generation-dynamical-environmental-conditions-using-high-throughput

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Northwest University

The laccase-CuO-nanowire mesocrystal hybrid materials were developed with a superior catalytic activity inspired by natural biocatalysis processes in living cells that highly resemble the metal ions activation and the well-organized spatial structure of the natural rough endoplasmic reticulum. The enzyme and nanobiocatalyst activities of the obtained hybrid material exhibited an approximate 10-fold and 2.2-fold increase than the free enzyme, surpassing the currently available nanobiocatalysts. The comprehensive catalytic performance of the hybrid materials has been further demonstrated using a prototype continuous-flow reactor for the bioremediation of 2,4-dichlorophenol-contaminated water, which showed a high degradation efficiency and remarkable reusability. These new highly efficient nanobiocatalysts are expected to be used for diverse applications in biotechnology, biosensing, and environmental remediation.

Enzyme-Nanowire Mesocrystal Hybrid Materials with an Extremely High Biocatalytic Activity.

Evaluating enzyme activity intracellularly on natural substrates is a significant experimental challenge in biomedical research. We report a label-free method for real-time monitoring of the catalytic behavior of class A, B, and D carbapenemases in live bacteria based on measurement of heat changes. By this means, novel biphasic kinetics for class D OXA-48 with imipenem as substrate is revealed, providing a new approach to detect OXA-48-like producers. This in-cell calorimetry approach offers major advantages in the rapid screening (10 min) of carbapenemase-producing Enterobacteriaceae from 142 clinical bacterial isolates, with superior sensitivity (97 %) and excellent specificity (100 %) compared to conventional methods. As a general, label-free method for the study of living cells, this protocol has potential for application to a wider range and variety of cellular components and physiological processes.

Label-Free Visualization of Carbapenemase Activity in Living Bacteria.

Abnormal rheological phenomena arising in Tris-borate-ethylenediaminetetraacetic acid solutions (believed to be Newtonian fluids) were observed in direct current electroosmotic flows within a nanocapillary with a diameter of 200 nm under a low electric field of tens of volts per meter. In solutions with different concentrations and pH values, the flow behavior indices of the power-law fluids were calculated on the basis of current-voltage relations. When the electric field intensity was below a critical value of 6.7 V/m, the fluids exhibited dilatant (shear thickening) effects. Fluid viscosity changed with electric field intensity because the near-wall shear rate of an electroosmotic flow changes with electric field intensity via a power-law relation. When the electric field intensity surpasses the critical electric field, the fluid again becomes Newtonian and has constant viscosity. The investigation shows that in nanocapillaries, fluids commonly believed to be Newtonian can become non-Newtonian near walls as a result of strong nanoscale interfacial effects. The results can also improve our understanding of electroosmosis-related transport phenomena in nanofluidics and biomedical science.

Abnormal Rheological Phenomena in Newtonian Fluids in Electroosmotic Flows in a Nanocapillary.

Genetic profiling would benefit from linearization of ssDNA through the exposure of the unpaired bases to gene-targeting probes. This is compromised by ssDNA's high flexibility and tendency to form self-annealed structures. Here, we demonstrate that self-annealing can be avoided through controlled coating with a cationic-neutral diblock polypeptide copolymer. Coating does not preclude site-specific binding of fluorescence labeled oligonucleotides. Bottlebrush-coated ssDNA can be linearized by confinement inside a nanochannel or molecular combing. A stretch of 0.32 nm per nucleotide is achieved inside a channel with a cross-section of 100 nm and a 2-fold excess of polypeptide with respect to DNA charge. With combing, the complexes are stretched to a similar extent. Atomic force microscopy of dried complexes on silica revealed that the contour and persistence lengths are close to those of dsDNA in the B-form. Labeling is based on hybridization and not limited by restriction enzymes. Enzyme-free labeling offers new opportunities for the detection of specific sequences.

Linearization and Labeling of Single-Stranded DNA for Optical Sequence Analysis.

Senile plaques are well-known hallmarks of Alzheimer's Diseases (AD). However, drugs targeting tangles of the protein tau and plaques of β-amyloid have no significant effect on disease progression, and the studies on the underlying mechanism of AD remain in high demand. Growing evidence supports the protective role of senile plaques in local inflammation driven by S100A9. We herein demonstrate that oleic acid (OA) micelles interact with hen egg white lysozyme (HEWL) and promote its amyloid formation. Consequently, SH-SY5Y cell line and mouse neural stem cells are rescued from OA toxicity by co-aggregation of OA and HEWL. Using atomic force microscopy in combination with fluorescence microscopy, we revealed that HEWL forms round-shaped aggregates in the presence of OA micelles instead of protofibrils of HEWL alone. These HEWL amyloids act as a sink for toxic OA micelles and their co-aggregate form large clumps, suggesting a protective function in amyloid and OA cytotoxicity.

