Mapping secretome-mediated interaction between paired neuron-macrophage single cells.

Neuron-immune interaction through secreted factors contributes significantly to the complex microenvironment in the central nervous system that could alter cell functionalities and fates in both physiological and pathological conditions, which remains poorly characterized at the single-cell level. Herein, using a spatially patterned antibody barcode microchip, we realized the mapping of 12 different secretomes, covering cytokines, neurotrophic factors (NFs), and neuron-derived exosomes (NDEs) from high-throughput, paired single cells (≥ 600) simultaneously under normal conditions and an Alzheimer's disease (AD) model induced with amyloid beta protein 1-42 (Aß1-42). We applied the platform to analyze the secretion profiles from paired neuron-macrophage and neuron-microglia single cells with human cell lines. We found that pairwise neuron-macrophage interaction would trigger immune responses and attenuate neuron cells' secretion, while neuron-microglia interaction generally results in opposite outcomes in secretion. When neuron cells are induced with Aß1-42 protein into the AD model, both neuron-macrophage and neuron-microglia interactions lead to increased cytokines and NDEs and decreased NFs. Further analysis of AD patients' serum showed that NDEs were significantly higher in patients' samples than in the control group, validating our observation from the interaction assay. Furthermore, we resolved previously undifferentiated heterogeneity underlying the secretions from single-neuron cells. We found that the NDE and NF secretion was less dependent on the paracrine signaling between one another and that secretions from neuron cells would attenuate after differentiation with Aß1-42. This study demonstrates the mapping of the different secretomes from paired neuron-immune single cells, providing avenues for understanding how neurons and immune cells interact through the complex secretome network.

Automated and parallel microfluidic DNA extraction with integrated pneumatic microvalves/pumps and reusable open-channel columns.

A novel microfluidic DNA extraction protocol based on integrated diaphragm microvalves/pumps and silica-deposited open-channel columns was developed specifically for automated and parallel DNA solid-phase extraction (SPE). The method uses microfluidic chips with a sandwiched structure containing three layers, which are the upper fluidic layer with surface-deposited silica on glass open channels as the extraction phase, the lower actuation layer with valve actuation channels on a glass wafer, and the middle poly(dimethylsiloxane) (PDMS) membrane for reversible bonding of the two glass substrates. These two glass substrates can be reused after thoroughly cleaning and the PDMS membrane can be replaced conveniently, which could effectively decrease the time and cost of chip manufacturing. The normally closed microvalves/pumps were used to automatically control all processes of the on-chip DNA SPE without cross-contamination and leakage, enabling the processing of multiple samples in parallel without changing the microvalve control module. Using the microchip device with integrated microvalves/pumps, automated, programmable, and simultaneous λ-DNA extractions from different samples could be attained, even from complex solutions such as human blood, and the silica-deposited open-channel columns could be reused stably and reliably. Results have demonstrated that most of the eluted λ-DNA was recovered in the second 2 µL of elution buffer with high-purity suitable for successful polymerase chain reaction amplification, making it possible for further integration into microfluidic devices for fully functional and high-throughput genetic analysis.

Comprehensive Evaluation of Dietary Exposure and Health Risk of Polychlorinated Naphthalenes.

Intake from food is considered an important route of human exposure to polychlorinated naphthalenes. To our knowledge, several studies have quantified dietary exposure but only in European countries and measuring only a few of the 75 congeners. In addition, the influence of source diversity on human exposure has seldom been assessed. We analyzed 192 composite food samples composed of 17,280 subsamples from 24 provinces in China to measure the concentrations of polychlorinated naphthalenes and estimate their daily intake and potential health risks on a national scale. The estimated cancer risk was in the range of 6.8 × 10-8 to 4.6 × 10-7. We compared our findings for 75 congeners with reports in the literature that quantified only 12 congeners. We estimate that these 12 congeners contribute only approximately 4% to the total mass daily intake of polychlorinated naphthalenes and 70% to the total toxic equivalent quantity, indicating underestimation of dietary exposure. The contributions of combustion-associated congeners to the total concentrations of polychlorinated naphthalenes were in the range of 31-52%, suggesting that the ongoing unintentional release of these compounds from industrial thermal processes is an important factor in polychlorinated naphthalene contamination and human exposure in China.

Multiplexed profiling of single-cell extracellular vesicles secretion.

