Ziyi He

Single-Cell Analysis Using Drop-on-Demand Inkjet Printing and Probe Electrospray Ionization Mass Spectrometry.

This study describes a novel method for single-cell analysis and lipid profiling by combining drop-on-demand inkjet cell printing and probe electrospray ionization mass spectrometry (PESI-MS). Through inkjet sampling of a cell suspension, droplets with single cells were generated, precisely dripped onto a tungsten-made electrospray ionization needle, and immediately sprayed under a high-voltage electric field. Lipid fingerprints of single cells were obtained by a mass spectrometry (MS) detector. A homemade magnetic stirring device was applied to the cell suspension reservoir, which controlled the homogeneous distribution of cells in liquid and improved the single-cell-droplet percentage by 43.8%. Eight types of single cells were screened in our platform and further differentiated by principal component analysis based on cellular surface phospholipids. Thus, this study successfully provides a facile method for the direct MS profiling of single-cell lipids by PESI-MS.

Engineering Cell-Compatible Paper Chips for Cell Culturing, Drug Screening, and Mass Spectrometric Sensing

Paper-supported cell culture is an unprecedented development for advanced bioassays. This study reports a strategy for in vitro engineering of cell-compatible paper chips that allow for adherent cell culture, quantitative assessment of drug efficiency, and label-free sensing of intracellular molecules via paper spray mass spectrometry. The polycarbonate paper is employed as an excellent alternative bioscaffold for cell distribution, adhesion, and growth, as well as allowing for fluorescence imaging without light scattering. The cell-cultured paper chips are thus amenable to fabricate 3D tissue construction and cocultures by flexible deformation, stacks and assembly by layers of cells. As a result, the successful development of cell-compatible paper chips subsequently offers a uniquely flexible approach for in situ sensing of live cell components by paper spray mass spectrometry, allowing profiling the cellular lipids and quantitative measurement of drug metabolism with minimum sample pretreatment. Consequently, the developed paper chips for adherent cell culture are inexpensive for one-time use, compatible with high throughputs, and amenable to label-free and rapid analysis.

Nephrocyte-neurocyte interaction and cellular metabolic analysis on membrane-integrated microfluidic device

Cell-cell interaction and cell metabolic analysis provide new opportunities for better understanding of critical biochemical processes. Advanced microfluidic technologies enable to create more realistic in vitro microenvironment by spatial and temporal control of cell growth and co-culture. In this work, we design a microfluidic device to achieve the co-culture of PC12 cells and 293 cells, and study in vitro cell-cell interaction via cell metabolic analysis by mass spectrometry. The membrane-integrated microfluidic device was firstly used for cell co-culture, and the cellular metabolite was further investigated by mass spectrometer (MS). Our results showed that the differentiation of PC12 cells could be successfully induced by mNGF and also greatly influenced by the microchannel treatment of fetal bovine serum (FBS) solution. The identification of cell morphology, microtubule-associated protein 2 (MAP-2) expression and viability of differentiated PC12 cells were conducted before 293 cells being introduced into the top microfluidic channels and stimulated to secrete cell metabolism products. The developed microfluidic device is a potentially useful tool for high throughput of cell-cell interaction study.

Gold nanoparticles modified porous silicon chip for SALDI-MS determination of glutathione in cells

The gold nanoparticles (Au NPs) modified porous silicon chip based surface assisted laser desorption/ionization mass spectrometry (SALDI-MS) was developed to capture and analyze glutathione (GSH) in cells. With silver-assisted chemical etching, Ag nanoparticles (Ag NPs) were generated and deposited on the silicon surface and the nanopores were etched on silicon substrate. Then Au NPs were in-situ synthesized on the ridges of silicon nanopores. This Ag-Au NPs modified porous silicon surface could specially capture and enrich thiol compounds through Au-S binding, and it could also function as matrix to assist ionization for SALDI-MS. The silicon chip was array patterned for high throughput SALDI-MS detection. GSH and cysteine could be distinguished without the interference from matrix signals. This approach was successfully applied to preconcentration and detection of GSH in Caco-2 cells. The GSH alterations in cells under drug stimulation were investigated. This invented silicon chip showed great potential for more efficient analysis of small thiol biomarkers in complex biological samples.

Efficient cell capture in an agarose–PDMS hybrid chip for shaped 2D culture under temozolomide stimulation

In this work, hybrid microfluidic devices were fabricated by assembling a polydimethylsiloxane (PDMS) mold with an agarose microarray to realize cell capture and patterning in precisely controlled spatial distribution. Microwells with diameter varying from 15 to 30 μm were formed on the agarose hydrogel surface at 15 μm to 40 μm spacing. Cells were efficiently captured in microwells with nearly 100% occupancy, thus achieving cell manipulation in a semi-quantitative manner. The size of the cell population captured on the microwell array is proportional to the patterning area. Further study revealed that the capture process was mainly regulated by fluid dynamics, where liquid was absorbed by the highly permeable agarose substrate and carried target cells into microwells. Our method spared the complex chemical modification steps, and the agarose substrate promised good biocompatibility. By designing PDMS channels with different geometrical layouts, various cell patterning geometries were easily created. Cell culture models with controllable pattern area and population size were successfully developed for a temozolomide stimulation study for as long as 2 days. This work should benefit the study of cancer developing niche and provide a powerful platform for the direct and continuous observation of cell dynamics under drug stimulation.