HEWL interacts with dissipated oleic acid micelles, and decreases oleic acid cytotoxicity.

Dynamical control of cellular microenvironments is highly desirable to study complex processes such as stem cell differentiation and immune signaling. We present an ultra-multiplexed microfluidic system for high-throughput single-cell analysis in precisely defined dynamic signaling environments. Our system delivers combinatorial and time-varying signals to 1500 independently programmable culture chambers in week-long live-cell experiments by performing nearly 10 pipetting steps, where single cells, two-dimensional (2D) populations, or 3D neurospheres are chemically stimulated and tracked. Using our system and statistical analysis, we investigated the signaling landscape of neural stem cell differentiation and discovered "cellular logic rules" that revealed the critical role of signal timing and sequence in cell fate decisions. We find synergistic and antagonistic signal interactions and show that differentiation pathways are highly redundant. Our system allows dissection of hidden aspects of cellular dynamics and enables accelerated biological discovery.

Ultra-multiplexed analysis of single-cell dynamics reveals logic rules in differentiation.

The ultrasonication-triggered interfacial assembly approach was developed to synthesize magnetic Janus amphiphilic nanoparticles (MJANPs) for cancer theranostic applications, where the biocompatible octadecylamine is used as a molecular linker to mediate the interactions between hydrophobic and hydrophilic nanoparticles across the oil-water interface. The obtained Co cluster-embedded FeO nanoparticles-graphene oxide (CCIO-GO) MJANPs exhibited superior magnetic heating efficiency and transverse relaxivity, 64 and 4 times higher than that of commercial superparamagnetic iron oxides, respectively. The methodology has been applicable to nanoparticles of various dimensions (5-100 nm), morphologies (sphere, ring, disk, and rod), and composition (metal oxides, noble metal and semiconductor compounds, etc.), thereby greatly enriching the array of MJANPs. In vivo theranostic applications using the tumor-bearing mice model further demonstrated the effectiveness of these MJANPs in high-resolution multimodality imaging and high-efficiency cancer therapeutics. The ubiquitous assembly approach developed in the current study pave the way for on-demand design of high-performance Janus amphiphilic nanoparticles for various clinical diagnoses and therapeutic applications.

Ultrasonication-Triggered Ubiquitous Assembly of Magnetic Janus Amphiphilic Nanoparticles in Cancer Theranostic Applications.

Given the great threat posed by clinical Carbapenemase-producing Enterobacteriaceae (CPE), its rapid and accurate detection is highly required to guide infection treatment and to prevent serious outbreaks. While the Carba NP test (CNPt) is a popular and methodologically simple colorimetric assay for detecting carbapenemase activity, its clinical application is limited by low throughput, poor sensitivity and obscure color interpretation due to the lack of quantitative information on time- and dose-dependent color changes. To address these limitations, we developed a multiplexed microfluidic device (1280 chambers) that is able to automatically generate 16 bacterial concentration gradients and uses green channel intensity to extract quantitative color changes as a function of time. Here we examined the dynamic color changing profiles of 3 reference and 8 clinical bacteria isolates. The results demonstrated linear correlations between color developments and time (within 0-120 min) or bacterial doses (within OD of 0.8-8.0), enabling accurate and reproducible detection of CPE with over 10 fold improved sensitivity for an NDM-producer (LOD of 2.4 × 10 cells) and over 10 fold improved sensitivity for an OXA-48-producer (LOD of 7.2 × 10 cells) as compared to the conventional CNPt method (LOD above 1.3 × 10 cells). As the first digital microfluidic platform for CPE, this on-chip CNPt compared favourably with the conventional bench-based method by significantly improved sensitivity, high throughput, low consumption and automatization, favouring its potential clinical applications for rapid screening of CPE with a low cost. Furthermore, the results also revealed dynamic signature associated with bacteria, which may hold promise for a new approach to study divergent bacterial responses to antibiotic treatments.

On-chip Carba NP test for accurate and high throughput detection of carbapenemase-producing Enterobacteriaceae.

Known for their capabilities in automated fluid manipulation, microfluidic devices integrated with pneumatic valves are broadly used for researches in life science and clinical practice. The application is, however, hindered by the high cost and overly complex fabrication procedure. Here, we present an approach for fabricating molds of active fluidic devices using a benchtop 3D printer and a simple 2-step protocol (i.e. 3D printing and polishing). The entire workflow can be completed within 6 h, costing less than US$ 5 to produce all necessary templates for PDMS replica molding, which have smooth surface and round-shaped pneumatic valve structures. Moreover, 3D printing can create unique bespoke on-off objects of a wide range of dimensions. The millimeter- and centimeter-sized features allow examination of large-scale biological samples. Our results demonstrate that the 3D-printed active fluidic device has valve control capacities on par with those made by photolithography. Controlled nutrients and ligands delivery by on-off active valves allows generation of dynamic signals mimicking the ever-changing environmental stimuli, and combinatorial/sequential drug inputs for therapeutic screening on liver tumor spheroid. We believe that the proposed methodology can pave the way for integration of active fluidic systems in research labs, clinical settings and even household appliances for a broad range of application.

