OsciDrop: A Versatile Deterministic Droplet Generator.

This paper describes OsciDrop, a versatile chip-free droplet generator used to produce size-tunable droplets on demand. Droplet generation is fundamental to miniaturized analysis. We designed OsciDrop to segment the fluid flowing out of the orifice of a disposable pipette tip into droplets by oscillating its distal end underneath an immiscible continuous phase. We described the theoretical model and investigated the effect of flow rate, oscillating amplitude, frequency, and waveform on droplet generation. Our study revealed a previously underexplored Weber number-dominated regime that leverages inertial force instead of viscous force to generate droplets. The same pipette tip allowed robust and deterministic generation of monodisperse droplets with programmable sizes ranging from 200 pL to 2 µL by asymmetrical oscillation. We validated this platform with two droplet-based nucleic acid amplification tests: a digital loop-mediated isothermal amplification assay for absolute quantification of African swine fever virus and a multi-volume digital polymerase chain reaction assay for the high dynamic range measurement of human genomic DNA. The OsciDrop method opens a facile avenue to miniaturization, integration, and automation, exhibiting full accessibility for digital molecular diagnostics.

Aliphatic organoimido derivatives of polyoxometalates containing a bioactive ligand.

A series of aliphatic organoimido derivatives of hexamolybdate based on amantadine, namely (nBu4 N)2 [Mo6 O18 (NC10 H15 )] (1), (nBu4 N)2 {cis-[Mo6 O17 (NC10 H15 )2 ]} (2), (nBu4 N)2 {trans-[Mo6 O17 (NC10 H15 )2 ]} (3), and (nBu4 N)2 [Mo6 O16 (NC10 H15 )3 ] (4), was synthesized in reasonable yield by dehydration with N,N'-dicyclohexylcarbodiimide (DCC). They were characterized by IR and UV/Vis spectroscopy, elemental analysis, ESI mass spectrometry, and single-crystal X-ray structure analysis. The spectral and structural similarities and differences between monosubstituted, cis-disubstituted, and trans-disubstituted organoimido derivatives were elucidated and may provide guidance for related work on organoimido-functionalized Lindqvist-type polyoxometalates. In addition, trans-disubstituted and polysubstituted derivatives containing aliphatic organoimido ligands have not yet been reported, and the crystal structure of the trans isomer may lead us to a deeper understanding of disubstituted derivatives. Furthermore, proliferation and morphology of MCF-7 cells were studied with compound 1. The present results show that the DCC-dehydrating protocol could be an efficient approach to covalently graft bioactive ligands such as amantadine onto POMs and enhance their application in clinical cancer treatment.

Wearable Microfluidic Sweat Chip for Detection of Sweat Glucose and pH in Long-Distance Running Exercise.

Traditional exercise training monitoring is based on invasive blood testing methods. As sweat can reveal abundant blood-related physiological information about health, wearable sweat sensors have received significant research attention and become increasingly popular in the field of exercise training monitoring. However, most of these sensors are used to measure physical indicators such as heart rate, blood pressure, respiration, etc., demanding a versatile sensor that can detect relevant biochemical indicators in body fluids. In this work, we proposed a wearable microfluidic sweat chip combined with smartphone image processing to realize non-invasive in situ analysis of epidermal sweat for sports practitioners. The polydimethylsiloxane (PDMS) based chip was modified with nonionic surfactants to ensure good hydrophilicity for the automatic collection of sweat. Besides, a simple, reliable, and low-cost paper-based sensor was prepared for high-performance sensing of glucose concentration and pH in sweat. Under optimized conditions, this proposed chip can detect glucose with low concentrations from 0.05 mM to 0.40 mM, with a pH range of 4.0 to 6.5 for human sweat. The ability of this microfluidic chip for human sweat analysis was demonstrated by dynamically tracking the changes in glucose concentration and pH in long-distance running subjects.

Alleviative Effects of Exercise on Bone Remodeling in Fluorosis Mice.

Fluorine is widely present in nature in the form of fluoride. Prolonged high-dose fluoride exposure can cause skeletal fluorosis, resulting in osteosclerosis, osteoporosis or osteomalacia. It has been proved that exercise is one of the important factors affecting the health of the bone and promoting bone formation. To investigate the effects of exercise on bone remodeling in fluorosis mice, 120 male 3-week-old ICR mice were randomly divided into four groups: control group (C), exercise group (E), fluoride group (F), fluoride plus exercise group (F + E). After 8-week physical exercise and/or fluoride exposure, we evaluated the content of fluorine, the histopathological structure and microstructure of femur, bone metabolism biochemical indexes and oxidative stress related parameters, and the mRNA and protein levels of genes in BMP-2/Smads and OPG/RANKL/RANK signaling pathways. Our results showed that 100 mg/L NaF exposure increased the accumulation of fluoride in bone, altered histology of bone, and enhanced the activities of ALP and TRACP. Meanwhile, excessive fluoride induced oxidative stress in bone tissue by increasing the content of ROS and MDA, and decreasing the activities of antioxidant enzymes. In addition, the results of qRT-PCR suggested that NaF significantly increased the mRNA expression of BMP-2, Smad-5, Col IA1, Col IA2, OPG, RANKL and RANK, as well as the elevated proteins of OPG, RANKL and RANK. However, these fluoride-induced changes were alleviated after moderate exercise. Taken together, these findings indicated that moderate exercise decreased the toxicity of fluoride by reducing the accumulation of fluorine in the body to relieve the bone damage caused by fluorosis.