A dual-functional microfluidic chip for on-line detection of interleukin-8 based on rolling circle amplification

Interleukin 8 (IL-8), also known as C-X-C motif ligand 8(CXCL8), is a proinflammatory chemokine functioned in neutrophil chemotaxis and activation. And it plays an important role in the process of glioma stem-like cell vascularization in the latest research. Herein, a dual-function microfluidic biosensor based on rolling circle amplification (RCA) was fabricated for cell culture and online IL-8 detection. A microfluidic chip was designed with two high passages connected by the vertical channels. One of the channels with immobilized capture antibody was prepared for IL-8 detection and another channel for cell culture. Immunoassays were achieved by a sandwich structure consisting of antibodies, IL-8, and aptamers. Signal amplification was mainly due to RCA and biotin-streptavidin linkage. The linear range for IL-8 was 7.5 -120pgmL-1 in this assay. Moreover, the developed method was successfully applied to detect the IL-8 in tumor-derived endothelial cells (TDEC) and Human Umbilical Vein Endothelial cells (HUVEC) under chemical hypoxia condition. Semi-quantitative detection of IL-8 consumption in HUVEC cells in low oxygen condition was also achieved. These results were in statistical agreement with those obtained by commercial assay of enzyme-linked immunoassay kit (ELISA). The microfluidic chip based biosensor reported hereby has a large prospect in the basic research and clinical diagnosis of cancer stem cell.

Shear Stress-Enhanced Internalization of Cell Membrane Proteins Indicated by a Hairpin-Type DNA Probe

Shear stress is an important mechanical stimulus that plays a critical role in modulating cell functions. In this study, we investigated the regulating effects of shear stress on the internalization of cell membrane proteins in a microfluidic chip. A hairpin-type DNA probe was developed and indiscriminately anchored to the cell surface, acting as an indicator for the membrane proteins. When cells were exposed to shear stress generated from fluid cell medium containing external proteins, strong fluorescence was emanated from intracellular regions. With intensive investigation, results revealed that shear stress could enhance the specific cell endocytosis pathway and promote membrane protein internalization. This process was indicated by the enhanced intracellular fluorescence, generated from the internalized and mitochondria accumulated DNA probes. This study not only uncovered new cellular mechanotransduction mechanisms but also provided a versatile method that enabled in situ and dynamic indication of cell responses to mechanical stimuli.

Quantitative determination of VEGF165 in cell culture medium by aptamer sandwich based chemiluminescence assay

In this work, we have developed a sensitive and selective chemiluminescence (CL) assay for vascular endothelial growth factor (VEGF165) quantitative detection based on two specific VEGF165 binding aptamers (Apt). VEGF is a predominant biomarker in cancer angiogenesis, and sensitive detection method of VEGF are highly demanded for both academic study and clinical diagnosis of multiple cancers. In our experiment, VEGF165 was captured in a sandwich structure assembled by two binding aptamers, one capture aptamer was immobilized on streptavidin-coated magnetic beads (MBs) and another VEGF-binding aptamer was labeled by biotin for further phosphatase conjunction. After Apt-VEGF-Apt sandwich was formed on MBs surface, alkaline phosphatase (ALP) was modified to the second aptamer to catalyze CL reaction. By applying 4-methoxy-4-(3-phosphatephenyl)-spiro-(1,2-dioxetane-3,2-adamantane) (AMPPD) as CL substrate, strong signal intensity was achieved. VEGF165 content as low as 1ng/mL was detected in standard spiked samples by our assay, and linear range of working curve was confirmed from 1 to 20ng/mL. Then our method was successfully applied for cell culture medium analysis and on-chip hypoxic HepG2-HUVEC co-culture model study with excellent accuracy equal to ELISA Kit. Our developed assay demonstrated an outstanding performance in VEGF165 quantification and may be further extended to clinical testing of important biomarkers as well as probing microchip cell culture model.

An on-chip intestine-liver model for multiple drugs absorption and metabolism behavior simulation

A double-layer microfluidic chip integrated with a hollow fiber (HF) was developed to reconstitute the intestine-liver functionality for studying the absorption and metabolism of combination drugs. Caco-2 cells were inoculated in the HF cavity at the top of the serpentine channel to simulate the intestinal tissue for drug absorption and transport studied, and HepG2 cells, seeded in the bottom chamber, were used to mimic the liver for metabolism-related studies. Genistein and dacarbazine were selected for combination drug therapy and its effects on cell viability, hepatotoxicity, and cell cycle arrest under drug-conditioned culture were investigated. The results suggested that the combined concentration below −100 μg/mL had no significant inhibitory effect on HepG2 cell viability, and therefore HepG2 cells maintained their drug metabolism ability. When the drug concentration was increased above 250 μg/mL, HepG2 cells underwent apoptosis. Detection of metabolites by mass spectrometry proved the effective metabolism in the microchip model. This dynamic, co-culture microchip successfully provided a podium for long-term observation of absorption, transport, and metabolism of combination drugs, and could be an effective in vitro simulation model for further clinical research.