Active fluidic chip produced using 3D-printing for combinatorial therapeutic screening on liver tumor spheroid.

In this study, we report the design and fabrication of a novel fluidic mixer. As proof-of-concept, the laminar flow in the main channel is firstly filled with small air-bubbles, which act as active stirrers inducing chaotic convective turbulent flow, and thus enhance the solutes mixing even at a low input flow rate. To further increase mixing efficiency, a design of neck constriction is included, which changes the relative positions of the inclusion bubbles significantly. The redistribution of liquid volume among bubbles then causes complex flow profile, which further enhances mixing. This work demonstrates a unique approach of utilizing air bubbles to facilitate mixing in bulk solution, which can find the potential applications in microfluidics, fast medical analysis, and biochemical synthesis.

Air-Bubble Induced Mixing: A Fluidic Mixer Chip.

The transition of amyloidogenic species into ordered structures (i.e., prefibrillar oligomers, protofibrils, mature fibrils, and amyloidogenic aggregates) is closely associated with many neurodegenerative disease pathologies. It is increasingly appreciated that the liquid-solid interface contributes to peptide aggregation under physiological conditions. However, much remains to be explored on the molecular mechanism of surface-directed amyloid formation. We herein demonstrate that physical environmental conditions (i.e., negatively charged surface) affect amyloid formation. Nontoxic amyloid aggregates quickly develop into intertwisting fibrils on a negatively charged mica surface. These fibrillar structures show significant cytotoxicity on both neuroblastoma cell-lines (SH-SY5Y) and primary neural stem cells. Our results suggest an alternative amyloid development pathway, following which Aβ peptides form large amyloidogenic aggregates upon stimulation, and later transit into neurotoxic fibrillar structures while being trapped and aligned by a negatively charged surface. Conceivably, the interplay between chemical and physical environmental conditions plays important roles in the development of neurodegenerative diseases.

Surface-Directed Structural Transition of Amyloidogenic Aggregates and the Resulting Neurotoxicity.

Inflammation is the primary pathological feature of neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease. Proinflammatory molecules (e.g., S100A9) play important roles during the progression of the diseases by regulating behavior and fate of multiple cell types in the nervous system. Our earlier studies reveal that S100A9 is toxic to neurons, and its interaction with Aβ peptides leads to the formation of large nontoxic amyloidogenic aggregates, suggesting a protective role of coaggregation with Aβ amyloids. We herein demonstrate that S100A9 interacts with neural stem cells (NSCs) and causes NSC differentiation. In the brain of transgenic AD mouse models, we found large quantities of proinflammatory S100A9, which colocalizes with the differentiated NSCs. NSC sphere formation, which is a representative character of NSC stemness, is also substantially inhibited by S100A9. These results suggest that S100A9 is a representative marker for the inflammatory conditions in AD, and it promotes NSC differentiation. Intriguingly, in contrast to the death of both stem and differentiated NSCs caused by high S100A9 doses, S100A9 at a moderate concentration is toxic only to the early differentiated NSCs but not the stem cells. We therefore postulate that, at the early stage of AD, the expression of S100A9 leads to NSC differentiation, which remedies the neuron damage. The application of drugs, which help maintain NSC stemness (e.g., the platelet-derived growth factor, PDGF), may help overcome the acute inflammatory conditions and improve the efficacy of NSC transplantation therapy.

Proinflammatory S100A9 Regulates Differentiation and Aggregation of Neural Stem Cells.

Mitochondria-targeting nanotherapy receives great attention these days for its capacity in disrupting mitochondria function and inducing tumor cell apoptosis through external magnetic and optical stimulations. However, the effect is significantly diminished when applied to animal models. The key factors include environmental complexity in vivo and intrinsic protective features of tumor tissues. To address these obstacles and reduce expenses on drug screening, we herein introduce a methodology for producing millimeter-sized spheroids with structural and functional characteristics of tumor tissues in vivo. The necessity of spheroid as a liver tumor model is demonstrated by comparing the effect of TPP-SPIONs (triphenylphosphonium cation-superparamagnetic iron oxide nanoparticles) on monolayer-cultured HepG2 cells and spheroids. Our study reveals that large-scale spheroid, in contrast to monolayer cells, reflects more in vivo tumor characters and is less responsive to TPP-SPIONs during magnetic hyperthermia treatment.

Liver Tumor Spheroid Reconstitution for Testing Mitochondrial Targeted Magnetic Hyperthermia Treatment.