Extracellular vesicles (EVs) are important intercellular mediators regulating health and diseases. Conventional methods for EV surface marker profiling, which was based on population measurements, masked the cell-to-cell heterogeneity in the quantity and phenotypes of EV secretion. Herein, by using spatially patterned antibody barcodes, we realized multiplexed profiling of single-cell EV secretion from more than 1,000 single cells simultaneously. Applying this platform to profile human oral squamous cell carcinoma (OSCC) cell lines led to a deep understanding of previously undifferentiated single-cell heterogeneity underlying EV secretion. Notably, we observed that the decrement of certain EV phenotypes (e.g., CD63+EV) was associated with the invasive feature of both OSCC cell lines and primary OSCC cells. We also realized multiplexed detection of EV secretion and cytokines secretion simultaneously from the same single cells to investigate the multidimensional spectrum of cellular communications, from which we resolved tiered functional subgroups with distinct secretion profiles by visualized clustering and principal component analysis. In particular, we found that different cell subgroups dominated EV secretion and cytokine secretion. The technology introduced here enables a comprehensive evaluation of EV secretion heterogeneity at single-cell level, which may become an indispensable tool to complement current single-cell analysis and EV research.

High-Throughput Single-Cell Extracellular Vesicle Secretion Analysis on a Desktop Scanner without Cell Counting.

Single-cell EV (extracellular vesicle) secretion analysis is emerging for a better understanding of non-genetic cellular heterogeneity regulating human health and diseases through intercellular mediators. However, the requirements of expensive and bulky instrumentations hinder its widespread use. Herein, by combining gold nanoparticle-enhanced silver staining and the Poisson distribution, we reported the use of a home-use scanner to realize high-throughput single-cell EV secretion analysis without cell counting. We applied the platform to analyze the secretions of different EV phenotypes with the human oral squamous cell carcinoma cell line and primary cells from patients, which generated single-cell results comparable with those of the immunofluorescence approach. Notably, we also realized the quantification of the number of EVs secreted from every single cell using their respective titration curves obtained from population samples, making it possible to directly compare different EV phonotypes in regard to their secretion number, secretion rate, and so forth. The technology introduced here is simple, easy to operate, and of low cost, which make it a potential, easily accessible, and affordable tool for widespread use in both basic and clinical research.

Single-Cell Secretion Analysis in the Engineered Tumor Microenvironment Reveals Differential Modulation of Macrophage Immune Responses.

It is increasingly recognized that the cellular microenvironment plays critical roles in regulating the fate and physiology of cells. Despite recent advancements in single-cell analysis technologies, engineering and integration of the microenvironment for single-cell analysis platforms remain limited. Here, we report a single-cell cytokine secretion analysis platform that integrated both the three-dimensional cell culture and the primary oral squamous cell carcinoma tumor cell co-culture to provide both physical and physiological cues for single cells to be analyzed. We apply the platform to investigate the immune responses of human macrophages stimulated with the ligand of toll-like receptor 4 lipopolysaccharide. Notably, we observe the differential modulation effect in cytokine secretions by the tumor microenvironment, in which antitumor cytokine TNF-a secretion was attenuated, and protumor cytokine IL-6 would increase. The differential modulation effect is conserved from cell line-derived macrophages to primary macrophages derived from healthy donors. Immunofluorescence staining further reveals that ∼50% of macrophage cells could be polarized from M1 to the M2 phenotype within 12 h in the engineered tumor microenvironment. This work demonstrates the significance of the cell microenvironment toward single-cell analysis, which could help to evaluate how immune cells will respond in the complex microenvironment more accurately.

PDMS Microwell Stencil Based Multiplexed Single-Cell Secretion Analysis.

Multiplexed single-cell protein secretion analysis provides an in-depth understanding of cellular heterogeneity in intercellular communications mediated by secreted proteins in both fundamental and clinical research. However, it has been challenging to increase the proteomic parameters co-profiled from every single cell in a facile way. Herein, a simple method to improve the multiplexed proteomic parameters of PDMS microwell based single-cell secretion analysis platform by sandwiching PDMS stencil in between two antibody-coated glass slides is introduced. Two different antibody panels can be immobilized easily by static coating, without using sophisticated fluid handling or bulky equipment. 5-plexed, 3-fluorescence color single-cell secretion assay is demonstrated with this platform to investigate human monocytic U937 cells in response to lipopolysaccharide and phorbol myristate acetate stimulation, which identified the existence of functional subsets dictated by different cytokine profiles. The technology introduced here is simple, easy to operate, which holds great potential to become a powerful tool for profiling multiplexed single-cell cytokine secretion at high throughput to dissect cellular heterogeneity in secretome signatures.