Study of Chitosan Ingestion Remitting the Bone Damage on Fluorosis Mice with Micro-CT.

Chronic excessive fluoride exposure may lead to fluorosis, which causes health problems like a decrease in bone mechanical strength. It was speculated that chitosan may combine with fluorine to form in vivo organic fluorine, and may reduce the damage caused by fluorine. Hence, it is necessary to conduct a study to investigate the influence of chitosan on fluorosis mice. To investigate this problem, forty-four 4-week-old male Kunming mice were randomly divided into four groups, the control group, the fluoride group, the fluoride plus chitosan group, and the chitosan group. After 100 days of feeding, the femurs were collected to scan the Micro-CT image. The ultimate load of the femur in the fluoride group was significantly lower than control group. The trabecular separation was increased in the fluoride group compared with the fluoride plus chitosan group and the chitosan group. The level of trabecular thickness was increased in the fluoride plus chitosan group compared with the fluoride group. Our findings suggest that chitosan ingestion can improve the condition of cancellous bone and cortical bone affected by fluorine.

Exercise Ameliorates Fluoride-induced Anxiety- and Depression-like Behavior in Mice: Role of GABA.

Fluoride exposure caused anxiety- and depression-like behavior in mice. Meanwhile, exercise contributes to relieve anxiety and depression. However, the effects of exercise on anxiety- and depression-like behavior in fluorosis mice remain unclear. In the current study, thirty-six Institute of Cancer Research (ICR) female mice were randomly assigned to four groups: control group (C, gavage with distilled water); exercise group (E, gavage with distilled water and treadmill exercise (speed, 10 m/min; time, 30 min/day)); fluoride group (F, gavage with 24 mg/kg sodium fluoride (NaF)); and exercise plus fluoride group (EF, gavage with 24 mg/kg NaF and treadmill exercise). All treatments lasted for 8 weeks. A number of entries into and time spent in the open zone in the elevated zero maze (EZM), resting time in the tail suspension test (TST) and levels of serotonin (5-HT) and gamma-aminobutyric acid (GABA), were significantly altered in F when compared to C. Meanwhile, the anxiety-like behavior in the EZM and the depression-like behavior in the TST were significantly improved in EF when compared to group F. Exercise significantly enhanced fluoride-induced low GABA level, with less effect on the concentration of 5-HT. Moreover, the mRNA and protein expressions of GABA synthesis and transport-related proteins of glutamic acid decarboxylase (GAD) 65 and GAD67 and vesicular GABA transporter (VGAT) were all strikingly decreased in F, while those in EF were increased. In conclusion, exercise ameliorates anxiety- and depression-like behavior in fluorosis mice through increasing the expressions of GABA synthesis and transport-related proteins, rather than 5-HT system.

Recent Progress in Wearable Biosensors: From Healthcare Monitoring to Sports Analytics.

Recent advances in lab-on-a-chip technology establish solid foundations for wearable biosensors. These newly emerging wearable biosensors are capable of non-invasive, continuous monitoring by miniaturization of electronics and integration with microfluidics. The advent of flexible electronics, biochemical sensors, soft microfluidics, and pain-free microneedles have created new generations of wearable biosensors that explore brand-new avenues to interface with the human epidermis for monitoring physiological status. However, these devices are relatively underexplored for sports monitoring and analytics, which may be largely facilitated by the recent emergence of wearable biosensors characterized by real-time, non-invasive, and non-irritating sensing capacities. Here, we present a systematic review of wearable biosensing technologies with a focus on materials and fabrication strategies, sampling modalities, sensing modalities, as well as key analytes and wearable biosensing platforms for healthcare and sports monitoring with an emphasis on sweat and interstitial fluid biosensing. This review concludes with a summary of unresolved challenges and opportunities for future researchers interested in these technologies. With an in-depth understanding of the state-of-the-art wearable biosensing technologies, wearable biosensors for sports analytics would have a significant impact on the rapidly growing field-microfluidics for biosensing.

Microfluidics cell sample preparation for analysis: Advances in efficient cell enrichment and precise single cell capture.

