanic cell system. The voltage was determined as 1.55–1.65 V for this built-in galvanic cell during the BC production, which can light the LED bulb (ESI, Fig. S2^†). Of course, it also can provide enough electricity to drive digital clocks, sensors, and other low-power devices in the whole incubation time. These results substantially proved that the conception of this work to produce electricity and promote BC production can be achieved via loading the built-in galvanic cell.The schematic mechanism diagram of acidification, pH adjustment, and electricity generation of the galvanic cell were illustrated with Fig. 5, the metabolic by-products, such as citric acid (CA), acetic acid (AC), gluconic acid (GLCA), and pyruvic acid (PA), are generally derived from the tricarboxylic acid (TCA) cycle, pentose phosphate pathway (HMP) and glycolytic pathway (EMP).²⁵ At the beginning of fermentation, glucose (GLC) in the medium was high enough to support the cell self-replication through the aerobic respiration of bacteria in the TCA cycle, which results in an amount of GLCA released to the medium. In this process, the by-products, such as CO₂, CA, and H₂O, also could be generated correspondingly. In addition, a considerable amount of AC and some PA can be released from the HMP and EMP, respectively.²⁴ These by-products, including GLCA, AC, CA, and PA or CO₂, were mainly responsible for medium acidosis, which inhibited bacteria activity and reduced the BC production as a consequence. Besides, the produced GLCA would inhibit the Acetobacter xylinum biosynthesis for BC.²⁶ The dissolved CO₂ in the medium also limited the aerobic respiration of bacteria, which will make the bacteria produce more CA and PA as a feedback regulation. This could create a vicious circle to deteriorate the medium pH and cease in BC production eventually. However, when the galvanic cell was built inside of the culture medium, those organic acids could provide the ionized H⁺ to maintain the galvanic reaction. The H⁺ would move to the Cu-electrode and be reduced as hydrogen gas by the acceptance of the electrons. The Mg-electrode would lose electrons and be oxidized to Mg²⁺, which can involve several cell life activities and potentially promote the BC yield (the effect of Mg²⁺ on BC production shown in ESI, Fig. S3^†). During this process, the directional transfer of electrons in the external circuit created the current, and the electric energy will be continuously output throughout the fermentation. It can image that this built-in galvanic cell can yield considerable power once a large-scale BC production was considered. Moreover, the conception of built-in galvanic cell in bioconversion processes that need pH control, such as anaerobic digestion of easy-acidification substrates, and ethanol fermentation by yeast can also be considered to apply this conception to maintain the stable and suitable pH.Open in a separate windowFig. 5The schematic mechanism diagram of acidification, pH adjustment, and electricity generation of the galvanic cell. GLC (glucose), GLCA (gluconic acid), CA (citric acid), AC (acetic acid), PA (pyruvic acid), GLC6P (glucose-6-phosphate), GLC1P (glucose-1-phosphate), UDPG (uridine diphosphoglucose), HMP (pentose phosphate pathway), FRU6P (fructose-6-phosphate), GAP (glyceraldehyde-3-phosphate), G3P (3-phosphoglycerate), PEP (phosphoenolpyruvate), PYR (pyruvate), ATP (adenosine triphosphate).     

Modulation for efficiency and spectra of non-doped white organic light emitting diodes by combining an exciplex with an ultrathin phosphorescent emitter

Herein, structured non-doped white organic light-emitting diodes (WOLEDs) were designed by combining the emission of a blue exciplex and orange-red phosphorescent ultrathin layer. The device efficiency and spectra were modulated successfully by adjusting the thickness of the exciplex layer and ultrathin layer, respectively. Meanwhile, high efficiency with external quantum efficiency (EQE) ranging from 15% to 22%, power efficiency from 33 lm W⁻¹ to 47 lm W⁻¹ and warm white emission with correlated color temperature (CCT) from 1600 K to 2600 K were realized. The energy transfer process and emission mechanism is also discussed, and the results reveal that the efficient charge trapping and recombination contribute to the improvement of device efficiency and reduce the roll-off efficiency.

Non-doped WOLED with modulated efficiency and spectra were designed by combining blue exciplex with orange-red phosphorescent ultrathin layer.     

Morphology,structural, thermal and rheological properties of wheat starch–palmitic acid complexes prepared during steam cooking

This work aimed to determine the changes in the morphology, complexation degree, the structural, thermal, and rheological properties of starch–fatty acid complexes during steam cooking. In this study, wheat starch with certain water and palmitic acid contents were steamed for 0.5, 1, 1.5, 2, and 2.5 h. The complexing index (CI) first decreased and then progressively increased with the prolonging of steam cooking time. The decrease in CI was associated with the decomposition of the complex layer formed on the granule surface at 0.5 h of steam cooking. The interaction between wheat starch and palmitic acid led to the change of starch crystal type. Prolonging treatment time promoted thermal stability and structural order degree. The type I and IIa complexes reached saturation and fatty acids in the interstitial space between helices increased with excessive treatment times. Rheological behavior analysis showed that the viscoelasticity and deformation degree of samples decreased and increased, respectively, with increasing steam cooking time. Results showed that the thermostability and order degree of the complex layer were lower than those of samples with long treatment times and complexing was effective during steam cooking.

The present paper introduces the formation and characteristics of wheat starch–palmitic acid complexes during long-term steam cooking.     

Dynamic fluorescence lifetime sensing with CMOS single-photon avalanche diode arrays and deep learning processors

Measuring fluorescence lifetimes of fast-moving cells or particles have broad applications in biomedical sciences. This paper presents a dynamic fluorescence lifetime sensing (DFLS) system based on the time-correlated single-photon counting (TCSPC) principle. It integrates a CMOS 192 × 128 single-photon avalanche diode (SPAD) array, offering an enormous photon-counting throughput without pile-up effects. We also proposed a quantized convolutional neural network (QCNN) algorithm and designed a field-programmable gate array embedded processor for fluorescence lifetime determinations. The processor uses a simple architecture, showing unparallel advantages in accuracy, analysis speed, and power consumption. It can resolve fluorescence lifetimes against disturbing noise. We evaluated the DFLS system using fluorescence dyes and fluorophore-tagged microspheres. The system can effectively measure fluorescence lifetimes within a single exposure period of the SPAD sensor, paving the way for portable time-resolved devices and shows potential in various applications.