Extracellular vesicles of carcinoma-associated fibroblasts creates a pre-metastatic niche in the lung through activating fibroblasts.

OBJECTIVES: Carcinoma-associated fibroblasts (CAFs) have been known to promote cancer progression by modifying the primary tumor microenvironment. We aimed to elucidate the intercellular communication between CAFs and secondary organs in salivary adenoid cystic carcinoma (SACC) metastasis. METHODS: Pre-metastatic and metastatic animal models of SACC were established using extracellular vesicles (EVs) from CAFs and SACC cells. Lung fibroblasts (LFs) were treated with EVs and their transcriptomic alterations were identified by RNA sequencing. ITRAQ were performed to analyze EV cargos. TC I-15 was used to inhibit EV uptake by LFs and SACC lung metastasis in vivo. RESULTS: Here, we show that CAF EVs induced lung pre-metastatic niche formation in mice and consequently increased SACC lung metastasis. The pre-metastatic niche induced by CAF EVs was different from that induced by SACC EVs. CAF EVs presented a great ability for matrix remodeling and periostin is a potential biomarker characterizing the CAF EV-induced pre-metastatic niche. We found that lung fibroblast activation promoted by CAF EVs was a critical event at the pre-metastatic niche. Integrin α2ß1 mediated CAF EV uptake by lung fibroblasts, and its blockage by TC I-15 prevented lung pre-metastatic niche formation and subsequent metastasis. Plasma EV integrin ß1 was considerably upregulated in the mice bearing xenografts with high risk of lung metastasis. CONCLUSIONS: We demonstrated that CAF EVs participated in the pre-metastatic niche formation in the lung. Plasma EV integrin ß1 might be a promising biomarker to predict SACC metastasis at an early stage. An integrated strategy targeting both tumor and stromal cells is necessary to prevent SACC metastasis.

A liver-chip-based alcoholic liver disease model featuring multi-non-parenchymal cells.

Non-parenchymal cells play a key role in the occurrence and development of alcoholic liver disease. However, this cellular behaviour has not been fully characterized, and it is inconvenient to observe in traditional in vitro alcoholic liver disease (ALD) models and animal models. Herein we developed a demountable liver-on-chip device for investigation of pathophysiological process of individual non-parenchymal cells in alcohol induced ALD. This liver-device comprised of HepG2, LX-2, EAhy926 and U937 cells, which were ordered in a physiological distribution under perfuse. This device allows improved HepG2 cells activities and maintained high liver functions which including albumin synthesis and urea secretion. This novel liver-device is able to recreate the damage process of hepatic non-parenchymal cell lines induced by alcohol, and to understand the intercellular communication between different types of hepatic cells during ALD by measuring multiple biomarkers of each types of hepatic non-parenchymal cell lines, including Ve-cadherin, eNOS, VEGF and α-SMA. The proposed liver-device is able to further studies of pathological analysis and drug- and toxicity-screening.

Paper-Based 3D Scaffold for Multiplexed Single Cell Secretomic Analysis.

Despite rapid progresses in single-cell analysis technologies, efforts to control the three-dimensional microenvironment for single cell measurements have been lacking. Here, we report a simple method to incorporate three-dimensional scaffolds, including polyvinylidene fluoride (PVDF) membranes and PVDF membrane replicated analog polydimethylsiloxane, into multiplexed single cell secretomic analysis platforms (including a microwell array and a single cell barcode microchip) to mimic the extracellular physical matrix and mechanical support for single cells. Applying this platform to brain tumor cell line U87 to investigate single cell protein secretion behavior on different substrates, we revealed that single cell protein secretions were regulated differently in three-dimensional (3D) microenvironments. This finding was further verified with intracellular cytokine staining, highlighting the significance of 3D single cell microenvironments. This new single cell biomimetic platform can be easily adaptable to other three-dimensional cell culture scaffolds or other single cell assays and may become a broadly applicable three-dimensional single cell analysis system to study the effect of microenvironment conditions on cellular functional heterogeneity in vitro.

High-performance InGaN-based green light-emitting diodes with quaternary InAlGaN/GaN superlattice electron blocking layer.