Single cell analysis has received increasing attention recently in both academia and clinics, and there is an urgent need for effective upstream cell sample preparation. Two extremely challenging tasks in cell sample preparation-high-efficiency cell enrichment and precise single cell capture-have now entered into an era full of exciting technological advances, which are mostly enabled by microfluidics. In this review, we summarize the category of technologies that provide new solutions and creative insights into the two tasks of cell manipulation, with a focus on the latest development in the recent five years by highlighting the representative works. By doing so, we aim both to outline the framework and to showcase example applications of each task. In most cases for cell enrichment, we take circulating tumor cells (CTCs) as the target cells because of their research and clinical importance in cancer. For single cell capture, we review related technologies for many kinds of target cells because the technologies are supposed to be more universal to all cells rather than CTCs. Most of the mentioned technologies can be used for both cell enrichment and precise single cell capture. Each technology has its own advantages and specific challenges, which provide opportunities for researchers in their own area. Overall, these technologies have shown great promise and now evolve into real clinical applications.

High-throughput in situ cell electroporation microsystem for parallel delivery of single guide RNAs into mammalian cells.

Arrayed genetic screens mediated by the CRISPR/Cas9 technology with single guide RNA (sgRNA) libraries demand a high-throughput platform capable of transfecting diverse cell types at a high efficiency in a genome-wide scale for detection and analysis of sophisticated cellular phenotypes. Here we developed a high-throughput in situ cell electroporation (HiCEP) microsystem which leveraged the superhydrophobic feature of the microwell array to achieve individually controlled conditions in each microwell and coupled an interdigital electrode array chip with the microwells in a modular-based scheme for highly efficient delivery of exogenous molecules into cells. Two plasmids encoding enhanced green and red fluorescent proteins (EGFP and ERFP), respectively, were successfully electroporated into attached HeLa cells on a 169-microwell array chip with transfection efficiencies of 71.6 ± 11.4% and 62.9 ± 2.7%, and a cell viability above 95%. We also successfully conducted selective electroporation of sgRNA into 293T cells expressing the Cas9 nuclease in a high-throughput manner and observed the four-fold increase of the GFP intensities due to the repair of the protein coding sequences mediated by the CRISPR/Cas9 system. This study proved that this HiCEP system has the great potential to be used for arrayed functional screens with genome-wide CRISPR libraries on hard-to-transfect cells in the future.

Label-free and real-time monitoring of single cell attachment on template-stripped plasmonic nano-holes.

Leveraging microfluidics and nano-plasmonics, we present in this paper a new method employing a micro-nano-device that is capable of monitoring the dynamic cell-substrate attachment process at single cell level in real time without labeling. The micro-nano-device essentially has a gold thin film as the substrate perforated with periodic, near-cm2-area, template-stripped nano-holes, which generate plasmonic extraordinary optical transmission (EOT) with a high sensitivity to refractive index changes at the metal-dielectric interface. Using this device, we successfully demonstrated label-free and real-time monitoring of the dynamic cell attachment process for single mouse embryonic stem cell (C3H10) and human tumor cell (HeLa) by collecting EOT spectrum data during 3-hour on-chip culture. We further collected the EOT spectral shift data at the start and end points of measurement during 3-hour on-chip culture for 50 C3H10 and 50 HeLa cells, respectively. The experiment results show that the single cell attachment process of both HeLa and C3H10 cells follow the logistic retarded growth model, but with different kinetic parameters. Variations in spectral shift during the same culture period across single cells present new evidence for cell heterogeneity. The micro-nano-device provides a new, label-free, real-time, and sensitive, platform to investigate the cell adhesion kinetics at single cell level.

Degradable Organically-Derivatized Polyoxometalate with Enhanced Activity against Glioblastoma Cell Line.

High efficacy and low toxicity are critical for cancer treatment. Polyoxometalates (POMs) have been reported as potential candidates for cancer therapy. On accounts of the slow clearance of POMs, leading to long-term toxicity, the clinical application of POMs in cancer treatment is restricted. To address this problem, a degradable organoimido derivative of hexamolybdate is developed by modifying it with a cleavable organic group, leading to its degradation. Of note, this derivative exhibits favourable pharmacodynamics towards human malignant glioma cell (U251), the ability to penetrate across blood brain barrier and low toxicity towards rat pheochromocytoma cell (PC12). This line of research develops an effective POM-based agent for glioblastoma inhibition and will pave a new way to construct degradable anticancer agents for clinical cancer therapy.

High-throughput superhydrophobic microwell arrays for investigating multifactorial stem cell niches.

Understanding the complex regulatory network that determines stem cell fates requires a high-throughput platform that can generate a large number of precisely controlled microenvironments representing multiple factors for stem cell culture and analysis. Here, we developed a superhydrophobic microwell array chip on which the culture conditions in each microwell can be spontaneously isolated by a grafted layer of superhydrophobic polymers. Simple steps for medium exchange were developed to facilitate the on-chip culture of both adherent and non-adherent cells for up to six days without compromising cell viability and functionality. The culture conditions in each microwell were facilely manipulated using a robotic spotter. Stem cell niches combining soluble factors, extracellular matrices and microtopographic cues were generated on a single 512-well SMARchip and their combinatorial effects on the fate of mouse Oct4-EGFP iPSCs were systematically probed. We observed significant differences in iPSC pluripotency and proliferation between adherent flat and suspended spherical cultures on our platform, which might provide insights into improvement of stem cell technologies.