Lipin 1 is an intracellular protein acting as a phosphatidic acid phosphohydrolase enzyme controlling lipid metabolism. Human recessive mutations in  cause recurrent, early-onset myoglobinuria, a condition normally associated with muscle pain and weakness. Whether and how lipin 1 deficiency in humans leads to peripheral neuropathy is yet unclear. Herein, two novel compound heterozygous mutations in  with neurological disorders, but no myoglobinuria were identified in an adult-onset syndromic myasthenia family. The present study sought to explore the pathogenic mechanism of  in muscular and neural development.  The clinical diagnosis of the proband was compared to the known 48 cases of  recessive homozygous mutations. Whole-exome sequencing was carried out on the syndromic myasthenia family to identify the causative gene. The pathogenesis of lipin 1 deficiency during somitogenesis and neurogenesis was investigated using the zebrafish model. Whole-mount  hybridization, immunohistochemistry, birefringence analysis, touch-evoke escape response and locomotion assays were performed to observe  the changes in muscles and neurons. The conservatism of the molecular pathways regulated by lipin 1 was evaluated in human primary glioblastoma and mouse myoblast cells by siRNA knockdown, drug treatment, qRT-PCR and Western blotting analysis.  The patient exhibited adult-onset myasthenia accompanied by muscle fiber atrophy and nerve demyelination without myoglobinuria. Two novel heterozygous mutations, c.2047A>C (p.I683L) and c.2201G>A (p.R734Q) in , were identified in the family and predicted to alter the tertiary structure of LPIN1 protein. Lipin 1 deficiency in zebrafish embryos generated by  morpholino knockdown or human  mutant mRNA injections reproduced the myotomes defects, a reduction both in primary motor neurons and secondary motor neurons projections, morphological changes of post-synaptic clusters of acetylcholine receptors, and myelination defects, which led to reduced touch-evoked response and abnormalities of swimming behaviors. Loss of lipin 1 function in zebrafish and mammalian cells also exhibited altered expression levels of muscle and neuron markers, as well as abnormally enhanced Notch signaling, which was partially rescued by the specific Notch pathway inhibitor DAPT.  These findings pointed out that the compound heterozygous mutations in human  caused adult-onset syndromic myasthenia with peripheral neuropathy. Moreover, zebrafish could be used to model the neuromuscular phenotypes due to the lipin 1 deficiency, where a novel pathological role of over-activated Notch signaling was discovered and further confirmed in mammalian cell lines.

Article	Year
Enzyme-Nanowire Mesocrystal Hybrid Materials with an Extremely High Biocatalytic Activity. Nano letters\| PubMed ID: 30136854	2018
Label-Free Visualization of Carbapenemase Activity in Living Bacteria. Angewandte Chemie (International ed. in English)\| PubMed ID: 30378725	2018
Abnormal Rheological Phenomena in Newtonian Fluids in Electroosmotic Flows in a Nanocapillary. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 30418030	2018
Linearization and Labeling of Single-Stranded DNA for Optical Sequence Analysis. The journal of physical chemistry letters\| PubMed ID: 30615463	2019
HEWL interacts with dissipated oleic acid micelles, and decreases oleic acid cytotoxicity. PloS one\| PubMed ID: 30794655	2019
Ultra-multiplexed analysis of single-cell dynamics reveals logic rules in differentiation. Science advances\| PubMed ID: 30949582	2019
Ultrasonication-Triggered Ubiquitous Assembly of Magnetic Janus Amphiphilic Nanoparticles in Cancer Theranostic Applications. Nano letters\| PubMed ID: 31140281	2019
On-chip Carba NP test for accurate and high throughput detection of carbapenemase-producing Enterobacteriaceae. Talanta\| PubMed ID: 31987207	2020
Active fluidic chip produced using 3D-printing for combinatorial therapeutic screening on liver tumor spheroid. Biosensors & bioelectronics\| PubMed ID: 31999576	2020
Air-Bubble Induced Mixing: A Fluidic Mixer Chip. Micromachines\| PubMed ID: 32074944	2020
Surface-Directed Structural Transition of Amyloidogenic Aggregates and the Resulting Neurotoxicity. ACS omega\| PubMed ID: 32095707	2020
Proinflammatory S100A9 Regulates Differentiation and Aggregation of Neural Stem Cells. ACS chemical neuroscience\| PubMed ID: 33079539	2020
Liver Tumor Spheroid Reconstitution for Testing Mitochondrial Targeted Magnetic Hyperthermia Treatment. ACS biomaterials science & engineering\| PubMed ID: 33405636	2019
Lipin 1 deficiency causes adult-onset myasthenia with motor neuron dysfunction in humans and neuromuscular junction defects in zebrafish. Theranostics\| PubMed ID: 33456573	2021

Ce Zhang

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