In this study, high-performance InGaN-based green light-emitting diodes (LEDs) with a quaternary InAlGaN/GaN superlattice electron blocking layer (QSL-EBL) have been demonstrated. The band structural simulation was employed to investigate the electrostatic field and carriers distribution, show that the efficiency and droop behavior can be intensively improved by using a QSL-EBL in LEDs. The QSL-EBL structure can reduce the polarization-related electrostatic fields in the multiple quantum wells (MQWs), leading to a smoother band diagram and a more uniform carriers distribution among the quantum wells under forward bias. In comparison with green LEDs with conventional bulk-EBL structure, the light output power of LEDs with QSL-EBL was greatly enhanced by 53%. The efficiency droop shows only 30% at 100 A/cm2 comparing to its peak value, suggesting that the QSL-EBL LED is promising for future white lighting with high performance.

Manual-slide-engaged paper chip for parallel SERS-immunoassay measurement of clenbuterol from swine hair.

Clenbuterol (CL), as a feed additive, has been banned in many countries due to its potential threat to human health. In detection of CL, a fast, low-cost technique with high accuracy and specificity would be ideal for its administrative on-field inspections. Among the attempts to pursue a reliable detection tool of CL, a technique that combines surface enhanced Raman spectroscopy (SERS) and immunoassay, is close to meet the requirements as above. However, multiple steps of interactions between CL analyte, antibody, and antigen are involved in this method, and under conventional setup, the operation of SERS/immunoassay were unwieldy. In this paper, to facilitate a more manageable sample manipulation for SERS-immunoassay measurement, a 3D paper chip was suggested. A switch-on-chip multilayered (abbreviated as SoCM-) microfluidic paper-based analysis device (µPad) was fabricated to provide operators with manual switches on the interactions between different microfluids. Besides, on a detection slip we made on the main body of our SoCM-µPad, antigen was anchored in pattern. With this architecture, multistep interactions between the CL analyte in swine hair extract and the SERS probe-modified antibody and antigen, were managed for on-chip SERS-immunoassay detection. This would be very attractive for fast, cheap, accurate, and on-site specific detection of CL from real samples.

Picomolar detection of carcinoembryonic antigen in whole blood using microfluidics and surface-enhanced Raman spectroscopy.

Carcinoembryonic antigen (CEA) is a wide-spectrum biomarker. Clinically, we generally use serum sample to detect CEA, which needs to be centrifuged to pretreat the raw blood sample. In this study, we realized direct CEA detection in raw blood samples exploiting microfluidics. The LOD was as low as 10(-12) M.

Easy-to-fabricate thin-film coating on PDMS substrate with super hydrophilicity and stability.

With the fast expansion of microfluidic applications, stable, and easy-to-fabricate PDMS surface coating with super hydrophilicity is highly desirable. In this study, we introduce a new kind of copolymer-based, single-layer thin-film coating for PDMS. The coating can exist in air at room temperature for at least 6 months without any noticeable deterioration in the super hydrophilicity (water contact angle ∼7°), resistance of protein adsorption, or inhibition of the EOF. In addition, this coating enables arbitrary patterning of cells on planar surfaces.

Direct measurement of beta-agonists in swine hair extract in multiplexed mode by surface-enhanced Raman spectroscopy and microfluidic paper.

Bare gold nanoparticles selectively enhance the Raman signal of beta-agnonists in swine hair extract at 780 nm, which enables analysis of beta-agonists in swine hair extract without chemical labeling, purification, or separation. The analysis is multiplexable and the LOD of beta-agonists is around ng/mL in the assistance of microfluidic paper.

Determination of beta-agonists in swine hair by µFIA and chemiluminescence.

ß-Agonists are a group of illegal feed additives. In this paper, it was found that the light emission produced by the oxidation of luminol by potassium ferricyanide was enhanced by the ß-agonists (ractopamine, salbutamol, and terbutaline). Based on chemiluminescence phenomenon, a novel, rapid, and sensitive microflow injection analysis system on a microfluidic glass chip was established for determination of the ß-agonists. The chip was fabricated from two glass plates (64 mm × 32 mm) with microchannels of 200 µm width and 100 µm depth. The detection limits were achieved at 2.0 × 10(-8) mol/L of ractopamine, 1.0 × 10(-8) mol/L of terbutaline and 5.0 × 10(-7) mol/L of salbutamol. In this report, our method was applied for determination of the ß-agonists in swine hair from three different sources with satisfactory results.

Target Analysis of Polychlorinated Naphthalenes and Nontarget Screening of Organic Chemicals in Bovine Milk, Infant Formula, and Adult Milk Powder by High-Resolution Mass Spectrometry.

Infant formula is intended as an effective substitute for breast milk but is the main source of polychlorinated naphthalenes (PCNs) to nonbreastfed infants. We performed target and nontarget analyses to determine PCNs and identify other organic contaminants in infant formula. The mean PCN concentrations in infant formula, milk powder, and bovine milk were 106.1, 88.8, and 78.2 µg kg-1 of dry weight, respectively. The PCN congener profiles indicated that thermal processes and raw materials were probably the main sources of PCNs in infant formula. A health risk assessment indicated that PCNs in infant formula do not pose health risks to infants. Using gas chromatography-Orbitrap mass spectrometry, 352, 372, and 161 organic chemicals were identified in the infant formula, milk powder, and bovine milk samples, respectively. Phthalate esters were detected in all four plastic-packed milk powder samples. The results indicated milk becomes more contaminated with organic chemicals during manufacturing, processing, and packaging.

Characteristics and degradation mechanisms of polychlorinated naphthalenes in surface soil in Yangtze River Delta, China.

Both thermal and environmental processes are significant factors influencing the existing characteristics, e.g., congener distributions, and existing levels, of polychlorinated naphthalenes (PCNs) in the environment. Soil plays an important role in the life cycle of PCNs, but degradation of PCNs in soils has never been reported. In this study, we collected surface soil samples from 13 cities in the Yangtze River Delta, which is one of the most crowded areas of China and analyzed the samples for 75 PCNs. The long-range transportation from polluted areas was the major source for PCNs in remote areas, but the PCN profiles in remote areas reported in our previous studies were different from those in human settlement in this study, indicating there is a transformation of PCNs after emissions from anthropogenic activities. Two experiments were then designed to reveal the degradation mechanisms, including influencing factors, products, and pathways, of PCNs in surface soils. Based on the experiments, we found that the major factor driving the losses of PCNs in surface soils was volatilization, followed by photo irradiation and microbial metabolism. Under photo-irradiation, the PCN structures would be destroyed through a process of dechlorination followed by oxidation. In addition, the dechlorination pathways of PCNs have been established and found to be significantly influenced by the structure-related parameters.

Vortexing-generated high throughput single-cell droplets for facile analysis of multiplexed microRNA dynamic secretion.

Discriminating secretory phenotypes provides a direct, intact, and dynamic way to evaluate the heterogeneity in cell states and activation, which is significant for dissecting non-genetic heterogeneity for human health studies and disease diagnostics. In particular, secreted microRNAs, soluble signaling molecules released by various cells, are increasingly recognized as a critical mediator for cell-cell communication and the circulating biomarkers for disease diagnosis. However, single-cell analysis of secreted miRNAs is still lacking due to the limited available tools. Herein, we realized three-plexed miRNA secretion analysis over four time points from single cells encapsulated in picoliter droplets with extreme simplicity, coupling vortexing-generated single-cell droplets with multiplexed molecular beacons. Notably, our platform only requires pipetting and vortexing steps to finish the assay setup within 5 min with minimal training, and customized software was developed for automatic data quantification. Applying the platform to human cancer cell lines and primary cells revealed previously undifferentiated heterogeneity and paracrine signaling underlying miRNA secretion. This platform can be used to dissect secretion heterogeneity and cell-cell interactions and has the potential to become a widely used tool in biomedical research.

A fast and low-cost spray method for prototyping and depositing surface-enhanced Raman scattering arrays on microfluidic paper based device.

In this study, a fast, low-cost, and facile spray method was proposed. This method deposits highly sensitive surface-enhanced Raman scattering (SERS) silver nanoparticles (AgNPs) on the paper-microfluidic scheme. The procedures for substrate preparation were studied including different strategies to synthesize AgNPs and the optimization of spray cycles. In addition, the morphologies of the different kinds of paper substrates were characterized by SEM and investigated by their SERS signals. The established method was found to be favorable for obtaining good sensitivity and reproducible results. The RSDs of Raman intensity of randomly analyzing 20 spots on the same paper or different filter papers depositing AgNPs are both below 15%. The SERS enhancement factor is approximately 2 × 10(7) . The whole fabrication is very rapid, robust, and does not require specific instruments. Furthermore, the total cost for 1000 pieces of chip is less than $20. These advantages demonstrated the potential for growing SERS applications in the area of environmental monitoring, food safety, and bioanalysis in the future.